HK1171378A

HK1171378A - Targeting pax2 for the treatment of breast cancer

Info

Publication number: HK1171378A
Application number: HK12112223.0A
Authority: HK
Inventors: 卡尔顿．D．唐纳德
Original assignee: 菲吉尼克斯公司
Priority date: 2009-08-24
Filing date: 2010-02-19
Publication date: 2013-03-28

Abstract

The present application provides methods of prevention and/or treatment of breast cancer in a subject by inhibiting expression of PAX2. In the certain embodiments, the method of inhibiting expression of PAX2 is to administrate the subject a nucleic acid encoding an siRNA for PAX2. A method of treating cancer in a subject by administering DEFB1 or by increasing expression of DEFB1 is also provided.

Description

Targeting PAX2 for treatment of breast cancer

This application claims priority to U.S. patent application No. 12/708,294, filed on 18/2/2010, and U.S. patent application No. 12/546,292, filed on 24/8/2009.

Background

Breast cancer is the most common cause of cancer in women, and is the second most common cause of cancer death in women in the united states. Although most primary breast cancers are diagnosed based on the finding of breast imaging abnormalities, changes in mass or breast tissue firmness may also be a warning signal for disease. Increased awareness of breast cancer risk over the past few decades has led to an increase in the number of women undergoing mammography screening, resulting in earlier detection of cancer and thus increased survival rates. Nevertheless, breast cancer is the most common cause of death in women between the ages of 45 and 55.

Many types of cancer are known to be caused by genetic aberrations, i.e., mutations. The accumulation of mutations and impaired cell control function lead to progressive phenotypic changes from normal tissues to early pre-cancerous, such as intraepithelial neoplasia (IEN) to increasingly severe IEN to superficial cancers and finally to invasive disease. This process typically occurs relatively slowly over a few years or even a decade, although in some cases it may be relatively rapid. Oncogene dependence is the physiological dependence of cancer cells on the continuous activation or overexpression of a single oncogene that maintains a malignant phenotype. This dependence occurs in the context of other changes that mark the progress of the tumor.

The long-term progression of invasive cancer provides opportunities for clinical intervention. Therefore, it is important to identify biomarkers indicative of pre-cancerous disease so that therapeutic measures can be taken to prevent or delay the progression of invasive cancer.

Summary of The Invention

One aspect of the present invention relates to a method of preventing or treating a breast disorder in an individual. The methods comprise administering to a breast tissue of the subject a composition that inhibits expression of PAX2 or activity of PAX 2.

In one embodiment, the breast disease is breast cancer or breast intraepithelial neoplasia (MIN).

In another embodiment, the inhibiting expression of PAX2 comprises administering to breast cancer tissue or MIN tissue of the individual a nucleic acid encoding a PAX2 siRNA.

In related embodiments, the siRNA comprises a sequence selected from the group consisting of: SEQ ID NO: 3-6 and 11-15.

In another embodiment, the composition comprises an oligonucleotide that inhibits binding of PAX2 to the DEFB1 promoter.

In related embodiments, the oligonucleotide comprises SEQ ID NO: 1.

in related embodiments, the oligonucleotide comprises an X1GGAACX2 sequence, wherein X1 and X2 are nucleotides that are identical to SEQ ID NO: 1 from 0 to 30 nucleotides complementary to the adjacent nucleotides of 1.

In related embodiments, the oligonucleotide comprises a sequence selected from the group consisting of: SEQ ID NO: 18-21, 25, 26, 28 and 29.

In another embodiment, the composition comprises a blocker of the RAS signaling pathway.

In another embodiment, the composition comprises an antagonist selected from the group consisting of: antagonists of angiotensin II, antagonists of angiotensin II receptors, antagonists of Angiotensin Converting Enzyme (ACE), antagonists of mitogen-activated protein kinase (MEK), antagonists of ERK1, 2 (extracellular signal-regulated kinase), and antagonists of signal transducer and activator of transcription 3(STAT 3).

Also disclosed are methods of treating breast cancer or MIN in an individual, comprising enhancing DEFB1 expression in breast cancer tissue or MIN tissue in an individual.

In one embodiment, enhancing DEFB1 expression comprises administering an effective amount of DEFB1 to breast cancer tissue or MIN tissue of an individual.

In another embodiment, enhancing DEFB1 expression comprises administering to breast cancer tissue or MIN tissue of an individual an effective amount of an expression vector encoding DEFB 1.

Also disclosed is a method for treating a breast disease in an individual, the method comprising (a) determining the expression rate of PAX 2-to-DEFB 1 in diseased breast tissue from the individual; (b) determining the ER/PR status of the diseased breast tissue from the individual; and (c) based on the results of (a) and (b), administering to the breast tissue of the subject a composition that (1) inhibits PAX2 expression or PAX2 activity, (2) expresses DEFB1, or (3) inhibits PAX2 expression or PAX2 activity and expresses DEFB 1.

Brief description of the drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the invention and together with the description, serve to explain the principles of the invention. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like elements of an embodiment.

FIGS. 1A to 1D show quantitative RT-PCR (QRT-PCR) analysis of β -defensin-1 (DEFB1) expression.

Fig. 2 shows the microscopic analysis of DEFB1 induced changes in membrane integrity and cell morphology. Black arrows indicate membrane fluctuations and white arrows indicate apoptotic bodies.

Fig. 3 shows a cytotoxicity assay of DEFB1 in prostate cancer cells. Prostate cell lines DU145, PC3 and LNCaP were treated with PonA to induce DEFB1 expression for 1-3 days, after which MTT assays were performed to determine cell viability. The results are expressed as mean ± standard deviation, n ═ 9.

Fig. 4A and 4B show that DEFB1 induces cell death of DU145 and PC3 cells.

FIG. 5 shows a pan-caspase (pan-caspase) assay after DEFB1 induction.

FIG. 6 shows silencing of partner cassette homolog 2(PAX2) protein expression following PAX2siRNA treatment.

Figure 7 presents an analysis of prostate cancer cell growth after treatment with PAX2 siRNA.

FIG. 8 presents an analysis of cell death following siRNA silencing of PAX 2. The results are expressed as mean ± standard deviation, n ═ 9.

FIG. 9 shows an analysis of caspase activity.

FIG. 10 presents an analysis of the apoptotic factors following PAX2siRNA treatment.

FIG. 11 shows a model for the binding of PAX2 to DNA recognition sequences.

Fig. 12 shows the DEFB1 reporter construct.

FIG. 13 shows that inhibition of PAX2 results in DEFB1 expression.

FIG. 14 shows that inhibition of PAX2 results in enhanced DEFB1 promoter activity.

Fig. 15 shows that DEFB1 expression causes a compromise in membrane integrity.

Figure 16 shows that PAX2 inhibition results in compromised membrane integrity.

FIGS. 17A and 17B show ChIP analysis of PAX2 binding to DEFB1 promoter. In FIG. 17A, lane 1 contains 100bp molecular weight markers. Lane 2 is a positive control representing the 160bp region of DEFB1 promoter amplified from DU145 prior to the crosslinking and immunoprecipitation reactions. Lane 3 is a negative control, representing PCR performed without DNA. Lanes 4 and 5 are negative controls representing PCR with IgG immunoprecipitation from cross-linked DU145 and PC3, respectively. PCR amplification of 25pg DNA (lanes 6 and 8) and 50pg DNA (lanes 7 and 9) immunoprecipitated with anti-PAX 2 antibody after cross-linking showed 160bp promoter fragments in DU145 and PC3, respectively. In FIG. 17B, lane 1 contains 100bp molecular weight markers. Lane 2 is a positive control representing the 160bp region of DEFB1 promoter amplified from DU145 prior to the crosslinking and immunoprecipitation reactions. Lane 3 is a negative control, representing PCR performed without DNA. Lanes 4 and 5 are negative controls representing PCR with IgG immunoprecipitation from cross-linked DU145 and PC3, respectively. PCR amplification of 25pg DNA (lanes 6 and 8) and 50pg DNA (lanes 7 and 9) immunoprecipitated with anti-PAX 2 antibody after cross-linking showed 160bp promoter fragments in DU145 and PC3, respectively.

FIG. 18 shows the predicted structures of PrdPD and PrdHD with DNA.

FIG. 19 shows a comparison of consensus sequences for different paired domains. At the top of the figure is shown a schematic representation of the proteins ± DNA contacts described in the crystallographic analysis of the Prd-pairing-domain ± DNA complex. Open boxes indicate alpha-helices, shaded boxes indicate beta-sheets, and bold lines indicate beta-turns. The amino acids contacted are given by the one letter code. Only the amino acids ± bases in direct contact are given. Empty circles indicate major sulcus contact, while red arrows indicate minor sulcus contact. The schematic is an alignment of all known consensus sequences of the paired domain proteins (only the sense strand is given). The vertical lines between consensus sequences indicate conserved base pairs. The position number is given at the bottom of the figure.

Figure 20 shows targeting PAX2 as a chemopreventive strategy.

Figure 21 shows the effect of angiotensin ii (ang ii) on PAX2 expression in DU145 cells.

Figure 22A shows the effect of Losartan (Losartan, Los) on PAX2 expression in DU145 cells.

FIG. 23 shows the effect of Los blockade of AngII on the expression of PAX2 in DU 145.

Figure 24 shows that AngII increases DU145 cell proliferation.

Fig. 25A, 25B and 25C show the effect of Los and MAP kinase inhibitors on PAX2 expression in DU145 cells. FIG. 25A shows that treatment of DU145 cells with losartan suppressed phospho-ERK 1/2 and PAX2 expression; figure 25B shows MEK kinase inhibitors and AICAR blocking PAX2 protein expression; figure 25C shows MEK kinase inhibitor and losartan suppressed phospho-STAT 3 protein expression.

FIGS. 26A and 26B show the effect of Los and MEK kinase inhibitors on PAX2 activation in DU145 cells.

Fig. 27 shows that AngII increases expression of PAX2 and decreases expression of DEFB1 in hPrEC cells.

Figure 28 gives a schematic of AngII signaling and PAX2 prostate cancer.

Figure 29 presents a schematic of blocking PAX2 expression as a prostate cancer therapy.

Fig. 30 gives a comparison of DEFB1 and PAX2 expression with Gleason scores.

FIGS. 31A and 31B show the ratio of PAX2-DEFB1 as predictors of prostate cancer progression.

FIG. 32 shows that the Donald Predictor (DPF) is based on relative expression rates of PAX2-DEFB 1.

FIGS. 33A and 33B present an analysis of hBD-1 expression in human prostate tissue.

FIGS. 34A and 34B present an analysis of hBD-1 expression in prostate cell lines. FIG. 34A shows hBD-1 expression levels in prostate cancer cell lines before and after hBD-1 induction compared to hPrEC cells. One asterisk represents statistically higher expression levels compared to hPrEC. The two asterisks represent statistically significant expression levels compared to the cell lines prior to hBD-1 induction (student t-test, p < 0.05). FIG. 34B shows the verification of abnormal hBD-1 expression in the prostate cancer cell line DU145 by immunocytochemistry. hBD-1 was stained with hPrEC cells as positive controls (A: DIC and B: fluorescence). DU145 cells were transfected with hBD-1 and induced for 18 hours (C: DIC and D: fluorescence). Scale bar 20 μ M.

FIG. 35 shows cytotoxicity assays for hBD-1 in prostate cancer cells. Each column represents the mean ± sem of three independent experiments performed in triplicate.

FIGS. 36A and 36B show QRT-PCR analysis of hBD-1 and cMYC expression in sections of normal, PIN and tumor LCM human prostate tissue. The expression of each gene was expressed as the expression rate compared to β -actin. FIG. 36A shows a comparison of hBD-1 expression levels in normal, PIN and tumor sections. Figure 36B gives a comparison of cMYC expression levels in normal, PIN and tumor sections.

FIG. 37 presents QRT-PCR analysis of hBD1 expression following knockdown of PAX2 with siRNA. hBD-1 expression levels are expressed as expression rates compared to β -actin. Asterisks represent statistically higher expression levels compared to cell lines before PAX2siRNA treatment (student t-test, p < 0.05).

FIGS. 38A and 38B show silencing of PAX2 protein expression following PAX2siRNA treatment. FIG. 38A shows detection of PAX2 expression in HPrEC prostate primary cells (lane 1) and DU145 (lane 2), PC3 (lane 3) and LNCaP (lane 4) prostate cancer cells by Western blot analysis. The blot was cleared and β -actin as an internal control was re-probed to ensure equal loading. Figure 38B presents Western blot analysis of all DU145, PC3 and LNCaP demonstrating knock-down of PAX2 expression after transfection with PAX2siRNA duplexes. Likewise, the blot was cleared and β -actin as an internal control was re-probed.

Figure 39 presents an analysis of prostate cancer cell growth after treatment with PAX2 siRNA. Scale bar 20 μm.

FIG. 40 presents an analysis of cell death following siRNA silencing of PAX 2. The results are expressed as mean ± standard deviation, n ═ 9.

FIG. 41 shows an analysis of caspase activity. Scale bar 20 μm.

Fig. 42A to 42C give an analysis of apoptotic factors after PAX2siRNA treatment. The results are expressed as mean ± standard deviation, n ═ 9. Asterisks indicate statistical differences (p < 0.05).

Detailed Description

One aspect of the invention provides a method of preventing or treating breast cancer in an individual. The method comprises administering to the subject a composition comprising an inhibitor of PAX2 expression or PAX2 activity, or an enhancer of DEFB-1 expression or DEFB-1 activity. In one embodiment, the individual is diagnosed with breast intraepithelial neoplasia (MIN).

In certain aspects, PAX2 of breast tissue is upregulated prior to MIN. Thus, the present application also provides methods of treating or preventing MIN in an individual. The method comprises administering to the subject a composition comprising an inhibitor of PAX2 expression or PAX2 activity, or an enhancer of DEFB-1 expression or DEFB-1 activity.

Protein "activity" includes, for example, transcription, translation, intracellular translocation, secretion, phosphorylation by kinases, cleavage by proteases, homophilic and heterophilic binding to other proteins, ubiquitination. In certain aspects, "PAX 2 activity" specifically refers to the binding of PAX2 to the DEFB-1 promoter.

Breast cancer

Methods commonly used for breast cancer screening include self and clinical breast examinations, x-ray mammography, and breast Magnetic Resonance Imaging (MRI). The latest technology for breast cancer screening is ultrasound computed tomography, which uses sound waves to form three-dimensional images and breast cancer to detect breast cancer without the dangerous radiation used in x-ray mammography. Genetic testing may also be used. Genetic testing for breast cancer typically involves testing for mutations in the BRCA gene. This is generally not a recommended technique except for those patients at increased risk for breast cancer.

The incidence of breast cancer, the leading cause of female mortality, has increased over the last thirty years in the united states. Although the pathogenesis of breast cancer is unclear, the transformation of normal mammary epithelial cells into a malignant phenotype may be the result of genetic factors, especially in women under 30 years of age. Recently, the discovery and characterization of BRCA1 and BRCA2 has expanded our understanding of genetic factors that promote familial breast cancer. Germ line mutations within these two loci are associated with a lifetime risk of 50-85% of breast and/or ovarian cancer. However, other non-genetic factors are likely to have a major impact on the etiology of the disease as well. Regardless of the cause, if undetected early in its progression, the incidence and mortality of breast cancer increases dramatically. Thus, much work has been focused on the early detection of cell transformation and tumor formation in breast tissue.

Currently, the primary means of identifying breast cancer is by detecting the presence of dense tumor tissue. This can be achieved to varying degrees of effectiveness by direct examination of the exterior of the breast or by mammography or other X-ray imaging methods. However, the latter approach comes at a considerable cost. Every time a mammography is performed, the patient is exposed to a lesser risk of having breast tumors induced by the ionizing properties of the radiation used during the test. Furthermore, the method is expensive and the subjective interpretation of the technician may lead to inaccuracies, e.g. one study shows that for a set of breast images interpreted individually by a set of radiologists under investigation, there is a major clinical divergence of about one third. In addition, many women find mammography a painful experience. Thus, the national cancer institute does not recommend mammography for women under 50 years of age because the population is less likely to develop breast cancer than older women. However, strikingly, although only about 22% of women under the age of 50 develop breast cancer, the data show that breast cancer is more aggressive in premenopausal women.

PAX2

The PAX genes are nine developmental regulatory gene families encoding nuclear transcription factors. They play an important role in embryogenesis and are expressed in a very ordered spatiotemporal pattern. They all contain 384 base pair "pairing box" regions encoding DNA binding domains that are highly conserved throughout evolution (Stuart, ET al 1994). The effect of the PAX gene on the developmental process has been demonstrated by a number of natural murine and human syndromes that can be directly attributed to the lack of heterozygosity of the PAX gene. The sequence of PAX2 has been given by Dressier et al (1990). The amino acid sequences of the human PAX2 protein and variants thereof, as well as the DNA sequences encoding these proteins, are set forth in SEQ ID NO: 39-59(SEQ ID NO: 39, amino acid sequence encoded by exon 1 of the human PAX2 gene, SEQ ID NO: 40, human PAX2 gene promoter and exon 1, SEQ ID NO: 41, amino acid sequence of human PAX2, SEQ ID NO: 42, human PAX2 gene, SEQ ID NO: 43, amino acid sequence of human PAX2 gene variant b, SEQ ID NO: 44, human PAX2 gene variant b, SEQ ID NO: 45, amino acid sequence of human PAX2 gene variant c, SEQ ID NO: 46, human PAX2 gene variant c, SEQ ID NO: 47, amino acid sequence of human PAX2 gene variant d, SEQ ID NO: 48, human PAX2 gene variant d, SEQ ID NO: 49, amino acid sequence of human PAX2 gene variant e, SEQ ID NO: 50, human PAX2 gene variant e). PAX2 was reported to repress DEFB-1 expression by binding to the DEFB-1 promoter (Bose SK et al, MoI Immunol.2009, 46: 1140-8.) at the 5 '-CCTTG-3' (SEQ ID NO: 1) recognition site immediately adjacent to the DEFB1 TATA box. In some documents, the binding site is also referred to as the 3 '-GTTCC-5' (SEQ ID NO: 1) or 5 '-CAAGG-3' (SEQ ID NO: 2) recognition site, with 5 '-CAAGG-3' (SEQ ID NO: 2) being the sequence on the opposite strand. Both sequences are involved in the PAX2 binding site on the DEFB1 promoter. Examples of cancers in which PAX2 expression has been detected are listed in Table 1.

Table 1: PAX2 expressing cancers

DEFB1

Beta-defensins are cationic peptides with broad spectrum antimicrobial activity that are products of epithelial and leukocyte cells. A single gene product with two exons is expressed on the epithelial surface and secreted at sites including skin, cornea, tongue, gingiva, salivary gland, esophagus, intestine, kidney, genitourinary tract, and respiratory epithelium. To date, five β -defensin genes of epithelial origin have been identified and characterized in humans: DEFB1(Bensch et al, 1995), DEFB2(Harder et al, 1997), DEFB3(Harder et al, 2001; Jia et al, 2001), DEFB4 and HE2/EP 2. The amino acid sequence of human DEFB1 and the gene sequence of human DEFB1 are respectively shown in SEQ ID NOS: 63 and 64.

The primary structure of each β -defensin gene product is characterized by a small size, six cysteine motifs, a high cationic charge and extreme diversity beyond these features. The most typical feature of defensin proteins is their six cysteine motifs which form three disulfide bridges. The three disulfide bonds of the β -defensin protein are located at C1-C5, C2-C4 and C3-C6. The most common spacings between adjacent cysteine residues are 6, 4, 9, 6, 0. The spacing between the cysteines of the β -defensin proteins may differ by one or two amino acids, except for C5 and C6 located closest to the carboxy terminus. In all known vertebrate β -defensin genes, the two cysteine residues are adjacent to each other.

The second feature of the β -defensin protein is its small size. The preproprotein encoded by each β -defensin gene ranged in size from 59 to 80 amino acids (average size 65 amino acids). The gene product is then cleaved by an unknown mechanism to form a mature peptide ranging in size from 36 to 47 amino acids (average size of 45 amino acids). The exception to these ranges is the EP2/HE2 gene product which contains the beta-defensin motif and is expressed in the epididymis.

The third characteristic of β -defensin proteins is the high concentration of cationic residues. The number of positively charged residues (arginine, lysine and histidine) in the mature peptide ranged from 6 to 14 (9 on average).

The last feature of the β -defensin gene product is its diverse primary structure but a clearly conserved tertiary structure. In all known members of the protein family, no amino acid is conserved at a given position, except for the six cysteines. However, there are conserved positions that appear to be important for secondary and tertiary structure and function.

Although the primary amino acid sequence of β -defensin proteins varies widely, limited data suggest that the tertiary structure of this family of proteins is conserved. The structural core is the three-strand antiparallel beta-sheet, exemplified by the proteins encoded by BNBD-12 and DEFB 2. The three beta strands are connected by beta turns and alpha hairpin loops, and the second beta strand also contains beta-bulge. When these structures fold into their characteristic tertiary structures, random sequences of cationic and hydrophobic residues apparently aggregate to both surfaces of globular proteins. One surface is hydrophilic and contains a number of positively charged side chains, while the other surface is hydrophobic. In solution, the HBD-2 protein encoded by the DEFB2 gene shows an alpha-helical segment near the N-terminus, which has not previously been considered to be the solution structure of alpha-defensin or beta-defensin BNBD-12. Between β -defensin proteins, the amino acids whose side chains point to the protein surface are less conserved, while the amino acid residues of the three β -strands of the core β -sheet are more highly conserved.

The β -defensin peptide is produced as a prepropeptide (pre-pro-peptides) and then cleaved to release the C-terminal active peptide fragment; however, the intracellular processing, storage and release pathways of human β -defensin peptides in airway epithelium are unknown.

Inhibitors of PAX2 expression or PAX2 activity

Functional nucleic acid

The inhibitor of the disclosed methods can be a functional nucleic acid that inhibits expression of PAX 2. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding to a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following categories, but are not intended to be limiting. For example, functional nucleic acids include antisense molecules, aptamers, ribozymes and triplex-forming molecules, RNAi and external guide sequences. Functional nucleic acid molecules can act as influencers, inhibitors, modulators, and stimulators of a particular activity possessed by a target molecule, or functional nucleic acid molecules can possess entirely new activities independent of any other molecule.

Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptide, or carbohydrate strands. Thus, functional nucleic acids may interact with the mRNA of PAX2 or the genomic DNA of PAX2, or they may interact with the polypeptide PAX 2. Functional nucleic acids are typically designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other cases, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather on the formation of a quaternary structure that allows specific recognition to occur.

Antisense molecules are designed to interact with a target nucleic acid molecule through conventional or unconventional base pairing. The interaction of the antisense molecule with the target molecule is designed to facilitate destruction of the target molecule by, for example, RNAseH mediated degradation of the RNA-DNA hybrid. Alternatively, antisense molecules are designed to block processing functions such as transcription or replication that normally occur on the target molecule. Antisense molecules can be designed based on the sequence of the target molecule. There are many ways to optimize antisense potency by finding the most susceptible region of the target molecule. Exemplary methods are in vitro selection assays and DNA modification studies using DMS and DEPC. Preferably, the antisense molecule binds to the target molecule with a dissociation constant (Kd) of less than or equal to 10^-6、10^-8、10^-10Or 10^-12。

Aptamers are molecules that preferably interact with a target molecule in a specific manner. Typically, aptamers are small nucleic acids ranging from 15 to 50 bases in length that can fold into defined secondary and tertiary structures, such as stem-loops or G-tetrads. Aptamers can bind small molecules such as ATP and theophylline, as well as large molecules such as reverse transcriptase and thrombin. The aptamer can interact with the target molecule with a Kd of less than 10^-12M binds very tightly. Preferably, the aptamer binds to the target molecule with a Kd of less than 10^-6、10^-8、10^-10Or 10^-12. Aptamers can bind specifically to a target molecule to a very high degree. For example, aptamers have been isolated that differ in binding affinity by more than a factor of 10,000 between the target molecule and another molecule that differs only in one position of the molecule. It is preferred that the Kd of the aptamer to the target molecule is at least 10, 100, 1000, 10,000 or 100,000 times lower than the Kd of the aptamer to the background binding molecule. For example, when performing polypeptide comparisons, the background molecules are preferably different polypeptides.

Ribozymes are nucleic acid molecules that are capable of catalyzing an intramolecular or intermolecular chemical reaction. Thus, ribozymes are nucleic acids that have catalytic activity. Ribozymes which catalyze intermolecular reactions are preferred. There are many different kinds of ribozymes catalyzing nuclease or nucleic acid polymerase type reactions based on the ribozymes that exist in natural systems, such as hammerhead ribozymes, hairpin ribozymes, and tetrahymena ribozymes. There are also a number of ribozymes which do not exist in the natural system but which are engineered to re-catalyze specific reactions. Preferred ribozymes cleave RNA or DNA substrates, more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to a target substrate and then cleaving. This recognition is usually based primarily on conventional or unconventional base pair interactions. Since recognition of the target substrate is based on the sequence of the target substrate, this feature makes ribozymes particularly suitable candidates for target-specific cleavage of nucleic acids.

Functional nucleic acid molecules that form triple helices are molecules that can interact with double-stranded or single-stranded nucleic acids. When a triple-helical molecule interacts with a target region, a so-called triple helix is formedIn which three DNA strands form a complex depending on Watson-Crick and Hoogsteen base pairing. Triple helix molecules are preferred because they are capable of binding the target region with high affinity and specificity. Preferably, the triple helix forming molecule binds to the target molecule with a Kd of less than 10^-6、10^-8、10^-10Or 10^-12。

An External Guide Sequence (EGS) is a molecule that binds to a target nucleic acid molecule to form a complex, and this complex is recognized by RNase P that cleaves the target molecule. EGS can be designed to specifically target selected RNA molecules. RNAse P aids in intracellular processing of transfer RNA (tRNA). By using EGS that results in the target RNA EGS complex mimicking the native tRNA substrate, bacterial RNAse P can complement the cleavage of virtually any RNA sequence. Likewise, eukaryotic EGS/RNAse P-directed RNA cleavage can be used to cleave a desired target in eukaryotic cells.

Gene expression can also be effectively silenced in a highly specific manner by RNA interference (RNAi). This silencing was initially observed by the addition of double stranded rna (dsrna). Once inside the cell, the dsRNA is cleaved by the RNase III-like enzyme Dicer into a double-stranded small interfering RNA (siRNA) of 21-23 nucleotides in length, which contains a 2-nucleotide overhang at the 3' end. In an ATP-dependent step, the siRNA is adapted to integrate into a multi-subunit protein complex commonly referred to as the RNAi-induced silencing complex (RISC), which directs the siRNA to a target RNA sequence. At some point, the siRNA duplex unravels and the antisense strand appears to remain associated with RISC and direct the degradation of complementary mRNA sequences by a combination of endonuclease and exonuclease. However, the action of irnas or sirnas or their use is not limited to any type of mechanism.

Short interfering RNAs (sirnas) are double-stranded RNAs that can induce sequence-specific post-transcriptional gene silencing, thereby reducing or even inhibiting gene expression. In one example, siRNA triggers specific degradation of a homologous RNA molecule (e.g., mRNA) within the region of sequence identity between the siRNA and the target RNA. For example, WO 02/44321 discloses that siRNA can be paired with a 3' overhang baseMethods for making these sirnas are incorporated herein by reference, which are capable of sequence-specific degradation of target mrnas. Sequence-specific gene silencing can be achieved in mammalian cells by using synthetic short double-stranded RNA to mimic siRNA produced by the enzyme dicer. The siRNA may be chemically synthesized or synthesized in vitro, or may be the result of short double-stranded hairpin-like RNA (shRNA) that can be processed intracellularly into siRNA. Synthetic sirnas are typically designed using algorithms and conventional DNA/RNA synthesizers. Vendors include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Vento, the Netherlands). siRNA may also be delivered using a kit such as Ambion' ssiRNA construction kit in vitro synthesis. Disclosed herein are any sirnas designed as described above based on the sequence of PAX 2.

More commonly, siRNA is generated from a vector by transcription of short hairpin RNA (shRNA). Kits for generating shRNA-containing vectors are available, for example, from GENESUPPRESSOR, e.g., Imgenex^TMConstruction kit and BLOCK-IT from Invitrogen^TMInducible RNAi plasmids and lentiviral vectors. Disclosed herein are any shRNA designed as described above based on the sequences of inflammatory mediators disclosed herein.

In certain embodiments, the functional nucleic acid sequence comprises an siRNA that inhibits expression of PAX2 (anti-PAX 2 siRNA). Examples of anti-PAX 2 sirnas include, but are not limited to, sirnas having the following sequences (5 'to 3' direction):

AUAGACUCGACUUGACUUCUU(SEQ ID NO：3)，

AUCUUCAUCACGUUUCCUCUU(SEQ ID NO：4)，

GUAUUCAGCAAUCUUGUCCUU(SEQ ID NO：5)，

GAUUUGAUGUGCUCUGAUGUU(SEQ ID NO：6)，

ACCCGACTATGTTCGCCTGG(SEQ ID NO：11)，

AAGCTCTGGATCGAGTCTTTG(SEQ ID NO：12)，

ATGTGTCAGGCACACAGACG(SEQ ID NO：13)，

GUCGAGUCUAUCUGCAUCCUU(SEQ ID NO：14)，

GGAUGCAGAUAGACUCGACUU (SEQ ID NO: 15), and

at least 10 nucleic acid fragments and conservative variants thereof; and combinations of the above.

In other embodiments, the functional nucleic acid comprises an antisense RNA of PAX2 and an oligonucleotide that interferes with or inhibits binding of PAX2 to the DEFB1 promoter. The oligonucleotide may be complementary to the sequence of PAX2 that binds the DEFB1 promoter. Alternatively, the oligonucleotide may interact with PAX2 in a manner that inhibits binding of PAX2 to DEFB 1. The interaction may be based on a three-dimensional structure rather than a primary nucleotide sequence.

PAX proteins are a family of transcription factors that are conserved during evolution and are capable of binding to specific DNA sequences via domains referred to as "pairing domains" and "homology domains". The Pairing Domain (PD) is a consensus sequence shared by certain PAX proteins, such as PAX2 and PAX 6. PD directs DNA binding to amino acids located within the α 3-helix of the DNA-protein complex. For PAX2, the amino acids within HD recognize and interact specifically with the CCTTG (SEQ ID NO: 1) DNA core sequence. Oligonucleotides comprising this sequence or its complement are expected to be inhibitors. The critical DNA region within the DEFB1 promoter for binding of the PAX2 protein has the sequence AAGTTCACCCTTGACTGTG (SEQ ID NO: 16).

In one embodiment, the oligonucleotide has the sequence V-CCTTG-W (SEQ ID NO: 17), wherein V and W are nucleotide sequences of 1 to 35 nucleotides. In certain embodiments, V or W or both comprise a contiguous nucleotide sequence that normally flanks the PAX2 binding site of the DEFB1 promoter. Alternatively, the nucleotide sequence of V and/or W may be independent of the DEFB1 promoter and randomly selected to avoid interference with the PAX2 recognition sequence.

Other examples of oligonucleotides that inhibit the binding of PAX2 to the DEFB1 promoter include, but are not limited to, oligonucleotides having the following sequences (5 'to 3' orientation):

CTCCCTTCAGTTCCGTCGAC(SEQ ID NO：18)，

CTCCCTTCACCTTGGTCGAC(SEQ ID NO：19)，

ACTGTGGCACCTCCCTTCAGTTCCGTCGACGAGGTTGTGC (SEQ ID NO: 20), and

ACTGTGGCACCTCCCTTCACCTTGGTCGACGAGGTTGTGC(SEQ ID NO：21)。

other inhibitors

In addition to functional nucleotides, inhibitors of PAX2 expression or PAX2 activity may be any small molecule that interferes with or inhibits binding of PAX2 to the DEFB1 promoter. Inhibitors of PAX2 expression or PAX2 activity may also be antagonists of angiotensin II, or antagonists of Angiotensin Converting Enzyme (ACE). For example, the inhibitor may be an antagonist of enalapril (enalapril) or/and angiotensin II type 1 receptor (AT 1R). The inhibitor may be valsartan (valsartan), olmesartan (olmesartan) or/and telmisartan (telmisartan). The inhibitor may be an antagonist of MEK, an antagonist of ERK1, 2, or/and an antagonist of STAT 3. In certain aspects, the disclosed inhibitors of PAX2 expression or activity are not AT1R receptor antagonists. The term "antagonist" refers to an agent that inhibits the activity of a target.

Antagonists of MEK and/or ERK1, 2 include U0126 and PD 98059. U0126 is a chemically synthesized organic compound, originally recognized as a cellular AP-1 antagonist, and was found to be a very selective and highly potent inhibitor of the mitogen-activated protein kinase (MAPK) cascade by inhibiting its direct upstream activators, mitogen-activated protein kinase kinases 1 and 2 (also known as MEK1 and MEK2, IC50 at 70nM and 60nM, respectively). U0126 inhibits activated and inactivated MEK1, 2, unlike PD98059 which inhibits activation of inactivated MEK only. Blockade of MEK activation prevents phosphorylation of many downstream factors including p62TCF (EIk-1), a component of the AP-1 complex, an upstream inducer of c-Fos and c-Jun. Inhibition of the MEK/ERK pathway by U0126 also blocks the full action of the oncogenes H-Ras and K-Ras, inhibits the partial action triggered by growth factors, and blocks the production of inflammatory cytokines and matrix metalloproteinases.

PD98059 has been shown to act in vivo as a highly selective inhibitor of MEK1 activation and the MAP kinase cascade. PD98059 binds to the inactive form of MEK1 and its activation is prevented by upstream activators such as c-Raf. PD98059 inhibited activation of MEK1 and MEK2 with IC50 values of 4 μ M and 50 μ M, respectively.

In certain embodiments, expression of PAX2 is inhibited by administering a blocker of the RAS signaling pathway to breast cancer tissue or MIN tissue of the individual.

In certain other embodiments, the inhibitor of PAX2 expression or PAX2 activity is conjugated to an antibody, receptor, or ligand to target tumor tissue.

Enhancer of DEFB-1 expression or DEFB-1 activity

An enhancer of DEFB-1 expression or DEFB-1 activity may be a vector that expresses DEFB-1 protein. Since PAX2 inhibits DEFB-1 expression, inhibitors of PAX2 expression or PAX2 activity are also enhancers of DEFB-1 expression.

Delivery system

There are a number of compositions and methods that can be used to deliver nucleic acids to cells in vitro or in vivo. These methods and compositions can be largely divided into two categories: viral-based delivery systems and non-viral-based delivery systems. For example, nucleic acids can be delivered by a number of direct delivery systems, such as electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or by transferring genetic material within cells or vectors such as cationic liposomes. Such methods are well known in the art and are suitable for use with the compositions and methods described herein. In some cases, the method will be modified to work specifically on large DNA molecules. Furthermore, by utilizing the targeting characteristics of the vector, these methods can be used to target certain diseases and cell populations.

Nucleic acid-based delivery system

Inhibitors of PAX2 expression or PAX2 activity and enhancers of DEFB1 expression or DEFB1 activity can be delivered to target cells using nucleic acid-based delivery systems such as plasmids and viral vectors. As used herein, a plasmid or viral vector is an agent that transports a disclosed nucleic acid, such as a PAX2siRNA, to a cell without degradation, comprising a promoter that produces expression of the gene in the cell to which it is delivered. In some embodiments, the vector is derived from a virus or retrovirus. Viral vectors are, for example, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, AIDS virus, neurotrophic virus (neuronic virus), Sindbis (Sindbis) virus and other RNA viruses, including those having an HIV backbone. Also preferred are any virus families that share the properties of these viruses that make them suitable for use as vectors. Retroviruses include murine moloney leukemia virus, MMLV, and retroviruses that express the desired properties of MMLV as a vector. Retroviral vectors are capable of carrying a larger genetic load, i.e., a transgene or marker gene, than other viral vectors, and are commonly used for this reason. However, they are not used in non-proliferating cells. Adenoviral vectors are relatively stable and easy to handle, have high titers, and can be delivered in aerosol formulations and can transfect non-dividing cells. Pox viral vectors are large and have several sites for insertion of genes, they are heat resistant and can be stored at room temperature. Viral vectors have a higher ability to transduce (ability to introduce genes) than chemical or physical methods, thereby introducing genes into cells. Typically, viral vectors contain nonstructural early genes, structural late genes, RNA polymerase III transcripts, inverted terminal repeats necessary for replication and packaging, and promoters that control transcription and replication of the viral genome. When engineered into a vector, the virus typically removes one or more of the early genes and inserts genes or gene/promoter cassettes into the viral genome in place of the removed viral DNA. This type of construct can transport up to about 8kb of exogenous genetic material. The essential function of the removed early gene is usually complemented by cell lines that are engineered to express the gene product of the early gene in trans.

The nucleic acid delivered to the cell typically comprises an expression control system. For example, inserted genes within viral and retroviral systems often contain promoters and/or enhancers to assist in controlling the expression of the desired gene product. Promoters are generally DNA sequences that function when the transcription start site is in a relatively fixed position. Promoters comprise core elements required for substantial interaction of RNA polymerase with transcription factors, and may comprise upstream and response elements.

Preferred promoters for controlling transcription of the vector in mammalian host cells may be obtained from a variety of sources, for example, the viral genome, such as polyoma virus, simian virus 40(SV40), adenovirus, retrovirus, hepatitis b virus, and most preferably cytomegalovirus, or from a heterologous mammalian promoter, such as the β -actin promoter.

Enhancers generally refer to DNA sequences that act at a variable distance from the transcription start site and may be located 5 'or 3' to the transcriptional unit. In addition, enhancers can be within introns, as well as within coding sequences. They are usually 10 to 300bp in length, and they act in cis. Enhancers function to increase transcription from adjacent promoters. Enhancers also typically contain response elements that mediate the regulation of transcription. Promoters also contain response elements that mediate transcriptional regulation. Enhancers generally determine the regulation of gene expression. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), for routine expression typically an enhancer from a eukaryotic cell virus will be used. Preferred examples are the SV40 enhancer downstream of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer downstream of the replication origin, and adenovirus enhancers.

Promoters and/or enhancers can be specifically activated by light or a specific chemical event that triggers their function. The system can be modulated by agents such as tetracycline and dexamethasone. There are also methods of enhancing viral vector gene expression by exposure to radiation (e.g., gamma radiation) or alkylating chemotherapeutic drugs.

In certain embodiments, the promoter and/or enhancer regions may function as constitutive promoters and/or enhancers to maximize expression of the transcriptional unit region to be transcribed. In certain constructs, the promoter and/or enhancer region is active in all eukaryotic cell types, even if expressed only in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are the SV40 promoter, cytomegalovirus (full-length promoter), and retroviral vector LTR.

It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that can be selectively expressed in specific cell types, such as melanoma cells. Glial Fibrillary Acidic Protein (GFAP) promoters have been used to selectively express genes in cells of glial origin.

Expression vectors for use in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells) may also contain sequences necessary for termination of transcription that can affect expression of the mRNA. These regions are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the tissue factor protein. The 3' untranslated region also contains a transcription termination site. Preferably, the transcription unit further comprises a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcriptional unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. Preferably, homologous polyadenylation signals are used in the transgene construct. In certain transcriptional units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcriptional unit contain other standard sequences that, alone or in combination with the above sequences, enhance expression of the construct or stability of the construct.

The viral vector may comprise a nucleic acid sequence encoding a marker product. The marker product is used to determine whether the gene has been delivered to the cell and is expressed upon delivery. Preferred marker genes are the E.coli lacZ gene, which encodes beta-galactosidase, and green fluorescent protein.

In some embodiments, the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin analog G418, hygromycin and puromycin. When such a selectable marker is successfully transferred to a mammalian host cell, the transformed mammalian host cell is viable if placed under selective pressure. There are two widely used different categories of options. The first is based on cellular metabolism and uses mutant cell lines that are unable to grow without reliance on supplemental media. Two examples are: CHO DHFR-cells and mouse LTK-cells. These cells are unable to grow without the addition of nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes necessary for the complete nucleotide synthesis pathway, they cannot survive without providing the missing nucleotides in the supplemental medium. An alternative to supplementing the medium is to introduce the complete DHFR or TK gene into cells lacking the respective gene, thus altering their growth requirements. Individual cells that are not transformed with the DHFR or TK gene will not survive in medium without supplementation.

The second category is dominant selection, which involves selection schemes used by any cell type and does not require the use of mutant cell lines. These protocols typically utilize drugs to prevent host cell growth. Those cells with the novel gene will express a protein that confers drug resistance and will survive selection. Examples of such dominant selection utilize the drugs neomycin, mycophenolic acid or hygromycin. These three examples use bacterial genes under eukaryotic control to deliver resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puromycin.

Non-nucleic acid based systems

Inhibitors of PAX2 expression or PAX2 activity and enhancers of DEFB1 expression or DEFB1 activity may also be delivered to target cells in a variety of ways. For example, the composition may be delivered by electroporation or by lipofection or by calcium phosphate precipitation. The choice of delivery modality depends in part on the cell type targeted and whether delivery occurs, for example, in vivo or in vitro.

Thus, the composition may comprise lipids such as liposomes, for example cationic liposomes (such as DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can also contain proteins to facilitate targeting to specific cells, if desired. Administration of a composition comprising the compound and cationic liposomes can be administered to target cells that have been either passed into the target organ by blood or inhaled into the respiratory tract. Furthermore, the compounds may be administered as components of microcapsules that may be targeted to a particular cell type, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsules is designed to a particular rate or dose.

In the methods described above that involve administration and uptake of exogenous DNA into cells of an individual (i.e., gene transduction or transfection), the compositions can be delivered to the cells in a variety of ways. As an example, delivery can be by liposomes, using commercially available liposome formulations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, inc., Gaithersburg, MD), SUPERFECT (Qiagen, inc. hilden, Germany) and TRANSFECTAM (Promega biotech, inc., Madison, WI), and other liposomes developed according to the standard of the art procedures. In addition, the disclosed nucleic acids or vectors can be delivered in vivo by electroporation (a technique available from Genetronics, Inc. (San Diego, CA)) as well as by sonophoration devices (ImaRx Pharmaceutical corp., Tucson, AZ).

The material may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells). These materials can be targeted to specific cell types by antibodies, receptors, or receptor ligands. Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs targeting colon cancer), receptors that target DNA mediated by cell-specific ligands, lymphocytes targeted to tumor targets, and highly specific therapeutic retroviruses targeted to murine glioma cells in vivo. Generally, receptors, either constitutive or ligand-induced, are involved in the endocytic pathway. These receptors aggregate in clathrin-coated pockets, enter the cell through clathrin-coated vesicles, pass through acidified endosomes (where the receptors are sorted), and are then either recycled to the cell surface, stored intracellularly, or degraded in lysosomes. The internalization pathway provides multiple functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, access opportunities for viruses and toxins, ligand separation and degradation, and receptor level regulation. Many receptors follow more than one intracellular pathway, depending on cell type, receptor concentration, ligand type, ligand potency, and ligand concentration.

Nucleic acids delivered to a cell and integrated into the host cell genome typically comprise integration sequences. These sequences are typically virus-related sequences, particularly when using virus-based systems. These viral integration systems may also incorporate the nucleic acid to be delivered using non-nucleic acid based delivery systems (e.g., liposomes) such that the nucleic acid contained in the delivery system can integrate into the host genome.

Other conventional techniques for integration into a host genome include, for example, systems designed to facilitate homologous recombination with the host genome. These systems typically rely on sequences flanking the nucleic acid to be expressed that have sufficient homology to a target sequence within the genome of the host cell where recombination occurs between the vector nucleic acid and the target nucleic acid, resulting in integration of the delivered nucleic acid into the host genome. These systems and the methods required to promote homologous recombination are well known to those skilled in the art.

Inhibitors of PAX2 expression or PAX2 activity and enhancers of DEFB1 expression or DEFB1 activity may be delivered to target cells in a variety of ways, may be administered in a pharmaceutically acceptable carrier, and may be delivered to individual cells in vivo and/or ex vivo by a variety of means well known in the art (e.g., naked DNA uptake, liposome fusion, intramuscular injection of DNA by gene gun, endocytosis, etc.).

If ex vivo methods are used, the cells or tissues may be removed and maintained in vitro according to standard protocols well known in the art. The composition may be introduced into the cell by any means of gene transfer such as, for example, calcium phosphate-mediated gene delivery, electroporation, microinjection, or proteoliposomes. The transduced cells can then be injected, e.g., in a pharmaceutically acceptable carrier) or orthotopically transplanted back into the individual according to standard methods for cell or tissue type. Standard methods for transplanting or injecting various cells into an individual are known.

Compositions and kits

Another aspect of the invention relates to compositions and kits for treating or preventing cancer. The compositions comprise an inhibitor of PAX2 expression or PAX2 activity and/or an enhancer of DEFB-1 expression or DEFB-1 activity, and a pharmaceutically acceptable carrier.

By "pharmaceutically acceptable" is meant not a biological or other unwanted material, i.e., a material that can be administered to an individual with a nucleic acid or vector without causing unwanted biological effects or interacting in a deleterious manner with any of the other components in which the pharmaceutical composition is contained. As is well known to those skilled in the art, the carrier should be chosen appropriately to minimize degradation of any active ingredient and to minimize any adverse side effects in the individual.

Suitable carriers and formulations thereof are described in Remington: the Science and practice of Pharmacy (Remington: pharmaceutical technology and practice, 19 th edition) ed.A.R.Gennaro, Mack Publishing Company, Easton, PA 1995. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation, so that the formulation is isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, ringer's solution, and dextrose solution. The solution pH is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred depending on, for example, the route of administration and the concentration of the composition being administered.

Pharmaceutical carriers are well known to those skilled in the art. These are most typically standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The composition may be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

The pharmaceutical compositions may contain, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surfactants, and the like. The pharmaceutical compositions may also contain one or more active ingredients such as antibacterial agents, anti-inflammatory agents, anesthetics, and the like.

Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, ethanol/water solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution or non-volatile oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose solution), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, water, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, powders or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Some compositions may be administered as a pharmaceutically acceptable acid or base addition salt formed by reaction with: inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid; the base addition salts are formed by reaction with: inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as monoalkyl, dialkyl, trialkyl, and aromatic amines and substituted ethanolamines.

The above-described materials, as well as other materials, can be packaged together in any suitable combination into a kit for performing or aiding in the practice of the disclosed methods. It is beneficial if the kit components in a given kit are designed and adapted for use with the disclosed methods. For example, kits for detecting, treating or preventing prostate cancer, PIN, breast cancer and MIN are disclosed. The kit comprises an inhibitor of PAX2 expression or PAX2 activity and/or an enhancer of DEFB1 expression or DEFB1 activity. In one embodiment, the kit comprises a peptide or antibody that specifically binds PAX2 or DEFB 1.

The compositions disclosed herein can be administered in a number of ways depending on whether local or systemic treatment is desired and on the site to be treated. For example, the compositions can be administered orally, parenterally (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection), by inhalation, extracorporeally, topically (including transdermal, ocular, vaginal, rectal, intranasal), and the like.

As used herein, "topical intranasal administration" means delivery of a composition to the nose or nasal cavity through one or both nostrils, and may include delivery by the spray or drip route, or by nebulization of a nucleic acid or carrier. Administration of the composition by inhalation may be by spray or drop delivery through the nose or mouth. It may also be delivered directly to any region of the respiratory system (e.g., the lungs) by intubation.

If parenteral administration of the composition is used, it is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolution in liquid to suspension prior to injection, or as emulsions. A more recently revised method of parenteral administration involves the use of slow release or sustained release systems in order to maintain a constant dose.

The precise amount of the composition required will vary from individual to individual, depending upon the species, age, weight, and general condition of the individual, the severity of the allergic condition to be treated, the particular nucleic acid or vector used, the manner of administration, and the like. Appropriate amounts can be determined by one of ordinary skill in the art using no more than routine experimentation in light of the teachings herein. Thus, effective dosages and schedules for administering the compositions can be determined empirically, and making such determinations is within the skill of the art. The dosage range at which the composition is administered should be large enough to be affected to produce the desired effect on the condition of the disorder. The dosage should not be so great as to cause adverse side effects such as unwanted cross-reactions, allergic reactions, and the like. In general, the dosage will vary with the age, sex, and extent of disease of the patient, the route of administration, or whether other drugs are included in the treatment regimen, and can be determined by one skilled in the art. The dosage may be adjusted by the individual physician if any indication of the contrary occurs. The dosage may vary and may be administered in one or more doses per day for one or more days. Guidance on the appropriate dosage for a given class of pharmaceutical products can be found in the literature.

For example, depending on the factors mentioned above, typical daily dosage ranges for the disclosed compositions alone may range from about 1 μ g/kg body weight to as much as 100mg/kg body weight or more per day. In certain embodiments, the method of treatment is modulated based on the expression rate (P/D ratio) of PAX 2-to-DEFB 1 and Estrogen Receptor (ER)/Progesterone Receptor (PR) status of the diseased tissue. Table 2 gives the treatment options based on P/D ratio and ER/PR status. There was a positive correlation between PAX2 status and ER status in normal breast tissue, MIN and low-grade breast cancer. PAX2 also regulates ERBB2 expression and subsequent Her2/neu expression through estrogen receptors. In contrast, there is an inverse relationship between PAX2 expression and high-grade (or invasive) breast cancer. Monitoring the expression level of PAX2 can therefore be used to predict drug response or resistance, as well as to identify patients that may be candidates for DEFB1 or anti-PAX 2 therapy. The term "anti-PAX 2 therapy" refers to a method of inhibiting expression of PAX2 or activity of PAX 2. The term "DEFB 1 therapy" refers to a method of increasing expression of DEFB 1. The term "DEFB 1 therapy" does not include methods of inhibiting expression of PAX2 or activity of PAX2, although such methods also result in increased expression of DEFB 1.

As shown in table 2, anti-PAX 2 therapy and/or DEFB1 therapy may be used in combination with one or more other breast cancer treatments, such as anti-hormonal treatment (e.g., Tamoxifen (Tamoxifen)), anti-ERBB 2 treatment (e.g., Herceptin (Herceptin)), anti-Her 2 treatment (e.g., Trastuzumab (Trastuzumab)), and anti-AIB-1/SRC-3 treatment.

Table 2: treatment of breast disorders using the ratio of PAX 2-to-DEFB 1

Comparison with the ratio of PAX2/DEFB1 in normal mammary epithelium

Expression rate of PAX 2-to-DEFB 1

The term "expression rate of PAX 2-to-DEFB 1" as used below refers to the ratio between the amount of functional PAX2 protein or variant thereof and the amount of functional DEFB1 protein or variant thereof in a given cell or tissue. The level of PAX2 and DEFB1 expression in a cell or tissue can be determined by any method known in the art. In certain embodiments, the level of PAX2 and DEFB1 expression in breast tissue is determined by determining the level of PAX2 and DEFB1 in cells obtained directly from the breast tissue.

The "expression rate of PAX 2-to-DEFB 1" can be measured directly at the protein level or indirectly at the RNA level. Protein levels can be measured using protein arrays, immunoassays, and enzyme assays. RNA levels can be measured, for example, using DNA arrays, RT-PCR and Northern blotting. In certain embodiments, the PAX 2-to-DEFB 1 expression rate is determined by determining the expression level of the PAX2 gene relative to the expression level of a control gene, determining the expression level of the DEFB1 gene relative to the expression level of the same control gene, and calculating the PAX 2-to-DEFB 1 expression rate based on the expression levels of PAX2 and DEFB 1. In one embodiment, the control gene is a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene.

Immunoassay method

The simplest and straightforward immunoassay is a binding assay involving the binding of an antibody to an antigen. Many types and formats of immunoassays are known and all are suitable for detecting the disclosed biomarkers. Examples of immunoassays are enzyme linked immunosorbent assay ELISA), Radioimmunoassay (RIA), radioimmunoprecipitation assay (RIPA), immuno-microsphere capture assay, Western blot, dot blot, gel shift assay, flow cytometry, protein arrays, multiplex bead arrays, magnetic capture, in vivo imaging, Fluorescence Resonance Energy Transfer (FRET), and fluorescence recurrence/localization after photobleaching (FRAP/FLAP).

In general, immunoassays comprise contacting a sample suspected of containing a molecule of interest (e.g., a biomarker as disclosed) with an antibody to the molecule of interest, or contacting an antibody to a molecule of interest (e.g., an antibody to a biomarker as disclosed) with a molecule capable of binding to the antibody, as the case may be, under conditions effective to allow the formation of an immune complex. In many forms of immunoassays, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, can be washed to remove any non-specifically bound antibody species, allowing detection of only those antibodies specifically bound within the primary immune complexes.

Radioimmunoprecipitation assay RIPA) is a sensitive assay that uses radiolabeled antigen to detect specific antibodies in serum. The antigen is allowed to react with the serum and then precipitated using a specialized reagent such as, for example, protein a agarose beads. The bound radiolabeled immunoprecipitates were then routinely analyzed by gel electrophoresis. Radioimmunoprecipitation assay RIPA) are often used as a validation test for diagnosing the presence of HIV antibodies. RIPA is also known in the art as Farr assay, precipitin assay, radioimmunoprecipitant assay, and radioimmunoprecipitation assay.

Also contemplated are immunoassays wherein a protein or protein-specific antibody is bound to a solid support (e.g., a tube, well, bead, or chamber) in combination with a method of detecting the protein or protein-specific antibody on the support, thereby capturing the antibody or protein of interest, respectively, from a sample. Examples of such immunoassays include Radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), flow cytometry, protein arrays, multiplex bead arrays, and magnetic capture methods.

Protein arrays are solid phase ligand binding assay systems that utilize immobilized proteins on surfaces, including glass, membranes, microtiter wells, mass spectrometer plates, and microbeads or other objects. Assays are highly parallel (multiplex) and are often miniaturized (microarrays, protein chips). Their advantages include rapidity and automation, high sensitivity, reagent savings and the generation of large amounts of data in one experiment. Bioinformatics support is important and data processing requires advanced software and comparative analysis of data. However, the software may be adapted according to the DNA array used, as may the hardware and detection system.

The capture array forms the basis of a diagnostic chip and array for expression profiling. They use high affinity capture reagents such as conventional antibodies, single domains, engineered scaffolds, peptides or nucleic acid aptamers to bind and detect specific target ligands in a high throughput manner. Antibody arrays are commercially available. In addition to conventional antibodies, Fab and scFv fragments from camelids or engineered human equivalent single V-domains (Domantis, Waltham, MA) can also be used in the array.

Non-protein capture molecules, in particular single-stranded nucleic acid aptamers that bind protein ligands with high specificity and affinity, can also be used in arrays (SomaLogic, Boulder, CO). Aptamers were passed through Selex^TMThe programs were selected from pools of oligonucleotides and their interaction with proteins could be enhanced by covalent attachment by incorporating bromodeoxyuridine and UV-activated cross-links (photoaptamers). Photocrosslinking with ligands reduces cross-reactivity of the aptamer due to specific steric requirements. Adaplet has a channelThe advantages of convenient production and stable and healthy DNA through automated oligonucleotide synthesis; on photoaptamer arrays, universal fluorescent protein staining can be used to detect binding.

Alternative capture molecule arrays are arrays prepared by "molecular printing" techniques, in which peptides (e.g. from the C-terminus of a protein) are used as templates to create structurally complementary, sequence-specific pores on a polymerisable matrix; the wells can then specifically capture (denatured) proteins (ProteinPrint) with the appropriate primary amino acid sequence^TM，Aspira Biosystems，Burlingame，CA)。

Another method that can be used in diagnosis and expression profiling isArrays (cipergen, Fremont, CA) in which solid phase chromatographic surfaces bind proteins with similar charge or hydrophobicity characteristics to mixtures such as plasma or tumor extracts and SELDI-TOF mass spectrometry is used to detect residual proteins.

Other useful methods include constructing large functional chips by immobilizing large amounts of purified proteins on the chip, as well as multiplex bead assays.

Antibodies

The term "antibody" is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers of those immunoglobulin molecules, as well as human or humanized forms of immunoglobulin molecules or fragments thereof, selected for their ability to interact with, for example, PAX2 or DEFB1 such that PAX2 is inhibited from interacting with DEFB 1. Also disclosed are antibodies that bind to a disclosed region of PAX2 or DEFB1 that is involved in the interaction of PAX2 and DEFB 1. The desired activity of the antibodies can be detected using the in vitro assays described herein or by similar methods, and then tested for their in vivo therapeutic and/or prophylactic activity according to known clinical detection methods.

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired antagonistic activity (see U.S. Pat. No. 4,816,567 and Morrison et al, Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

The term "antibody" as used herein also refers to human and/or humanized antibodies. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are natural antigens of humans and thus can produce an unwanted immune response when administered to humans. Thus, the use of human or humanized antibodies in methods is useful for reducing the chance that an antibody administered to a human will elicit an unwanted immune response. Methods for humanizing non-human antibodies are well known in the art.

DNA array

A DNA or oligonucleotide microarray consists of an array of multiple oligonucleotide microspots called "features", each spot containing a small, typically picomolar, amount of a particular oligonucleotide sequence. The specific oligonucleotide sequence may be a small portion of a gene or other oligonucleotide element that can be used as a probe to hybridize a cDNA or cRNA sample under high stringency conditions. Hybridization of probe targets is typically detected and quantified based on fluorescence detection of fluorophore-labeled targets to determine the relative abundance of nucleic acid sequences in the targets.

The probes are typically attached to the solid surface by covalent bonds (via epoxy-silane, amino-silane, lysine, polyacrylamide, etc.) to the chemistry matrix. The solid surface may be a glass or silicon wafer or beads. Oligonucleotide arrays differ from other types of microarrays only in that they measure nucleotides or use oligonucleotides as part of their detection system.

To detect gene expression in a target tissue or cell using an oligonucleotide array, a nucleic acid of interest is purified from the target tissue or cell. The nucleotides may be all RNA used for expression profiling, DNA used for comparative hybridization, or DNA/RNA binding to specific proteins used for immunoprecipitation (ChIP-on-ChIP) for epigenetic or regulatory studies.

In one embodiment, total RNA (referred to as total because it is in the nucleus and cytoplasm) is isolated by guanidinium isothiocyanate-phenol-chloroform extraction (e.g., Trizol). The purified RNA can be used for qualitative (e.g., by capillary electrophoresis) and quantitative (e.g., by using a nanodrop spectrophotometer) analysis. Total RNA is reverse transcribed into DNA using polyT primers or random primers. The DNA product can be optionally amplified by PCR. Labels are added to the amplification products during the RT step or other steps after amplification, if any. The label may be a fluorescent label or a radioactive label. The labeled DNA product is then hybridized to a microarray. The microarray is then washed and scanned. The expression level of the gene of interest is determined based on the hybridization results using methods well known in the art.

Pharmacogenomics

In another embodiment, PAX2 and/or DEFB1 expression profiles are used to determine pharmacogenomics of breast cancer. Pharmacogenomics refers to the relationship between the genotype of an individual and the response of that individual to foreign compounds or drugs. Metabolic differences in treatment can lead to severe toxicity or therapeutic failure by altering the relationship between the dose of the pharmacologically active drug and the blood concentration. Thus, a physician or clinician can consider the information obtained in the relevant pharmacogenomic studies to determine whether to administer an anti-cancer drug, as well as to adjust the dosage and/or treatment regimen for treatment with an anti-cancer drug.

Pharmacogenomic processing responds clinically to significant genetic variation of drugs due to altered drug disposition and aberrant action in affected humans. In general, two types of pharmacogenomic states can be distinguished. The gene status is transmitted as a single factor that changes the way the drug acts on the body (changes the drug action), or the gene status is transmitted as a single factor that changes the way the body acts on the drug (changes the drug metabolism). These pharmacogenomic states can arise as rare gene deletions or naturally occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common genetic enzymopathy in which the major clinical complications are hemolysis after absorption of oxidant drugs (anti-malarial drugs, sulfa drugs, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomic approach to identifying genes that predict drug response (so-called "whole genome association") relies primarily on high-resolution maps of the human genome consisting of known gene-associated sites (e.g., a genetic marker map of a "biallelic" consisting of 60,000-100,000 polymorphic or variable sites of the human genome, each with two variants). Such high resolution gene maps can be compared to the individual genomic maps of a statistically significant number of individuals involved in phase II/III drug trials to identify genes associated with a particular observed drug response or side effect. Alternatively, such high resolution maps may be formed from a combination of tens of millions of known Single Nucleotide Polymorphisms (SNPs) of the human genome. The term "SNP" as used herein is a common change in a single nucleotide base that occurs in a DNA segment. For example, a SNP may occur every 1,000 bases of DNA. SNPs may be associated with disease progression. However, most SNPs may be unrelated to disease. Given that genetic maps are based on the occurrence of such SNPs, individuals can be classified into genetic classes that depend on the specific pattern of SNPs in their individual genomes. In this manner, treatment regimens may be adjusted for such genetically similar groups of individuals, taking into account genetic characteristics that may be shared among the genetically similar individuals. Thus, mapping PAX2 and/or DEFB1 to SNP maps of breast patients may allow these genes to be more easily identified according to the genetic methods described herein.

Alternatively, a method known as the "alternative gene method" may be used to identify genes that are predictive of drug response. According to this method, if the gene encoding the drug target is known, all common variants of that gene can be identified quite easily in the human population, and it can be determined whether a gene with one form is associated with a particular drug response relative to another gene.

As an exemplary embodiment, the activity of a drug metabolizing enzyme is a major determinant of the intensity and duration of drug action. The discovery of genetic polymorphisms in drug metabolizing enzymes, such as N-acetyltransferase 2(NAT 2) and the cytochrome P450 enzymes CYP2D6 and CYPZC19, has provided an explanation as to why some individuals do not achieve the expected drug effect or show excessive drug response and severe toxicity after taking standard and safe doses of the drug. These polymorphisms are expressed in a population of two phenotypes-pan metabolizers and poor metabolizers. The common undesirable metabolizer phenotype varies among different populations. For example, the gene encoding CYP2D6 is highly polymorphic, and several mutations have been identified in poor metabolizers, all of which result in the deletion of functional CYP2D 6. Adverse metabolizers of CYP2D6 and CYP2C19, receiving standard doses, suffer from excessive drug response and side effects very frequently. If the metabolite is the active therapeutic moiety, the malmetabolizer does not show a therapeutic response, as evidenced by its analgesic effect mediated by the codeine's CYP2D 6-formed metabolite morphine. The other extreme is the so-called ultra-fast metabolizer, which is not responsive to standard doses. Recently, it has been identified that the molecular basis for ultra-fast metabolism is due to CYP2D6 gene proliferation.

Alternatively, a method known as "gene expression profiling" can be used to identify genes that are predictive of drug response. For example, gene expression in an animal administered a drug can give an indication of whether a gene pathway associated with toxicity has been opened.

Information generated by more than one pharmacogenomic approach can be used to determine the appropriate dose and treatment regimen for prophylactically or therapeutically treating an individual. When applied to dosage or drug selection, this information may avoid adverse reactions or therapeutic failure, thus enhancing therapeutic or prophylactic efficacy in treating individuals with breast disorders.

In one embodiment, the PAX2 and/or DEFB1 expression profile, and the ER/PR status of the subject are used to determine an appropriate treatment regimen for the subject having a breast disorder.

In another embodiment, the expression level of PAX2 (typically determined with reference to a control gene such as the actin gene or GAPDH gene) is used in patients with triple negative breast cancer (i.e., Estrogen Receptor (ER) negative, Progesterone Receptor (PR) negative, human epidermal growth factor receptor 2(HER2) negative) to measure the effectiveness of cancer therapy, determine the course of treatment, or monitor cancer recurrence.

The invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all documents, patents and published patent applications cited in this application, as well as the figures and tables, are incorporated herein by reference.

Example 1: human beta-defensin-1 is cytotoxic to advanced prostate cancer and is also prostate Plays a role in adenocarcinoma tumor immunity

In this example, DEFB1 was cloned into an inducible expression system to examine how it affects normal prostate epithelial cells as well as androgen receptor positive (AR +) and androgen receptor negative (AR-) prostate cancer cell lines. Induction of DEFB1 expression resulted in reduced cell growth of AR-cells DU145 and PC3, but had no effect on the growth of AR + prostate cancer cells LNCaP. DEFB1 also causes rapid induction of caspase-mediated apoptosis. The data presented here provide for the first time evidence that DEFB1 plays a role in natural tumor immunity and suggests that its impairment will contribute to tumor progression in prostate cancer.

Materials and methods

Cell line: cell line DU145 was cultured in DMEM medium, PC3 was cultured in F12 medium, and LNCaP was cultured in RPMI medium (Life Technologies, inc. Growth media for all three cell lines was supplemented with 10% (v/v) fetal bovine serum (Life Technologies). In the prostate epitheliumhPrEC cells were cultured in cell basal medium (Cambrex BioScience, inc., Walkersville, MD). All cell lines were maintained at 37 ℃ and 5% CO₂The following steps.

Tissue samples and laser capture microdissection: prostate tissue from patients with informed consent for radical prostatectomy was obtained from the Hollings cancer center tumor bank according to protocols approved by the institutional review Board. This includes guidelines for sample processing, sectioning, histological characterization, RNA purification, and PCR amplification. After pathological examination of frozen tissue sections, Laser Capture Microdissection (LCM) was performed to ensure that the tissue samples examined consisted of a pure population of benign prostate cells. For each tissue section analyzed, LCM was performed on three different areas containing benign tissue, and the collected cells were then pooled.

Prostate tissue was obtained from patients who had provided informed consent prior to radical prostatectomy. Samples were obtained from the Hollings cancer center tumor pool according to protocols approved by the institutional review Board. This includes guidelines for sample processing, sectioning, histological characterization, RNA purification, and PCR amplification. The surgeon obtains a prostate specimen and the pathologist quickly freezes in the OCT compound. Each OCT block was cut to produce a series of sections to be stained and detected. Regions containing benign cells, Prostate Intraepithelial Neoplasia (PIN) and cancer were identified and used to guide us in selecting regions from unstained slides using the Arcturus PixCell II system (Sunnyvale, CA). Caps containing capture material were exposed to 20 μ l lysate from Arcturus Pico PureRNA isolation kit and immediately processed. RNA quantification and characterization were assessed using a primer set that produced the 5' amplicon. The primer set included primers for ribosomal protein L32 (298 bases apart 3 'and 5' amplicons), for glucose phosphate isomerase (391 bases apart), and for glucose phosphate isomerase (842 bases apart). These primer sets routinely achieve ratios of 0.95 to 0.80 using samples from a variety of prepared tissues. Other tumor and normal samples were roughly cut by the pathologist, snap-frozen in liquid nitrogen and evaluated for hBD-1 and cMYC expression.

Cloning of DEFB1 gene: DEFB1 cDNA was generated from RNA by reverse transcription PCR. The PCR primers were designed to contain ClaI and KpnI restriction sites. DEFB1 PCR product was digested with ClaI and KpnI and ligated into the TA cloning vector. The TA/DEFB1 vector was then transfected into E.coli by heat shock, and a single clone was selected and amplified. Plasmids were isolated by Cell Culture DNA midi prep (Qiagen, Valencia, CA) and sequence integrity was verified by automated sequencing. Then, the DEFB1 gene fragment was ligated into pTRE2 (used as an intermediate vector for targeting purposes) digested with ClaI and KpnI. The pTRE2/DEFB1 construct was then digested with ApaI and KpnI to excise the DEFB1 insert and this insert was ligated into the pIND vector (Invitrogen, Carlsbad, Calif.) of the ecdysteroid inducible expression system, which was also double digested with ApaI and KpnI. The constructs were transfected into E.coli again, and single clones were selected and amplified. The plasmid was isolated and the sequence integrity of pIND/DEFB1 was again verified by automated sequencing.

Transfection: cells (1X 10)⁶) Inoculated into 100-mm petri dishes and grown overnight. Cells were then co-transfected with 1 μ g of pVgRXR plasmid (ecdysone receptor expressing heterodimers) and 1 μ g of pIND/DEFB1 vector construct or empty pIND control vector in Opti-MEM medium (Life Technologies, inc., grand island, NY) using Lipofectamine 2000(Invitrogen, Carlsbad, CA).

RNA isolation and quantitative RT-PCR: to verify DEFB1 protein expression in cells transfected with DEFB1 construct, RNA was collected after 24 hours of induction with pinsterone a (pon a). Briefly, the SV Total RNA isolation System (Promega, Madison, Wis.) was used to isolate approximately 1X10 from fractions collected by trypsinization⁶Total RNA was isolated from the cells. At this point, the cells were lysed and total RNA was isolated by centrifugation on a spin column. To collect cells by LCM, total RNA was isolated using picopurer RNA isolation kit (Arcturus Biosciences, mt. Total RNA from two sources (0.5. mu.g per reaction) was reverse transcribed using random primers (Promega)cDNA. AMV reverse transcriptase II (500 units per reaction; Promega) was used for first strand synthesis and Tf1 DNA polymerase was used for second strand synthesis (500 units per reaction; Promega) according to the manufacturer's manual. In each case 50pg of cDNA was used each time to ensure the PCR reaction. Reverse transcription System and method Using the same from TaqManMultisribe reverse transcriptase from Green PCR MasterMix (Applied Biosystems) performs two-step QRT-PCR on the resulting cDNA.

A primer pair for DEFB1 was generated from the published DEFB1 sequence (GenBank accession No. U50930). The primer sequences are as follows:

QRT-PCR primer sequence

Sense (5 '-3')

β -actin 5'-CCTGGCACCCAGCACAAT-3' SEQ ID NO: 51

DEFB1 5’-GTTGCCTGCCAGTCGCCATGAGAACTTCCTAC-3’SEQ ID NO：53

Antisense (5 '-3')

β -actin 5'-GCCGATCCACACGGAGTACT-3' SEQ ID NO: 52

DEFB1 5’-TGGCCTTCCCTCTGTAACAGGTGCCTTGAATT-3’SEQ ID NO：54

Under standard conditions, 40 PCR cycles were performed using an annealing temperature of 56 ℃. In addition, β -actin (table 2) was amplified as a housekeeping gene to normalize the initial content of total cDNA. DEFB1 expression was calculated as the relative expression rate between DEFB1 and β -actin, and compared to cell lines that induced and uninduced DEFB1 expression, as well as LCM benign prostate tissue. As a negative control, a QRT-PCR reaction without cDNA template was also performed. All reactions were run in triplicate.

MTT cell viability assay: to examine the effect of DEFB1 on cell growth, the metabolite 3- (4, 5-dimethylthia-t-hiazole) was performedOxazol-2 yl) -2, 5-diphenyltetrazolium bromide (MTT) assay. PC3, DU145 and LNCaP cells co-transfected with the pVgRXR plasmid and pIND/DEFB1 construct or empty pIND vector were seeded in 96-well plates at 1-5X10 per well³A cell. 24 hours after inoculation, DEFB1 expression was induced by daily addition of fresh growth medium containing 10. mu.M ponasterone A for 24, 48, and 72 hours, after which MTT assays were performed according to the manufacturer's instructions (Promega). Reactions were performed in triplicate three times.

Flow cytometry: PC3 and DU145 cells co-transfected with DEFB1 expression system were cultured in 60-mm dishes and induced with 10 μ M ponasterone a for 12, 24 and 48 hours. After each incubation period, the media was collected from the plate (to retain any isolated cells) and mixed with PBS used to wash the plate. The remaining adherent cells were collected by trypsinization and mixed with detached cells and PBS. The cells were then pelleted by centrifugation at 4 deg.C (500Xg) for 5 minutes, washed twice with PBS, and resuspended in 100ul of 1-fold annexin binding buffer (0.1M Hepes/NaOH, pH7.4, 1.4M NaCl, 25mM CaCl) containing 5. mu.l annexin V-FITC and 5. mu.l PI₂) In (1). Cells were incubated in the dark at RT for 15 min, then diluted with 400 μ l of 1-fold annexin binding buffer and analyzed by FACscan (Becton Dickinson, San Jose, CA). All reactions were carried out in triplicate.

And (3) microscopic analysis: cell morphology was analyzed by phase contrast microscopy. DU145, PC3 and LNCaP cells containing no vector, either empty plasmid or DEFB1 plasmid were seeded into 96-well culture plates (BD Falcon, USA). The following day plasmid containing cells were induced with medium containing 10 μ M pinsterone a for 48 hours, while control cells received fresh medium. The cells were then observed under an inverted Zeiss IM35 microscope (Carl Zeiss, Germany). Phase contrast maps of the cellular field of view were obtained using a SPOT Instrument Mosaic4.2 Camera (Diagnostic Instruments, USA). Cells were examined by phase contrast microscopy at 32 x magnification, and digital images were saved as uncompressed TIFF files and transferred to Photoshop CS software (Adobe Systems, San Jose, CA) for image processing and hard copy display.

Caspase detection: using APO LOGIX^TMCarboxyfluorescein caspase assay kit (Cell Technology, Mountain View, CA) was used for caspase activity detection in prostate cancer Cell lines. Active caspases are detected by using FAM-VAD-FMK inhibitors that irreversibly bind to the active caspases. Briefly, DU145 and PC3 cells (1.5-3X 10) containing the DEFB1 expression system⁵) Seeded on 35mm glass-bottom microwell dishes (Matek, Ashland, MA) and treated with medium alone or with PonA-containing medium for 24 hours as previously described. Next, 10. mu.l of 30-fold working dilution of carboxyfluorescein-labeled peptide fluoromethyl ketone (FAM-VAD-FMK) was added to 300. mu.l of the medium and added to each 35mm dish. Then 5% CO at 37 deg.C₂Cells were incubated for 1 hour. Next, the medium was aspirated and the cells were washed twice with 2ml of 1-fold working dilution wash buffer. Cells were observed under differential interference phase contrast DIC) or 488nm laser excitation. Fluorescence signals were analyzed using a confocal microscope (Zeiss LSM 5 Pascal) with a Vario 2 RGB laser scanning assembly and a 63-fold DIC oil mirror.

Statistical analysis: statistical differences were assessed using student t-test for unpaired values. P values were determined by bilateral calculation and were considered statistically significant to be less than 0.05.

Results

DEFB1 expression in prostate tissues and cell lines: the expression levels of DEFB1 were measured by QRT-PCR in benign and malignant prostate tissue, hPrEC prostate epithelial cells, and DU145, PC3 and LNCaP prostate cancer cells. DEFB1 expression was detected in all benign clinical samples. The average amount of DEFB1 relative expression was 0.0073. Furthermore, the relative expression of DEFB1 in hPrEC cells was 0.0089. There was no statistical difference in DEFB1 expression between the benign prostate tissue samples tested and the hPrEC (fig. 1A). Analysis of relative DEFB1 expression levels in prostate cancer cell lines showed significantly lower levels in DU145, PC3 and LNCaP. As a further reference point, expression of relative DEFB1 in adjacent malignant sections of prostate tissue from patient #1215 was measured. There was no significant difference in the observed expression levels of DEFB1 in the three prostate cancer cell lines compared to malignant prostate tissue from patient #1215 (fig. 1B). In addition, the expression levels in all four samples were close to the negative control without template (confirming that there was little endogenous DEFB1 expression, data not shown). QRT-PCR was also performed on prostate cancer cell lines transfected with the DEFB1 expression system. After 24 hours induction period, relative expression levels: DU145 is 0.01360, PC3 is 0.01503, and LNCaP is 0.138. The amplification product was verified by gel electrophoresis.

QRT-PCR was performed on LCM tissue regions containing benign, PIN and cancer. DEFB1 relative expression: the benign region was 0.0146, compared to 0.0009 for the malignant region (fig. 1C). This represents a 94% reduction, which again confirms a significant down-regulation of expression. Furthermore, analysis of PIN showed DEFB1 expression levels of 0.044, which was a 70% reduction. Comparing the expression in patient #1457 to the mean expression levels seen for benign regions in six other patients (fig. 1A), a ratio of 1.997 is shown, indicating almost two-fold more expression (fig. 1D). However, compared to the average expression level in benign tissues, the expression rate: PIN 0.0595, and malignant tissue 0.125.

DEFB1 causes cell membrane permeability and fluctuations: induction of DEFB1 in prostate cancer cell lines resulted in a significant reduction in the number of DU145 and PC3 cells, but had no effect on cell proliferation of LNCaP (fig. 2). As a negative control, cell proliferation was monitored in all three cell lines containing empty plasmids. No change in cell morphology was observed in DU145, PC3 or LNCaP cells after PonA addition. In addition, DEFB1 induced changes in cell morphology leading to DU145 and PC 3. At this point the cells appeared more rounded and showed membrane fluctuations indicative of cell death. Apoptotic bodies also appeared in both cell lines.

DEFB1 expression results in decreased cell viability: MTT assay showed that DEFB1 in PC3 and DU145 cells resulted in decreased cell viability, but had no significant effect on LNCaP cells (fig. 3). After 24 hours, relative cell viability: DU145 is 72%, and PC3 is 56%. Analysis after 48 hours of induction showed 49% cell viability for DU145 and 37% cell viability for PC 3. DEFB1 resulted in relative cell viability of 44% and 29% for DU145 and PC3 cells, respectively, after 72 hours of expression.

DEFB1 causes rapid caspase-mediated apoptosis in advanced prostate cancer cells: to determine whether DEFB1 effects on PC3 and DU145 were cytostatic or cytotoxic, FACS analysis was performed. Under normal growth conditions, more than 90% of PC3 and DU145 cultures survived and were non-apoptotic (lower left quadrant) and had no annexin V or PI staining. After induction of DEFB1 expression in PC3 cells, the number of apoptotic cells (lower right and upper right quadrants) totaled: 10% for 12 hours, 20% for 24 hours, and 44% for 48 hours (fig. 4B). For DU145 cells, the number of apoptotic cells totaled: 12 hours, 34% at 24 hours, and 59% at 48 hours after induction (fig. 4A). No increase in apoptosis was observed after induction of cells containing the empty plasmid with PonA (data not shown).

Caspase activity was determined by confocal laser microscopy analysis (figure 5). Expression of DEFB1 was induced in DU145 and PC3 cells and activity was monitored based on binding of green-fluorescing FAM-VAD-FMK to caspase in actively apoptotic cells. Analysis of the cells under DIC showed the presence of survival control DU145 (panel A), PC3 (panel E) and LNCaP (panel I) cells at 0 hours. Excitation at 488nm by confocal laser did not produce detectable green staining, indicating no caspase activity in DU145 (panel B), PC3 (panel F) or LNCaP (panel J). After 24 hours of induction, DU145 (panel C), PC3 (panel G) and LNCaP (panel K) cells were again visible under DIC. Confocal analysis under fluorescence showed green staining in DU145 (panel D) and PC3 (panel H) cells, indicating caspase activity. However, LNCaP (panel L) cells did not stain green, indicating no DEFB 1-induced apoptosis.

In summary, this study provides a functional role for DEFB1 in prostate cancer. Furthermore, these findings indicate that DEFB1 is part of the innate immune system involved in tumor immunity. The data presented here demonstrate that DEFB1 expressed at physiological levels is cytotoxic to AR-hormone refractory prostate cancer cells, but not to AR + hormone sensitive prostate cancer cells and to normal prostate epithelial cells. Given that DEFB1 is constitutively expressed in normal prostate cells, without cytotoxicity, it is likely that advanced AR-prostate cancer cells with different phenotypic characteristics render them susceptible to DEFB1 cytotoxicity. Thus, DEFB1 is a viable therapeutic agent for advanced prostate cancer, and possibly other cancer treatments.

Example 2: SiRNA-mediated knockdown of PAX2 expression results in P53-independent status Of prostate cancer cell death

This example measures the effect of inhibiting expression of PAX2 by RNA interference in prostate cancer cells with different p53 gene status. The results demonstrate that inhibition of PAX2 results in cell death independent of the p53 state, suggesting the presence of other tumor suppressor genes or cell death pathways inhibited by PAX2 in prostate cancer.

Materials and methods

siRNA silencing of PAX 2: to achieve efficient gene silencing, four pools of complementary short interfering ribonucleotides (sirnas) targeted to human PAX2mRNA (accession No. NM — 003989.1) were synthesized (Dharmacon Research, Lafayette, CO, USA). The second pool of four siRNAs served as an internal control for testing the specificity of the PAX2 siRNA. Two of the synthesized sequences targeted GL2 luciferase mRNA (accession number X65324), and two were non-sequence specific (table 3). For annealing of siRNA, 35M single strands were incubated in annealing buffer (100mM potassium acetate, 30mM HEPES-KOH, pH7.4, 2mM magnesium acetate) for 1 min at 90 ℃ followed by 1 h at 37 ℃.

TABLE 3 PAX2siRNA sequences

Inhibition of PAX2 protein expression using four siRNA pools

Sense (5 '-3')

Sequence a 5'-GAAGUCAAGUCGAGUCUAUUU-3' SEQ ID NO: 7

Sequence B5'-GAGGAAACGUGAUGAAGAUUU-3' SEQ ID NO: 8

Sequence C5'-GGACAAGAUUGCUGAAUACUU-3' SEQ ID NO: 9

Sequence D5'-CAUCAGAGCACAUCAAAUCUU-3' SEQ ID NO: 10

Antisense (5 '-3')

Sequence a 5'-AUAGACUCGACUUGACUUCUU-3' SEQ ID NO: 3

Sequence B5'-AUCUUCAUCACGUUUCCUCUU-3' SEQ ID NO: 4

Sequence C5'-GUAUUC AGC AAUCUUGUCCUU-3' SEQ ID NO: 5

Sequence D5'-GAUUUGAUGUGCUCUGAUGUU-3' SEQ ID NO: 6

Western analysis: briefly, cells were collected by trypsinization and washed twice with PBS. Lysis buffer was prepared according to the manufacturer (Sigma) instructions and then added to the cells. After incubation for 15 minutes at 4 ℃ on an orbital shaker, cell lysates were collected and pelleted with cell debris by centrifugation at 12000x g for 10 minutes. The protein-containing supernatant was collected and quantified. Next, 25. mu.g of the protein extract was loaded onto 8-16% gradient SDS-PAGE (Novex). After electrophoresis, the proteins were transferred to PVDF membrane and then blocked with 5% skimmed milk powder in TTBS (0.05% Tween20 and 100mM Tris-Cl) for 1 hour. Blots were probed with rabbit anti-PAX 2 primary antibody (Zymed, San Francisco, Calif.) diluted 1: 2000. After washing, the membranes were incubated with horseradish peroxidase (HRP) -conjugated anti-rabbit antibody (dilution 1: 5000; Sigma) and signal detection was visualized on AlphaInotech Fluorchem 8900 using a chemiluminescent reagent (Pierce). As a control, the blot was cleared and re-probed with mouse anti- β -actin primary antibody (1: 5000; Sigma-Aldrich) and HRP-conjugated anti-mouse secondary antibody (1: 5000; Sigma-Aldrich) and signal detection was visualized again.

Phase contrast microscopy: the effect of PAX2 knockdown on cell growth was analyzed by phase contrast microscopy as described in example 1.

MTT cytotoxicity assay: DU145, PC3 and LNCaP cells (1X 10) were transfected with 0.5. mu.g PAX2siRNA pools or control siRNA pools using Codebraker transfection reagent according to the manufacturer's manual (Promega)⁵). Next, the cell suspension was diluted and concentrated at 1-5X10 per well³Cells were seeded in 96-well plates and allowed to grow for 2, 4 or 6 days. Following incubation, cell viability was determined by measuring the conversion of 3- (4, 5-dimethylthiazol-2 yl) -2, 5-diphenyltetrazolium bromide, mtt, (promega), to the colored formazan product. The absorbance was read at 540nm on a multi-well scanning spectrophotometer.

Detecting pan-caspase: detection of caspase activity in prostate cancer cell lines was performed as described in example 1.

Quantitative real-time RT-PCR: to verify gene expression after PAX2siRNA treatment of PC3, DU145 and LNCaP cell lines, quantitative real-time RT-PCR was performed as described in example 1. Primer pairs for GAPDH (control gene), BAX, BID and BAD were as follows:

sense (5 '-3')

GAPDH 5’-CCACCCATGGCAAATTCCATGGCA-3’SEQ ED NO：55

BAD 5’-CTCAGGCCTATGCAAAAAGAGGA-3’SEQ ED NO：57

BID 5’-AACCTACGCACCTACGTGAGGAG-3’SEQ ED NO：59

BAX 5’-GACACCTGAGCTGACCTTGG-3’SEQ DD NO：61

Antisense (5 '-3')

GAPDH 5’-TCTAGACGGCAGGTCAGGTCAACC-3’SEQ ID NO：56

BAD 5’-GCCCTCCCTCCAAAGGAGAC-3’SEQ ID NO：58

BID 5’-CGTTCAGTCCATCCCATTTCTG-3’SEQ ID NO：60

BAX 5’-GAGGAAGTCCAGTGTCCAGC-3’SEQ ID NO：62

Reactions were performed on MicroAmp Optical 96-well reaction plates (PE Biosystems). Under standard conditions, 40 PCR cycles were performed using an annealing temperature of 60 ℃. Quantification was determined by the number of cycles at which exponential amplification started (threshold) and the average obtained from three replicates. There is an inverse relationship between the signal level and the threshold. In addition, GAPDH serves as a housekeeping gene to normalize the initial content of total cDNA. Gene expression was calculated as the relative expression rate between the pro-apoptotic gene and GAPDH. All reactions were performed in triplicate.

Results

siRNA inhibition of PAX2 protein: to confirm that the siRNA effectively targets PAX2mRNA, Western analysis was performed to monitor the expression levels of PAX2 protein during the 6 day treatment. One round of cells were transfected with PAX2siRNA pools. Specific targeting of PAX2mRNA was confirmed by showing the results of PAX2 protein knockdown in day 4 DU145 (fig. 6, panel a) and day 6 PC3 (fig. 6, panel B).

Knock-down of PAX2 inhibited prostate cancer cell growth: cells were analyzed after 6 days of treatment with medium alone, negative control non-specific siRNA or PAX2siRNA (fig. 7). DU145 (panel a), PC3 (panel D) and LNCaP (panel G) cells all reached at least 90% confluence in medium-only culture dishes. Treatment of DU145 (panel B), PC3 (panel E) and LNCaP (panel H) with negative control non-specific siRNA had no effect on cell growth, and cells reached confluence again after 6 days. However, treatment with PAX2siRNA resulted in a significant reduction in cell number. DU145 cells were approximately 15% confluent (panel C), and PC3 cells were only 10% confluent (panel F). LNCaP cells were 5% confluent after siRNA treatment.

Cytotoxicity assay: after 2, 4 and 6 days of exposure, cell viability was measured and expressed as the ratio of the absorbance at 570-630nm of the treated cells divided by the absorbance of the untreated control cells (FIG. 8). Relative cell viability after 2 days of treatment: LNCaP 77%, DU145 82%, and PC3 78%. After 4 days, relative cell viability: LNCaP 46%, DU145 53%, and PC3 63%. After 6 days of treatment, the relative cell viability decreased to: LNCaP 31%, PC3 37%, and DU145 53%. As a negative control, cell viability was measured 6 days after treatment with negative control non-specific siRNA or transfection reagent alone. For both conditions, the change in viability of the cells was not statistically significant compared to normal growth medium.

Detecting pan-caspase: caspase activity was detected by confocal laser microscopy analysis. DU145, PC3 and LNCaP cells were treated with PAX2siRNA and activity was monitored based on the binding of FAM-labeled peptides to caspases in actively apoptotic cells (green fluorescence). Analysis of medium-only treated cells under DIC showed the presence of viable DU145(a), PC3(E) and lncap (i) cells at 0 hours (fig. 9). Excitation at 488nm by confocal laser did not produce detectable green staining, indicating no caspase activity in untreated DU145(B), PC3(F) or lncap (j). After 4 days of treatment with PAX2siRNA, DU145(C), PC3(G) and LNCaP (K) cells were visualized again under DIC. Under fluorescence, treated DU145(D), PC3(H) and lncap (l) cells showed green staining indicating caspase activity.

The effect of PAX2 inhibition on pro-apoptotic factors: DU145, PC3 and LNCaP cells were treated with siRNA against PAX2 for 6 days and pro-apoptotic gene expression dependent and independent of p53 transcriptional regulation was measured to monitor the cell death pathway. For BAX: a 1.81 fold increase in LNCaP; increase 2.73 times in DU 145; and a 1.87 fold increase in PC3 (fig. 10, panel a). Increased expression levels of BID: LNCaP is 1.38 times; and DU145 by a factor of 1.77 (fig. 10, panel B). However, after treatment, BID expression levels in PC3 decreased 1.44-fold (fig. 10, panel C). BAD analysis showed increased expression: LNCaP is 2.0 times; DU145 is 1.38 times and PC3 bit is 1.58 times.

These results demonstrate that the survival of prostate cancer cells is dependent on PAX2 expression. After activation of p53 by PAX2 knockdown in p 53-expressing cell line LNCaP, p 53-mutated cell line DU145 and p 53-deleted cell line PC3, caspase activity was detected in all three cell lines, indicating initiation of programmed cell death. BAX expression in all three cell lines was independent of p53 status upregulation. Following inhibition of PAX2, expression of the pro-apoptotic factor BAD was also increased in all three cell lines. BID expression increased in LNCaP and DU145 but decreased in PC3 after treatment with PAX2 siRNA. These results indicate that the cell death observed in prostate cancer is affected but independent of p53 expression. Initiation of apoptosis of prostate cancer cells by a different cell death pathway unrelated to p53 status suggests PAX2 inhibits other tumor suppressors.

Example 3: inhibition of the PAX2 oncogene results in DEFB1- Mediated death

The identification of tumor-specific molecules for use as targets for the development of novel cancer drugs is considered a major goal of cancer research. Example 1 demonstrates that there is a high frequency of impaired DEFB1 expression in prostate cancer, and that induction of DEFB1 expression leads to rapid apoptosis in androgen receptor negative stages of prostate cancer. These data suggest a role for DEFB1 in prostate tumor suppression. Furthermore, given that it is a naturally occurring component of the immune system of normal prostate epithelial cells, DEFB1 is expected to be a viable therapeutic agent with little to no side effects. Example 2 demonstrates that inhibition of PAX2 expression results in p 53-independent prostate cancer cell death. These data indicate the presence of other pro-apoptotic factors or tumor suppressors inhibited by PAX 2. In addition, the data indicate that the oncogene factor PAX2 overexpressed in prostate cancer is a transcriptional repressor of DEFB 1. The objective of this study was to determine whether impaired DEFB1 expression was due to aberrant expression of the PAX2 oncogene, and whether inhibition of PAX2 could lead to DEFB1 expression and DEFB 1-mediated cell death (fig. 11).

Materials and methods

RNA isolation and quantitative RT-PCR: RNA isolation and quantitative RT-PCR of DEFB1 were performed as described in example 1.

Production of DEFB1 reporter construct: pGL3 luciferase reporter plasmid was used to monitor DEFB1 reporter activity. Here, a region of 160 bases upstream of the transcription start site of DEFB1 and includes the DEFB1 TATA box. The region also includes the CCTTG (SEQ ID NO: 1) sequence necessary for PAX2 binding. PCR primers were designed containing Kpn1 and Nhe1 restriction sites. The DEFB1 promoter PCR product was restriction digested with Kpn1 and Nhe1 and ligated to the similarly restriction digested pGL3 plasmid (FIG. 12). The constructs were transfected into E.coli, and single clones were selected and amplified. Plasmids were isolated and the sequence integrity of the DEFB1/pGL3 construct was verified by automated sequencing.

Luciferase reporter assay: at this time, 1 μ g of DEFB1 reporter construct or control pGL3 plasmid was transfected to 1X10⁶DU145 cells. Then, 0.5x10³Cells were seeded into each well of a 96-well plate and allowed to grow overnight. Fresh medium containing PAX2siRNA or medium alone was then added and cells were incubated for 48 hours. Luciferase was detected by the BrightGlo kit according to the manufacturer's manual (Promega) and the plates were read with a Veritas automated 96-well luminescence detector. Promoter activity is expressed as relative luminescence.

Membrane permeability analysis: acridine Orange (AO)/ethidium bromide (EtBr) double staining was performed to identify changes in cell membrane integrity, and apoptotic cells were identified by staining condensed chromatin. AO stained live cells as well as early apoptotic cells, whereas EtBr stained late apoptotic cells with impaired membrane permeability. Briefly, cells were seeded on 2-chamber culture slides (BD Falcon, USA). Cells transfected with empty pIND plasmid/pvgRXR or pINDDEFB1/pvgRXR were induced for 24 or 48 hours in medium containing 10. mu.M pinsterone A. Control cells were supplied with fresh medium at 24 and 48 hours. To determine the effect of PAX2 inhibition on membrane integrity, individual slides containing DU145, PC3 and LNCaP were treated with PAX2siRNA and incubated for 4 days. After this, the cells were washed once with PBS and stained for 5 minutes with a mixture (1: 1) of 2ml AO (Sigma, USA) and EtBr (Promega, USA) (5ug/ml) solutions. After staining, the cells were washed again with PBS. Fluorescence was observed by a Zeiss LSM 5 Pascal Vario 2 laser scanning confocal microscope (Carl Zeiss Jena, Germany). The excitation color wheel contains BS505-530 (green) and LP560 (red) filter assemblies that allow green light emitted from the AO to be split into the green channel and red light from the EtBr to be split into the red channel. The laser power output and gain control settings were the same for each independent experiment between control and DEFB 1-induced cells. The excitation wavelengths provided by a Kr/Ar mixed gas laser were 543nm (for AO) and 488nm (for EtBr). The slides were analyzed at 40 x magnification and the digital images were saved as uncompressed TIFF files and exported to Photoshop CS software (Adobe Systems, San Jose, CA) for image processing and hard copy display.

ChIP analysis of PAX 2: chromatin immunoprecipitation (ChIP) allows the identification of binding sites for DNA-binding proteins based on the in vivo occupancy of promoters by transcription factors and enrichment of transcription factors bound to chromatin by immunoprecipitation. Using the modification described by Farnham laboratories; also online address http:// mcardle. oncology. wisc. edu/farnham /). DU145 and PC3 cell lines over-expressed PAX2 protein, but did not express DEFB 1. Cells were incubated with 1.0% formaldehyde in PBS for 10 minutes to crosslink protein and DNA. The samples were then sonicated to generate DNA of average length 600 bp. Sonicated chromatin pre-cleared with protein a Dynabead was incubated with PAX 2-specific antibody or "no antibody" control [ isotype matched control antibody ]. The washed immunoprecipitates were then collected. After reversal of the cross-linking, DEFB1 was determined to be present in the PAX 2-immunoprecipitated samples by PCR analysis of DNA using promoter-specific primers. Primers were designed to amplify a 160bp region just upstream of the DEFB1 mRNA start site, which comprises the DEFB1 TATA box and a functional CCTTG (SEQ ID NO: 1) PAX2 recognition site. For these studies, positive controls included PCR of aliquots of input chromatin (prior to immunoprecipitation, but with cross-linking reversed). All steps are carried out in the presence of a protease inhibitor.

Results

siRNA inhibition of PAX2 increased DEFB1 expression: QRT-PCR analysis of DEFB1 expression prior to siRNA treatment showed relative expression levels: DU145 is 0.00097, PC3 is 0.00001, and LNCaP is 0.00004 (fig. 13). After siRNA knockdown of PAX2, relative expression: DU145 is 0.03294 (338 fold increase), PC3 is 0.00020 (22.2 fold increase), and LNCaP is 0.00019 (4.92 fold increase). As a negative control, the PAX 2-deficient human prostate-level cell line (hPrEC) showed expression levels: 0.00687 before treatment and 0.00661 after siRNA treatment confirmed that DEFB1 expression was not statistically changed.

siRNA inhibition of PAX2 enhanced DEFB1 promoter activity: FIG. 14 shows that inhibition of PAX2 results in enhanced DEFB1 promoter activity. The PC3 promoter/pGL 3 and DU145 promoter/pGL 3 constructs were generated and transfected into PC3 and DU145 cells, respectively. The promoter activity before and after inhibition of PAX2 by siRNA treatment was compared. After treatment, DEFB1 promoter activity was enhanced: DU145 by a factor of 2.65 and PC3 by a factor of 3.78.

DEFB1 causes cell membrane permeability: membrane integrity was monitored by confocal analysis. As shown in fig. 15, intact cells stained green due to membrane-permeable AO. In addition, cells with damaged cytoplasmic membranes stained red with membrane-impermeable EtBr. Uninduced DU145(a) and PC3(D) cells stained positive for AO and emitted green, but no EtBr staining. However, DEFB1 induction in DU145(B) and PC3(E) resulted in EtBr aggregation in the cytoplasm at 24 hours, as indicated by red staining. At 48 hours, DU145(C) and PC3(F) had condensed nuclei and appeared yellow due to the presence of green and red stains from AO and EtBr aggregates, respectively.

Inhibition of PAX2 resulted in membrane permeability: cells were treated with PAX2siRNA for 4 days and again membrane integrity was monitored by confocal analysis. As shown in fig. 16, DU145 and PC3 had condensed nuclei and showed yellow color. However, both the cytoplasm and nucleus of LNCaP cells remained green after siRNA treatment. The red staining around the cells indicates that the integrity of the cell membrane is maintained. These findings indicate that inhibition of PAX2 results in specific DEFB 1-mediated cell death of DU145 and PC3, but not LNCaP cell death. The observed death in LNCaP was due to transactivation of wild-type p53 in LNCaP after PAX2 inhibition.

Binding of PAX2 to DEFB1 promoter: ChIP analysis was performed on DU145 and PC3 cells to determine whether the PAX2 transcriptional repressor binds to the DEFB1 promoter (fig. 17). Lane 1 contains 100bp molecular weight markers. Lane 2 is a positive control representing the 160bp region of DEFB1 promoter amplified from DU145 prior to the crosslinking and immunoprecipitation reactions. Lane 3 is a negative control, representing PCR performed without DNA. Lanes 4 and 5 are negative controls representing PCR with IgG immunoprecipitation from cross-linked DU145 and PC3, respectively. PCR amplification of 25pg DNA (lanes 6 and 8) and 50pg DNA (lanes 7 and 9) immunoprecipitated with anti-PAX 2 antibody after cross-linking showed 160bp promoter fragments in DU145 and PC3, respectively.

FIG. 18 shows the predicted structures of PrdPD and PrdHD and DNA. The synergy of the structures that PrdPD binds to DNA (Xu et al, 1995) and PrdHD binds to DNA (Wilson et al, 1995) was used to construct a model for the binding of both domains to the PH0 site. As shown, the binding sites are adjacent to each other in a particular orientation. The RED domain is oriented based on the PrdPD crystal structure.

These results demonstrate that the oncogene factor PAX2 suppresses DEFB1 expression. Repression occurs at the transcriptional level. Furthermore, computational analysis of the DEFB1 promoter revealed the presence of a CCTTG (SEQ ID NO: 1) DNA binding site for the PAX2 transcriptional repressor in close proximity to the DEFB1 TATA box (FIG. 1). One characteristic of defensin cytotoxicity is the disruption of membrane integrity. These results indicate that abnormal expression of DEFB1 in prostate cancer cells results in impaired membrane potential due to damage to the cell membrane. The same phenomenon was observed after inhibition of PAX2 protein expression. Thus, repression of PAX2 expression or function results in the re-establishment of DEFB1 expression and subsequent DEFB 1-mediated cell death. Likewise, the present results establish the use of DEFB1 as a targeted therapy for prostate cancer treatment by innate immunity, and possibly other cancer treatments.

Example 4: effect of DEFB1 expression on engraftment of tumor cells

The anti-tumor capacity of DEFB1 was assessed by injecting DEFB 1-overexpressing tumor cells into nude mice. DEFB1 was cloned into a pBI-EGFP vector (which has a bi-directional tetracycline responsive promoter). The Tet-Off cell line was generated by transfecting pTet-Off into DU145, PC3 and LNCaP cells and selecting with G418. The pBI-EGFP-DEFB1 plasmid was co-transfected with pTK-Hyg into a Tet-off cell line and selected with hygromycin. Only single cell suspensions with > 90% viability were used. Each animal received approximately 500,000 cells and was administered subcutaneously to the right flank of female nude mice. There were two groups, the control group injected with vector-only clones, and one with clones overexpressing DEFB 1. 35 mice per group were determined by a statistician. Animals were weighed twice weekly, tumor growth was monitored by calipers, and tumor volume was determined using the following equation: volume 0.5x (width) 2x long. When the tumor size reaches 2mm³Or six months after implantation, all animals passed excess CO₂Sacrifice; tumors were excised, weighed and preserved in neutral buffered formalin for pathological examination. The difference in tumor growth between the two groups was characterized by a summary statistical table and graphical display. Statistical significance was assessed using a t-test or non-parametric equivalent.

Example 5: effect of PAX2siRNA on Implantation of tumor cells

Hairpin PAX2siRNA template oligonucleotides utilized in vitro studies were used to detect effects on the upregulation of DEFB1 expression in vivo. The sense and antisense strands (see Table 3) were annealed and cloned into the pSilencer 2.1U 6 hygro siRNA expression vector (Ambion) under the control of the human U6RNA pol III promoter. The cloned plasmids were sequenced, validated and transfected into PC3, DU145 and LNCap cell lines. Scrambled shRNA was cloned and used as a negative control for this study. Clones resistant to hygromycin were selected. Cells were introduced subcutaneously into mice and tumor growth was monitored as described above.

Example 6: effect of small molecule inhibitors binding to PAX2 on engraftment of tumor cells

The DNA recognition sequence to which PAX2 binds is located between nucleotides-75 and-71 (+1 means transcription initiation site) of the DEFB1 promoter. Short oligonucleotides complementary to the PAX2 DNA-binding domain are provided. Examples of such oligonucleotides include 20-mer and 40-mer oligonucleotides containing recognition sequences for CCTTG (SEQ ID NO: 1) provided below. These lengths are randomly selected and other lengths are expected to be effective as blockers of binding. As a negative control, an oligonucleotide having a scrambled sequence (CTCTG) (SEQ ID NO: 22) was designed to verify specificity. The oligonucleotides were transfected into prostate cancer cells and HPrEC cells using lipofectamine reagent or Codebreaker transfection reagent (Promega, Inc). For confirming the DNA-protein interaction, the double-stranded oligonucleotide [ 2 ]³²P]dCTP was labeled and subjected to electrophoretic mobility measurement. In addition, DEFB1 expression was monitored by QRT-PCR and Western analysis after treatment with oligonucleotides. Finally, cell death was detected by MTT assay and flow cytometry as previously described.

Recognition sequence # 1: CTCCCTTCAGTTCCGTCGAC (SEQ ID NO: 18)

Recognition sequence # 2: CTCCCTTCACCTTGGTCGAC (SEQ ID NO: 19)

Out-of-order sequence # 1: CTCCCTTCACTCTGGTCGAC (SEQ ID NO: 23)

Recognition sequence # 3:

ACTGTGGCACCTCCCTTCAGTTCCGTCGACGAGGTTGTGC(SEQ ID NO：20)

recognition sequence # 4:

ACTGTGGCACCTCCCTTCACCTTGGTCGACGAGGTTGTGC(SEQ ID NO：21)

out-of-order sequence # 2:

ACTGTGGCACCTCCCTTCACTCTGGTCGACGAGGTTGTGC(SEQ ID NO：24)

other examples of oligonucleotides of the invention include:

recognition sequence # 1: 5'-AGAAGTTCACCCTTGACTGT-3' (SEQ ID NO: 25)

Recognition sequence # 2: 5'-AGAAGTTCACGTTCCACTGT-3' (SEQ ID NO: 26)

Sequence # 1: 5'-AGAAGTTCACGCTCTACTGT-3' (SEQ ID NO: 27)

Recognition sequence # 3:

5’-TTAGCGATTAGAAGTTCACCCTTGACTGTGGCACCTCCC-3’(SEQ ID NO：28)

recognition sequence # 4:

5’-GTTAGCGATTAGAAGTTCACGTTCCACTGTGGCACCTCCC-3’(SEQ ID NO：29)

out-of-order sequence # 2:

5’-GTTAGCGATTAGAAGTTCACGCTCTACTGTGGCACCTCCC-3’(SEQ ID NO：30)

the set of selectable inhibitory oligonucleotides represents the recognition sequences for the PAX2 binding domain and homeobox. These include the actual sequence from the DEFB1 promoter.

The PAX2 gene is essential for the growth and survival of various cancer cells, including the prostate. In addition, inhibition of PAX2 expression resulted in cell death mediated by the innate immune component DEFB 1. Repression of DEFB1 expression and activity was achieved by binding of the PAX2 protein to the CCTTG (SEQ ID NO: 1) recognition site of the DEFB1 promoter. Thus, this pathway provides a viable therapeutic target for prostate cancer treatment. In the present method, the sequence binds to the PAX2 DNA binding site and blocks binding of PAX2 to the DEFB1 promoter, thereby allowing DEFB1 expression and activity. The oligonucleotide sequences and experiments described above are examples of other PAX2 inhibitor drugs and demonstrate models for the design of other PAX2 inhibitor drugs.

Whereas the CCTTG (SEQ ID NO: 1) sequence exists in interleukin-3, interleukin-4, insulin receptor, etc. PAX2 also regulates their expression and activity. Thus, the PAX2 inhibitors disclosed herein are useful in a number of other diseases, including those directly associated with inflammation, including prostatitis and Benign Prostatic Hyperplasia (BPH).

Example 7: impaired DEFB1 expression leads to increased tumorigenesis

Generation of functionally impaired mice: the Cre/loxP system is useful in elucidating the underlying molecular mechanisms of prostate carcinogenesis. At this time, DEFB1 Cre conditional KO was used for induced disruption in the prostate. DEFB1 Cre conditional KO involves generating a targeting vector containing a loxP site flanking the DEFB1 coding exon, targeting ES cells with the vector, and generating germ line chimeric mice from these targeted ES cells. The hybrids were mated to the prostate-specific Cre transgene and the hybrid hybrids were used to generate prostate-specific DEFB1 KO mice. Four genotoxic compounds have been found to induce rodent prostate cancer: N-methyl-N-nitrosourea (MNU), N-nitrosobis 2-oxypropylamine (BOP), 3, 2X-dimethyl-4-amino-biphenyl (MAB), and 2-amino-1-methyl-6-phenylimidazo-4, 5-bipyridine (PhIP). DEFB1 transgenic mice were treated with these carcinogenic compounds by intragastric administration or intravenous injection for prostate adenoma and adenocarcinoma induction studies. Differences in tumor growth and changes in gene expression in prostate samples were studied by histology, immunohistology, mRNA and protein analysis.

Generation of GOF mice: for PAX 2-induced GOF mice, 5-week-old PAX2 GOF (double transgene) and wild-type (single transgene) littermate mouse doxycycline (Dox) were administered to induce prostate-specific PAX2 expression. Briefly, PROBASIN-rtTA single transgenic mice (prostate cell-specific expression of tetracycline-dependent rtTA inducer) were crossed with our PAX2 transgenic induction line (responder line). For induction, double transgenic mice were fed Dox (500mg/L, freshly prepared, twice a week) by drinking water. Initial experiments utilized transgenic established lines (founder lines) in double transgenic mice, demonstrating low background levels, good inducibility, and cell type-specific expression of PAX2 and the EGFP reporter. With respect to the size of the experimental groups, 5-7 individuals of matched age and gender in each group (wild type and GOF) were statistically significant. For all animals in this study, prostate tissue was initially collected every other week for analysis and comparison to determine the time parameter for carcinogenesis.

PCR genotyping, RT-PCR and qPCR: the PROBASIN-rtTA transgenic mice were genotyped using the following PCR primers and conditions:

PROBASIN5 (Forward) 5'-ACTGCCCATTGCCCAAACAC-3'

(SEQ ID NO：31)；

RTTA3 (reverse) 5'-AAAATCTTGCCAGCTTTCCCC-3'

(SEQ ED NO：32)；

Denaturation at 95 ℃ for 5 min; then, 30 cycles were performed at 95 ℃ for 30 seconds, 57 ℃ for 30 seconds, and 72 ℃ for 30 seconds; followed by extension at 72 ℃ for 5 minutes, resulting in a 600bp product. The PAX 2-inducible transgenic mice were genotyped using the following PCR primers and conditions:

PAX2For 5’-GTCGGTTACGGAGCGGACCGGAG-3’

(SEQ ID NO：33)；

Rev5’IRES 5’-TAACATATAGACAAACGCACACCG-3’

(SEQ ID NO：34)；

denaturation at 95 ℃ for 5 min; then, 34 cycles of 95 ℃, 30 seconds, 63 ℃, 30 seconds, 72 ℃ and 30 seconds are carried out; followed by extension at 72 ℃ for 5 min, yielding a 460bp product.

Genotyping of immortalized mouse hemizygous molecules was performed using the following PCR primers and conditions: immol1, 5'-GCGCTTGTGTC GCCATTGTATTC-3' (SEQ ID NO: 35); immol2, 5'-GTCACACCACAGAAGTAAGGTTCC-3' (SEQ ID NO: 36); 30 cycles at 94 ℃ for 30 seconds, 58 ℃ for 1 minute, 72 ℃ for 1 minute and 30 seconds yielded a transgenic band of-lkb. For genotyping of PAX2 knockout mice, the following PCR primers and conditions were used:

PAX2 For 5’-GTCGGTTACGGAGCGGACCGGAG-3’(SEQ ID NO：37)；

PAX2 Rev 5’-C AC AGAGC ATTGGCG ATCTCG ATGC-3’(SEQ ID NO：38)；

36 cycles of 94 ℃ for 1 min, 65 ℃ for 1 min, 72 ℃ for 30 sec gave bands of-280 bp.

DEFB1 peptide animal studies: by 10⁶Viable PC3 cells were injected subcutaneously into scapulae in six-week-old male athymic (nude) mice (purchased from Charles River Laboratories). One week after injection, animals were randomly assigned to one of three groups: group I-control; group II-In peritoneal injection of DEFB1, 100 μ g/day, 5 days a week for 2-14 weeks; group III-In P.I. injection of DEFB1 at 100 mg/day for 5 days a week for 8-14 weeks. Animals were housed in a sterile room, four cages, and observed daily. Every 10 days, tumors were measured by using calipers, and tumor volumes were calculated by the following formula: v ═ 2 (LxW 2).

Example 8: targeting PAX2 expression for chemoprevention of intraepithelial neoplasia and cancer

Cancer chemoprevention is defined as the prevention of cancer or treatment at a precancerous state or earlier. The long-term progression of invasive cancer is a major scientific opportunity, but is also an economic barrier to show the clinical benefit of alternative chemopreventive drugs. Therefore, an important component of the development of chemopreventive agents in recent years is the identification of endpoints or biomarkers that accurately predict the clinical benefit of an agent at an earlier stage (than cancer) or reduce the effects of the incidence of cancer. In many cancers, IEN is an early endpoint such as prostate cancer. Given that the PAX2/DEFB1 pathway may be uncontrolled during IEN, and perhaps at earlier histopathological states, this makes it a powerful predictive biomarker and excellent target for cancer chemoprevention. A number of compounds are given that suppress PAX2 and increase DEFB1 expression, which may have utility as a prostate cancer chemopreventive agent.

As shown in table 1, the PAX2 gene is expressed in many cancers. In addition, several cancers have been shown to have aberrant PAX2 expression (fig. 20). Angiotensin ii (angii) is a major regulator of blood pressure and cardiovascular homeostasis, and is considered to be a potent mitogen. AngII mediates its biological effects through binding to two subtypes of the receptor, angiotensin type I receptor (AT1R) and angiotensin type II receptor (AT2R), which belong to the superfamily of G-protein coupled receptors but have different tissue distribution and intracellular signal transduction pathways. In addition to its effects on blood pressure, AngII has also been shown to play a role in a variety of pathological conditions including tissue reconstruction such as wound healing, myocardial hypertrophy and development. In fact, recent studies have shown the local expression of several components of the renin-angiotensin system (RAS) in various cancer cells or tissues, including the prostate. Upregulation of AT1R provides advantages for cancer cells that have learned to evade apoptotic and growth regulatory elements. A large number of cancers have been shown to express PAX2 aberrantly to date. Chemoprevention by targeting expression of PAX2 may have a significant impact on cancer-related death.

Materials and methods

Cell culture: cell lines DU145, LnCap and PC3 were cultured as described in example 1. On the basis of prostate epithelial cellshPrEC cells were cultured in media (Cambrex Bio Science, Inc., Walkersville, Md.) and maintained at 37 ℃ and 5% CO₂The following steps.

Reagents and treatment: cells were treated with 5 or 10uM AngII, 5uM ATR1 antagonist Los, 5uM matr2 antagonist PD123319, 25uM MEK inhibitor U0126, 20uM MEK/ERK inhibitor PD98059, or 250 μ M AMP kinase inducer AICAR.

Western analysis: western blots were performed as described in example 2. Primary blots were probed with (anti-PAX 2, anti-phospho-PAX 2, anti-JNK, anti-phospho-JNK, anti-ERK 1/2 or anti-phospho-ERK 1/2) (Zymed, San Francisco, Calif.) diluted 1: 1000-2000. After washing, the membranes were incubated with horseradish peroxidase (HRP) -conjugated anti-rabbit antibody (dilution 1: 5000; Sigma) and signal detection was visualized on Alpha Inotech Fluorchem 8900 using a chemiluminescent reagent (Pierce). As a control, the blot was cleared and re-probed with mouse anti- β -actin primary antibody (1: 5000; Sigma-Aldrich) and HRP-conjugated anti-mouse secondary antibody (1: 5000; Sigma-Aldrich) and signal detection was visualized again.

QRT-PCR analysis: to verify the gene expression changes following PAX2 knockdown in PC3 and DU145 prostate cancer cell lines and hPrEC normal prostate epithelial cells, quantitative real-time RT-PCR was performed as described in example 1. Under standard conditions, 40 PCR cycles were performed using an annealing temperature of 60 ℃. Quantification was determined by the number of cycles at which exponential amplification started (threshold) and the average obtained from three replicates. There is an inverse relationship between the signal level and the threshold. In addition, GAPDH was used as a housekeeping gene to normalize the initial content of total cDNA. Relative expression was calculated as the ratio between each gene and GAPDH. All reactions were performed in triplicate.

Thymidine incorporation method: by mixing³H]Thymidine nucleotide ([ 2 ]³H]TdR) incorporated into DNA to determine cell proliferation. Mix 0.5x10⁶DU145 cell suspension of cells/well was seeded into its appropriate medium. Cells were incubated for 72 hours in the presence or absence of the indicated concentrations of AngII. Exposure of cells to the same Medium37kBq/ml [ methyl-³H]Thymidine for 6 hours. Adherent cells were fixed by 5% trichloroacetic acid and lysed in SDS/NaOH lysis buffer overnight. Radioactivity was measured by a Beckman LS3801 scintillation counter (Canada). Suspension cell cultures were collected by a cell harvester (Packard instrument co., Meriden, CT) and radioactivity was measured by a 1450microbeta liquid scintillation counter (PerkinElmer Life Sciences).

Results

To investigate the effect of AngII on PAX2 expression in DU145 prostate cancer cells, PAX2 expression was measured after 30 minutes to 48 hours of treatment with AngII. As shown in fig. 21, PAX2 expression gradually increased over time after AngII treatment. Blocking RAS signaling by treatment of DU145 with Los significantly reduced PAX2 expression. At this time, PAX2 was expressed after Los treatment compared to untreated control DU145 cells: 37% at 48 hours, and 50% at 72 hours (fig. 22A). The AT2R receptor is known to antagonize the effects of AT 1R. Thus, the effect of blocking the AT2R receptor on PAX2 expression was examined. Treatment of DU145 with the AT2R blocker PD123319 resulted in increased PAX2 expression: 7 times for 48 hours, and 8 times for 96 hours (fig. 22B). Collectively, these findings demonstrate that PAX2 expression is regulated by the ATR1 receptor.

AngII is known to directly affect the proliferation of prostate cancer cells through AT 1R-mediated activation of MAPK and STAT3 phosphorylation. Treatment of DU145 with AngII resulted in a2 to 3 fold increase in proliferation rate (fig. 23). However, treatment with Los reduced the proliferation rate by 50%. Furthermore, blockade of AT1R receptor by pre-treatment with Los for 30 min suppressed the effect of AngII on proliferation.

In order to further test the role of AT1R signal transduction in regulating the expression and activation of PAX2, the influence of various components blocking the MAP kinase signal transduction pathway on the expression of PAX2 was tested. At this time, treatment of DU145 cells with MEK inhibitor U0126 resulted in a significant reduction in PAX2 expression (fig. 24). Furthermore, treatment with the MEK/ERK inhibitor PD98059 also resulted in a reduction in PAX 2. Treatment of DU145 cells with Los had no effect on ERK protein levels, but decreased the amount of phospho-ERK (fig. 25A). However, treatment of DU145 with Los resulted in a significant reduction in PAX2 expression. Similar results were observed with U0126 and PD98059 (fig. 25B). PAX2 expression is also known to be regulated by STAT3, STAT3 being a downstream target of ERK. Treatment of DU145 with Los, U0126 and PD98059 reduced the level of phospho-STAT 3 protein (fig. 25C). These results demonstrate that PAX2 is regulated by AT1R in prostate cancer cells.

In addition, the effect of AT1R signaling on PAX2 activation by JNK was examined. Treatment of DU145 with Los, U0126 and PD98059 all resulted in a significant decrease or repression of phosphorylated-PAX 2 protein levels (fig. 26A). However, Los and U0126 did not reduce the level of phosphorylated-JNK protein (fig. 26B). Thus, the decrease in phosphorylated-PAX 2 appears to be due to a decrease in PAX2 levels, but not to a decrease in phosphorylation.

5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is widely used as an AMP-kinase activator, which regulates energy balance and responds to metabolic stress. Recent reports have shown that pharmacological agents or AMPK overexpression are utilized to activate the anti-proliferative and pro-apoptotic effects of AMPK. AMPK activation has been shown to induce apoptosis in human gastric, lung, prostate, pancreatic and liver cancer cells, and to enhance oxidative stress-induced apoptosis in mouse neuroblastoma cells by various mechanisms including inhibition of the fatty acid synthesis pathway and induction of stress kinase and caspase 3. In addition, treatment of PC3 prostate cancer cells increased p21, p27, and p53 protein expression, and inhibited the PDK-Akt pathway. All these pathways are regulated directly or indirectly by PAX 2. Treatment of prostate cancer cells with AICAR resulted in expression of PAX2 (fig. 25B) and repression of its activated form, phosphorylated-PAX 2 (fig. 26A). In addition, phosphorylation-STAT 3, which regulates expression of PAX2, was also repressed (fig. 25C).

Finally, aberrant RAS signaling leading to up-regulation and overexpression of PAX2 is presumed to suppress expression of DEFB1 tumor suppressor genes. To investigate this, normal prostate epithelial primary cultures hPrEC were treated with AngII and tested for expression levels of PAX2 and DEFB 1. An inverse relationship was found between DEFB1 and PAX2 expression in normal prostate cells versus prostate cancer cells. As shown in figure 27, untreated hPrEC showed 10% relative PAX2 expression compared to expression in PC3 prostate cancer cells. In contrast, untreated PAX2 showed only 2% relative DEFB1 expression compared to expression in hPrEC. DEFB1 expression was reduced by 35% compared to untreated hPrEC after 72 hours of treatment with 10uM AngII and DEFB1 expression was reduced by 50% compared to untreated hPrEC cells at 96 hours. However, at 72 hours, PAX2 expression was increased by 66%, and PAX2 expression was increased by 79% at 96 hours, compared to untreated hPrEC cells. Furthermore, after 72 hours, the increase in PAX2 expression in hPrEC was 77% of the observed PAX2 levels in PC3 prostate cancer cells. After 96 hours of AngII treatment, PAX2 was expressed as 89% of the expression of PAX2 in PC 3. These results demonstrate that uncontrolled RAS signaling suppresses DEFB1 expression by upregulation of PAX2 expression in prostate cells.

Inhibition of apoptosis is a key pathophysiological factor contributing to cancer development. Despite the major advances in cancer therapy, little progress has been made in treating advanced disease. Given that carcinogenesis is the process of a multi-stage, multi-pathway disease for many years, chemical prevention by the use of drugs or other agents to inhibit, slow down or reverse this process has been considered a very promising area in cancer research. Successful drug therapy for prostate cancer chemoprevention requires the use of therapies that have specific effects on target cells while keeping clinical impact on the host to a minimum with the overall goal of suppressing cancer development. Therefore, understanding the mechanism of early carcinogenesis is critical to determining the efficacy of a particular treatment. The importance of aberrant PAX2 expression and its elimination of apoptosis (which subsequently contributes to tumor formation) suggests that it may be a suitable target for prostate cancer therapy. PAX2 was regulated by AT1R in prostate cancer (fig. 28). Among these, deregulated RAS signaling regulation results in increased expression of the PAX2 oncogene and decreased expression of the DEFB1 tumor repressor. Thus, use of AT1R antagonist reduced PAX2 expression and resulted in increased prostate cancer cell death by re-expression of DEFB1 (fig. 29). These results provide a novel discovery that targeting PAX2 expression through the renin-angiotensin signaling pathway, AMP kinase pathway, or other methods involving inactivation of PAX2 protein (i.e., anti-PAX 2 antibody immunization) may be a viable target for cancer prevention (table 4).

TABLE 4 Compounds used for chemopreventive inhibition of PAX2 expression

This study demonstrates that upregulation of the PAX2 oncogene in prostate cancer is due to deregulated RAS signal transduction. PAX2 expression is regulated by the ERK1/2 signaling pathway mediated through angiotensin type 1 receptors. In addition, blocking AT1R with losartan (Los) suppressed PAX2 expression. In addition, the AMPK activator AICAR has been shown to be a potential inhibitor of PAX 2. Taken together, these studies strongly suggest that these classes of drugs act as potential repressors of PAX2 expression and may ultimately be used as novel chemopreventive agents.

Example 9: PAX2-DEFB1 expression level as a grading tool for prostate tissue and predictor of prostate cancer progression

Materials and methods

QRT-PCR analysis: prostate sections were collected from patients undergoing radical prostatectomy. Following pathological examination, laser capture microdissection was performed to isolate normal, Proliferative Intraepithelial Neoplasia (PIN) and cancerous tissue regions. QRT-PCR was performed as described previously to estimate expression. DEFB1 and PAX2 in each region were expressed, and GAPDH was used as an internal control.

Blood collection and RNA isolation: for QRT-PCR, blood (2.5ml) from each individual was collected into PAX gene according to the manufacturer's manual^TMBlood RNA tube (QIAGEN). Whole blood was mixed thoroughly with PAX gene stabilizing reagent and stored at room temperature for 6 hours prior to RNA extraction. Then use PAXgene according to the manufacturer's instructions^TMTotal RNA was extracted using Blood RNA kit (QIAGEN). To remove contaminating genomic DNA, adsorption to PAXgene was incubated with DNase I (QIAGEN) at 25 deg.C^TM20 min of total RNA sample from Blood RNA system centrifugal columnClock to remove genomic DNA. Total RNA was eluted, quantified and QRT-PCR performed as previously described to compare PAX2 and DEFB1 expression ratios.

Results

QRT-PCR analysis of LCM normal tissue confirmed that patients with relative DEFB1 expression levels greater than 0.005 had lower Gleason scores compared to those patients with relative DEFB1 expression levels less than 0.005 (figure 30). Thus, there is an inverse relationship between DEFB1 expression and Gleason score. In contrast, there was a positive correlation between PAX2 expression and Gleason score in malignant prostate tissue and PIN (fig. 30, panel B).

PAX2 and DEFB1 expression levels in normal, PIN and cancer tissues from each patient were calculated and compared (fig. 31A and 31B). Overall, the expression level range of PAX2 relative to the GAPDH internal control: normal (benign) tissue was 0 to 0.2, PIN was 0.2 to 0.3, and cancer (malignant) tissue was 0.3 to 0.5 (fig. 32). For DEFB1, there is an inverse relationship compared to PAX 2. At this time, the expression level range of DEFB1 relative to the GAPDH internal control: normal (benign) tissue of 0.06 to 0.005, PIN of 0.005 to 0.003, and cancerous (malignant) tissue of 0.003 to 0.001. Thus, a pre-measurement table is disclosed, named Donald Predictor Factor (DPF), which utilizes the PAX2-DEFB1 expression ratio as predictors of benign, pre-cancerous (PIN) and malignant prostate tissue. Based on DPF, tissues with PAX2-DEFB1 ratios of 0 to 39 represent normal (pathologically benign). Tissues with a PAX2-DEFB1 ratio of 40 to 99 represent PIN (pre-cancerous) based on the DPF scale. Finally, tissues with a PAX2-DEFB1 ratio of 100 to 500 represent malignant (low-grade to high-grade cancer).

There is an urgent need for predictive biomarkers for the development of prostate cancer. Prostate cancer is known to have developed long before it can be detected by current screening methods, such as PSA tests or digital rectal examination. It is believed that a reliable test capable of monitoring prostate cancer progression and early onset should greatly reduce mortality through more effective disease management. Disclosed herein are predictors that allow physicians to learn the pathological state of the prostate ahead of time. The DPF assessed a reduction in the expression rate of PAX2-DEFB1 associated with the progression of prostate disease. This robust assessment not only predicts the likelihood of a patient developing prostate cancer, but also confirms early onset before cancer progression. Finally, the tool may allow the physician to isolate patients with more severe disease from those patients who are not.

The identified cancer-specific markers have been used to aid in the identification of Circulating Tumor Cells (CTCs). There is also new evidence that the detection of disseminated tumor cells in peripheral blood can provide important clinical data for tumor staging, prognosis and identification of surrogate markers for the purpose of early assessment of the efficacy of adjuvant therapy. Furthermore, by comparing the gene expression profiles of all circulating cells, the expression of DEFB1 and PAX2 genes, which play a role in "immune surveillance" and "cancer survival", respectively, can be detected, as predictors of early detection of prostate cancer.

Example 10: functional analysis of host defense peptide human beta-defensin-1: it is in cancer Novel understanding of potential Effect

Materials and methods

Cell culture: prostate cancer cell lines were cultured as described in example 1. hPrEC primary cultures were obtained from Cambrex Bio Science, inc. (Walkersville, MD) and cells were grown in prostate epithelial cell basal media.

Tissue samples and laser capture microdissection: prostate tissue was obtained from patients who had provided informed consent prior to radical prostatectomy. Samples were obtained from the Hollings cancer center tumor pool according to protocols approved by the institutional review Board. This includes guidelines for sample processing, sectioning, histological characterization, RNA purification, and PCR amplification. Prostate specimens were received from the surgeon and the pathologist quickly frozen in the OCT compound. Each OCT block was cut to produce a series of sections to be stained and detected. Regions containing benign cells, Prostate Intraepithelial Neoplasia (PIN) and cancer were identified and used to guide us in selecting regions from unstained slides using the Arcturus PixCell II system (Sunnyvale, CA). Caps containing capture material were exposed to 20 μ l of lysate from Arcturus Pico Pure RNA isolation kit and immediately processed. RNA quantification and characterization were assessed using a primer set that produced the 5' amplicon. The primer set included primers for ribosomal protein L32 (298 bases apart 3 'and 5' amplicons), for glucose phosphate isomerase (391 bases apart), and for glucose phosphate isomerase (842 bases apart). These primer sets routinely achieve ratios of 0.95 to 0.80 using samples from a variety of prepared tissues. Other tumor and normal samples were roughly cut by the pathologist, snap-frozen in liquid nitrogen and evaluated for hBD-1 and cMYC expression.

Cloning of hBD-1 Gene: hBD-1 cDNA was generated by reverse transcription PCR using primers generated from the published hBD-1 sequence (accession number U50930) (Ganz, 2004). The PCR primers were designed to contain ClaI and KpnI restriction sites. The hBD-1 PCR product was digested with ClaI and KpnI and ligated into the TA cloning vector. The TA/hBD-1 vector was then transfected into E.coli XL-I Blue strain by heat shock, and single clones were selected and amplified. Plasmids were isolated by Cell Culture dnamidi (Qiagen, Valencia, CA) and sequence integrity was verified by automated sequencing. Then, the hBD-1 gene fragment was ligated into pTRE2 (used as an intermediate vector for targeting purposes) digested with ClaI and KpnI. The hBD-1 insert was excised by digesting the pTRE2/hBD-1 construct with ApaI and KpnI. The insert was ligated into pIND vector (Invitrogen, Carlsbad, CA) of ecdysone-inducible expression system, also double digested with ApaI and KpnI. The constructs were transfected into E.coli and single clones were selected and amplified. The plasmid was isolated and the sequence integrity of pIND/hBD-1 was again verified by automated sequencing.

Transfection: cells (1X 10)⁶) Inoculated into 100-mm petri dishes and incubated overnight. Next, using Lipofectamine 2000(Invitrogen, Carlsbad, Calif.), 1. mu.g of pVgRXR plasmid (expressing heterodimeric ecdysone receptor) and 1. mu.g of pIND/hBD-1 vector construct or pIND/betaGalactosidase (. beta. -gal) control vector co-transfectants cells in Opti-MEM medium (Life technologies, Inc.). Transfection efficiency was determined by inducing β -gal expression with ponasterone a (pona) and staining cells with β -galactosidase detection kit (Invitrogen). As confirmed by counting transfection efficiency assessments of positively stained (blue) clones, 60-85% of the cells in the cell line expressed β -galactosidase.

Immunocytochemistry: to verify hBD-1 protein expression, DU145 and hPrEC cells were plated at 1.5-2X10 per chamber⁴Cells were seeded on 2-chamber culture slides (BD Falcon, USA). DU145 cells transfected with either pvgRXR alone (control) or with the hBD-1 plasmid were induced with medium containing 10 μ M Pon a for 18 hours, while untransfected cells received fresh growth medium. After induction, cells were washed with 1-fold PBS and fixed with 4% paraformaldehyde at room temperature. Cells were then washed six times with 1-fold PBS and blocked in 1-fold PBS supplemented with 2% BSA, 0.8% normal sheep serum (Vector Laboratories, inc., Burlingame, CA), and 0.4% Triton-X100 for 1 hour at room temperature. Next, the cells were incubated overnight in primary rabbit anti-human BD-1 polyclonal antibody (PeproTech Inc., Rocky Hill, N.J.) diluted 1: 1000 in blocking solution. After this, the cells were washed six times with blocking solution and incubated for 1 hour at room temperature in Alexa Fluor 488 goat anti-rabbit IgG (H + L) secondary antibody diluted 1: 1000 in blocking solution. After washing the cells six times with blocking solution, coverslips were sealed with Gel Mount (Biomeda, Foster City, CA). Finally, cells were observed under Differential Interference Contrast (DIC) and laser excitation at 448 nm. The fluorescence signal was analyzed by confocal microscopy (Zeiss LSM 5 Pascal) with a Vario 2 RGB laser scanning assembly using a 63-fold DIC oil mirror. The digital images were transferred to Photoshop CS software (Adobe Systems) for image processing and hard copy display.

RNA isolation and quantitative RT-PCR: QRT-PCR was performed as previously described (Gibson et al, 2007). Briefly, total RNA from tissue sections (0.5. mu.g per reaction) was reverse transcribed into cDNA using random primers (Promega). MultiScriptube reverse transcriptase from TaqMan reverse transcriptase System andgreen PCR Master Mix (applied biosystems) performed a two-step QRT-PCR on the generated cDNA. Primer pairs for hBD-1 and c-MYC were generated from published sequences (Table 5). 40 PCR cycles were performed under standard conditions using an annealing temperature of 56.4 deg.C (for hBD-1 and c-MYC) and 55 deg.C (for PAX 2). In addition, β -actin (table 5) was amplified as a housekeeping gene to normalize the initial content of total cDNA. Gene expression in benign prostate tissue samples was calculated as the expression rate compared to β -actin. The hBD-1 expression levels of malignant prostate tissue, hPrEC prostate primary cultures, and prostate cancer cell lines before and after induction were calculated as the average relative hBD-1 expression in hPrEC cells. As a negative control, a QRT-PCR reaction without cDNA template was also performed. All reactions were performed a minimum of three times.

TABLE 5 QRT-PCR primer sequences

MTT cell viability assay: to examine the effect of hBD-1 on cell growth, a 3- (4, 5-dimethylthiazol-2 yl) -2, 5-diphenyltetrazolium bromide (MTT) metabolite assay was performed. DU145, LNCaP, PC3 and PC3/AR + cells co-transfected with the pVgRX plasmid and pIND/hBD-1 construct or control pVgRX plasmid were seeded in 96-well plates at 1-5X10 per well³A cell. 24 hours after inoculation, hBD-1 expression was induced by daily addition of fresh growth medium containing 10. mu.M Pon A for 24, 48 and 72 hours, after which MTT assays were performed according to the manufacturer's instructions (Promega). Reactions were performed in triplicate three times.

And (3) membrane integrity analysis: acridine Orange (AO)/ethidium bromide (EtBr) double staining was performed to identify changes in cell membrane integrity, and apoptotic cells were identified by staining condensed chromatin. AO stained live cells as well as early apoptotic cells, whereas EtBr stained membrane damaged late apoptotic cells. Briefly, PC3, DU145 and LNCaP cells were seeded on 2-chamber culture slides (BDFalcon). Cells transfected with either the empty plasmid or the hBD-1 plasmid were induced for 24 or 48 hours with medium containing 10. mu.M Pon A, while control cells received fresh growth medium at each time point. After induction, cells were washed once with PBS and stained with a mixture of 2ml AO (Sigma, St. Louis, MO) and EtBr (Promega) (5. mu.g/ml) solution (1: 1) for 5 minutes, and again washed with PBS.

Fluorescence was observed by a Zeiss LSM 5 Pascal Vario 2 laser scanning confocal microscope (Carl Zeiss). The excitation color wheel contains BS505-530 (green) and LP560 (red) filter assemblies that allow green light emitted from the AO to be split into the green channel and red light from the EtBr to be split into the red channel. The laser power output and gain control settings were the same for each independent experiment between control and hBD-1 induced cells. The excitation wavelengths provided by a Kr/Ar mixed gas laser were 543nm (for AO) and 488nm (for EtBr). Slides were analyzed at 40 x magnification and digital images were saved as uncompressed TIFF files and exported to Photoshop CS software (Adobe Systems) for image processing and hard copy display.

Flow cytometry: PC3 and DU145 cells transfected with the hBD-1 expression system were cultured in 60-mm dishes and induced with 10. mu.M Pon A for 12, 24 and 48 hours. Collected as described in example 1 and analyzed by flow cytometry.

Caspase detection: detection of caspase activity in prostate cancer cell lines was performed as described in example 1.

siRNA silencing of PAX 2: SiRNA knockdown and validation was performed as described in example 2.

Results

hBD-1 expression in prostate tissue: 82% of the prostate cancer frozen tissue sections analyzed showed little or no hBD-1 expression (Donald et al, 2003). To compare hBD-1 expression levels, QRTPCR analysis was performed on normal prostate tissue obtained by LCM of normal prostate tissue near a gross cut or randomly selected malignant region. At this time, hBD-1 was detected in all roughly cut normal clinical samples with a range of expression representing approximately 6.6-fold difference in expression levels (FIG. 33A). LCM capture of normal tissue samples expressing hBD-1 at levels representing expression that differed by a factor of 32 (FIG. 33B). Matching of sample numbers with corresponding patient characteristics showed that in most cases, the expression level of hBD-1 was higher in patient samples with a Gleason score of 6 than in patient samples with a Gleason score of 7. In addition, a comparison of the hBD-1 expression levels in tissues obtained by rough dissection and LCM from the same patient #1343 confirmed a 854-fold difference in expression between the two isolation techniques. Thus, these results indicate that LCM provides a more sensitive technique for assessing hBD-1 expression in prostate tissue.

hBD-1 expression in prostate cell lines: to verify upregulation of hBD-1 in prostate cancer cell lines following transfection with the hBD-1 expression system, QRTPCR was performed. In addition, a negative control without template was also performed, and the amplification product was verified by gel electrophoresis. At this time, hBD-1 expression was significantly lower in prostate cancer cell lines compared to hPrEC cells. After 24 hours of induction, the relative expression levels of hBD-1 in DU145, PC3 and LNCaP were significantly increased compared to the cell lines before hBD-1 induction (FIG. 34A).

Next, protein expression of hBD-1 in DU145 cells transfected with the hBD-1 expression system was verified by immunocytochemistry techniques after induction with Pon A. As a positive control, hPrEC prostate epithelial cells expressing hBD-1 were also detected. Cells were stained with primary anti-hBD-1 antibody and protein expression was monitored based on green fluorescence of the secondary antibody (FIG. 34B). Cell staining under DIC confirmed that hPrEC and DU145 cells induced hBD-1 expression at 18 hours in the presence of hPrEC and DU cells. Excitation by confocal laser at 488nm showed green fluorescence, indicating the presence of hBD-1 protein in hPrEC as a positive control. However, no green fluorescence was detected in control DU145 cells and empty plasmid-induced DU145 cells, indicating no hBD-1 expression. Confocal analysis of DU145 cells inducing hBD-1 expression showed green fluorescence, indicating the presence of hBD-1 protein following induction with Pon A.

hBD-1 expression resulted in decreased cell viability: MTT assays were performed to assess the effect of hBD-1 expression on the relative cell viability of DU145, PC3, PC3/AR + and LNCaP prostate cancer cell lines. MTT analysis with empty vector showed no statistically significant change in cell viability. Relative cell viability 24 hours after hBD-1 induction: DU145 cells were 72% and PC3 cells were 56%, while after 48 hours, cell viability decreased to: DU145 cells were 49% and PC3 cells were 37% (fig. 35). After 72 hours of hBD-1 induction, the relative cell viability was further reduced to: DU145 cells were 44%, and PC3 cells were 29%. In contrast, there was no significant effect on LNCaP cell viability. To assess whether the observed resistance to hBD-1 cytotoxicity in LNCaP was due to the presence of Androgen Receptor (AR), hBD-1 cytotoxicity was examined in PC3 cells (PC3/AR +) with aberrant AR expression. At this time, there was no difference between PC3/AR + and PC3 cells. Thus, the data indicate that hBD-1 is cytotoxic specifically to advanced prostate cancer cells.

To determine whether the effect of hBD-1 on PC3 and DU145 was cytostatic or cytotoxic, FACS analysis was performed to measure cell death. Under normal growth conditions, more than 90% of PC3 and DU145 cultures survived and did not apoptosis (lower left quadrant) and did not stain with annexin V or PI. After induction of hBD-1 expression from PC3 cells, the number of cells undergoing early apoptosis and late apoptosis/necrosis (lower right and upper right quadrants, respectively) totaled: 10% for 12 hours, 20% for 24 hours, and 44% for 48 hours (fig. 4B). For DU145 cells, the number of cells undergoing early apoptosis and late apoptosis/necrosis totaled: 12% after 12 hours of induction, 34% at 24 hours, and 59% at 48 hours (fig. 4A). No increase in apoptosis was observed in cells containing the empty plasmid after induction with Pon a. Annexin V and propidium iodide uptake studies have demonstrated that hBD-1 has cytotoxic activity against DU145 and PC3 prostate cancer cells, and the results indicate that apoptosis is the mechanism of cell death.

hBD-1 caused changes in membrane integrity and caspase activity: study whether the cell death observed in prostate cancer cells following hBD-1 induction was caspase-mediated apoptosis. To better understand the cellular mechanisms involved in hBD-1 expression, confocal laser microscopy analysis was performed on hBD-1 expression-inducing DU145 and LNCaP cells (FIG. 5). Pan-caspase activation was monitored based on the binding and dissociation of green-fluorescent FAM-VAD-FMK to caspase in actively apoptotic cells. Cell analysis under DIC showed the presence of viable control DU145 (panel a) and LNCaP (panel E) cells at 0 hours. Excitation at 488nm by confocal laser produced no detectable green staining, indicating no caspase activity in DU145 (panel B) or LNCaP (panel F) control cells. After 24 hours of induction, DU145 (panel C) and LNCaP (panel G) cells were again visible under DIC. Confocal analysis under fluorescence showed green staining in DU145 (panel D) cells, indicating pan caspase activity after induction of hBD-1 expression. However, there was no green staining in LNCaP (panel H) cells that induced hBD-1 expression. Thus, the cell death observed after induction of hBD-1 was caspase-mediated apoptosis.

The proposed mechanism of defensin peptide antibacterial activity is the disruption of microbial membranes due to pore formation (Papo and Shai, 2005). To determine whether hBD-1 expression altered membrane integrity, EtBr uptake was detected by confocal analysis. Intact cells stained green due to membrane permeation of AO, while only cells with damaged cytoplasmic membranes stained red due to incorporation of EtBr that is not membrane permeable. Control DU145 and PC3 cells stained positively with AO and emitted green, but not with EtBr. However, as indicated by red staining, hBD-1 induced 24 hours of DU145 and PC3 resulting in EtBr accumulation in the cytoplasm. At 48 hours, DU145 and PC3 had condensed nuclei and yellow color appeared due to co-localization of green and red from AO and EtBr, respectively. In contrast, there was no observable change in membrane integrity of LNCaP cells as indicated by positive green fluorescence with AO but no red EtBr fluorescence after 48 hours of induction. This finding suggests that membrane integrity and permeability are altered between early and late stage prostate cancer cells in response to differences in hBD-1 expression.

Comparison of hBD-1 and cMYC expression levels: QRT-PCR analysis was performed on LCM prostate tissue from three patients (FIG. 34). In patient #1457, hBD-1 expression showed: a 2.7-fold decrease from normal to PIN, a 3.5-fold decrease from PIN to tumor, and a 9.3-fold decrease from normal to tumor (fig. 36A). Likewise, cMYC expression of patient #1457 follows a similar expression pattern, where: expression decreased 1.7-fold from normal to PIN, 1.7-fold from PIN to tumor, and 2.8-fold from normal to tumor (fig. 36B). Furthermore, the expression of cMYC in the other two patients showed a statistically significant decrease. Patient #1569 decreased 2.3-fold from normal to PIN, whereas patient #1586 decreased 1.8-fold from normal to PIN, 4.3-fold from PIN to tumor, and 7.9-fold from normal to tumor.

Induction of hBD-1 expression following inhibition of PAX 2: to further examine the effect of PAX2 in regulating hBD-1 expression, siRNA was used to knock down PAX2 expression and QRT-PCR was performed to monitor hBD-1 expression. hPrEC cells treated with PAX2siRNA showed no effect on hBD-1 expression (FIG. 37). However, PAX2 knockdown resulted in increased expression of hBD-1 compared to untreated cells: LNCaP is 42 times, PC3 is 37 times, and DU145 is 1026 times. As a negative control, cells treated with non-specific siRNA had no significant effect on hBD-1 expression.

Example 11: inhibition of PAX2 expression results in alternating prostate columns with different P53 status Cell death pathway of adenocarcinoma cells

Materials and methods

Cell line: cancer cell lines PC3, DU145 and LNCaP with different mutation status of p53 (table 6) were cultured as described in example 1. The prostate epithelial cell line HPrEC was obtained from Cambrex BioScience, inc. (walker ville, MD) and cultured in prostate epithelial cell basal medium. The cells were maintained at 37 ℃ and 5% CO₂The following steps.

TABLE 6 mutation of p53 Gene in prostate cancer cell line species

siRNA silencing of PAX 2: siRNA silencing of PAX2 was performed as described in example 2.

Western analysis: western blots were performed as described in example 2. Blots were probed with rabbit anti-PAX 2 primary antibody (Zymed, San Francisco, Calif.) at a 1: 1000 dilution. After washing, the membranes were incubated with horseradish peroxidase (HRP) -conjugated anti-rabbit antibody (dilution 1: 5000; Sigma) and signal detection was visualized on Alpha Inotech Fluorchem 8900 using a chemiluminescent reagent (Pierce). As a control, the blot was cleared and re-probed with mouse anti- β -actin primary antibody (1: 5000; Sigma-Aldrich) and HRP-conjugated anti-mouse secondary antibody (1: 5000; Sigma-Aldrich) and signal detection was visualized again.

Phase contrast microscopy: the effect of PAX2 knockdown on cell number was analyzed by phase contrast microscopy as described in example 1.

MTT cytotoxicity assay: MTT cytotoxicity assays were performed as described in example 1.

Pan caspase assay: detection of caspase activity in prostate cancer cell lines was performed as described in example 1.

Quantitative real-time RT-PCR to verify changes in gene expression in PC3, DU145 and LNCaP cell lines after PAX2 knockdown, quantitative real-time RT-PCR was performed as described in example 1. Primer pairs for BAX, BID, BCL-2, AKT and BAD were generated from published sequences (Table 7). Reactions were performed on Micro Amp Optical 96-well reaction plates (PE Biosystems). Under standard conditions, 40 PCR cycles were performed using an annealing temperature of 60 ℃. Quantification was determined by the number of cycles at which exponential amplification started (threshold) and the average obtained from three replicates. There is an inverse relationship between the signal level and the threshold. In addition, GAPDH was used as a housekeeping gene to normalize the initial content of total cDNA. Relative expression was calculated as the ratio between each gene and GAPDH. All reactions were performed in triplicate.

TABLE 10 quantitative RT-PCR primers

Membrane permeability measurement: the membrane permeability assay was performed as described in example 3.

Results

Analysis of PAX2 protein expression in prostate cells: PAX2 protein expression was detected by Western analysis in HPrEC prostate primary cultures and LNCaP, DU145 and PC3 prostate cancer cell lines. At this time, PAX2 protein was detected in all prostate cancer cell lines (fig. 38A). However, no PAX2 protein was detected by HPrEC. Blots were cleared and reprobed with β -actin as an internal control to ensure equal loading. PAX2 protein expression was also monitored after selective targeting and inhibition by PAX2 specific siRNA in DU145, PC3 and LNCaP prostate cancer cell line. Cells were transfected one round with PAX2siRNA pools during 6 days of treatment. PAX2 protein was expressed in control cells treated with medium only. Specific targeting of PAX2mRNA was confirmed by observing knock-down of PAX2 protein in all three cell lines (fig. 38B).

Effect of PAX2 knockdown on prostate cancer cell growth: the effect of PAX2siRNA on cell number and cell viability was analyzed using light microscopy and MTT assay. To examine the effect of PAX2siRNA on cell number, PC3, DU145 and LNCaP cell lines were transfected with either non-specific siRNA or PAX2siRNA in culture medium only for 6 days. Each cell line reached 80-90% confluence in a 60mm dish containing medium only. Treatment of HprEC, DU145, PC3, and LNCaP cells with non-specific siRNA appeared to have little to no effect on cell growth compared to cells treated with medium alone (fig. 39, fig. A, C and E, respectively). Treatment of HprEC, a PAX 2-deficient cell line, with PAX2siRNA, appeared to have no significant effect on cell growth (fig. 39, panel B). However, treatment of prostate cancer cell lines DU145, PC3 and LNCaP with PAX2siRNA resulted in a significant reduction in cell number (fig. 39, fig. D, F and H, respectively).

Effect of PAX2 knockdown on prostate cancer cell viability: cell viability was measured after 2, 4 and 6 days of exposure. The percent viability was calculated as the ratio of the absorbance at 630nm 570 for cells treated with PAX2siRNA divided by the absorbance at 630nm 570 for untreated control cells. As a negative control, cell viability was measured after each treatment with negative control non-specific siRNA or transfection with reagent only. Relative cell viability was calculated by dividing the percent viability after PAX2siRNA treatment by the percent viability after treatment with non-specific siRNA (figure 40). After 2 days of treatment, relative cell viability: DU145 is 116%, PC3 is 81%, and LNCaP is 98%. After 4 days of treatment, the relative cell viability decreased to: DU145 is 69%, PC3 is 79%, and LNCaP is 80%. Finally, by day 6, relative cell viability: DU145 is 63%, PC3 is 43% and LNCaP is 44%. In addition, cell viability was also measured after transfection treatment with the agent alone. At this time, each cell line showed no significant decrease in cell viability.

Detection of pan activity: caspase activity was detected by confocal laser microscopy analysis. LNCaP, DU145 and PC3 cells were treated with PAX2siRNA and activity was monitored based on the binding of FAM-labeled peptides to caspases in actively apoptotic cells (green fluorescence). Analysis of cells treated with medium only showed the presence of viable LNCaP, DU145 and PC3 cells, respectively. Excitation at 488nm by confocal laser did not produce detectable green staining, indicating no caspase activity in the untreated cells (fig. 41, panels A, C and E, respectively). LNCaP, DU145 and PC3 cells, which showed green staining under fluorescence after 4 days of treatment with PAX2siRNA, showed caspase activity (fig. 41, fig. B, D and F, respectively).

The effect of PAX2 inhibition on apoptotic factors: LNCaP, DU145 and PC3 cells were treated with siRNA against PAX2 for 4 days and expression of pro-and anti-apoptotic factors was measured by QRTPCR. Following PAX2 knockdown, BAD analysis showed: LNCaP was 2 times, DU145 was 1.58 times, and PC3 was 1.375 times (fig. 42A). Expression level of BID: LNCaP increased 1.38-fold, and DU145 increased 1.78-fold, but there were no statistically significant differences observed in BID in PC3 after repression of PAX2 expression (fig. 42B). Analysis of the anti-apoptotic factor AKT after treatment showed: LNCaP expression was reduced 1.25 fold and DU145 expression was reduced 1.28 fold, whereas no change in PC3 was observed (fig. 42C).

Membrane integrity and necrosis analysis: the membrane integrity of LNCaP, DU145 and PC3 cells was monitored by confocal analysis. At this time, the intact cells stained green due to membrane-permeable AO; cells with damaged cytoplasmic membranes stained red due to incorporation of membrane-impermeable EtBr within the cytoplasm; and yellow due to co-localization of AO and EtBr in the nucleus. Untreated LNCaP, DU145 and PC3 cells stained positively with AO and emitted green, but not with EtBr. Following PAX2 knockdown, there was no observable change in membrane integrity of LNCaP cells as indicated by positive green fluorescence with AO without red EtBr fluorescence. These findings further indicate that LNCaP cells can undergo apoptosis, but not necrotic cell death, following PAX2 knockdown. In contrast, PAX2 knockdown in DU145 and PC3 caused EtBr to accumulate in the cytoplasm, as indicated by red staining. In addition, DU145 and PC3 have condensed nuclei that appear yellow due to co-localization of green and red from AO and EtBr, respectively. These results indicate that DU145 and PC3 are undergoing an alternate cell death pathway involving necrotic cell death compared to LNCaP.

Example 12: breast cancer cell lines and breast groups with ductal or lobular intraepithelial neoplasia Expression of PAX2 and DEFB-1 in tissues

Determining PAX2 and DEFB-1 expression in breast biopsy samples of ductal or lobular intraepithelial neoplasia and in the following breast cancer cell lines:

BT-20: isolation from primary invasive ductal carcinoma; the cells express E-cadherin, ER, EGFR, and uPA.

BT-474: isolation from primary invasive ductal carcinoma; the cells express E-cadherin, ER, PR, and have increased HER 2/neu.

Hs 578T: isolation from primary invasive ductal carcinoma; a cell line, also established from normal adjacent tissues, was designated Hs578 Bst.

MCF-7: established by pleural effusion. The cells express ER and are the most common example of estrogen-responsive breast cancer cells.

MDA-MB-231: established by pleural effusion. Cells were ER-negative, E-cadherin negative, and the in vitro assay was highly invasive.

MDA-MB-361: established from brain metastases. The cells express ER, PR, EGFR and HER 2/neu.

MDA-MB-435: established by pleural effusion. The cells were ER-negative, E-cadherin negative, and highly infiltrating and metastatic in immunodeficient mice.

MDA-MB-468: established by pleural effusion. The cells have increased EGFR and are ER-negative.

SK-BR-3: established by pleural effusion. The cells have increased HER/2neu, express EGFR and are ER-negative.

T-47D: established by pleural effusion. The cells remain expressing E-cadherin, ER and PR.

ZR-75-1: established from ascites. The cells express ER, E-cadherin, HER2/neu and VEGF.

The PAX 2-to-DEFB expression rate was determined using the method described in example 9.

Example 13: DEFB1 expression in breast cancer cells

DEFB1 was expressed in breast cancer cells using the method described in example 1. Cell viability and caspase activity were determined as described in example 1.

Example 14: inhibition of PAX2 expression in breast cancer cells

The siRNA described in example 2 was used to inhibit PAX2 expression in breast cancer cells. Expression levels, cell viability and caspase activity of pro-apoptotic genes such as BAX, BID and BAD were determined as described in example 2.

Example 15: effect of DEFB1 expression on tumor growth in vivo

The anti-tumor capacity of DEFB1 was assessed by injecting DEFB 1-overexpressing breast cancer cells into nude mice. Breast cancer cells were transfected with an expression vector carrying the DEFB1 gene. Cells expressing the exogenous DEFB1 gene were selected and cloned. Only single cell suspensions with > 90% viability were used. Each animal received approximately 500,000 cells and was administered subcutaneously to the right flank of female nude mice. There were two groups, the control group injected with vector-only clones, and one with clones overexpressing DEFB 1. 35 mice per group were determined by a statistician. Animals were weighed twice weekly, tumor growth was monitored by calipers, and tumor volume was determined using the following equation: volume 0.5x (width) 2x long. The tumor size reaches 2mm³Or six months after implantation by excess CO₂All animals were sacrificed; tumors were excised, weighed and preserved in neutral buffered formalin for pathological examination. The difference in tumor growth between the two groups was characterized by a summary statistical table and graphical display. Statistical significance was assessed using a t-test or non-parametric equivalent.

Example 16: effect of PAX2siRNA on tumor growth in vivo

Hairpin PAX2siRNA template oligonucleotides utilized in vitro studies were used to detect the effect of up-regulation of DEFB1 expression in vivo. The sense and antisense strands (see Table 3) were annealed and cloned into the pSilencer 2.1U 6 hygro siRNA expression vector (Ambion) under the control of the human U6RNA pol III promoter. The cloned plasmids were sequenced, verified and transfected into breast cancer cell lines. Scrambled shRNA was cloned and used as a negative control for this study. Clones resistant to hygromycin were selected, cells were introduced subcutaneously into mice, and tumor growth was monitored as described above.

Example 17: effect of small molecule inhibitors of PAX2 binding on Breast cancer cells

The optional inhibitory oligonucleotides described in example 6 were transfected into breast cancer cells using lipofectamine reagent or Codebreaker transfection reagent (Promega, Inc). To confirm the DNA-protein interaction, use³²P]dCTP was labeled with a double-stranded oligonucleotide and subjected to electrophoretic mobility measurement. DEFB1 expression was monitored by QRT-PCR and Western analysis after treatment with oligonucleotides. Finally, cell death was detected by MTT assay and flow cytometry as previously described.

The above description is intended only to teach one of ordinary skill in the art how to practice the invention and is not intended to detail those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the invention which is defined by the following claims. The claims are intended to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

Claims

1. A method for treating a breast condition in a subject, the method comprising administering to a breast tissue of the subject a composition that inhibits expression of PAX2 and/or activity of PAX2,

wherein the composition comprises one or more ingredients selected from the group consisting of: polynucleotides encoding PAX2 sirnas, polynucleotides that inhibit the binding of PAX2 to the DEFB1 promoter, antagonists of angiotensin II receptors, antagonists of Angiotensin Converting Enzyme (ACE), antagonists of mitogen-activated protein kinase (MEK), antagonists of extracellular signal-regulated kinases 1, 2(ERK1, 2), antagonists of signal transducer and activator of transcription 3(STAT3), and blockers of the RAS signaling pathway.

2. The method of claim 1, wherein the breast disease is breast cancer or breast intraepithelial neoplasia (MIN).

3. The method of claim 1, wherein the composition comprises a polynucleotide encoding an siRNA comprising a sequence selected from the group consisting of SEQ ID NOs: SEQ ID NO: 3-6 and 11-15.

4. The method of claim 1, wherein the composition comprises a polynucleotide comprising a forward or reverse orientation of the sequence set forth in SEQ ID NO: 1.

5. the method of claim 4, wherein the polynucleotide comprises a V-CCTTG-W sequence and its complement, wherein V and W are contiguous nucleotide sequences of 1 to 35 nucleotides flanking the PAX2 binding site of the DEFB1 promoter.

6. The method of claim 5, wherein the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: SEQ ID NO: 18-21, 25, 26, 28 and 29.

7. The method of claim 1, wherein said composition comprises enalapril.

8. The method of claim 1, wherein the composition comprises valsartan, olmesartan or telmisartan.

9. The method of claim 1, wherein the composition comprises U0126 or PD 98059.

10. A method of treating breast cancer or MIN in an individual, the method comprising enhancing DEFB1 expression in breast cancer tissue or MIN tissue of the individual.

11. The method of claim 10, wherein the enhancing DEFB1 expression comprises administering an effective amount of DEFB1 to breast cancer tissue or MIN tissue of the individual.

12. The method of claim 10, wherein the enhancing DEFB1 expression comprises administering to the breast cancer tissue or MIN tissue of the individual an effective amount of an expression vector encoding DEFB 1.

13. The method of claim 1, further comprising the step of administering to the individual an effective amount of an anti-hormonal agent.

14. The method of claim 13, wherein the anti-hormonal agent is tamoxifen.

15. The method of claim 1, further comprising the step of administering to the individual an effective amount of an anti-ERBB-2 agent.

16. The method of claim 15, wherein the anti-ERBB-2 agent is herceptin.

17. The method of claim 1, further comprising the step of administering to the individual an effective amount of an anti-Her-2 agent.

18. The method of claim 17, wherein the anti-Her-2 agent is trastuzumab.

19. The method of claim 1, further comprising the step of administering to the individual an effective amount of an anti-AIB-1/SRC-3 agent.

20. The method of claim 1, wherein the composition further comprises one or more agents selected from the group consisting of: anti-hormonal agents, anti-ERBB-2 agents, anti-Her-2 agents, and anti-AIB-1/SRC-3 agents.

21. A method for treating a breast condition in a subject, the method comprising:

(a) determining the expression rate of PAX 2-to-DEFB 1 in diseased breast tissue from the individual;

(b) determining the ER/PR status of the diseased breast tissue from the individual; and

(c) based on the results of (a) and (b), administering to the breast tissue of the subject a first composition that (1) inhibits PAX2 expression or PAX2 activity, (2) expresses DEFB1, or (3) inhibits PAX2 expression or PAX2 activity and expresses DEFB 1.

22. The method of claim 21, wherein the breast disease is breast cancer or MIN.

23. The method of claim 21, wherein the first composition comprises a polynucleotide encoding an siRNA comprising a sequence selected from the group consisting of seq id nos: SEQ ID NO: 3-6 and 11-15.

24. The method of claim 21, wherein the first composition comprises a polynucleotide comprising the sequence of SEQ ID NO: 1.

25. the method of claim 24, wherein the polynucleotide comprises a V-CCTTG-W sequence and its complement, wherein V and W are contiguous nucleotide sequences of 1 to 35 nucleotides flanking the PAX2 binding site of the DEFB1 promoter.

26. The method of claim 25, wherein the polynucleotide comprises a sequence selected from the group consisting of seq id nos: SEQ ID NO: 18-21, 25, 26, 28 and 29.

27. The method of claim 21, wherein the first composition comprises an antagonist selected from the group consisting of: antagonists of angiotensin II, antagonists of angiotensin II receptors, antagonists of Angiotensin Converting Enzyme (ACE), antagonists of mitogen-activated protein kinase (MEK), antagonists of extracellular signal-regulated kinases 1, 2(ERK1, 2), antagonists of signal transducer and activator of transcription 3(STAT 3).

28. The method of claim 21, wherein the first composition comprises a blocker of the RAS signaling pathway.

29. The method of claim 21, wherein the first composition is an anti-PAX 2 agent conjugated to an antibody, receptor, or ligand to target tumor tissue in the subject.

30. The method of claim 21, wherein the first composition is a small molecule anti-PAX 2 agent comprising a compound that interferes with or inhibits binding of PAX2 to the DEFB1 promoter.

31. The method of claim 21, wherein step (c) further comprises administering a second composition comprising an anti-hormonal agent.

32. The method of claim 31, wherein the anti-hormonal agent is tamoxifen.

33. The method of claim 21, wherein step (c) further comprises administering a second composition comprising an anti-ERBB-2 agent.

34. The method of claim 33, wherein the anti-ERBB-2 agent is herceptin.

35. The method of claim 21, wherein step (c) further comprises administering a second composition comprising an anti-Her-2 agent.

36. The method of claim 35, wherein the anti-Her-2 agent is trastuzumab.

37. The method of claim 21, wherein step (c) further comprises administering a second composition comprising an anti-AIB-1/SRC-3 agent.