Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling
Hua Xiong
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Search for more papers by this authorZhao-Fei Chen
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Search for more papers by this authorQin-Chuan Liang
Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi’an, China
Search for more papers by this authorWan Du
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Search for more papers by this authorHui-Min Chen
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Search for more papers by this authorWen-Yu Su
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Search for more papers by this authorGuo-Qiang Chen
Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine, Luwan, Shanghai, China
Search for more papers by this authorZe-Guang Han
Shanghai-Ministry Key Laboratory of Disease and Health Genomics, Chinese National Human Genome Center, Shanghai, China
Search for more papers by this authorCorresponding Author
Jing-Yuan Fang
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Correspondence to: Jing-Yuan FANG, M.D., Ph.D., Shanghai Institute of Digestive Disease, Ren-Ji Hospital, 145 Middle Shandong Road, Shanghai, 200001, China.Tel.: 0086-21-63200874Fax: 0086-21-63266027E-mail: [email protected]Search for more papers by this authorHua Xiong
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Search for more papers by this authorZhao-Fei Chen
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Search for more papers by this authorQin-Chuan Liang
Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi’an, China
Search for more papers by this authorWan Du
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Search for more papers by this authorHui-Min Chen
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Search for more papers by this authorWen-Yu Su
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Search for more papers by this authorGuo-Qiang Chen
Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine, Luwan, Shanghai, China
Search for more papers by this authorZe-Guang Han
Shanghai-Ministry Key Laboratory of Disease and Health Genomics, Chinese National Human Genome Center, Shanghai, China
Search for more papers by this authorCorresponding Author
Jing-Yuan Fang
Department of Gastroenterology, Shanghai Jiao-Tong University School of Medicine Ren-Ji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China
Correspondence to: Jing-Yuan FANG, M.D., Ph.D., Shanghai Institute of Digestive Disease, Ren-Ji Hospital, 145 Middle Shandong Road, Shanghai, 200001, China.Tel.: 0086-21-63200874Fax: 0086-21-63266027E-mail: [email protected]Search for more papers by this authorAbstract
DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2′-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16ink4a, p21waf1/cip1 and p27kip1. Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.
References
- 1 Lubbert M. DNA methylation inhibitors in the treatment of leukemias, myelodysplastic syndromes and hemoglobinopathies: clinical results and possible mechanisms of action. Curr Top Microbiol Immunol. 2000; 249: 135–64.
- 2 Momparler RL, Ayoub J. Potential of 5-aza-2’-deoxycytidine (Decitabine) a potent inhibitor of DNA methylation for therapy of advanced non-small cell lung cancer. Lung Cancer. 2001; 34: S111–5.
- 3 Issa JP, Kantarjian H. Azacitidine. Nat Rev Drug Discov. 2005: S6–7.
- 4 Wijermans P, Lubbert M, Verhoef G, et al . Low-dose 5-aza-2’-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome: a multicenter phase II study in elderly patients. J Clin Oncol. 2000; 18: 956–62.
- 5 Aparicio A, Eads CA, Leong LA, et al . Phase I trial of continuous infusion 5-aza-2′-deoxycytidine. Cancer Chemother Pharmacol. 2003; 51: 231–9.
- 6 Lyons J, Bayar E, Fine G, et al . Decitabine: development of a DNA methyltransferase inhibitor for hematological malignancies. Curr Opin Investig Drugs. 2003; 4: 1442–50.
- 7 Issa JP, Garcia-Manero G, Giles FJ, et al . Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2′-deoxycytidine (decitabine) in hematopoietic malignancies. Blood. 2004; 103: 1635–40.
- 8 Balch C, Yan P, Craft T, et al . Antimitogenic and chemosensitizing effects of the methylation inhibitor zebularine in ovarian cancer. Mol Cancer Ther. 2005; 4: 1505–14.
- 9 Niwa Y, Kanda H, Shikauchi Y, et al . Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma. Oncogene. 2005; 24: 6406–17.
- 10 Lacronique V, Boureux A, Valle VD, et al . A TEL-JAK2 fusion protein with constitutive kinase activity in human leukemia. Science. 1997; 278: 1309–12.
- 11 Zhang F, Li C, Halfter H, et al . Delineating an oncostatin M-activated STAT3 signaling pathway that coordinates the expression of genes involved in cell cycle regulation and extracellular matrix deposition of MCF-7 cells. Oncogene. 2003; 22: 894–905.
- 12 Alvarez JV, Greulich H, Sellers WR, et al . Signal transducer and activator of transcription 3 is required for the oncogenic effects of non-small-cell lung cancer-associated mutations of the epidermal growth factor receptor. Cancer Res. 2006; 66: 3162–8.
- 13 Shen Y, Devgan G, Darnell JE Jr, et al . Constitutively activated Stat3 protects fibroblasts from serum withdrawal and UV-induced apoptosis and antagonizes the proapoptotic effects of activated Stat1. Proc Natl Acad Sci USA. 2001; 98: 1543–8.
- 14 Mora LB, Buettner R, Seigne J, et al . Constitutive activation of Stat3 in human prostate tumors and cell lines: direct inhibition of Stat3 signaling induces apoptosis of prostate cancer cells. Cancer Res. 2002; 62: 6659–66.
- 15 Ma XT, Wang S, Ye YJ, et al . Constitutive activation of Stat3 signaling pathway in human colorectal carcinoma. World J Gastroenterol. 2004; 10: 1569–73.
- 16 Lassmann S, Schuster I, Walch A, et al . STAT3 mRNA and protein expression in colorectal cancer: effects on STAT3-inducible targets linked to cell survival and proliferation. J Clin Pathol. 2007; 60: 173–9.
- 17 Lin Q, Lai R, Chirieac LR, et al . Constitutive activation of JAK3/STAT3 in colon carcinoma tumors and cell lines: inhibition of JAK3/STAT3 signaling induces apoptosis and cell cycle arrest of colon carcinoma cells. Am J Pathol. 2005; 167: 969–80.
- 18 Xiong H, Zhang ZG, Tian XQ, et al . Inhibition of JAK1, 2/STAT3 signaling induces apoptosis, cell cycle arrest, and reduces tumor cell invasion in colorectal cancer cells. Neoplasia. 2008; 10: 287–97.
- 19 Han Y, Amin HM, Franko B, et al . Loss of SHP1 enhances JAK3/STAT3 signaling and decreases proteosome degradation of JAK3 and NPM-ALK in ALK+ anaplastic large-cell lymphoma. Blood. 2006; 108: 2796–803.
- 20 Chim CS, Fung TK, Cheung WC, et al . SOCS1 and SHP1 hypermethylation in multiple myeloma: implications for epigenetic activation of the Jak/STAT pathway. Blood. 2004; 103: 4630–5.
- 21 Buettner R, Mora LB, Jove R. Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention. Clin Cancer Res. 2002; 8: 945–54.
- 22 Khoury JD, Rassidakis GZ, Medeiros LJ, et al . Methylation of SHP1 gene and loss of SHP1 protein expression are frequent in systemic anaplastic large cell lymphoma. Blood. 2004; 104: 1580–1.
- 23 Galm O, Yoshikawa H, Esteller M, et al . SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma. Blood. 2003; 101: 2784–8.
- 24 He B, You L, Uematsu K, et al . SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer. Proc Natl Acad Sci USA. 2003; 100: 14133–8.
- 25 Wu C, Guan Q, Wang Y, et al . SHP-1 suppresses cancer cell growth by promoting degradation of JAK kinases. J Cell Biochem. 2003; 90: 1026–37.
- 26 Jegalian AG, Wu H. Regulation of Socs gene expression by the proto-oncoprotein GFI-1B: two routes for STAT5 target gene induction by erythropoietin. J Biol Chem. 2002; 277: 2345–52.
- 27 Valentino L, Pierre J. JAK/STAT signal transduction: regulators and implication in hematological malignancies. Biochem Pharmacol. 2006; 71: 713–21.
- 28 Rakesh K, Agrawal DK. Controlling cytokine signaling by constitutive inhibitors. Biochem Pharmacol. 2005; 70: 649–57.
- 29 Wu C, Sun M, Liu L, et al . The function of the protein tyrosine phosphatase SHP-1 in cancer. Gene. 2003; 306: 1–12.
- 30 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25: 402–8.
- 31 Lu R, Wang X, Chen ZF, et al . Inhibition of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway decreases DNA methylation in colon cancer cells. J Biol Chem. 2007; 282: 12249–59.
- 32 Morita Y, Naka T, Kawazoe Y, et al . Signals transducers and activators of transcription (STAT)-induced STAT inhibitor-1 (SSI-1)/suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor alpha-induced cell death in fibroblasts. Proc Natl Acad Sci USA. 2000; 97: 5405–10.
- 33 Wang YY, Zhou GB, Yin T, et al . AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec. Proc Natl Acad Sci USA. 2005; 102: 1104–9.
- 34 Hecht M, Papoutsi M, Tran HD, et al . Hepatocyte growth factor/c-Met signaling promotes the progression of experimental human neuroblastomas. Cancer Res. 2004; 64: 6109–18.
- 35 Luczak MW, Jagodzinski PP. The role of DNA methylation in cancer development. Folia Histochem Cytobiol. 2006; 44: 143–54.
- 36 Hennessy BT, Garcia-Manero G, Kantarjian HM, et al . DNA methylation in haematological malignancies: the role of decitabine. Expert Opin Investig Drugs. 2003; 12: 1985–93.
- 37 Karpf AR, Moore BC, Ririe TO, et al . Activation of the p53 DNA damage response pathway after inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine. Mol Pharmacol. 2001; 59: 751–7.
- 38 Zhu WG, Hileman T, Ke Y, et al . 5-aza-2′-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation. J Biol Chem. 2004; 279: 15161–6.
- 39 Fang JY, Chen YX, Lu J, et al . Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116. Cell Res. 2004; 14: 217–26.
- 40 Paling NR, Welham MJ. Role of the protein tyrosine phosphatase SHP-1 (Src homology phosphatase-1) in the regulation of interleukin-3-induced survival, proliferation and signalling. Biochem J. 2002; 368: 885–94.
- 41 Kazansky AV, Kabotyanski EB, Wyszomierski SL, et al . Differential effects of prolactin and src/abl kinases on the nuclear translocation of STAT5B and STAT5A. J Biol Chem. 1999; 274: 22484–92.
- 42 Yoshikawa H, Matsubara K, Qian GS, et al . SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet. 2001; 28: 29–35.
- 43 Sakai I, Takeuchi K, Yamauchi H, et al . Constitutive expression of SOCS3 confers resistance to IFN-alpha in chronic myelogenous leukemia cells. Blood. 2002; 100: 2926–31.
- 44 Cho-Vega JH, Rassidakis GZ, Amin HM, et al . Suppressor of cytokine signaling 3 expression in anaplastic large cell lymphoma. Leukemia. 2004; 18: 1872–8.
- 45 Takeuchi K, Sakai I, Narumi H, et al . Expression of SOCS3 mRNA in bone marrow cells from CML patients associated with cytogenetic response to IFN-alpha. Leuk Res. 2005; 29: 173–8.
- 46 Brender C, Nielsen M, Kaltoft K, et al . STAT3-mediated constitutive expression of SOCS-3 in cutaneous T-cell lymphoma. Blood. 2001; 97: 1056–62.
- 47 Mowen KA, Tang J, Zhu W, et al . Arginine methylation of STAT1 modulates IFNalpha/beta-induced transcription. Cell. 2001; 104: 731–41.
- 48 Lee DY, Teyssier C, Strahl BD, et al . Role of protein methylation in regulation of transcription. Endocr Rev. 2005; 26: 147–70.
- 49 Komyod W, Bauer UM, Heinrich PC, et al . Are STATS arginine-methylated? J Biol Chem. 2005; 280: 21700–5.
- 50 Schmelz K, Wagner M, Dorken B, et al . 5-Aza-2′-deoxycytidine induces p21WAF expression by demethylation of p73 leading to p53-independent apoptosis in myeloid leukemia. Int J Cancer. 2005; 114: 683–95.
- 51 Han Y, Amin HM, Frantz C, et al . Restoration of shp1 expression by 5-AZA-2′-deoxycytidine is associated with downregulation of JAK3/STAT3 signaling in ALK-positive anaplastic large cell lymphoma. Leukemia. 2006; 20: 1602–9.
- 52 Esteller M, Tortola S, Toyota M, et al . Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status. Cancer Res. 2000; 60: 129–33.
- 53 Fornaro M, Plescia J, Chheang S, et al . Fibronectin protects prostate cancer cells from tumor necrosis factor-alpha-induced apoptosis via the AKT/survivin pathway. J Biol Chem. 2003; 278: 50402–11.
- 54 Kunnumakkara AB, Nair AS, Ahn KS, et al . Gossypin, a pentahydroxy glucosyl flavone, inhibits the transforming growth factor beta-activated kinase-1-mediated NF-kappaB activation pathway, leading to potentiation of apoptosis, suppression of invasion, and abrogation of osteoclastogenesis. Blood. 2007; 109: 5112–21.
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