BRPI1011929B1 - METHOD FOR TESTING A SUBJECT WHO IS CONSIDERED TO BE PREDISPOUSED TO HAVING METASTATIC CANCER, METHOD FOR DETERMINING A SUBJECT'S RESPONSE TO ANTI-METASTATIC CANCER THERAPY, AND METHOD FOR SCREENING POTENTIAL ANTIMESTASTATIC COMPOUNDS - Google Patents

Abstract

METHOD FOR TESTING A SUBJECT THOUGHT TO BE PREDISPOUSED TO HAVING METASTATIC CANCER USING DELTA133P53BETA. The present invention relates to a method for testing a subject who is thought to be predisposed to having metastatic cancer, comprising the steps of: (i) analyzing a biological sample from said subject for the determination of the presence of a p53 isoform, selected from the group consisting of (delta)133p53, (delta)133p53y and (delta)133p53(beta), the presence of said isoform being indicative of a metastatic cancer.

Description

Field of Invention

[0001] The present invention relates to a method for testing a subject who is thought to be predisposed to having metastatic cancer, preferably breast cancer or colon cancer.

Background of the Invention

[0002] Despite efforts to improve the treatment and management of cancer patients, survival rates for many cancer types have not improved over the past two decades. A critical event during tumor genesis is the conversion of a localized primary tumor into an invasive metastasis. Most patients with metastasis die from it. Only 35% of patients detected early did not develop metastasis.

[0003] The invasive process is a consequence of morphological changes in cells that begin with the loss of adhesion capacity due to the disruption of epithelial junctions. Cells then acquire migration capacity through actin remodeling in the cytoskeleton, which involves the Rho family of small GTPases. Tumor cells can employ two modes of migration: mesenchemic migration, which requires integrin-mediated adhesion dynamics and surface proteases for degradation of the extracellular matrix (ECM), or amoeboid migration, which is more effective and does not require ECM digestion or integrin-mediated adhesion but involves the RhoA/ROCK pathway. The tumor suppressor p53 prevents cancer progression toward invasiveness by modulating Rho GTPase-mediated cell motility.

[0004] Optimal therapy will be based on a combination of diagnostic and prognostic information. An accurate, diagnostic, and reproducible test is necessary to provide an assessment that will provide specific information regarding survival.

[0005] In breast cancer, the clinical and biological variables commonly used to predict survival from primary surgical treatments are regional lymph node invasion, histological grades, and hormone receptor expression. All of these parameters are well-recognized prognostic and predictive factors.

[0006] However, these variables do not allow a specific and complete survival prognosis to be established. In fact, there is significant heterogeneity among patients suffering from the same type of breast cancer.

[0007] Thus, there is an existing need for the provision of other biological markers that will strengthen the prediction of survival in breast cancer patients.

[0008] Biological markers are useful in biology to distinguish between a normal medical state and a pathological state, or to assess the progression of a disease. Technologies that enable early cancer detection thanks to biological markers should be very beneficial in cancer research and clinical care.

[0009] The purpose of a tumor marker is to establish, evaluate and validate the molecular classification of human tumors, thus enabling, from the diagnosis onwards: to better assess the infra-clinical potential of cancer metastasis, thus preventing over-treatment of patients presenting a low risk and allowing the isolation of patients presenting a high risk of progression; to shift from a group assessment to a personal assessment, thus making personalized therapies possible; to direct patients presenting a high risk of progression and not, or barely, sensitive to conventional treatments to therapeutic innovations; and to allow the evaluation of the efficiency of alternative therapeutic molecules.

[00010] Breast cancer affects about one million women each year and is the most common form of cancer in women, affecting approximately 10% of all women at some stage in their lives in the Western world. Men can also develop breast cancer, although their risk is less than 1 in 1000.

[00011] The World Health Organization (WHO) has predicted that by 2010 cancers will be the leading cause of death worldwide, ahead of cardiovascular disease. The latest incidence and mortality data in France show the same trend (Communication 2008 from Francim, the Institut de Veille Sanitaire, the Hospice Civils of Lyon and the Institut National du Cancer).

In breast cancer, it is essential to treat patients based on the biopathological identity of their tumors. The goal is therefore to provide reliable predictors for choosing targeted chemotherapy only for chemotherapy-sensitive patients and for choosing specific drugs based on tumor-specific biological markers. Thus, the effectiveness of chemotherapy depends on cellular characteristics and the presence of sensitivity to biological markers, such as hormone receptors, and the expression of UPA, PAI1, Her2, and topoisomerase II α. The tumor suppressor p53 is the most frequently mutated gene in human tumors. Nevertheless, mutations affecting the p53 gene must be interpreted carefully, especially since its predictive value depends on the type of breast cancer.

[00013] The inventors now teach that the expression pattern of p53 tumor suppressor isoforms due to aberrant splicing is associated with a poor prognosis.

[00014] In other words, the inventors have revealed markers that are specifically associated with metastatic cancers. Consequently, these p53 isoforms allow for the differentiation of metastatic cancers from non-metastatic cancers.

[00015] The present invention relates to a method for testing a subject who is thought to be predisposed to having cancer which comprises the step of (i) analyzing a biological sample from said subject to detect the presence of a p53 isoform selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β, the presence of said p53 isoform being indicative of cancer, preferably of a metastatic cancer.

[00016] Preferably, said method is a method of testing a subject who is thought to be predisposed to having metastatic cancer.

[00017] In a specific embodiment, the presence of a p53 isoform corresponds to determining the level of expression of the p53 isoform.

[00018] The presence of the p53 isoform can be assessed by detecting a polypeptide, or a fragment thereof, of a p53 isoform or by detecting RNA of a p53 isoform or a fragment thereof.

[00019] Furthermore, said method may include an additional step of comparing the detected expression level of the p53 isoform with a threshold value. Said threshold value may correspond to a positive or negative control.

[00020] For example, a negative control may be the p53 isoform expression of a subject who has non-metastatic cancer or the p53 isoform expression level of a healthy subject.

[00021] Preferably a higher expression level than a negative control will be indicative of a metastatic cancer.

[00022] For example, a positive control may be the expression level of the p53 isoform of a subject who has metastatic cancer.

[00023] Preferably, the methods according to the invention are in vitro methods.

[00024] Preferably said cancer is a metastatic cancer.

[00025] In another aspect, the invention relates to a method for measuring the aggressiveness of cancer in a subject, comprising the step of (i) determining the presence of a p53 isoform selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β in a biological sample from the subject, the presence of said isoform being indicative of an aggressive cancer, preferably said isoform being indicative of a metastatic cancer.

[00026] In another aspect, the present invention relates to a method for determining the response of a subject to an anti-cancer therapy, said subject being or having received therapy in relation to a condition associated with cancer, comprising the steps of: (i) determining the level of expression of a dep53 isoform selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β in a biological sample from the subject, and (ii) comparing the same to a threshold value.

[00027] Said threshold value may correspond to a positive or negative control as defined above, or the expression level measured in a sample from the patient before said patient has received therapy.

[00028] Preferably, this method includes an additional step of determining whether the subject responds to therapy.

[00029] For example, the presence of said isoform is indicative of the subject's response to therapy when the expression level of said p53 isoform is lower than the expression level measured in a sample from the subject before said subject received therapy.

[00030] Alternatively, the presence of said p53 isoform is indicative of the subject's response to therapy when the level of expression of said p53 isoform is equal to or lower than the level of expression measured in a sample from a subject who does not have cancer.

[00031] The above method of the invention can be used in combination with other methods of cancer diagnosis or prognosis.

[00032] As used herein, in this patent application the term "p53 isoform" refers to a polypeptide or an mRNA that is different from the p53 polypeptide of the sequence SEQ ID NO: 7 or from the p53 mRNA having a cDNA sequence SEQ ID NO: 8, respectively, due to a splicing pattern. The p53 isoform of the present invention is selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β.

[00033] Preferably, said p53 isoform is Δ133p53β.

[00034] Preferably, the Δ133p53β polypeptide is represented by the sequence SEQ ID NO: 1, and the Δ133p53β mRNA has a cDNA sequence SEQ ID NO: 2.

[00035] Preferably, the Δ133p53 polypeptide is represented by the sequence SEQ ID NO: 3 and the Δ133p53 mRNA has the cDNA sequence SEQ ID NO: 4.

[00036] Preferably, the Δ133p53y polypeptide is represented by the sequence SEQ ID NO: 5 and the Δ133p53y mRNA has the cDNA sequence SEQ ID NO: 6.

[00037] "Cancer" includes a malignant neoplasm characterized by unregulated or uncontrolled cell growth. The term "cancer" includes primary malignant tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the original tumor site) and secondary malignant tumors (e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the original tumor site).

[00038] In the methods of the present invention, this cancer is preferably selected from the group consisting of breast cancer, ovarian cancer, digestive cancers and throat cancer, specifically of a human subject, more preferably a breast cancer or a colon cancer, and even more preferably a breast cancer.

[00039] The term splicing process consists of the elimination of introns in pre-messenger RNAs to produce mature messenger RNAs that can be used by the cell's translational mechanism (SHARP, Cell, vol. 77, p. 805-815, 1994). In the case of alternative splicing, the same precursor can be the origin of messenger RNAs that code for proteins with distinct functions (BLACK, Annu. Rev. Biochem. vol. 72, p. 291-336, 2003).

[00040] "Default splicing" means an abnormal splicing process that leads to the formation of abnormal isoforms.

[00041] The term "aggressive" (or "invasive") as used herein in this patent application with respect to cancer refers to the ability of a tumor to expand beyond its borders into adjacent tissue, or the tumor's ability to metastasize. Invasive cancer can be contrasted with cancer confined to an organ. For example, a basal cell carcinoma of the skin is a non-invasive or minimally invasive tumor, confined to the primary tumor site and expanding in size but not metastasizing. In contrast, melanoma cancer is highly invasive of adjacent and distal tissues. The invasive nature of a tumor is almost always accompanied by the production of proteolytic enzymes such as collagenases, which degrade the matrix material and the membrane scaffolding material to allow the tumor to expand beyond the confines of the capsule, and beyond the confines of the specific tissue in which that tumor is located.

[00042] The terms "metastasis" or "metastatic" as used herein in this patent application refer to the conditions of spread of cancer from the organ of origin to additional distal sites in the patient. The process of tumor metastasis is multistep involving local invasion and destruction of the intercellular matrix, intravasation into blood vessels, lymphatics or other transport channels, survival in the circulation, extravasation out of the vessel at the secondary site, and growth at the new location.

[00043] The term "subject" includes mammals, such as humans, dogs, cows, horses, kangaroos, pigs, sheep, goats, cats, mice, rabbits, rats, and non-human transgenic animals, preferably human subjects and most preferably a woman.

[00044] The term "biological samples" includes both solid samples and body fluids. Biological samples of the present invention may include cells, protein, blood or biological fluids such as bone marrow, ascites fluid, or brain fluid (e.g., cerebrospinal fluid).

[00045] Examples of solid biological samples include samples taken from feces, rectum, central nervous system, bone, breast tissue, kidney tissue, uterine cervix, endometrium, neck/head tissue, gallbladder, parotid tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, urinary bladder, adipose tissue, lymph node tissue, uterus, ovary, adrenal tissue, testicular tissue, tonsils, and thymus. Examples of "body fluid samples" include samples taken from blood, serum, semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bone marrow, lymph, and tears. Samples for use in the analyses of the invention can be obtained through standard methods, including venipuncture and surgical biopsy. In one embodiment, the biological sample is a breast tissue sample obtained by needle biopsy.

[00046] In a preferred embodiment, the biological sample used in the methods of the present invention is selected from the group consisting of bone marrow, serum, plasma, blood, lymph, or cells from cancerous or suspected cancerous tissue or tissue adjacent thereto, preferably a bone marrow sample.

[00047] The terms "expression level" or "level" mean the concentration of an expression product, such as a polypeptide or mRNA, in a sample.

[00048] As described in more detail below, the detection methods of the invention can be used for the detection of the mRNA of a p53 isoform of the present invention, the polypeptide of a p53 isoform of the present invention or a specific fragment thereof, in a biological sample in vitro. For example, in vitro techniques for the detection of the mRNA of said p53 isoform include Northern hybridizations and in situ hybridization. In vitro techniques for the detection of the p53 isoform, and more specifically of the Δ133p53β polypeptide, include immunohistochemistry, quantitative PCR, enzyme-linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.

[00049] In a preferred embodiment, the invention relates to the methods according to the present invention, in which the p53 isoform is Δ133p53β.

[00050] When the methods according to the present invention are based on the detection or quantification of an mRNA of a p53 isoform, it is also preferably that the determination of the presence of said mRNA comprises an additional step of: amplifying the mRNA or cDNA of the p53 isoform, the complementary sequence thereof or a fragment thereof that is at least 10 nucleotides in length that is specific to said p53 isoform.

[00051] As used herein, in this patent application, the term "mRNA" refers to mature mRNA, that is, one that has already undergone the division event.

[00052] As used herein in this patent application, the term "cDNA" shall refer to either a copy of DNA or mRNA.

[00053] It is also preferred that the amplification step of the Δ133p53β mRNA or cDNA is performed by PCR or RT-PCR reaction.

[00054] Such detection can be achieved by isolating mRNA from a sample. When mRNA detection is used, the method can be performed by converting the isolated mRNA to cDNA according to standard methods, treating the converted cDNA with amplification reaction reagents (such as cDNA amplification reagents) in a vessel together with an appropriate mixture of nucleic acid primers, reacting the contents of the vessel to produce amplification products, and analyzing the amplification products to detect the presence of a p53 isoform-specific nucleic acid of the present invention in the biological sample.

[00055] Preferably, said primers have the sequence SEQ ID NO: 9 and SEQ ID NO: 10.

[00056] The term "primer" as used herein in this patent application refers to an oligonucleotide, whether naturally occurring (such as in a purified restriction digest) or synthetically produced, that is capable of initiating the synthesis of a strand complementary to a nucleic acid when placed under appropriate conditions, i.e., in the presence of nucleotides and an inducing agent, such as a DNA polymerase, and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be long enough to initiate the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend on many factors, including temperature, sequence and/or primer homology, and the method used. For example, in diagnostic applications, the oligonucleotide primer typically contains 10 to 25 or more nucleotides, depending on the complexity of the target sequence, although it may contain fewer nucleotides.

[00057] The primers herein, in this patent application, are selected to be "substantially" complementary to specific targeted DNA sequences. This means that the primers must be sufficiently complementary to hybridize to their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment (i.e., one containing a restriction site) may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases from longer sequences may be interspersed within the primer, as long as the primer sequence is sufficiently complementary to the sequence to hybridize to it and form the template for the synthesis of the extension product.

[00058] "Amplifying" refers to template-dependent processes and vector-mediated propagation that result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal. As used herein, in this patent application, the term template-dependent process is intended to refer to a process involving the template-dependent expansion of a primer molecule.

[00059] The term template-dependent process refers to the synthesis of a nucleic acid from an RNA or DNA molecule in which the sequence of the newly synthesized nucleic acid strand is determined by the well-known rules of complementary base pairing (see, e.g., Watson, J. D. et al., In: Molecular Biology of the Gene, 4th Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1987)). Typically, vector-mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Maniatis T. et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982.

[00060] A number of template-dependent processes are available for amplifying target sequences of present interest in a sample. One of the best known amplification methods is the polymerase chain reaction (PCR) which is described in detail in Mullis et al., U.S. Patent No. 4,683,195, Mullis et al., U.S. Patent No. 4,683,202, and Mullis et al., U.S. Patent No. 4,800,159, and in Innis et al., PCR Protocols, Academic Press, Inc., San Diego, Calif. Briefly, in PCR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleotide triphosphate is added to the reaction mixture along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target, and the polymerase will extend the primers along the target sequence through the addition of nucleotides. By increasing and decreasing the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products. Excess primers will bind to the target and the reaction products, and the process is repeated. Preferably, a reverse transcriptase PCR amplification procedure can be performed to quantify the amount of amplified mRNA. Polymerase chain reaction methodologies are well known in the art.

[00061] Still other amplification methods described in GB Patent Application No. 2,202,328 and PCT Patent Application No. PCT/US 89/01025 may be used in accordance with the present invention. In the former Patent Application, "modified" primers are used in a PCR such as template-dependent and enzyme-dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detection moiety (e.g., an enzyme). In the latter Patent Application, an excess of labeled probes is added to a sample. In the presence of the target sequence, the probe binds and is catalytically cleaved. After cleavage, the target sequence is released intact to be bound by the excess probe. Cleavage of the labeled probe signals the presence of the target sequence.

[00062] Following amplification, the presence of the amplification product can be detected. The amplified product can be sequenced by any method known in the art.

[00063] In another embodiment, the invention relates to methods according to the present invention, in which the determination of the presence of a p%$ isoform selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β is performed through a probe capable of specifically hybridizing with said p53 isoform mRNA, complementary sequence thereof or a fragment thereof that is at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length that is specific to said p53 isoform, preferably hybridizing with human Δ133p53β mRNA and even more preferably with Δ133p53β mRNA having the sequence SEQ ID NO: 2.

[00064] The probe is advantageously chosen from the group consisting of the sequences SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, preferably SEQ ID NO: 11.

[00065] In a specific embodiment, the invention relates to methods according to the present invention in which the determination of the presence of p53 isoform mRNA, more specifically Δ133p53β mRNA, is performed by the following method, which comprises the steps of: (a) contacting a nucleic acid probe specific for a p53 isoform selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β under hybridization conditions with the biological test sample, comprising either: - RNA molecules isolated from a biological sample of the human subject, wherein the biological sample is suspected of containing tumor cells, or - nucleic acid molecules synthesized from isolated RNA molecules, such as DNA, wherein the nucleic acid probe has a nucleotide sequence comprising either fragments of length of at least 15 nucleotides or fragments of length of at least 15 nucleotides. the sequence of the p53 isoform or a fragment thereof or its complement, and(b) detecting the formation of hybrids of the nucleic acid probe and the test sample, wherein the presence of hybrids indicates the presence of tumor cells in the tissue obtained from the human subject.

[00066] Probes based on the sequence of a nucleic acid molecule of the invention can be used for the detection of transcripts that correspond to the mRNA of p53 isoforms selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β. The nucleic acid probe can be, for example, a full-length cDNA or a fragment thereof such as an oligonucleotide that has a length sufficient to hybridize specifically under stringent conditions to mRNA of the p53 isoform of the invention. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed. In one embodiment, the probe includes a labeling group attached thereto, such as a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor.

[00067] In one format, the mRNA is immobilized on a solid surface and contacted with a probe, e.g., by running the mRNA on an agar gel and transferring the mRNA from the gel to a membrane, such as microcellulose. In an alternative format, the probe(s) is immobilized on a solid surface and the mRNA is contacted with the probe(s), e.g., on an Affymetrix gene chip array. One skilled in the art can readily adapt known mRNA detection methods for use in detecting the level of expression of the mRNA encoded by the markers of the present invention.

[00068] When the methods according to the present invention are based on the detection or quantification of the level of expression of a polypeptide of a p53 isoform, it is also preferred that the determination of the presence of said p53 isoform be carried out by an immunohistochemical method or immunoanalysis using an antibody capable of specifically recognizing said p53 isoform.

[00069] In one embodiment, determining the presence of a p53 isoform selected from the group consisting of Δ133p53, Δ133p53Y, and Δ133p53β is performed by contacting the biological sample with an antibody specific for a p53 isoform selected from the group consisting of a polypeptide of Δ133p53, Δ133p53Y, and Δ133p53β, or a fragment thereof, and determining binding of the antibody to the biological sample.

[00070] "Antibody" includes immunoglobulin molecules and immunobiologically active determinants of immunoglobulin molecules, that is, molecules that contain an antigen-binding site (epitope) that specifically binds (immunoreacts with) an antigen. Binding specificity in the large and diverse set of antibodies is found in the variable (V) determinant of the H and L chains. Antibodies include polyclonal antibodies, monoclonal antibodies, whole immunoglobulins, and antigen-binding fragments of immunoglobulins.

[00071] The binding sites of the proteins that make up an antibody, i.e., the antigen-binding functions, are located by analyzing fragments of a naturally occurring antibody. Thus, antigen-binding fragments encompassed within the term antibody include Fab fragments consisting of the VL, VH, CL, and Cm domains; an Fd fragment consisting of the VH and CHI domains; an Fv fragment consisting of the VL and VH domains of a single branch of an antibody, an isolated complementary determining region (CDR); and an F(ab')3 fragment, a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge in the hinge region. These antibody fragments are obtained using techniques well known to those skilled in the art, and the fragments are screened for utility in the same manner as an intact antibody. The term "antibody" is also intended to include bispecific and chimeric molecules that have at least one antigen-binding determinant derived from an antibody molecule.

[00072] In the diagnostic and prognostic analyses of the invention, the antibody may be a polyclonal antibody or a monoclonal antibody, and monoclonal antibodies are preferred.

[00073] Preferably, said antibody is labeled.

[00074] Polyclonal antibodies are produced by immunizing animals, usually a mammal, by multiple subcutaneous or intraperitoneal injections of an immunogen (antigen) and an adjuvant as appropriate. As an illustrative embodiment, animals are typically immunized against a protein, peptide, or immune response, along with an enhancing vehicle preparation, such as complete Freund's adjuvant, or an aggregating agent such as alum, and injecting the composition intradermally at multiple sites. The animals are later boosted with at least one subsequent administration of a lower amount, such as 1/5 or 1/10 of the original amount of the immunogen in incomplete Freund's adjuvant (or other suitable adjuvant) by subcutaneous injections at multiple sites. The animals are then bled, the serum is analyzed for the specific antibody titer, and the animals are again boosted and analyzed until the antibody titer no longer increases (i.e., plateau).

[00075] This population of antibody molecules is referred to as "polyclonal" because the population comprises a large pool of antibodies each of which is specific for one of the very different epitopes found on the immunogen, and each is characterized by a specific affinity for that epitope. An epitope is the smallest antigen-forming determinant which for a protein may comprise a peptide of six to eight residues in length (Berzofsky J. and Berkower I. (1993) in Paul, W., Ed., Fundamental Immunology, Raven Press, N.Y., p. 246). Affinities vary from low, e.g., 10-6 M, to high, e.g., 10-11.

[00076] The polyclonal antibody fraction collected from mammalian serum is isolated by well-known techniques, such as by chromatography with an affinity matrix that selectively binds immunoglobulin molecules such as protein A to obtain the IgG fraction. To increase antibody purity and specificity, specific antibodies can be further purified by immunoaffinity chromatography using solid-phase-fixed immunogen. The antibody is contacted with the solid-phase-fixed immunogen for a period of time sufficient for the immunogen to react with the antibody molecules to form a solid-phase-fixed immunocomplex. The bound antibodies are eluted from the solid phase by standardized techniques, such as by using buffers of decreasing pH or increasing ionic valence. The eluted fractions are analyzed, and those containing the specific antibodies are combined.

[00077] "Monoclonal antibody" as used herein in this patent application refers to an antibody molecule preparation of individual molecular composition. A monoclonal antibody composition exhibits a unique binding specificity and affinity for a specific epitope. Monoclonal antibodies may be prepared using a technique that provides for the production of antibody molecules through the continuous growth of cells in culture. These techniques include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature, 256: 495-497; see also Brown et al., 1981, J. Immunol, 127: 539-46; Brown et al., 1980, J. Biol. Chem., 255: 4980-83; Yeh et al., 1976, PNAS 76: 2927-31; and Yeh et al., 1982, Int. J. Cancer, 29: 269-75) and the more recent human B-cell hybridoma technique (Kozbor et al., 1983, Immunol. Today 4: 72), EBV hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96), and trioma techniques. The technology for producing hybridomas is well known (see generally, Current Protocols in Immunology, Coligan et al. ed., John Wiley & Sons, New York, 1994). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening hybridoma culture supernatants for antibodies that bind the polypeptide of interest, such as by using a standard ELISA assay.

[00078] A monoclonal antibody can be produced through the following steps. In all procedures, an animal is immunized with an antigen such as a protein (or peptide thereof) as described above with respect to the preparation of a polyclonal antibody. Immunization is typically accomplished by administering the immunogen to an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to produce an immune response. Preferably, the mammal is a rodent such as a rabbit, rat, or mouse. The mammal is then maintained on a booster schedule for a period of time sufficient for the mammal to generate high-affinity antibody molecules as described. After a time sufficient for the generation of high-affinity antibodies, the animal (e.g., a mouse) is sacrificed, and antibody-producing lymphocytes are obtained from one or more of the lymph nodes, spleen, and peripheral blood. Spleen cells are preferred and can be mechanically separated into individual cells in a physiological medium using methods well known to one skilled in the art. Antibody-producing cells are immortalized through fusion with a mouse myeloma cell line.

[00079] Mouse lymphocytes give a high percentage of stable fusions with homologous mouse myelomas, however rat, rabbit, and frog somatic cells can also be used. Spleen cells from animals producing the desired antibodies are immortalized by fusion with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol. Any number of suitable myeloma cell lines as fusion partners can be used with standard techniques, for example, the P3-NS1/1-Ag4-1, P3-x63-Ag8.653, or Sp2/0-Agl4 myeloma lines, available from the American Type Culture Collection (ATCC), Rockville, MD.

[00080] The fusion product cells, which include the desired hybridomas, are cultured in a selective medium such as HAT medium, designed to eliminate the original myeloma cells, lymphocytes, or unfused spleen cells. The hybridoma cells are selected and cultured under limited culture conditions to obtain isolated clones. The supernatants from each clonal hybridoma are screened for the production of antibodies of the desired specificity and affinity, such as by immunoassay techniques for the determination of the desired antigen such as those used for immunization. The monoclonal antibody is isolated from the producer cell cultures by conventional techniques such as ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography (Zola et al., Monoclonal Hybridoma Antibodies: Techniques And Applications, Hurell (ed.), pp. 51-52, CRC Press, 1982).

[00081] Hybridomas produced according to these methods can be propagated in culture in vitro or in vivo (in ascites fluid) using techniques well known to those skilled in the art.

[00082] As an alternative to preparing antibody-secreting monoclonal hybridomas, a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a combinatorial recombinant immunoglobulin pool (e.g., an antibody phage display pool) with the polypeptide of interest. Kits for generating and screening phage display pools are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SutfZ4P Phage Display Kit, Catalog No. 240612). Additional examples of methods and reagents specifically suited for use in generating and screening an antibody display pool can be found, for example, in U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas, 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J., 12: 725-734.

[00083] In addition, recombinant antibodies, such as humanized monoclonal chimeric antibodies, comprising both human and non-human parts, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such humanized and chimeric monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using methods described in PCT Publication No. WO 87/02671; European Patent Application 0 184 187; European Patent Application 0 171 496; European Patent Application 0 173 494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application 0 125 023; Better et al. (1988) "Labelled antibody" as used herein in this patent application includes antibodies that are labeled by a device that can be detected and includes antibodies labeled enzymatically, radioactively, fluorescently, chemiluminescently, and/or bioluminescently by any of the many different methods known to those skilled in the art.

[00084] One way in which an antibody can be detectably labeled is by binding it to an enzyme. This enzyme, when later exposed to its substrate, will react with the substrate in such a way as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric, or visual means. Enzymes that can be used for detectably labeling an antibody specific for a p53 isoform include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-V-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[00085] Detection may be achieved using any of a variety of immunoassays. For example, by radiolabeling an antibody, the antibody may be detected using immunoradiation assays. A description of an immunoradiation assay (RIA) may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work T. S. et al., North Holland Publishing Company, NY (1978), with specific reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard T. The radioactive isotope may be detected by such devices as a gamma counter or scintillation counter or by audio radiography. Isotopes that are specifically useful for the purpose of the present invention are: 3H, 13I1, 35S, 14C, and preferably 125I.

[00086] It is also possible to label an antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycocriterin, phycocyanin, allophycocyanin, ophthaldehyde, and fluorescamine.

[00087] An antibody may also be detectably labeled using fluorescently emitting metals, such as 152Eu and others of the lanthanide series. Such metals may be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (DTPA) or ethylenediamine tetraacetic acid (EDTA),

[00088] An antibody may also be detectably labeled by coupling it to a chemically luminescent compound. The presence of the chemically luminescently labeled antibody is then determined by detecting the presence of luminescence that appears during the course of a chemical reaction. Examples of specifically useful chemically luminescently labeled compounds are luminol, luciferin, isoluminol, acrimidinium theromatic ester, imidazole, acrimidinium salt, and oxalate ester.

[00089] Similarly, a bioluminescent compound may be used to label an antibody of the present invention. Bioluminescence is a type of chemical luminescence found in biological systems in which a catalytic protein increases the efficiency of the chemical luminescence reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important luminescent compounds for labeling purposes are luciferin, luciferase, and acquorin.

[00090] In the detection analyses of the invention, the amount of binding of the antibody to the biological sample can be determined by the intensity of the signal emitted by the labeled antibody and/or by the number of cells in the biological sample bound to the labeled antibody.

[00091] Detection of the expression level of a p53 isoform selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β in a biological sample can be determined by means of an immunoradio analysis, an immunoradiometric analysis and/or an enzyme immunoanalysis.

[00092] An "immunoradiation assay" is a technique for detecting and measuring the concentration of an antigen using a labeled (i.e., radioactively labeled) form of the antigen (i.e., the Δ133p53β polypeptide). Examples of radioactive labels for antigen include 3H, 14C, and 125I. The concentration of the p53 isoform in a biological sample is measured by causing the antigen in the sample to compete with a labeled (i.e., radioactively labeled) antigen for the binding of an antibody to the antigen. To ensure competitive binding between the labeled antigen and the unlabeled antigen, the labeled antigen is present in a concentration sufficient to saturate the antibody binding sites. The higher the concentration of the antigen in the sample, the lower the concentration of the labeled antigen that will bind to the antibody.

[00093] In an immunoradioassay to determine the concentration of labeled antigen bound to an antibody, the antigen-antibody complex must be separated from free antigen. One method for separating the antigen-antibody complex from free antigen is by precipitating the antigen-antibody complex with an anti-isotype antiserum. Another method for separating the antigen-antibody complex from free antigen is by precipitating the antigen-antibody complex with formalin-killed S. aureus. Yet another method for separating the antigen-antibody complex from free antigen is by performing a "solid phase radioimmunoassay" in which the antibody is bound (i.e., covalently) to Sepharose beads, polystyrene wells, polyvinyl chloride wells, or microtiter wells. By comparing the concentration of labeled antigen bound to the antibody to a standard curve based on samples having a known antigen concentration, the concentration of the antigen in the biological sample can be determined.

[00094] An "immunoradiometric analysis" (IRMA) is an immunoassay in which the antibody reagent is labeled with radioactivity. Insofar as IRMA requires the production of a multivalent antigen conjugate through techniques such as conjugation to a protein, such as rabbit serum albumin (RSA). The multivalent antigen conjugate must have at least 2 antigen residues per molecule, and the antigen residues must be separated by a sufficient distance to allow binding by at least two antibodies to the antigen. For example, in an IRMA, the multivalent antigen conjugate may be bound to a solid surface such as a plastic bead.

[00095] An unlabeled "sample" antigen and an antibody to the antigen that is radioactively labeled are added to a test tube containing the multivalent antigen conjugate-coated bead. The antigen in the sample competes with the multivalent antigen conjugate for the antigen-binding sites on the antibody. After an appropriate incubation period, unbound reagents are removed by washing and the amount of radioactivity on the solid phase is determined. The amount of bound radioactive antibody is inversely proportional to the concentration of antigen in the sample.

[00096] The most common enzyme immunoassay is the "Enzyme-Linked Immunosorbent Assay (ELISA)." The "Enzyme-Linked Immunosorbent Assay (ELISA)" is a technique for detecting and measuring the concentration of an antigen using a labeled (i.e., enzyme-linked) form of an antibody.

[00097] In a "sandwich ELISA" an antibody (i.e., the Δ133p53β peptide) is bound to a solid phase (i.e., a microtiter plate) and exposed to a biological sample containing the antigen (i.e., the Δ133p53β peptide). The solid phase is then washed to remove unbound antigen. A labeled (i.e., enzyme-linked) antibody is then bound to the bound antigen (if present), forming an antibody-antigen-antibody sandwich. Examples of enzymes that can be bound to the antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease, and β-galactosidase. The enzyme-linked antibody reacts with a substrate to generate a colored reaction product that can be analyzed.

[00098] In a "competitive ELISA," the antibody is incubated with a sample containing the antigen (i.e., a p53 isoform peptide). The antigen-antibody mixture is then contacted with a solid phase coated with the antigen (i.e., a microtiter plate). The more antigen present in the sample, the less free antibody will be available to bind to the solid phase. A secondary (i.e., enzyme-linked) antibody is then added to the solid phase to determine the amount of primary antibody found on the solid phase.

[00099] In an "immunohistochemical analysis" a tissue section is tested for specific proteins by exposing the tissue to antibodies that are specific for the protein being analyzed. The antibodies are then visualized by any of a number of methods for determining the presence and amount of the protein present. Examples of methods used for visualizing antibodies are, for example, by enzymes linked to the antibodies (i.e., luciferase, alkaline phosphatase, horseradish peroxidase, or β-galactosidase), or chemical methods (e.g., DAB/substrate chromogen), or gold, fluorescent, or labeled antibodies by one of many different methods known to those skilled in the art.

[000100] In another aspect the present invention relates to a method for screening potential anticancer, and preferably antimetastatic, compounds, comprising the steps of: (a) optionally measuring in a cell known to express a p53 isoform selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β, the level of expression of said p53 isoform; (b) contacting the compound to be tested with said cell; (c) determining the level of expression of said p53 isoform by a method for selecting said p53 isoform as described above; and (d) selecting said compound as a potential anticancer compound if the p53 isoform is not expressed in the cell, or has a lower level of expression than before step (a). Optionally, said screening method may include an additional step (e) of testing the compound selected in step (d) on cells, preferably on metastatic cells, for confirmation of the anticancer properties, preferably the antimetastatic properties, of the selected compound.

[000101] For example, said step (c) may correspond to the method described in International Patent Application WO 2006/134305, that is, a method comprising the step of: contacting tumor cells that do not express E-cadherin in their cell membranes with the selected compound and determining the presence of E-cadherin on the cell surface, said presence being indicative of an antimetastatic activity.

[000102] Said step (c) may also correspond to the method described in Smith HW, Marra P, Marshall CJJ Cell Biol. 2008 Aug 25;182(4):777-90, i.e., a method for testing the invasion of tumor cells into a three-dimensional collagen matrix in the presence of the selected compound.

[000103] Preferably the compound to be tested is antisense RNA or interfering RNA (iRNA).

[000104] In another aspect, the present invention is directed to a method for producing polyclonal antibodies specifically recognizing a p53 isoform polypeptide selected from the group consisting of a Δ133p53, Δ133p53Y, and Δ133p53β polypeptide, said method comprising the steps of: (a) immunizing a mammalian animal with an immunologically effective amount of a p53 isoform polypeptide selected from the group consisting of Δ133p53, Δ133p53Y, and Δ133p53β, or against a specific fragment of an epitope of at least 9 amino acids in length of said polypeptide, optionally with an enhancing vehicle preparation; (b) optionally determining in vitro the presence of specific antibodies in the serum or plasma of the animal; and (c) purifying or isolating the specific anti-Δ133p53, anti-Δ133p53Y and anti-Δ133p53β polypeptide from the animal serum or plasma.

[000105] Also part of the present invention is a method for producing a hybridoma cell capable of secreting monoclonal antibodies that specifically recognize a p53 isoform selected from the group consisting of a Δ133p53, Δ133p53Y, and Δ133p53β polypeptide, said method comprising the steps of: (a) immunizing a mammalian animal with an immunologically effective amount of a p53 isoform polypeptide selected from the group consisting of Δ133p53, Δ133p53Y, and Δ133p53β, or against a fragment of an epitope of at least 9 amino acids in length of said polypeptide, optionally with an enhancing vehicle preparation; (b) isolating anti-p53 isoform peptide antibodies from lymphocytes producing the same; from the spleen, lymph nodes or peripheral blood of that mammalian animal; and (c) immortalize the polypeptide antibodies of anti-Δ133p53, anti-Δ133p53Y and anti-Δ133p53β producing lymphocytes through the fusion of said lymphocytes with cells of the same myeloma line of the same mammalian species.

[000106] Also part of the present invention is a method for the production of monoclonal antibodies that specifically recognize a p53 isoform selected from the group consisting of a polypeptide of Δ133p53, Δ133p53Y and Δ133p53β, which comprises the steps of: (a) producing a hybridoma cell capable of secreting monoclonal antibodies that specifically recognize a p53 isoform selected from the group consisting of a polypeptide of Δ133p53, Δ133p53Y and Δ133p53β, according to the method of the invention above; (b) culturing such hybridoma cell in an appropriate culture medium and under culture conditions; (c) purifying or isolating from such culture medium the monoclonal antibodies that are secreted.

[000107] In another part, the present invention comprises polyclonal or monoclonal antibodies that can be obtained through the method for producing polyclonal or monoclonal antibodies according to the present invention in which said antibodies are specifically recognizing a p53 isoform selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β.

[000108] In another aspect, the present invention relates to a kit for the detection or quantification of a p53 isoform selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β, or polypeptide, wherein said kit comprises: (a) a monoclonal or polyclonal antibody according to the invention; or (b) a pair of primers selected from the group consisting of a pair of primers capable of amplifying the sequences SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or a fragment thereof of at least 10 nucleotides in length; or (c) a probe that has a nucleotide sequence comprising either a fragment of at least 10 nucleotides in length of a sequence of a p53 isoform selected from the group consisting of Δ133p53, Δ133p53Y, and Δ133p53β, or the complement thereof.

[000109] For antibody-based kits, the kit comprises, for example, (1) a first antibody, e.g., in solution or fixed to a solid support) that binds to a p53 isoform polypeptide according to the invention; and optionally (2) a second, different antibody conjugate with respect to a detectable marker that binds both to the p53 isoform peptide and to the first antibody.

[000110] For oligonucleotide-based kits, the kit may comprise, for example, (1) an oligonucleotide, such as a detectably labeled oligonucleotide, that hybridizes to a p53 isoform nucleic acid sequence (mRNA or cDNA, or specific fragments thereof) or (2) a pair of primers useful for amplifying a p53 isoform nucleic acid molecule of the present invention. The kit may also comprise, for example, a buffering agent, a preservative, or a protein stabilizing agent. The kit may further comprise components necessary to detect the detectable label (such as an enzyme or a substrate). The kit may also contain a control sample or a series of control samples that can be analyzed and compared to the biological sample. Each component of the kit may be included within an individual container and all of the various containers may be within a single package, along with instructions for interpreting the results of analyses performed using the kit.

[000111] The present invention also relates to a method for complementing a morphological diagnosis by detecting or quantifying a p53 isoform selected from the group consisting of Δ133p53, Δ133p53Y and Δ133p53β, in a mammalian biological sample, by the methods mentioned above or by a method well known to a person skilled in the art, such as immunohistochemistry or immunoanalysis for the detection of the peptide defined above or by any method that detects the nucleotide sequence defined above (such as, for example, RT-PCR, quantitative RT-PCR, FISH, etc.) in cancer of any type (such as, for example, breast, colon, pancreas, head and neck, etc.).

DESCRIPTION OF FIGURES

[000112] Figure 1 shows disease-free survival (Figure 1a) and survival functions (Figure 1b) over time in patients expressing or not expressing Δ133p53β.

[000113] Figure 2 shows disease-free survival (figure 2a) and survival functions (figure 2b) over time in patients depending on Δ133p53β and ER expression.

[000114] Figure 3 shows the percentage of invasion (figure 3a) and the percentage of migration (figure 3b) depending on the expression of Δ133p53β. Figure 3c is a schematic presentation of p53 and Δ133p53β isoform (NLS: Nuclear Localization Signal).

[000115] Figure 4 shows the quantitative analysis of blebbing cells versus adherent cells in the GFP positive cells.

[000116] Figures 4 b and c show Western blot analysis of the expansion of myc-tagged constructs in cells used for Western blot analysis of E-cadherin and beta 1-integrin. Adh: cells that are still adherent to the support, bleb: cells that exhibit "blebbing" movement and have detached from the support. Normalization was performed using a GAPDH antibody,

[000117] The following examples and figures are given for the purpose of illustrating various embodiments of the invention and are not intended to limit the present invention in any way.

EXAMPLES Example 1: Materials and methods DNA constructs, reagents and antibodies:

[000118] Human p53 isoform constructs were kindly provided by J.C. Bourdon. They were subcloned into the EcoR1 and BamH1 sites of pEGFPCl (Clonetech) to give GFP-tagged proteins and into the BamH1 and EcpR1 sites of pLCnyc to give myc-tagged proteins. The constructs were amplified using the Nucleobond PC 500 kit (Macherey-Nagel) according to the manufacturer's instructions.

[000119] Y27632 was purchased from Calbiochem and was used at 10 μM in all experiments.

[000120] Mouse anti-adherin (clone 56), mouse anti-beta 1 integrin, mouse anti-ROCK 1, and mouse anti-ROCK II antibodies were purchased from BD-Transduction Laboratories and were diluted at 1/400°, 1/2500°, 1/250°, and 1/250°, respectively. Mouse anti-RhoA and rabbit anti-ETC2 antibodies were purchased from Santa Cruz (26C4 and C-20, respectively) and were diluted at 1/500 and 1/200, respectively. Mouse anti-GEP-H1 antibody was provided by K. Mater and was diluted at 1/50°. Rabbit anti-p53 antibodies (CM1) were kindly provided by J.C. Bourdeon and were diluted at 1/1000°.

[000121] Horseradish Peroxidase (HRP) conjugated anti-IgG antibodies were purchased from GE-Healthcare and were diluted to 1/5000°.

[000122] Western Lightning Chemiluminescence (ECL) reagents were purchased from PerkinElmer.

Cell culture and transfections:

[000123] HCct116 cells were kindly provided by B. Vogelstein and were cultured at 37°C in the presence of 5% CO2 in McCOY'5A medium (Sigma) supplemented with 10% fetal bovine serum (FSC).

[000124] Transient transfections of isoforms were performed using the JetPei kit (Qbiogen) according to the manufacturer's instructions: 9 ug of DNA was used for the transfection of a 100 mm dish of 70% confluent cells. After 24 hours of transfection, the cells were diluted to 1/2 a 100 mm dish and all experiments were performed 48 hours after transfection. Transient transfections of 50 nM siRNA were performed using Interferin (Polyplus) according to the manufacturer's instructions.

[000125] Image formation time lapse:

[000126] Time-lapse "nomasrsky" microscopy was performed on an inverted Leica DMIRE2 microscope with an automatic shutter and GFP filter set, a 63x oil immersion objective (HC x PL APO 1.32-0.6 CS oil), sample heater (37°C), and a home-made CO2 incubation chamber. Images were captured with a micromax CCD camera (1300Y/HS) and imaging software, converted to TIFF files, and edited and compiled with Metamorf. The exposure time was 500 ms for GFP and 300 ms for light. Images were captured every 3 seconds for 5 minutes or every 4 minutes for 12 hours.

FACS:

[000127] Cells transfected with GEP-tagged p53 isoforms that were not used for invasion analyses were spun at 1200 rpm for 5 minutes and fixed by adding 1 ml of 70% EtOH at -20°C. Propidium iodide staining was performed and the number of cells expressing GEP compared to the total cells in each condition was quantified by FACS using CellQuest software.

Western blotting cell extracts.

[000128] Media containing blebbing cells was spun at 1200 rpm for 5 minutes, and the pellet of invasive blebbing cells was lysed. The remaining adherent cells were gently scraped off in lysis buffer. The two cell populations were analyzed separately for each condition (see figure legends). The total amount of proteins in each extract was quantified using a BCA kit (Promega). 8% SDS-PAGE gels were used to detect E-cadherin, beta 1-integrin, ROCK 1, ROCK 2, ECT2, and GEF-H1, and 12% SDS-PAGE gels were used to detect Rho-A. An equal amount (30 μg) of proteins was loaded in each lane. The proteins were then transfected by electroporation onto nitrocellulose (for p53, E-cadherin, beta 1-integrin, ROCK 1, ROCK 2, ECT2, and GEF-H1) or PVDF membrane (for GTP-RhoA). The membranes were blocked in TBS/0.1% Tween 20 containing 3% milk for 1 hour and then incubated overnight with primary antibodies diluted in TBS/0.1% Tween 20 containing 3% milk. After several washes in TBS/Tween, the membranes were incubated with HRP-linked anti-rabbit or anti-mouse Ig antibodies. The membranes were developed with ECL according to the manufacturer's instructions.

[000129] The scanned autoradiographs were quantified using the AINDA/2D densitometry software.

RhoA activity analysis:

[000130] For RhoA activity analysis, cells were lysed in 30 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 500 mM NaCl, 10 mM MgCl2, 1 mM PMSF, and protease inhibitor cocktail. Cleared lysates were incubated with 23 μg of a commercial GST fusion protein containing the Rho-A binding domain of Rhotekin beads (GST-RBD, cytoskeleton) for 30 minutes at 4°C. Precipitated complexes were washed four times in Tris buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, 0.1 mM PMSF, eluted in SDS sample buffer, immunoblotted, and analyzed with antibodies specific for RhoA; An aliquot of the total lysate used for precipitation was combined to quantify the total RhoA present in the cell lysates. Scanned autoradiographs were quantified using the AINDA/2D densitometry software and normalized as a function of RhoA protein expression.

Invasion analysis:

[000131] Quantification of cell invasion was performed in Transwell cell culture chambers, which contained fluorescence-blocking polycarbonate porous membrane inserts (Fluoroblock; #351152; BD Biosciences; 8 μm pore size). 100 μl of 2mg/ml Matrigel with reduced growth factors (a commercially reconstituted BM prepared from Englebreth-Holm-Swarm tumors, #354230; BD Biosciences) in a Transwell. Cells were transfected and treated or untreated with Y27632 as monolayers prior to trypsinization and plating (10,104) in 2% PCS containing medium as a thick (around 500 μm) layer of Matrigel contained within the upper chamber of a Transwell. Controls were left untreated. The upper and lower chambers were then filled with medium containing 2% FSC and medium containing 10% FSC, respectively, establishing a soluble gradient of chemical attractant that allowed cell invasion through the Marigel. Cells were allowed to invade through the gel at 37°C and 5% CO2 before being fixed for 15 minutes in 3.7% formaldehyde. Cells that had invaded through the Matrigel were detected on the underside of the filter by GFP fluorescence and counted for cell number. The entire filter surface was counted, and each analysis was performed twice in triplicate for each condition.

Breast cancer patients

[000132] Primarily, previously untreated breast cancer from 171 Caucasian women (age range 24 to 89 years, mean age 64 years) with sufficient tumor tissue beyond that required for diagnosis and complete clinical and pathological data were analyzed. Tumor tissues were macro-dissected by a breast pathologist and immediately frozen in liquid nitrogen before being stored at -80°C. Normal breast tissue was obtained from patients undergoing reduction mammoplasty who had no personal or family history of breast cancer. Samples were examined following approval from the Local Research Ethics Committee under the delegated authority of the Tayside Tissue Bank.

RT-PCR analysis.

[000133] Approximately 10 mg of tumor tissue (> 40% tumor cells) was homogenized in 700 ul of QIAzol lysis reagent (Qiagen Ltd, Crawley, West Sussex, UK) and the quality of the extracted RNA was confirmed using the BioAnalyzer 2100™ (Agilent Technologies, Palo Alto, CA, USA), demonstrating 28S/18S > 1.5 with two sharp peaks confirmed prior to RT-PCR

[000134] The o53 isoform cDNA is too long to be specifically quantified by RT-qPCR, so a semi-quantitative RT-PCR method requiring high-quality total RNA with a 28S/18S ratio > 1.5 was used; After reverse transcription of 500 ng of total RNA using random primers, the actin cDNA was amplified by PCR to confirm the efficiency of reverse transcription. 0.5 ug of total RNA from each tumor sample was reverse transcribed (AMV RT, 45C, random primers) and the quality of the cDNA was confirmed by PCR amplification of actin for 30 cycles. The p53 isoform cDNA was amplified by 30-cycle nested 2-PCR using primers specific for each isoform as previously described (Bourdon et al., 2005a). Tumors were considered to express each p53 isoform after sequencing of the corresponding PCR fragment.

p53 Mutation Analysis AmpliChip p53 Test

[000135] The AmpliChip p53 Test is a product currently under development at Roche Molecular Systems, Inc. (Pleasanton, California, USA). A 10-micron section of formalin-fixed, paraffin-embedded tumor tissue or 100 ng of genomic DNA purified from fresh-frozen tumor are required for this test. In this study, 100 ng of genomic DNA extracted from frozen homogenized tissue was used for the amplification of products encompassing the coding regions of the p53 gene (A and B). Reaction A amplifies the sequences of exons 2, 5, 8 and exon 4 upstream with an internal control, while Reaction B amplifies the sequences of exons 3, 6, 7, 9, 11, exon 4 downstream with the same internal control. The PCR products generated from reactions A and B were combined for DNase I cleavage to generate small DNA fragments with an average size of 50 to 100 nucleotides. The fragmented DNA applicons were then labeled with biotin using Terminal Transferase. The biotin-labeled p53 DNA fragments were added to the hybridization buffer. The mixture was hybridized to the oligonucleotides located on the AmpliChip p53 Microarray using the Affymetrix GeneChip Fluidics Station 450Dx and a specific AmpliChip p53 protocol. The hybridized AmpliChip p53 Microarray was washed and labeled with a fluorescent dye conjugated to streptavidin (phycoerythrin). After labeling, the AmpliChip p53 Microarray was scanned on an Affymetrix GeneChip Scanner 3000Dx using a laser that excites the fluorescent label bound to the target hybridized p53 DNA fragments. The amount of light emitted is proportional to the target DNA bound at each location on the microprobe array.

Chip Design and Micro Array Signal Data Analysis.

[000136] The AmpliChip p53 Microarray designed by Roche Molecular Systems consists of more than 33,000 probe sets of more than 230,000 individual oligonucleotides, grown against a total of 1268 nucleotide positions of the coding regions of exons 2-11. A single probe set against a base position for interrogation includes five probes, one probe to hybridize to wild-type, three probes to detect three possible mutations in a single base pair, and one probe to detect a single deletion. There are at least 24 probe sets for each nucleotide position, including both sense and antisense probe sequences. AmpliChip p53 Microarrays are manufactured by Affymetrix using technology that combines photolithographic methods and combinatorial chemistry. The p53 mutation status was determined using a p53 Mutation Detection Algorithm developed by Roche Molecular System, which is designed to detect single base pair substitutions and single base pair deletions of a sample against a background of wild-type p53 DNA probe intensities.

Statistical analysis.

[000137] Statistical analysis was performed using SPSS statistical software (needs reference) for Chi square, Fisher's 2-tailed exact test and Kaplan-Meier analysis. Results were judged significant at a confidence level greater than 95% (P < 0.05).

[000138] Example 2: Analysis of Δ133p53β expression in breast cancer patients.

[000139] Primarily, previously untreated operable breast cancer from 171 Caucasian women (age range 24 to 89 years, mean age 64 years) with sufficient tumor tissue beyond that required for diagnosis and complete clinical and pathological data were analyzed.

Results

[000140] In 171 breast cancers, expression of Δ133p53 in 50/171 (29%), Δ133p53β in 20/171 (11%) and Δ133p53Y in 27/171 (16%) was identified, whereas none of the three isoforms was detected in normal breast tissue.

[000141] Δ133p53β expression was associated with axillary lymph node metastasis (1-tailed exact Mann-Whitney analysis, p<0.036) and consistent with this, patients with tumors expressing Δ133p53β had significantly worse disease-free survival (log-rank, Cox-Mantel, p<0.025) (figure 1a) and overall survival (log-rank, Cox-Mantel, p<0.025) (figure 1b).

[000142] Δ133p53β was significantly associated with low ER expression (x2 = 4.1, x d.f, p< 0.043, respectively) specifically with respect to invasive carcinoma of the ductus arteriosus (142/171 cases), the most common histological type of breast cancer (paired t-test, p< 0.015).

[000143] Figure 2a shows that ER-positive patients expressing Δ133p53β had significantly worse disease-free survival than ER-positive patients free of Δ133p53β expression (log rank, Cox-Mantel, p<0.0002, pairwise comparison Δ133p53β+ ER+ vs Δ133p53β-ER+, p<0.003). Furthermore, Figure 2 shows that ER-positive patients expressing Δ133p53β unexpectedly had poorer survival compared to ER-negative patients (survival: log rank, Cox-Mantel, p<0.0003, pairwise comparison Δ133p53β+ER+ vs ER-, p<0.658).

[000144] For this reason Δ133p53β has been associated with poor prognostic features (axillary node metastasis, ER negative), a shorter disease-free survival and a worse prognosis in primary breast cancer.

Example 3: Main role of a Δ133p53β isoform in the invasion process.

[000145] First, the effects of expression of the Δ133p53, Δ133p53Y, and Δ133p53β isoforms on the invasion of colon and breast carcinoma cells that retain epithelial characteristics and express p53 (hctl l6 for colon cancer and MCF7 for breast cancer) were tested using invasion assays (see methods).

[000146] Isolated GFP-transfected cells were used as negative controls. The cells were plated on dishes on top of a thick layer of matrigel that mimics the physiological basement membrane. This in vitro analysis aims to mimic the progress of tumor cells crossing basement membranes.

[000147] Figure 3a shows that invasiveness was significantly increased by expression of the Δ133p53β isoform. Similar results were obtained with MCF7 (data not shown). This suggests that overexpression of the ectopic Δ133p53β isoform may overcome the antimigratory activity of endogenous p53.

[000148] To determine which migration mode was used by p53 isoform-expressing cells to invade through the matrigel, GFP-tagged isoform-expressing HCT 115 cells were filmed. Two cell populations were observed: cohesive epithelial cells adherent to the glass and rounded cells that were above the cohesive cells, in a higher focus. Cells transfected with the GFP-tagged Δ133p53β isoform generated dynamic "bleb"-like structures on their surfaces. During the video, some rounded cells moved and some adherent cohesive cells progressively detached from the epithelium and became rounded (data not shown). The loss of adhesive structures and the concomitant acquisition of rounded-blebbing movement is remarkably similar to the epithelial-amoeboid transition. A control by FACS that these blebbing movements were not due to apoptosis was performed (data not shown).

[000149] To quantify the penetrance of the phenotype, rounded cells were separated from cohesive cells and GFP-positive cells were counted in both populations by FACS: for Δ133p53 and Δ133p53Y, the number of blebbing cells was 2.5- and 2.25-fold higher than the number of adherent cells, respectively, while for Δ133p53β the number of blebbing cells was 4-fold higher than the number of adherent cells (figure 4a). In conclusion, this phenotype is significantly penetrant, i.e., cells expressing the GFP-tagged Δ133p53β isoform largely adopt a rounded morphology and cell separation from the epithelium.

[000150] During EAT, tight junctions are first disassociated, then adherens junctions disappear afterwards. Since E-cadherin is the main component of adherens junctions, it is widely used as a specific marker of the integrity of such junctions. In p53-expressing cells, E-cadherin was expressed at a high level in cohesive adherens cells, whereas E-cadherin expression was lost in blebbing cells (Figure 4b), confirming that blebbing cells have lost a key epithelial characteristic.

[000151] Tumor cells subjected to EAT express low levels of beta 1-integrin and thus adopt amoeba-like migration independent of the integrin adhesion process. The expression of beta 1-integrin in het116 cells expressing the Δ133p53, Δ133p53Y and Δ133p53β isoforms was evaluated. Regarding E-cadherin, blebbing cells do not express beta 1-integrin (Figure 4c). The same results were observed using myc- or GFP-tagged isoforms. The expression of myc-tagged isoforms was controlled by Western blot using an antimyc antibody (data not shown).

[000152] These results show that the expression of p53 isoforms with the N-terminus deleted promotes the epithelial-amoeboid transition in hct116 cells, which consequently migrate through rounded blebbing movements. This mode of migration is more effective than mesenchemic migration, indicating that the expression of these Δ133p53β variants in tumors may be associated with a poor prognosis for patients. This reinforces the idea that failure to properly regulate the expression of p53 isoforms can have dramatic consequences on tumor progression and metastasis.

[000153] Since the rounded motility mode associated with blebbing is dependent on ROCK (Rho Kinase) signaling, invasion analyses in the presence of the ROCK inhibitor, Y27632, were performed. The protocol was as described above.

[000154] The results showed that treatment with Y27632 highly reduced the invasiveness of the transfected cells indicating that ROCK activity is required for the invasiveness provided through the expression of the Δ133p53β isoform.

[000155] Recently, it was reported that the two ROCK homologs may not be functionally equivalent. To determine whether both ROCK isoforms have identical effects on associated movements in p53 isoform-dependent rounded blebbing, the expression of ROCK I and ROCK II in transfected cells was studied. The results show that ROCK I expression was increased in adherent cells (compared to control T cells) but significantly decreased in blebbing cells. Conversely, ROCK II was expressed at high levels in all situations and was maintained specifically in blebbing cells. This suggests that ROCK II is preferentially required for blebbing movement, while ROCK I may be involved in the early steps of the epithelial-amoeboid transition. Despite their high sequence homologies (65% overall identity and 92% kinase domain identity), ROCK I and ROCK II do not function redundantly; there is no compensation for the loss of ROCK II by ROCK I in knockout mice, and the two isoforms regulate different aspects of myosin II activity. This demonstrated for the first time that these ROCK isoforms have different implications for amoeboid-like migration and, consequently, for invasiveness.

[000156] Actin reorganization during round-blebbing motility is largely dependent on the Rho GTPase family protein RhoA.

[000157] To investigate the mechanisms through which p53 isoforms exert their effects on ROCK signaling, RhoA expression in adherent or attached cells expressing the three Δ133 isoforms of p53 was examined.

Total RhoA levels were identical when cells did or did not express the Δ133-p53 isoforms (data not shown). Expression was maintained in blebbing cells. However, the protein expression level did not reflect kinase activity. RhoA activity in adherent cells versus blebbing cells expressing or not expressing the Δ133 isoforms was compared using knockdown assays that capture only the GFP-bound active form of RhoA. We first observed basal RhoA activity in adherent epithelial cells. However, this activity was highly increased in blebbing cells. Interestingly, the increase obtained with Δ133γ-p53 correlated with the significant ability of cells expressing Δ133β-p53 to lose surface adhesion.

[000159] The question arises as to whether RhoA activation in cells expressing the Δ133-p53 isoform was due to overexpression of Guanine Exchange Activating Factors (GEFs). The study was focused on two RhoA GEFs that were both involved in mediating p53-dependent RhoA regulation: GEF-H1 that was found to be activated by mutant p53 and whose expression strongly correlated with the p53 status in cancer cells, and ECT2 that is known to be a transcriptional target of wt p53 and that is involved in the regulation of the epithelial junction.

GEF-H1 was reported to be overexpressed in blebbing cells expressing all three p53 isoforms. In contrast, ECT2 expression was decreased in blebbing cells compared to adherent cells. This downregulation of ECT2 in blebbing cells is consistent with its known function as a regulator of epithelial junctions, but does not account for RhoA activation, which may depend on GEF-H1 upregulation. This indicates distinct roles for GEF-H1 and ECT2 during the epithelial-amoeboid transition. The results are consistent with the transforming ability of GEF-H1 when transfected into mouse fibroblasts and tumor induction when GEF-H1-transfected cells are injected into nude mice. Two opposing pathways downstream of RhoA function in the regulation of adherens junctions: a ROCK-dependent pathway involved in epithelial junction disruption and a Dia-dependent pathway that promotes cadherin-catenin complex formation and junction stabilization. One possibility is that these two pathways are activated by two different RhoA GEFs: ECT2 may activate the RhoA-Dia pathway to promote adherens functions as this GEF regulates epithelial polarity, and GEF-H1 may activate the RhoA-ROCK pathway to disrupt cell-cell junctions and promote epithelial-amoeboid transition. p53 is the upstream regulator of these two pathways, and the expression of p53 Δ133 isoforms disrupts the balance between the two pathways during tumorigenesis.

[000161] The data show that unregulated expression of Δ133p53 isoforms (isoforms deleted in the N-terminal domain) confers increased motility and invasiveness to carcinoma cells. This indicates that changing the ratio of p53 isoforms favors increased tumor aggressiveness and enhanced metastatic ability of cancer cells, and highlights the importance of cellular division events during cancer progression.

Claims (10)

1. A method for testing a subject who is considered to be predisposed to metastatic cancer, comprising the steps of: (1) analyzing a biological sample selected from the group consisting of bone marrow, serum, plasma, blood, lymph, or cancerous or cancer-suspect tissue cells or tissue adjacent thereto, from said subject to detect the presence of a p53 isoform selected from the group consisting of Δ133p53β, Δ133p53, and Δ133p53Y, the presence of said isoform being indicative of a predisposition to metastatic cancer. 2. A method for determining the response of a subject to metastatic cancer therapy, wherein said subject is receiving or has received therapy for a condition associated with metastatic cancer, comprising the steps of: (i) determining whether or not a p53 isoform selected from the group consisting of Δ133p53β, Δ133p53, and Δ133p53Y is present in a biological sample selected from the group consisting of bone marrow, serum, plasma, blood, lymph, or cells from cancerous or suspected cancerous tissue or tissue adjacent thereto, from the subject, (ii) comparing the same to a threshold value. 3. Method according to claim 1 or 2, characterized in that the p53 isoform is Δ133p53β. 4. Method according to any one of claims 1 to 3, characterized in that the expression level of a p53 isoform selected from the group consisting of Δ133p53β, Δ133p53 and Δ133p53Y is assessed by detecting a polypeptide of the p53 isoform or a fragment thereof, or by detecting the mRNA of a p53 isoform or a fragment thereof. 5. Method according to any one of claims 1 to 4, characterized in that it comprises an additional step of amplifying said mRNA or cDNA of the p53 isoform, the complementary sequence thereof or a fragment thereof that has at least a length of 10 nucleotides that is specific to said p53 isoform. 6. Method according to any one of claims 1 to 5, characterized in that the determination of the presence of a p53 isoform selected from the group consisting of Δ133p53β, Δ133p53 and Δ133p53Y is carried out by a probe capable of specifically hybridizing with the mRNA of said p53 isoform, complementary sequences thereof or a fragment thereof, said probe being selected from the group consisting of the sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, preferably the sequence SEQ ID NO: 11. 7. The method of any one of claims 1 to 5, wherein the determination of the presence of mRNA of a p53 isoform selected from the group consisting of Δ133p53β, Δ133p53, and Δ133p53Y is carried out by the following method, which comprises the steps of: (a) contacting a nucleic acid probe selected from the group consisting of the sequences SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, preferably sequence SEQ ID NO: 11, under hybridization conditions with the biological sample under test comprising either: - RNA molecules isolated from a biological sample of the human subject, wherein the biological sample is suspected of containing tumor cells, or - nucleic acid molecules synthesized from isolated RNA molecules, such as cDNA, and (b) detecting the formation of hybrids of the nucleic acid probe and the biological sample under test. test, in which the presence of hybrids indicates the presence of tumor cells in the tissue obtained from the human subject. 8. Method according to any one of claims 1 to 7, characterized in that the determination of the presence of a p53 isoform selected from the group consisting of Δ133p53β, Δ133p53 and Δ133p53Y is performed by contacting the biological sample with an antibody specific for the p53 isoform polypeptide or a fragment thereof and determining the binding of the antibody to the biological sample. 9. A method for screening potential antimetastatic compounds comprising the steps of: (a) contacting the compound to be tested with a cell known to express a p53 isoform selected from the group consisting of Δ133p53β, Δ133p53, and Δ133p53Y; (b) determining the presence of said p53 isoform by a method for detecting said p53 isoform as defined in any one of claims 1 to 8; and (c) selecting said compound as a potential anticancer compound if the p53 isoform is not present in the cell. 10. The method of any one of claims 1 to 9, wherein the cancer is breast cancer or colon cancer.