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WO2008046964A2 - Nouveaux inhibiteurs utiles - Google Patents

Nouveaux inhibiteurs utiles Download PDF

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WO2008046964A2
WO2008046964A2 PCT/FI2007/050557 FI2007050557W WO2008046964A2 WO 2008046964 A2 WO2008046964 A2 WO 2008046964A2 FI 2007050557 W FI2007050557 W FI 2007050557W WO 2008046964 A2 WO2008046964 A2 WO 2008046964A2
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smad3
smad7
cells
hypoxia
phosphorylation
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WO2008046964A8 (fr
WO2008046964A3 (fr
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Panu Jaakkola
Pekka Heikkinen
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to methods and assays for identifying compounds that inhibit the interaction of Smad3, Smad7 and PP2A and/or in- hibit hypoxia-induced dephosphorylation of Smad3. Such compounds are useful in treating, preventing and/or alleviating disorders related to hypoxia and/or TGF- ⁇ signalling.
  • hypoxia Reduced oxygen availability i.e. hypoxia is a key component in de- velopmental regulation, wound healing and progression of several diseases. These include cardiovascular and inflammatory diseases as well as carcinogenesis. For example in solid tumors, which have hypoxic regions due to aberrant or limited amount of vasculature, hypoxia promotes cell migration and invasion. Hypoxia is also known to select for cells that are deficient in apoptosis. In most tumors, including colon and pancreatic cancer, as well as head and neck squamous cell carcinoma (HNSCC), hypoxia correlates with poor prognosis and cause resistance to radiation therapy and chemotherapy.
  • HNSCC head and neck squamous cell carcinoma
  • hypoxia-inducible factor 1 and 2 hypoxia-inducible factor 1 and 2
  • HIF-1 and HIF-2 hypoxia-inducible factor 1 and 2
  • PDD prolyl hydroxylase domain proteins
  • HPH HIF prolyl hydroxylases
  • Egl-9 homologues Egl-9 homologues
  • the hydroxylated HIF is recognized by the von Hippel-Lindau tumor suppressor protein (pVHL) that subsequently leads to ubiquitination and proteosomal destruction of HIF- ⁇ .
  • pVHL von Hippel-Lindau tumor suppressor protein
  • HIF-1 ⁇ is expressed in most of the common human cancer types. The expression correlates, together with the downstream targets, with poor prognosis and resistance to treatment.
  • TGF- ⁇ 1 transforming growth factor- ⁇
  • TGF- ⁇ 1 is a well-studied prototype of the superfamily. It can restrict cell growth and proliferation and induce apoptosis of epithelial and endothelial cells.
  • cancer cells can become resistant to the growth inhibitory properties of TGF- ⁇ and use TGF- ⁇ as a growth promoting cytokine. While mutations in the TGF- ⁇ signalling pathway have been characterised, the mechanism behind this remain elusive.
  • TGF- ⁇ 1 ligand functions by inducing formation of heterotetrameric TGF- ⁇ re- ceptor complexes on the cell surface and subsequent phosphorylation of TGF- ⁇ receptor I.
  • R-Smads bind to a common Smad4 mediator, are imported to nucleus and activate transcriptional responses. Smad2 and -3 elicit at least partially different responses. The knockouts show different fenotype. Smad2-/- fenotype is embryonic lethal and Smad3-/- viable. In MEFs the deletion of Smad2 or -3 demonstrate different transcriptional responses. Smad2 and -3 can also activate phosphorylation of a different set of proteins.
  • the TGF- ⁇ signaling through Smad2/3 is antagonized by inhibitory Smad7.
  • Smad7 has been reported to inhibit Smad2 and -3 induced signaling at least by three ways. First, it may bind to TGF- ⁇ receptor and sterically inhibit the binding of R-Smads to TGF- ⁇ receptor. Second, in conjugation with E3- ubiquitin ligases (Smurfi and -2 ⁇ Smad7 can export Smad2/3 from the nucleus and ubiquitylate TGF- ⁇ receptors leading to receptor degradation. Third, Smad7 may recruit phosphatases, which dephosphorylate the receptor complex.
  • Smad7 expression has been shown to occur by several extracellular stimuli. These include TGF- ⁇ that generates an autoinhibitory loop, but also several cytokines and growth factors as well as UV-irradiation. However, the mechanism of elevated Smad7 expression in cancer and inflammatory diseases has remained enigmatic as has the mechanism by which Smad7 causes malignant lesions.
  • TGF- ⁇ functions as a cell growth suppressing factor in healthy tissue, but becomes a growth promoting factor in hyperproliferative disorders and cancers, especially at later stages, by unknown mechanisms.
  • This dual function of TGF- ⁇ is a problem when developing specific, therapeutically active compounds that have use in treating, preventing and/or alleviating disorders related to TGF- ⁇ signalling, such as cancer and other hyperproliferative diseases, as well as autoimmune diseases. There is thus an identified need of further elucidating the mechanism by which TGF- ⁇ becomes cell growth and invasion promoting factor.
  • the present invention relates to a method for identifying a therapeutic lead compound comprising the steps of a) transfecting cells of a cell culture with a DNA encoding a tagged bait protein selected from the group consisting of Smad3, Smad7 and PP2A, b) expressing said bait protein in said cells, c) treating said cells with a compound suspected to inhibit the interaction of Smad3, Smad7 and PP2A, d) lyzing said cells, e) collecting said tagged bait protein and proteins attached thereto from said lysate, f) determining the pres- ence and optionally the identity of said attached proteins, and g) identifying said compound as a therapeutic lead compound if the determination in step f) is positive.
  • the present invention further relates to an assay for screening therapeutic lead compounds comprising the following steps: a) providing a cell culture comprising hypoxic cells, b) adding a candidate compound to said cell culture, said candidate compound suspected to inhibit hypoxia-induced dephosphorylation of Smad3, but not Smad2, c) adding TGF- ⁇ to stimulate said hypoxic cells, d) determining the level of Smad3 phosphorylation, and e) identifying said candidate compound as an inhibitor of hypoxia-induced dephosphorylation of Smad3 if the level of Smad3 phosphorylation in step d) is statistically significantly higher compared to the level of Smad3 phosphorylation in non-treated control cells.
  • FIG. 1A - 1D shows that Smad7 gene is hypoxia-inducible.
  • FIG. 1B Quantitative real time PCR (Q-RT-PCR) for SMAD7 and GLUT1 expression in HeLa cells exposed to either hypoxia or CoCI 2 for 6 or 16 hours. SMAD7 and GLUT1 expression were normalized against EF-1 ⁇ in each treatment and fold chances were calculated against normoxic / non-treated control.
  • FIG. 1C Western blot analysis of SMAD7 protein in HeLa cells exposed to hypoxia or CoCI 2 for 6 and 16 hours. HIF-1 ⁇ is shown as control for hypoxia and ⁇ -actin as a loading control.
  • FIG. 2A - 2D shows that hypoxic induction of SMAD7 is HIF- and pVHL-dependent.
  • SMAD7 and GLUT1 genes are induced by exogenous HIF-1 ⁇ expression in normoxia.
  • HeLa cells were transfected with control (pcDNA3 empty vector), wild type HIF-1 ⁇ (HIF-I ⁇ -WT), or HIF-2 ⁇ constructs. After 24 hours of transient transfection gene expression levels were analyzed by Q-RT-PCR.
  • FIG. 2B HeLa cells were transfected with either non-target siRNA or siRNA against HIF-1 ⁇ followed by hypoxic exposure and analysis of endogenous Smad7 and GLUT-1 mRNA levels by Q-RT-PCR.
  • Figure 3A - 3C shows that SMAD7 gene is upregulated in head and neck squamous cell carcinomas and correlates with tumour hypoxia.
  • Fig. 3A 13 normal and 28 HNSCC patient samples were analysed by Q-RT-PCR for SMAD7, SMAD4 and GLUT1 mRNA levels. Results were normalized to EF-1 ⁇ levels in each sample. Each dot represents the expression in one patient sam- pie. Differences between control and tumour samples were statistically analysed using Wilcoxon two sample test. For each gene the p-value is shown.
  • Fig. 3B SMAD7 (grey bars) and GLUT1 (white bars) expression plotted against each other.
  • Fig. 3C Statistical analysis (Spearmann correlation coefficient and p- values) for the data from Fig. 3B. Asterisks show statistically significant correlation.
  • FIG. 4A - 4H shows that SMAD3 phosphorylation decreases in hypoxia but other R-Smads are not affected.
  • Fig. 4A HeLa and
  • Fig. 4B Ha- CaT cells were serum starved for 8 h and exposed to either ambient oxygen (21% O 2 ) or hypoxia (1% O 2 ) for 16 hours. This was followed by TGF- ⁇ 1 treatment (5 ng / ml) for indicated time. Protein expression levels were studied using phospho specific antibodies ⁇ P-Smad1/3 and P-Smad2) by western blotting. HIF-1 ⁇ was used for monitoring hypoxia and ⁇ -actin for loading control. Both cell lines show TGF- ⁇ 1 activated Smad2 and -3 phosphorylation (0 vs. 15 min).
  • FIG. 4C Quantification of the difference between nor- moxic and hypoxic Smad2 and -3 phosphorylation at indicated time points of TGF- ⁇ stimulation. For each data point the normoxic value was subtracted from corresponding hypoxic value. The means and standard error from four independent experiments are shown.
  • FIG. 4D HaCaT cell were serum starved and exposed to hypoxia as in Figures 4A and 4B. This was followed by BMP-2 treatment (5 ng / ml) for indicated time and protein was detected by P-Smad1/3 antibody. BMP-2 induced Smad phosphorylation (0 vs. 15 min).
  • Fig. 4E HeLa cells were transfected with a non-target or two different Smad7-specific double-stranded oligonucleotide si RNAs (SMAD7-1 and -2). Cells were ex- posed to 16 hour hypoxia, followed by detection of Smad7 and GLUT-1 mRNA expression by Q-RT-PCR.
  • Fig. 4F Hypoxic block of SMAD3 phosphorylation is Smad7-dependent.
  • the siRNAs used in panel E were transfected to HeLa cells, which were exposed to chemical hypoxia (100 ⁇ M CoCI 2 for 16 hours) followed by TGF- ⁇ 1 (5 ng / ml) treatment for 30 and 60 minutes. Phos- phospecific Smad3 was detected by western blotting. HIF-1 ⁇ was used to monitor hypoxia and ⁇ -actin as a loading control.
  • Fig. 4G Hypoxia does not affect total Smad3 levels. HeLa cells were exposed to either ambient oxygen (21% O 2 ) or hypoxia (1% O 2 ) for 16 hours followed by TGF- ⁇ 1 treatment for indicated times. Total Smad3 levels were detected by western blot analysis (Fig. 4H) Smad7 knockdown does not affect total Smad3 levels. HaCaT cells were transfected with indicated siRNAs, exposed to chemical hypoxia (100 ⁇ M CoCI 2 for 16 hours) followed by TGF- ⁇ 1 treatment and analysis of total Smad3 levels by western blot.
  • FIG. 5A - 5E shows that PP2A interacts and dephosphorylates Smad3 under hypoxia.
  • Fig. 5A HeLa cells were left untreated or exposed to 16 hour hypoxia and 1 hour TGF- ⁇ , followed by analysis of Smad3 phosphory- lation. Treatment with okadaic acid (OA) for 1 hour prior to TGF- ⁇ (last lane) reversed the hypoxia-induced inhibition in Smad3 phosphorylation.
  • FIG. 5B HeLa cells transfected with an empty vector or Strep-PR65 (Str-PR65) fusion protein were exposed to normoxia (N) or hypoxia (H) followed by TGF- ⁇ treat- ment. Stre ⁇ -PR65 was captured on streptavidin columns.
  • Figure 6 is a schematic representation of the model of hypoxic dephosphorylation of Smad3.
  • the present invention is based on the elucidation of the mechanism by which Smad7 is elevated in abnormally proliferating tissue, such as cancer, and autoimmune diseases in hypoxic conditions. Furthermore, the present invention describes a mechanism by which TGF- ⁇ can become cell growth and invasion promoting factor. Understanding the molecular mechanism behind TGF- ⁇ signalling provides novel means to restrict cancer cell proliferation and tumour growth.
  • the present invention thus provides means for developing therapeutics for cancer and other proliferative diseases, as well as autoimmune dis- eases.
  • hypoxia upregulates Smad7 in a HIF- and pVHL-dependent manner.
  • the hypoxia-activated Smad7 recruits protein phosphatase 2A (PP2A) leading to dephosphorylation of TGF- ⁇ -activated Smad3.
  • PP2A protein phosphatase 2A
  • hypoxia or PP2A does not affect the TGF- ⁇ induced phosphoryla- tion of Smad2, nor BMP-induced phosphorylation of Smadi.
  • the present invention relates to a method of identifying therapeutic lead compounds that inhibit the interaction between Smad3, Smad7 and PP2A. Such a compound is identified through its capability of breaking down the protein-protein interactions within the said complex.
  • a method according to the present invention may comprise the following steps: a) transfecting cells of a cell culture with a DNA encoding a tagged bait protein selected from the group consisting of Smad3, Smad7 and PP2A, b) expressing said tagged bait protein in said cells, c) treating said cells with a candidate compound, d) fyzing said cells, e) collecting said tagged bait protein and proteins bound or attached thereto from said lysate, f) determining the presence and optionally the identity of said attached proteins, and g) identifying said candidate compound as a therapeutic lead compound if the determination in step f) is positive.
  • the bait protein may be tagged with any suitable tag known in the art such as Myc-tag, Haemagglutin-tag, GST-tag or any
  • any of the proteins Smad3, Smad7 or PP2A may be used as bait.
  • the proteins bound or attached to the bait protein may be detected and identi- fled by any suitable method such as western blot or immunoassays. If no specifically binding proteins such as Smad7, Smad3 or subunits of PP2A are present in step f), or the amount of said specifically binding proteins in step f) is statistically significantly decreased compared to the amount of corresponding specifically binding proteins obtained from non-treated control cells, the candi- date compound is identified to be an inhibitor of the interaction between Smad3, Smad7 and PP2A.
  • the present invention also relates to a method for producing a pharmaceutical composition, said method comprising a method for identifying compound(s) that inhibit the interaction between Smad3, Smad7 and PP2A, and mixing the compound identified with a pharmaceutically acceptable excipi- ent.
  • excipients are readily available to those skilled in the art.
  • the present invention further relates to an assay for screening and identifying therapeutic lead compounds that inhibit hypoxia-induced depho- shorylation of Smad3 but do not affect Smad2 phosphorylation.
  • a method may comprise the following steps: a) providing a cell culture comprising hypoxic cells, b) adding a candidate compound to said cell culture, c) adding TGF- ⁇ to stimulate said hypoxic cells, d) determining the level of Smad3 phosphorylation, and e) identifying said candidate compound as an inhibitor of hypoxia-induced dephosphorylation of Smad3 if the level of Smad3 phosphoryla- tion in step d) is statistically significantly higher compared to the level of Smad3 phosphorylation in non-treated control cells.
  • Hypoxic cells may be obtained by culturing cells in vitro under reduced pressure of oxygen, such as 1% O 2 , for e.g. 16 hours.
  • hypoxic cells may be obtained by treating the cells with a suitable chemical hy- poxia mimetic, such as cobalt chloride.
  • Step c) may be performed by adding TGF- ⁇ , such as TGF- ⁇ 1, TGF- ⁇ 2 or TGF- ⁇ 3, preferably TGF- ⁇ 1 , to hypoxic cells for e.g. 1 hour prior to step d).
  • TGF- ⁇ such as TGF- ⁇ 1, TGF- ⁇ 2 or TGF- ⁇ 3, preferably TGF- ⁇ 1
  • steps b) and c) may be performed simultaneously.
  • Determining the level of Smad3 phosphorylation in step d) may be performed by any suitable method known in the art, such as mass spectrome- try or an immunoassay including but not limited to enzyme-linked immunoassays (ELISA), radioimmunoassays (RIA) or fluoroimmunoassays (FIA), or western blotting.
  • ELISA enzyme-linked immunoassays
  • RIA radioimmunoassays
  • FFA fluoroimmunoassays
  • Smad3 phosphorylation refers to phosphorylation of C-terminal serines of Smad3.
  • the present invention further relates to a method for producing a pharmaceutical composition, said method comprising identifying compound(s) that inhibit hypoxia-induced dephoshorylation of Smad3, and mixing the compound identified with a pharmaceutically acceptable excipient.
  • excipients are readily available to those skilled in the art.
  • identification is performed by an assay described above.
  • a compound capable of inhibiting dephosphorylation of Smad3 is identified based on its ability to restore phosphorylation of Smad3 upon hypoxia. This restoration may be determined by comparing the level of Smad3 phophorylation in cells treated with a candidate compound to the level of Smad3 phosphorylation in non-treated control cells. When said restoration is statistically significant, the candidate compound is regarded as a compound capable of inhibiting dephosphorylation of Smad3.
  • Compounds or therapeutic lead compounds that may be identified according to the embodiments of the present invention include natural and synthetic chemical compounds, small molecules, natural and synthetic proteins and peptides, as well as vectors encoding any suitable peptide or protein.
  • the therapeutic lead compounds identifiable according to the embodiments of the present invention are useful in preventing, treating and/or alleviating conditions associated with hypoxia including infarction of brain or heart and related ischemic diseases; other ischemic diseases such as complications of arterial blockage; rejection in organ transplantation; delayed or impaired wound healing; various autoimmune diseases such as inflammatory bowel disease; hyperproliferative disorders such as psoriasis or keloids; and diverse types of cancer, including carcinomas of pancreas, gastrointestinal tract, liver, breast, lung, head and neck, and ovaries, as well as sarcomas and brain cancer (glioblastoma).
  • hypoxia including infarction of brain or heart and related ischemic diseases; other ischemic diseases such as complications of arterial blockage; rejection in organ transplantation; delayed or impaired wound healing; various autoimmune diseases such as inflammatory bowel disease; hyperproliferative disorders such as psoriasis or keloids; and diverse types of cancer, including carcinomas of pancreas, gastrointestinal tract, liver,
  • the present invention thus also relates to a method for diagnosing cancer, especially head and neck cancer.
  • a method for diagnosing cancer may comprise the following steps: a) determining the level of Smad7 expression in a sample taken from said mammal, said sample suspected to comprise malignant cells, b) comparing the determined expression level from step a) with the expression level of Smad7 in a non-malignant control sample, and c) determining the presence of malignant changes on the basis of the expression level of Smad7 in said sample being at least 2 when compared to the expression level of Smad7 in said control sample.
  • the expression level of Smad7 may be determined on nriRNA level or protein level by any suitable method known in the art. Such methods include northern blot and immunoassays.
  • the present invention is thus also directed to a method of diagnos- ing cancer or detecting a malignant change in a mammal.
  • a method may comprise the following steps: a) determining the level of Smad3 phosphorylation in a sample taken from a mammal, said sample suspected to comprise malignant cells, b) comparing the determined level of Smad3 phosphorylation from step a) with the level of Smad3 phosphorylation in a non-malignant con- trol sample, and c) determining the presence of malignant changes on the basis of the presence of reduced level of Smad3 phosphorylation.
  • the level of phosphorylated Smad3 in said sample is at least two-fold lower compared to the level of Smad3 phosphorylation in said control sample.
  • Smad3 phosphorylation refers to the phosphorylation in the C-terminal serines of Smad3.
  • TGF- ⁇ pathway regulated by hypoxia To identify genes of the TGF- ⁇ pathway regulated by hypoxia, we compared gene expression of normoxic hepatocellular carcinoma HepG2 cells (ATTC) with cells exposed to 16 hour hypoxia using a custom made cDNA mi- croarray containing 4000 genes.
  • HepG2 cells were grown in Dulbecco's minimum essential eagle modified medium (Sigma-Aldrich) supplemented with 10% fetal calf serum, 20 U/ml penicillin, 50 ⁇ g/ml streptomycin and 2 mM L-glutamin. Hypoxic treatments were performed in Invivo2 400 incubator (Ruskinn technologies, UK) in 1% O 2 , 5 % CO 2 , 90 % moisture. Oxygen was replaced with 99,5% pure N 2 (AGA, Finland).
  • Hybridization was carried out over night in +65°C in a mixture containing 5 ⁇ g COT-1 DNA, 10 ⁇ g poly A DNA and 2,1 ⁇ g Yeast t-RNA.
  • RT-PCR real-time PCR
  • HeLa cells were grown in Dulbecco's minimum essential eagle modified medium (Sigma-Aldrich) supplemented with 10% fetal calf serum, 20 U/ml penicillin, 50 ⁇ g/ml streptomycin and 2 mM L-glutamin.
  • Dulbecco's minimum essential eagle modified medium Sigma-Aldrich
  • 10% fetal calf serum 20 U/ml penicillin, 50 ⁇ g/ml streptomycin and 2 mM L-glutamin.
  • cell culture media was supplemented with 100 ⁇ M CoCI 2 (Sigma- Aldrich) for 6 to 16 hours. Hypoxic treatment was performed as described above.
  • RNA samples were lyzed and mRNA extracted as described above followed by mRNA quantification by Q-RT-PCR.
  • Q-RT-PCR TAQMAN
  • cDNA was prepared from 0,5 ⁇ g of total RNA.
  • First total RNA was combined with 0,5 ⁇ g of oligo-dT-primer (Promega) and filled with RNase free water (Ep- pendorf) to 18,25 ⁇ l.
  • RNase free water Ep- pendorf
  • To denature RNA sample was heated to 70 0 C for 5 minutes and transferred on ice for 5 minutes. After cooling 5 ⁇ l 5 x reaction buffer (Promega), 100U of M-MMLV RT (H-) (Promega) and NTP-mix (Fermentas) 0,5 mM final concentration.
  • cDNA synthesis was carried out in +40 0 C for 1 hour followed by inactivation in 7O 0 C for 15 minutes.
  • Each cDNA reaction was diluted 1 :9 in MQ-water prior to TAQMAN pipetting.
  • Master mix for TAQMAN was prepared for each gene containing 5 ⁇ l universal Master Mix (Applied Bio- systems) gene specific forward and reverse primers and probe 1 ⁇ l each. Final concentrations and sequences are listed in Table I.
  • HaCaT Immortalized keratinocytes
  • Dulbecco's minimum essential eagle modified medium Sigma-Aldrich
  • 10% fetal calf serum 20 U/ml penicillin, 50 ⁇ g/ml streptomycin and 2 mM L-glutamin.
  • Human umbical vein endothelial cells (HUVEC) were kindly provided by Dr. E.
  • HUVEC cells plastic cell culture bottles or plates (BectonDickson) were first coated with gelatin (BD). Cultured cells were exposed to hypoxia and mRNA levels were determined using Q-RT-PCR as described above. Immortalized keratinocytes (HaCaT) and primary endothelial cells
  • Smad7 Hypoxia-induced protein expression of Smad7.
  • the expression of Smad7 by hypoxia was further studied at protein level from hypoxic and nor- moxic HeLa samples.
  • the cells were lysed in SDS-sample buffer (2% SDS 1 10 % glycerol, 0,01% BPB, 62,5 mM TRIS-HCI pH 7,5 and 5 % ⁇ -merkapto- ethanol in MQ-water). Protein samples were run on SDS-PAGE in a mini-gel chamber (Biorad) and transferred on PVDF-membrane (Millipore). Western blots were done in 5% BSA in TBS with 0,1% Tween-20 using rabbit polyclonal Smad7 antibody (Imgenex) dilution 1:2500.
  • hypoxic gene expression occurs through HIF- and pVHL-dependent pathway.
  • HIF dimer binds to a hypoxic response element (HRE) on the promoters of HIF- activated genes.
  • HRE hypoxic response element
  • Cells were transfected with a plasmid construct containing wild type HIF-Ia described by Wang GL et al. in Proc Natl Acad Sci U S A, 92(12):5510-4, 1995. After two days in culture, cells were lysed, mRNA isolated and analysed for Smad7 and GLUT-1 expression as described above.
  • HIF-1 ⁇ Overexpression of wild-type HIF-1 ⁇ caused a 4-fold elevation in Smad7 mRNA. This was comparable with GLUT-1 expression. Lower, but clear induction was observed for HIF-2 ⁇ as well, indicating that the Smad7 gene is under control of HIF.
  • HIF-1 ⁇ siRNA which have previously shown to specifically downregulate HIF-1 ⁇ , to study Smad7 expression in HeLa (Fig. 2B) and HaCaT (Fig. 2C) cells exposed to 16 hour hypoxia.
  • double-stranded siRNA oligos were diluted to 1 ⁇ M concentration and used 60 nM final concentration on cell culture plate two days prior to any treatment.
  • Transfections were performed with Trans-IT-TKO (Mirus) following manufacturers protocol.
  • Smad7 mRNA levels were measured using Q-RT-PCR as describe above.
  • HIF-1 ⁇ siRNA markedly downregulated or abolished the hypoxic Smad7 induction as compared to non-target siRNA.
  • VHL gene Deletion or mutations in VHL gene are the cause of VHL tumor syndrome. These lead to constitutive stabilization of HIF-1 ⁇ and are seen for example in 80% of sporadic renal clear cell carcinomas (RCC).
  • RCC4- sporadic renal clear cell carcinomas
  • RCC+pVHL fuctional pVHL
  • Fig. 2D sporadic renal clear cell carcinomas
  • RCC4- is a naturally occuring renal clear cell carcinoma cell line. The generation of RCC+pVHL has been described elsewhere. Both cell lines are readily available from European Collection of Cell Culture (ECACC accession numbers, RCC4-: 03112702, RCC+pVHL: 03112703).
  • This example shows the effect of hypoxia on the TGF- ⁇ induced phosphorylation of Smad2 and -3.
  • Serum starved HeLa cells were pre- exposed to normoxia or 16 hour hypoxia followed by addition of TGF- ⁇ 1 (5 ng/ml) for 30 to 120 minutes.
  • TGF- ⁇ 1 treatment cells were either serum starved or grown in growth factor stripped serum for 8 hours prior to addition of TGF- ⁇ 1 (Promocell) at 5 ng/ml final concentration.
  • Phosphorylation of Smad2 and -3 was subsequently analysed by western blotting as described above in Example 1 using phospho-spesific antibodies P-SMAD3 and P-SMAD2 described by Leivonen, S. et al. in J. Biol. Chem.
  • hypoxia was routinely controlled by detection of HIF-Ia using mouse monoclonal HlF-Ia antibody (Tran- duction laboratories) at 1 :2500 (Fig 4A).
  • Smad2 and -3 phosphorylation was activated by TGF- ⁇ 1 as expected, and peaked at 60 minutes.
  • the TGF- ⁇ 1 induced Smad3 phosphorylation was abolished or markedly decreased at all time points studied up to two hours. Surprisingly however, hypoxia had no effect on the TGF- ⁇ 1 induced Smad2 phosphorylation.
  • the phospho-Smad3 antibody used in the study has been reported to recognize phosphorylated Smadi as well.
  • the recognition of Smadi is unlikely, since TGF- ⁇ 1 should not induce Smadi phosphorylation in epithelial cells.
  • BMP2 bone morphogenetic protein 2
  • Fig. 4D bone morphogenetic protein 2
  • BMP2 Induced robust phosphorylation of the protein recognised by the phosphoS- mad1/3 antibody.
  • no changes in the phosphorylation status was de- tected upon hypoxia.
  • hypoxia specifically blocks the Smad3 phosphorylation induced by TGF- ⁇ 1, but does not effect TGF- ⁇ 1 -induced Smad2 phosphorylation and furthermore, does not influence BMP-induced Smadi phosphorylation.
  • siRNA oligos used were: SMAD7 Oligol 5'- AGGUCACCACCAUCCCCACdTdT-3' (5) (Eurogentec); SMAD7 Oligo2 5'- GGACGCUGUUGGUACACAAdTdT-3' (MWG Biotech). Two days after trans- fection, mRNA was extracted and Smad7 mRNA determined by Q-RT-PCR.
  • siRNAs suppressed Smad7 expression from 70 to 80% but did not have marked effect on GLUT-1 expression (Fig. 4E).
  • the siRNAs were next used in an experiment where HeLa cells were first transfected with siRNAs, then treated with the hypoxia-mimetic cobalt chloride for 16 hours fol- lowed by TGF- ⁇ 1 exposure for 30 to 60 minutes.
  • Cobalt chloride suppressed the phosphorylation of Smad3 as expected, when exposed to a non-target siRNA, but when exposed to either of the Smad7 siRNAs, the cobalt chloride-induced suppression was completely reversed (Fig. 4F). This demonstrated that Smad7 is required for the hypoxic downregulation of TGF- ⁇ activated Smad3 phosphorylation.
  • Smad7 is known to bind TGF- ⁇ receptors and thereby block subsequent Smad2/3 phosphorylation in normoxic conditions. In the case of hypoxia- induced SmadZ this did not occur since Smad2 phosphorylation was not af- fected. Another possibility was that Smad7 causes ubiquitylation-dependent degradation of Smad3.
  • OA a widely used chemical that blocks the enzymatic activity of protein phosphatases. At high concentration it abrogates both protein phosphatase 1 and 2 (PP1 and PP2) activity, but at lower concentration ( ⁇ 50 mM) OA is specific for PP2A. Pre-treatment of cells with 20 mM OA reversed the hypoxic blockade on Smad3 phosphorylation (Fig. 5A) and did not affect the total Smad3 protein levels. This implied that PP2A could dephosphorylate Smad3 under hypoxic conditions.
  • PP2A is a multiprotein complex consisting of regulatory, catalytic and scaffold (PR65) subunits.
  • PR65 regulatory, catalytic and scaffold
  • the cell lysate was then transferred into 100 ⁇ l STREP-purification column (IBA, www.iba-go.com). The column was washed 5 times with 200 ⁇ l of STREP-wash-buffer. Next, the sample was eluted with STREP-elution-buffer in 6 fractions of 100 ⁇ l. The fractions were either combined and concentrated or run separately on PAGE followed by western blotting using endogenous Smad2 or -3.
  • HeLa cells were transfected with either non- target or PR65 specific siRNA oligonucleotides. siRNA treatment knocked down the PR65 expression to 20 - 40% of that seen with non-target siRNA (not shown). The next day cells were subjected to either normoxia or 16 hour hypoxia followed by 60 min TGF- ⁇ 1 treatment. In control cells TGF- ⁇ elicited phosphorylation of Smad3, which was suppressed by hypoxia. In the cells with downregulated PR65 expression, the hypoxic suppression of Smad3 phosphorylation was abolished or markedly reduced (Fig. 5D). Smad3 phosphorylation was quantified from three independent experiments (Fig. 5E). Together these data indicated that PP2A is required for dephosphorylation of Smad3 under hypoxia.
  • Solid tumors suffer from hypoxia, which is known to activate a number of genes enhancing tumor progression. We hypothezied that hypoxia could cause overexpression of Smad7 in human tumors.
  • GLUT-1 is a prototype of hypoxia-activated gene and is upregulated in a number of carcinomas including HNSCC.

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Abstract

La présente invention concerne un procédé et un essai permettant d'identifier des têtes de série thérapeutiques utiles pour traiter, prévenir et/ou soulager des troubles associés à une hypoxie et/ou un signal TGF-β. L'invention concerne en outre des compositions pharmaceutiques et des procédés permettant de détecter des modifications malignes.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009047532A3 (fr) * 2007-10-12 2009-07-02 Cancer Rec Tech Ltd Loci de sensibilité au cancer
US8648186B2 (en) 2003-04-02 2014-02-11 Nogra Pharma Limited Antisense oligonucleotides (ODN) against SMAD7 and uses thereof in medical field
EP3527213A1 (fr) * 2012-10-26 2019-08-21 The Chinese University Of Hong Kong Traitement du cancer au moyen d'un inhibiteur smad3

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* Cited by examiner, † Cited by third party
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US6998240B2 (en) * 2001-06-11 2006-02-14 Wisconsin Alumni Research Foundation Screen for selective inhibitors or activators of Smad protein function
WO2004064770A2 (fr) * 2003-01-17 2004-08-05 Government Of The United States Of America As Represented By The Secretary, Department Of Health Andhuman Services Utilisation d'un inhibiteur de smad3 dans le traitement d'une fibrose dependant de la transition epithelium-mesenchyme comme dans l'oeil et le rein

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US9382541B2 (en) 2003-04-02 2016-07-05 Nogra Pharma Limited Antisense oligonucleotides (ODN) against SMAD7 and uses thereof in medical field
US10036022B2 (en) 2003-04-02 2018-07-31 Nogra Pharma Limited Antisense oligonucleotides (ODN) against Smad7 and uses thereof in medical field
US8907078B2 (en) 2003-04-02 2014-12-09 Nogra Pharma Limited Antisense oligonucleotides (ODN) against SMAD7 and uses thereof in medical field
US9006418B2 (en) 2003-04-02 2015-04-14 Nogra Pharma Limited Antisense oligonucleotides (ODN) against Smad7 and uses thereof in medical field
US9096854B1 (en) 2003-04-02 2015-08-04 Nogra Pharma Limited Antisense oligonucleotides (ODN) against SMAD7 and uses thereof in medical field
US9279126B2 (en) 2003-04-02 2016-03-08 Nogra Pharma Limited Antisense oligonucleotides (ODN) against SMAD7 and uses thereof in medical field
US8648186B2 (en) 2003-04-02 2014-02-11 Nogra Pharma Limited Antisense oligonucleotides (ODN) against SMAD7 and uses thereof in medical field
US9605264B2 (en) 2003-04-02 2017-03-28 Nogra Pharma Limited Antisense oligonucleotides (ODN) against Smad7 and uses thereof in medical field
US10738309B2 (en) 2003-04-02 2020-08-11 Nogra Pharma Limited Antisense oligonucleotides (ODN) against SMAD7 and uses thereof in medical field
US9951334B2 (en) 2003-04-02 2018-04-24 Nogra Pharma Limited Antisense oligonucleotides (ODN) against SMAD7 and uses thereof in medical field
US9518264B2 (en) 2003-04-02 2016-12-13 Nogra Pharma Limited Antisense oligonucleotides (ODN) against SMAD7 and uses thereof in medical field
US10633660B2 (en) 2003-04-02 2020-04-28 Nogra Pharma Limited Antisense oligonucleotides (ODN) against SMAD7 and uses thereof in medical field
WO2009047532A3 (fr) * 2007-10-12 2009-07-02 Cancer Rec Tech Ltd Loci de sensibilité au cancer
EP3527213A1 (fr) * 2012-10-26 2019-08-21 The Chinese University Of Hong Kong Traitement du cancer au moyen d'un inhibiteur smad3
US11666565B2 (en) 2012-10-26 2023-06-06 The Chinese Universitv of Hong Kong Treatment of cancer using a SMAD3 inhibitor

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