ABSTRACTSilencing of microRNAs (miRNAs) by promoter CpG island methylation may be an important mechanism in prostate carcinogenesis. To screen for epigenetically silenced miRNAs in prostate cancer (PCa), we treated prostate normal epithelial and carcinoma cells with 5-aza-20-deoxycytidine (AZA) and subsequently examined expression changes of 650 miRNAs by megaplex stemloop reverse transcription -- quantitative PCR. After applying a selection strategy, we analyzed the methylation status of CpG islands upstream to a subset of miRNAs by methylation-specific PCR. The CpG islands of miR-18b, miR-132, miR-34b/c, miR-148a, miR-450a and miR-542-3p showed methylation patterns congruent with their expression modulations in response to AZA. Methylation analysis of these CpG islands in a panel of 50 human prostate carcinoma specimens and 24 normal controls revealed miR-132 to be methylated in 42% of human cancer cases in a manner positively correlated to total Gleason score and tumor stage. Expression analysis of miR-132 in our tissue panel confirmed its downregulation in methylated tumors. Re-expression of miR-132 in PC3 cells induced cell detachment followed by cell death (anoikis). Two pro-survival proteins-heparin-binding epidermal growth factor and TALIN2-were confirmed as direct targets of miR-132. The results of this study point to miR-132 as a methylation-silenced miRNA with an antimetastatic role in PCa controlling cellular adhesion.
1 Nelson WG, De Marzo AM, Yegnasubramanian S. Epigenetic alterations in human prostate cancers. Endocrinology 2009; 150: 3991 -- 4002.
2 Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis 2010; 31: 27 -- 36.
3 Rodrı´guez-Paredes M, Esteller M. Cancer epigenetics reaches mainstream oncology. Nat Med 2011; 17: 330 -- 339.
4 Cooper CS, Foster CS. Concepts of epigenetics in prostate cancer development. Br J Cancer 2009; 100: 240 -- 245.
5 Park JY. Promoter hypermethylation in prostate cancer. Cancer Control 2010; 4: 245 -- 255.
6 Fabbri M, Calin GA. Epigenetics and miRNAs in human cancer. Adv Genet 2010; 70: 87 -- 99.
7 Zhang B, Pan X, Cobb GP, Anderson TA. MicroRNAs as oncogenes and tumor suppressors. Dev Biol 2007; 302: 1 -- 12.
8 Ambs S, Prueitt R, Yi M, Hudson R, Howe T, Petrocca F et al. Genomic profiling of microRNA and messenger RNA reveals deregulated microRNA expression in prostate cancer. Cancer Res 2008; 68: 6162 -- 6170.
9 Coppola V, de Maria R, Bonci D. MicroRNAs and prostate cancer. Endocr Relat Cancer 2010; 17: F1--F17.
10 Ozen M, Creighton CJ, Ozdemir M, Ittmann M. Widespread deregulation of microRNA expression in human prostate cancer. Oncogene 2008; 27: 1788 -- 1793.
11 Lujambio A, Esteller M. CpG island hypermethylation of tumor suppressor microRNAs in human cancer. Cell Cycle 2007; 6: 1455 -- 1459.
12 Lujambio A, Calin GA, Villanueva A, Ropero S, Sa´nchez-Ce´spedes M, Blanco D et al. A microRNA DNA methylation signature for human cancer metastasis. Proc Natl Acad Sci USA 2008; 105: 13556 -- 13561.
13 Kozaki K, Imoto I, Mogi S, Omura K, Inazawa J. Exploration of tumor-suppressive microRNAs silenced by DNA hypermethylation in oral cancer. Cancer Res 2008; 68: 2094 -- 2105.
14 Rauhala HE, Jalava SE, Isotalo J, Bracken H, Lehmusvaara S, Tammela TL et al. miR- 193b is an epigenetically regulated putative tumor suppressor in prostate cancer. Int J Cancer 2010; 127: 1363 -- 1372.
15 Suh SO, Chen Y, Zaman MS, Hirata H, Yamamura S, Shahryari V et al. MicroRNA- 145 is regulated by DNA methylation and p53 gene mutation in prostate cancer. Carcinogenesis 2011; 32: 772 -- 778.
16 Pavicic W, Perkio¨ E, Kaur S, Peltoma¨ ki P. Altered methylation at microRNAassociated CpG islands in hereditary and sporadic carcinomas: MS-MLPA-based approach. Mol Med 2011; 17: 726 -- 735.
17 Kalimutho M, Di Cecilia S, Del Vecchio Blanco G, Roviello F, Sileri P, Cretella M et al. Epigenetically silenced miR-34b/c as a novel faecal-based screening marker for colorectal cancer. Br J Cancer 2011; 104: 1770 -- 1778.
18 Yoon S, Choi YC, Lee S, Jeong Y, Yoon J, Baek K. Induction of growth arrest by miR- 542-3p that targets survivin. FEBS Lett 2010; 584: 4048 -- 4052.
19 Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, Yi M et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 2006; 9: 189 -- 198.
20 Anand S, Majeti BK, Acevedo LM, Murphy EA, Mukthavaram R, Scheppke L et al. MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis. Nat Med 2010; 16: 909 -- 914.
21 Wong TS, Liu XB, Chung-Wai Ho A, Po-Wing Yuen A, Wai-Man Ng R, Ignace Wei W. Identification of pyruvate kinase type M2 as potential oncoprotein in squamous cell carcinoma of tongue through microRNA profiling. Int J Cancer 2008; 123: 251 -- 257.
22 Gougelet A, Pissaloux D, Besse A, Perez J, Duc A, Dutour A et al. Micro-RNA profiles in osteosarcoma as a predictive tool for ifosfamide response. Int J Cancer 2010; 129: 680 -- 690.
23 Lages E, Guttin A, El Atifi M, Ramus C, Ipas H et al. MicroRNA and target protein patterns reveal physiopathological features of glioma subtypes. PLoS ONE 2011; 6: e20600.
24 Zhang S, Hao J, Xie F, Hu X, Liu C, Tong J et al. Downregulation of miR-132 by promoter methylation contributes to pancreatic cancer development. Carcinogenesis 2011; 32: 1183 -- 1189.
25 Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS et al. Integrative genomic profiling of human prostate cancer. Cancer Cell 2010; 18: 11--22.
26 Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A et al.Accurate normalization of real-time quantitative RT--PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3: RESEARCH0034.
27 Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH et al. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ 2009; 16: 1093 -- 1107.
28 Bartkowiak D, Ho¨ gner S, Baust H, Nothdurft W, Ro¨ ttinger EM. Comparative analysis of apoptosis in HL60 detected by Annexin-V and fluorescein-diacetate. Cytometry 1999; 37: 191 -- 196.
29 Cooper CR, Pienta KJ. Cell adhesion and chemotaxis in prostate cancer metastasis to bone: a minireview. Prostate Cancer Prostatic Dis 2000; 3: 6 -- 12.
30 Chu K, Cheng CJ, Ye X, Lee YC, Zurita AJ, Chen DT et al. Cadherin-11 promotes the metastasis of prostate cancer cells to bone. Mol Cancer Res 2008; 6: 1259 -- 1267.
31 Boissier S, Magnetto S, Frappart L, Cuzin B, Ebetino FH, Delmas PD et al. Bisphosphonates inhibit prostate and breast carcinoma cell adhesion to unmineralized and mineralized bone extracellular matrices. Cancer Res 1997; 57: 3890 -- 3894.
32 Matsunaga S, Iguchi K, Usui S, Hirano K. Incadronate induces cell detachment and apoptosis in prostatic PC-3 cells. Anticancer Res 2007; 27: 927 -- 932.
33 Miyamoto S, Yagi H, Yotsumoto F, Horiuchi S, Yoshizato T, Kawarabayashi T et al. New approach to cancer therapy: heparin binding-epidermal growth factor-like growth factor as a novel targeting molecule. Anticancer Res 2007; 27: 3713 -- 3721.
34 Tørring N, Jørgensen PE, Sørensen BS, Nexø E. Increased expression of heparin binding EGF (HB-EGF), amphiregulin, TGF alpha and epiregulin in androgenindependent prostate cancer cell lines. Anticancer Res 2000; 20: 91 -- 95.
35 Adam RM, Kim J, Lin J, Orsola A, Zhuang L, Rice DC et al. Heparin-binding epidermal growth factor-like growth factor stimulates androgen-independent prostate tumor growth and antagonizes androgen receptor function. Endocrinology 2002; 143: 4599 --4608.
36 Monkley SJ, Pritchard CA, Critchley DR. Analysis of the mammalian talin2 gene TLN2. Biochem Biophys Res Commun 2001; 286: 880 -- 885.
37 Zhang X, Jiang G, Cai Y, Monkley SJ, Critchley DR, Sheetz MP. Talin depletion reveals independence of initial cell spreading from integrin activation and traction. Nat Cell Biol 2008; 10: 1062 -- 1068.
38 Sakamoto S, McCann RO, Dhir R, Kyprianou N. Talin1 promotes tumor invasion and metastasis via focal adhesion signaling and anoikis resistance. Cancer Res 2010; 70: 1885 -- 1895.
39 Papadopoulos GL, Alexiou P, Maragkakis M, Reczko M, Hatzigeorgiou AG. DIANAmirPath: integrating human and mouse microRNAs in pathways. Bioinformatics 2009; 25: 1991 -- 1993.
40 Mestdagh P, Feys T, Bernard N, Guenther S, Chen C, Speleman F et al. Highthroughput stem -- loop RT -- qPCR miRNA expression profiling using minute amounts of input RNA. Nucleic Acids Res 2008; 36: e143.
41 Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann F, Speleman F et al. A novel and universal method for microRNA RT --qPCR data normalization. Genome Biol 2009; 10: R64.
42 Brandes JC, Carraway H, Herman JG. Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter. Oncogene 2007; 26: 6229 -- 6237.
43 Lena AM, Shalom-Feuerstein R, Rivetti di Val Cervo P, Aberdam D, Knight RA, Melino G et al. miR-203 represses ‘stemness’ by repressing DeltaNp63. Cell Death Differ 2008; 15: 1187 -- 1195.
44 Viticchie` G, Lena AM, Latina A, Formosa A, Gregersen LH, Lund AH et al. miR-203 controls proliferation, migration and invasive potential of prostate cancer cell lines. Cell Cycle 2011; 10: 1121 -- 1131.