||Tumor-associated macrophage promotes DNMT1-mediated GSN silencing in gastric cancer cell
||Institute of Clinical Medicine
DNA methyltransferase 1 (DNMT1)
Tumor-associated macrophage (TAM)
Metastasis is one of the major difficulties in current therapy of gastric cancer. In this study, we expected to discover mechanisms of metastasis in order to raise possibility in disruption of metastasis. Gelsolin, encoded by GSN, is suggested a possible metastasis suppressor which is silenced by hypermethylation in various cancers. Immunohistochemistry staining of clinical specimen demonstrated that gelsolin expression decreased in gastric cancer tissue compared with normal tissue. By treating gastric cell lines with demethylation agent, 5-aza, up-regulation of gelsolin expression was observed which suggestes that aberrant DNA methylation is also involved in gastric cancer. DNA methylation is catalyzed by DNA methyltranseferase family (DNMTs). DNMT1, one of the families, has been discovered up-regulation in many cancers. We found expression of DNMT1 inversely correlated with gelsolin from immunohistochemistry staining and DNMT1 silencing contributed to elevated gelsolin indicates that the major role of DNMT1 in aberrant methylation of gelsolin. Chronic inflammation is a crucial event in progression of gastric cancer, and increasing DNA hypermethylation followed by chronic inflammation has been identified. However, the mechanism within events is still unknown. Here we proposed that tumor-associated macrophage (TAM) in chronic inflammation plays a causal role to activate DNA methyltransferase 1 (DNMT1) in gastric cancer cell, and further methylation of target gene, GSN. Staining of CD204, a marker of TAM, shows the infiltration of TAM in tumor, and DNMT1 expressed tumor cells are surrounded by TAM via immunofluoresence double staining. Coculture gastric cancer cell line with U937 indeed elevated expression of DNMT1, and thus down-regulated expression of gelsolin. Results suggest that tumor-assaociated macrophage up-regulates DNMT1 expression and consequently silenced GSN by hypermethylation.
Epidemiology of gastric cancer 1
Metastasis in cancer progression 2
Role of gelsolin in metastasis 3
Epigenetic regulation: DNA methylation and DNA methyltransferase 4
Chronic inflammation and DNA methylation 5
Role of tumor-associated macrophage (TAM) in inflammation-related cancer 6
Research motive 7
Material and methods 9
Cell culture 9
Treatment of 5-Aza-2'-deoxycytidine (5-aza) 9
RNA interence 10
Immunohistochemistry (IHC) and immunofluorescence (IF) 10
Protein extraction 12
Western blotting (WB) 12
RNA extraction 13
Reverse transcriptase-polymerase chain reaction (RT-PCR) 13
Real-time polymerase chain reaction (qPCR) 14
DNA extraction 15
Bisulfite conversion 15
Migration assay 17
Cytokine array 17
Statistical analysis 18
Gelsolin is down-regulated in clinical gastric cancer patients. 19
Relatively low expression level of gelsolin in human gastric cancer cell lines compared with normal tissue from clinical specimen. 19
Methylation status of CpG island in GSN promoter. 20
Gelsolin expression is silenced via promoter hypermethylation. 20
Up-regulation of DNMT1 in gastric cancer correlates with poor survival. 21
Expression of DNMT1 shows inverse correlation with gelsolin in gastric cancer specimen. 22
DNMT1 mediates transcriptional repression of gelsolin in gastric cancer. 22
Infiltration of tumor-associated macrophage (TAM) positively correlates with expression of DNMT1 in gastric cancer cell. 23
Co-culture system provides an in vitro microenviroment to mimic infiltration of TAM in gastric cancer cell. 24
Up-regulation of DNMT1 and subsequently transcriptional depression of gelsolin in gatric cancer cell lines after co-cultured with U937. 25
TAM mainly down-regulates expression of gelsolin through a DNMT1-dependent mechanism. 25
TAM promotes cell migration through a DNMT1-dependent mechanism. 26
Figure 1. Clinical evidence indicated that gelsolin is down-regulated in gastric cancer. 37
Figure 2. Expression level of gelsolin in human gastric cancer cell lines. 38
Figure 3. Methylation status of CpG island in GSN promoter. 39
Figure 4. Restoration of gelsolin by treated with 5’-aza-2’deoxycytidine (5-aza), a demethylation agent, in gastric cancer cell lines indicated that gelsolin is down-regulated by DNA methylation. 41
Figure 5. Clinical evidence indicated that DNMT1 is up-regulated in gastric cancer. 43
Figure 6. Dislocalization of DNMT1 and geloslin in gastric cancer cells. 44
Figure 7. Expression level of DNMT1 in human gastric cancer cell lines. 45
Figure 8. DNMT1 knockdown in two gastric cancer cell lines, AGS and AZ521, contributes to up-regulation of gelsolin in transcription level. 46
Figure 9. Infiltration of tumor-associated macrophage (TAM) in gastric cancer. 48
Figure 10. DNMT1 expressed tumor cells are surrounded by TAM in close juxtaposition. 49
Figure 11. Schema presents a co-culture system that provides an in vitro microenviroment to mimic infiltration of TAM in gastric cancer cell. 50
Figure 12. Up-regulation of M2 marker and cytokines proved that U937 tends to differentiated into M2 type after co-culture with gastric cancer cell lines. 52
Figure 13. Up-regulation of DNMT1 in gatric cancer cell lines, AGS and HR, and subsequently transcriptional depression of gelsolin after co-cultured with U937. 53
Figure 14. U937 inhibits expression of gelsolin through a DNMT1-dependent mechanism. 55
Figure 15. U937 promotes cell migration through a DNMT1-dependent mechanism. 57
Table 1. Grading of gelsolin expression by IHC staining of human gastric cancer patients. 59
Table 2. Grading of DNMT1 expression by IHC staining of human gastric cancer patients. 60
Table3. Grading of CD204 expression by IHC staining of human gastric cancer patients. 61
Appendix 1. List of antibody 62
Appendix 2. List of primer 64
Appendix 3. Cytokine array of co-culture 65
Curriculum vitae 67
Abbaszadegan, M. R., O. Moaven, et al. (2008). "p16 promoter hypermethylation: a useful serum marker for early detection of gastric cancer." World J Gastroenterol 14(13): 2055-2060.
Alvarez, H., J. Opalinska, et al. (2011). "Widespread hypomethylation occurs early and synergizes with gene amplification during esophageal carcinogenesis." PLoS Genet 7(3): e1001356.
Aoki, E., H. Ohashi, et al. (2003). "Expression levels of DNA methyltransferase genes do not correlate with p15INK4B gene methylation in myelodysplastic syndromes." Leukemia 17(9): 1903-1904.
Bingle, L., N. J. Brown, et al. (2002). "The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies." J Pathol 196(3): 254-265.
Chen, W., N. Gao, et al. (2010). "Hypermethylation downregulates Runx3 gene expression and its restoration suppresses gastric epithelial cell growth by inducing p27 and caspase3 in human gastric cancer." J Gastroenterol Hepatol 25(4): 823-831.
Chen, Y., G. J. Gorelik, et al. (2010). "Decreased ERK and JNK signaling contribute to gene overexpression in "senescent" CD4+CD28- T cells through epigenetic mechanisms." J Leukoc Biol 87(1): 137-145.
Clements, E. G., H. P. Mohammad, et al. (2012). "DNMT1 modulates gene expression without its catalytic activity partially through its interactions with histone-modifying enzymes." Nucleic Acids Research.
Condeelis, J. and J. W. Pollard (2006). "Macrophages: Obligate Partners for Tumor Cell Migration, Invasion, and Metastasis." Cell 124(2): 263-266.
Conti, I. and B. J. Rollins (2004). "CCL2 (monocyte chemoattractant protein-1) and cancer." Semin Cancer Biol 14(3): 149-154.
Dicken, B. J., D. L. Bigam, et al. (2005). "Gastric adenocarcinoma: review and considerations for future directions." Ann Surg 241(1): 27-39.
Dong, Y., H. L. Asch, et al. (2002). "Molecular mechanism of transcriptional repression of gelsolin in human breast cancer cells." Exp Cell Res 276(2): 328-336.
Dunn, G. P., A. T. Bruce, et al. (2002). "Cancer immunoediting: from immunosurveillance to tumor escape." Nat Immunol 3(11): 991-998.
Eads, C. A., K. D. Danenberg, et al. (1999). "CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression." Cancer Res 59(10): 2302-2306.
Elgert, K. D., D. G. Alleva, et al. (1998). "Tumor-induced immune dysfunction: the macrophage connection." J Leukoc Biol 64(3): 275-290.
Espada, J., H. Peinado, et al. (2011). "Regulation of SNAIL1 and E-cadherin function by DNMT1 in a DNA methylation-independent context." Nucleic Acids Research.
Eun, D. W., S. H. Ahn, et al. (2007). "PKCepsilon is essential for gelsolin expression by histone deacetylase inhibitor apicidin in human cervix cancer cells." Biochem Biophys Res Commun 354(3): 769-775.
Foran, E., M. M. Garrity-Park, et al. (2010). "Upregulation of DNA methyltransferase-mediated gene silencing, anchorage-independent growth, and migration of colon cancer cells by interleukin-6." Mol Cancer Res 8(4): 471-481.
Forman, D., D. G. Newell, et al. (1991). "Association between infection with Helicobacter pylori and risk of gastric cancer: evidence from a prospective investigation." BMJ 302(6788): 1302-1305.
Fox, J. G. and T. C. Wang (2007). "Inflammation, atrophy, and gastric cancer." The Journal of Clinical Investigation 117(1): 60-69.
Friedl, P. and K. Wolf (2003). "Tumour-cell invasion and migration: diversity and escape mechanisms." Nat Rev Cancer 3(5): 362-374.
Fujita, H., F. Okada, et al. (2001). "Gelsolin functions as a metasatsis suppressor in B16-BL6 mouse melanoma cells and requirement of the carboxyl-terminus for its effect." International Journal of Cancer 93(6): 773-780.
Gay, F., Y. Estornes, et al. (2008). "In colon carcinogenesis, the cytoskeletal protein gelsolin is down-regulated during the transition from adenoma to carcinoma." Human Pathology 39(10): 1420-1430.
Grange, J. M., B. Krone, et al. (2011). "Infection, inflammation and cancer." Int J Cancer 128(9): 2240-2241.
Grivennikov, S. I., F. R. Greten, et al. (2010). "Immunity, inflammation, and cancer." Cell 140(6): 883-899.
Gupta, G. P. and J. Massague (2006). "Cancer metastasis: building a framework." Cell 127(4): 679-695.
Hahn, M. A., T. Hahn, et al. (2008). "Methylation of polycomb target genes in intestinal cancer is mediated by inflammation." Cancer Res 68(24): 10280-10289.
Heinrich, P. C., I. Behrmann, et al. (1998). "Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway." Biochem J 334 ( Pt 2): 297-314.
Hoshikawa, Y., H. J. Kwon, et al. (1994). "Trichostatin A induces morphological changes and gelsolin expression by inhibiting histone deacetylase in human carcinoma cell lines." Exp Cell Res 214(1): 189-197.
Hur, K., T. Niwa, et al. (2011). "Insufficient role of cell proliferation in aberrant DNA methylation induction and involvement of specific types of inflammation." Carcinogenesis 32(1): 35-41.
Issa, J. P., N. Ahuja, et al. (2001). "Accelerated age-related CpG island methylation in ulcerative colitis." Cancer Res 61(9): 3573-3577.
Janunger, K. G., L. Hafstrom, et al. (2002). "Chemotherapy in gastric cancer: a review and updated meta-analysis." Eur J Surg 168(11): 597-608.
Jemal, A., M. M. Center, et al. (2010). "Global Patterns of Cancer Incidence and Mortality Rates and Trends." Cancer Epidemiology Biomarkers & Prevention 19(8): 1893-1907.
Joyce, J. A. and J. W. Pollard (2009). "Microenvironmental regulation of metastasis." Nat Rev Cancer 9(4): 239-252.
Katayama, Y., M. Takahashi, et al. (2009). "Helicobacter pylori causes runx3 gene methylation and its loss of expression in gastric epithelial cells, which is mediated by nitric oxide produced by macrophages." Biochemical and Biophysical Research Communications 388(3): 496-500.
Keating, G. M. (2012). "Azacitidine: a review of its use in the management of myelodysplastic syndromes/acute myeloid leukaemia." Drugs 72(8): 1111-1136.
Kelly, T. K., D. D. De Carvalho, et al. (2010). "Epigenetic modifications as therapeutic targets." Nat Biotechnol 28(10): 1069-1078.
Kim, H., Y. M. Kwon, et al. (2006). "Elevated mRNA levels of DNA methyltransferase-1 as an independent prognostic factor in primary nonsmall cell lung cancer." Cancer 107(5): 1042-1049.
Kim, T. Y., H. S. Jong, et al. (2004). "DNA hypermethylation in gastric cancer." Alimentary Pharmacology & Therapeutics 20: 131-142.
Kuper, H., H. O. Adami, et al. (2000). "Infections as a major preventable cause of human cancer." J Intern Med 248(3): 171-183.
Kwon, H. J., M. Yoshida, et al. (1997). "Suppression of morphological transformation by radicicol is accompanied by enhanced gelsolin expression." Oncogene 15(21): 2625-2631.
Lee, C. F., D. S. Ou, et al. (2010). "hNaa10p contributes to tumorigenesis by facilitating DNMT1-mediated tumor suppressor gene silencing." J Clin Invest 120(8): 2920-2930.
Li, G. H., P. D. Arora, et al. (2010). "Multifunctional roles of gelsolin in health and diseases." Medicinal Research Reviews: n/a-n/a.
Libby, P. (2002). "Inflammation in atherosclerosis." Nature 420(6917): 868-874.
Lin, R. K., Y. S. Hsieh, et al. (2010). "The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients." J Clin Invest 120(2): 521-532.
Liu, W. T., H. L. Jiao, et al. (2007). "[Correlation of E-cadherin hypermethylation to tumorigenesis and development of gastric cancer]." Ai Zheng 26(11): 1199-1203.
Mielnicki, L. M., A. M. Ying, et al. (1999). "Epigenetic Regulation of Gelsolin Expression in Human Breast Cancer Cells." Experimental Cell Research 249(1): 161-176.
Milne, A. N., F. Carneiro, et al. (2009). "Nature meets nurture: molecular genetics of gastric cancer." Hum Genet 126(5): 615-628.
Mutze, K., R. Langer, et al. (2011). "DNA methyltransferase 1 as a predictive biomarker and potential therapeutic target for chemotherapy in gastric cancer." European Journal of Cancer 47(12): 1817-1825.
Ni, X. G., L. Zhou, et al. (2008). "The ubiquitin-proteasome pathway mediates gelsolin protein downregulation in pancreatic cancer." Mol Med 14(9-10): 582-589.
Niwa, T., T. Tsukamoto, et al. (2010). "Inflammatory processes triggered by Helicobacter pylori infection cause aberrant DNA methylation in gastric epithelial cells." Cancer Res 70(4): 1430-1440.
Noske, A., C. Denkert, et al. (2005). "Loss of Gelsolin expression in human ovarian carcinomas." European Journal of Cancer 41(3): 461-469.
O'Hagan, H. M., W. Wang, et al. (2011). "Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands." Cancer Cell 20(5): 606-619.
Pantel, K. and R. H. Brakenhoff (2004). "Dissecting the metastatic cascade." Nat Rev Cancer 4(6): 448-456.
Parsonnet, J., G. D. Friedman, et al. (1991). "Helicobacter pylori infection and the risk of gastric carcinoma." N Engl J Med 325(16): 1127-1131.
Pollard, J. W. (2004). "Tumour-educated macrophages promote tumour progression and metastasis." Nat Rev Cancer 4(1): 71-78.
Rhee, I., K. E. Bachman, et al. (2002). "DNMT1 and DNMT3b cooperate to silence genes in human cancer cells." Nature 416(6880): 552-556.
Rogler, G., K. Brand, et al. (1998). "Nuclear factor kappaB is activated in macrophages and epithelial cells of inflamed intestinal mucosa." Gastroenterology 115(2): 357-369.
Sarkar, S., A. L. Abujamra, et al. (2011). "Histone deacetylase inhibitors reverse CpG methylation by regulating DNMT1 through ERK signaling." Anticancer Res 31(9): 2723-2732.
Soria, G. and A. Ben-Baruch (2009). The CCL5/CCR5 Axis in Cancer
Chemokine Receptors in Cancer. A. M. Fulton, Humana Press: 109-130.
Suerbaum, S. and P. Michetti (2002). "Helicobacter pylori infection." N Engl J Med 347(15): 1175-1186.
Tanaka, H., R. Shirkoohi, et al. (2006). "siRNA gelsolin knockdown induces epithelial-mesenchymal transition with a cadherin switch in human mammary epithelial cells." International Journal of Cancer 118(7): 1680-1691.
Tanaka, M., L. Müllauer, et al. (1995). "Gelsolin: A Candidate for Suppressor of Human Bladder Cancer." Cancer Research 55(15): 3228-3232.
Thompson, G. B., J. A. van Heerden, et al. (1993). "Adenocarcinoma of the stomach: are we making progress?" Lancet 342(8873): 713-718.
Ushijima, T. and M. Sasako (2004). "Focus on gastric cancer." Cancer Cell 5(2): 121-125.
Vakkila, J. and M. T. Lotze (2004). "Inflammation and necrosis promote tumour growth." Nat Rev Immunol 4(8): 641-648.
Waugh, D. J. and C. Wilson (2008). "The interleukin-8 pathway in cancer." Clin Cancer Res 14(21): 6735-6741.
Wehbe, H., R. Henson, et al. (2006). "Interleukin-6 contributes to growth in cholangiocarcinoma cells by aberrant promoter methylation and gene expression." Cancer Res 66(21): 10517-10524.
Winston, J. S., H. L. Asch, et al. (2001). "Downregulation of Gelsolin Correlates with the Progression to Breast Carcinoma." Breast Cancer Research and Treatment 65(1): 11-21.
Yang, J., X. Wei, et al. (2011). "Clinical significance of the expression of DNA methyltransferase proteins in gastric cancer." Mol Med Report 4(6): 1139-1143.
Yilmaz, M. and G. Christofori (2009). "EMT, the cytoskeleton, and cancer cell invasion." Cancer Metastasis Rev 28(1-2): 15-33.
Zhang, Q., H. Y. Wang, et al. (2006). "STAT3 induces transcription of the DNA methyltransferase 1 gene (DNMT1) in malignant T lymphocytes." Blood 108(3): 1058-1064.