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01-01-2009 | Preclinical Study

RUNX3 inactivation by frequent promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression

Authors: Manish Mani Subramaniam, Jason Yongsheng Chan, Richie Soong, Kosei Ito, Yoshiaki Ito, Khay Guan Yeoh, Manuel Salto-Tellez, Thomas Choudary Putti

Published in: Breast Cancer Research and Treatment | Issue 1/2009

Abstract

Background We had previously established that inactivation of RUNX3 occurs by frequent promoter hypermethylation and protein mislocalization in invasive ductal carcinomas (IDC) of breast. Here, we hypothesize that inactivation of RUNX3 occurring in ductal carcinoma in situ (DCIS) represent early event in breast carcinogenesis. Methods The study cohort of 40 patients included 17 pure DCIS cases and 23 cases of DCIS with associated IDC (DCIS-IDC). The DCIS and IDC components of mixed cases were manually microdissected to permit separate evaluation. All the 63 samples including 17 pure DCIS, 23 samples each of DCIS and IDC of DCIS-IDC cases were analyzed for RUNX3 protein expression using R3-6E9 monoclonal antibody as well as promoter methylation status by methylation specific PCR. Results Compared to matched normal breast samples (4 of 40, 10%), DCIS (35 of 40, 88%) and IDC (21 of 23, 91%) exhibited significant RUNX3 inactivation (P < 0.001) in the form of negative or weak nuclear staining. In contrast to normal breast tissues (1/10, 10%), promoter hypermethylation of RUNX3 was significantly higher in the neoplastic breast samples (46 of total 61, 75%) including 30 of 40 (75%) DCIS and 16 of 21 (76%) IDC samples (P = 0.009). Overall, promoter hypermethylation correlated with RUNX3 inactivation in 42 of 46 (91%) methylated samples (P = 0.03). Mislocalized cytoplasmic expression also accounted for RUNX3 inactivation in majority of DCIS (33/40, 83%) and IDC (20/23, 87%) samples independent of promoter hypermethylation. Conclusion Our data suggest that RUNX3 inactivation by promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression.

Dua RS, Isacke CM, Gui GP (2006) The intraductal approach to breast cancer biomarker discovery. J Clin Oncol 24:1209–1216PubMedCrossRef

Agrawal A, Murphy RF, Agrawal DK (2007) DNA methylation in breast and colorectal cancers. Mod Pathol 20:711–721PubMedCrossRef

Jackson K, Yu MC, Arakawa K, Fiala E, Youn B, Fiegl H, Muller-Holzner E, Widschwendter M, Ehrlich M (2004) DNA hypomethylation is prevalent even in low-grade breast cancers. Cancer Biol Ther 3:1225–1231PubMedCrossRef

Chekhun VF, Kulik GI, Yurchenko OV, Tryndyak VP, Todor IN, Luniv LS, Tregubova NA, Pryzimirska TV, Montgomery B, Rusetskaya NV, Pogribny IP (2006) Role of DNA hypomethylation in the development of the resistance to doxorubicin in human MCF-7 breast adenocarcinoma cells. Cancer Lett 231:87–93PubMedCrossRef

Li QL, Ito K, Sakakura C, Fukamachi H, Inoue K, Chi XZ, Lee KY, Nomura S, Lee CW, Han SB, Kim HM, Kim WJ, Yamamoto H, Yamashita N, Yano T, Ikeda T, Itohara S, Inazawa J, Abe T, Hagiwara A, Yamagishi H, Ooe A, Kaneda A, Sugimura T, Ushijima T, Bae SC, Ito Y (2002) Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell 109:113–124PubMedCrossRef

Schulmann K, Sterian A, Berki A, Yin J, Sato F, Xu Y, Olaru A, Wang S, Mori Y, Deacu E, Hamilton J, Kan T, Krasna MJ, Beer DG, Pepe MS, Abraham JM, Feng Z, Schmiegel W, Greenwald BD, Meltzer SJ (2005) Inactivation of p16, RUNX3, and HPP1 occurs early in Barrett’s-associated neoplastic progression and predicts progression risk. Oncogene 24:4138–4148PubMed

Park WS, Cho YG, Kim CJ, Song JH, Lee YS, Kim SY, Nam SW, Lee SH, Yoo NJ, Lee JY (2005) Hypermethylation of the RUNX3 gene in hepatocellular carcinoma. Exp Mol Med 37:276–281PubMed

Takahashi T, Shivapurkar N, Riquelme E, Shigematsu H, Reddy J, Suzuki M, Miyajima K, Zhou X, Bekele BN, Gazdar AF, Wistuba II (2004) Aberrant promoter hypermethylation of multiple genes in gallbladder carcinoma and chronic cholecystitis. Clin Cancer Res 10:6126–6133PubMedCrossRef

Tozawa T, Tamura G, Honda T, Nawata S, Kimura W, Makino N, Kawata S, Sugai T, Suto T, Motoyama T. Promoter hypermethylation of DAP-kinase is associated with poor survival in primary biliary tract carcinoma patients. Cancer Sci 95:736–740

10.

Wada M, Yazumi S, Takaishi S, Hasegawa K, Sawada M, Tanaka H, Ida H, Sakakura C, Ito K, Ito Y, Chiba T (2004) Frequent loss of RUNX3 gene expression in human bile duct and pancreatic cancer cell lines. Oncogene 23:2401–2407PubMedCrossRef

11.

Goel A, Arnold CN, Tassone P, Chang DK, Niedzwiecki D, Dowell JM, Wasserman L, Compton C, Mayer RJ, Bertagnolli MM, Boland CR (2004) Epigenetic inactivation of RUNX3 in microsatellite unstable sporadic colon cancers. Int J Cancer 112:754–759PubMedCrossRef

12.

Ku JL, Kang SB, Shin YK, Kang HC, Hong SH, Kim IJ, Shin JH, Han IO, Park JG (2004) Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines. Oncogene 23:6736–6742PubMedCrossRef

13.

Imamura Y, Hibi K, Koike M, Fujiwara M, Kodera Y, Ito K, Nakao A (2005) RUNX3 promoter region is specifically methylated in poorly-differentiated colorectal cancer. Anticancer Res 25:2627–2630PubMed

14.

Lau QC, Raja E, Salto-Tellez M, Liu Q, Ito K, Inoue M, Putti TC, Loh M, Ko TK, Huang C, Bhalla KN, Zhu T, Ito Y, Sukumar S (2006) RUNX3 is frequently inactivated by dual mechanisms of protein mislocalization and promoter hypermethylation in breast cancer. Cancer Res 66:6512–6520PubMedCrossRef

15.

Yanada M, Yaoi T, Shimada J, Sakakura C, Nishimura M, Ito K, Terauchi K, Nishiyama K, Itoh K, Fushiki S (2005) Frequent hemizygous deletion at 1p36 and hypermethylation downregulate RUNX3 expression in human lung cancer cell lines. Oncol Rep 14: 817–822PubMed

16.

Salto-Tellez M, Peh BK, Ito K, Tan SH, Chong PY, Han HC, Tada K, Ong WY, Soong R, Voon DC, Ito Y (2006) RUNX3 protein is overexpressed in human basal cell carcinomas. Oncogene 25:7646–7649PubMedCrossRef

17.

Kato N, Tamura G, Fukase M, Shibuya H, Motoyama T (2003) Hypermethylation of the RUNX3 gene promoter in testicular yolk sac tumor of infants. Am J Pathol 163:387–391PubMed

18.

Ito K, Liu Q, Salto-Tellez M, Yano T, Tada K, Ida H, Huang C, Shah N, Inoue M, Rajnakova A, Hiong KC, Peh BK, Han HC, Ito T, Teh M, Yeoh KG, Ito Y (2005) RUNX3, a novel tumor suppressor, is frequently inactivated in gastric cancer by protein mislocalization. Cancer Res 65:7743–7750PubMedCrossRef

19.

Kim TY, Lee HJ, Hwang KS, Lee M, Kim JW, Bang YJ, Kang GH (2004) Methylation of RUNX3 in various types of human cancers and premalignant stages of gastric carcinoma. Lab Invest 84:479–484PubMedCrossRef

20.

Suzuki M, Shigematsu H, Shames DS, Sunaga N, Takahashi T, Shivapurkar N, Iizasa T, Frenkel EP, Minna JD, Fujisawa T, Gazdar AF (2005) DNA methylation-associated inactivation of TGFbeta-related genes DRM/Gremlin, RUNX3, and HPP1 in human cancers. Br J Cancer 93:1029–1037PubMedCrossRef

21.

Chen W, Salto-Tellez M, Palanisamy N, Ganesan K, Hou Q, Tan LK, Sii LH, Ito K, Tan B, Wu J, Tay A, Tan KC, Ang E, Tan BK, Tan PH, Ito Y, Tan P (2007) Targets of genome copy number reduction in primary breast cancers identified by integrative genomics. Genes Chromosomes Cancer 46:288–301PubMedCrossRef

22.

Sneige N, Lagios MD, Schwarting R, Colburn W, Atkinson E, Weber D, Sahin A, Kemp B, Hoque A, Risin S, Sabichi A, Boone C, Dhingra K, Kelloff G, Lippman S (1999) Interobserver reproducibility of the Lagios nuclear grading system for ductal carcinoma in situ. Hum Pathol 30:257–262PubMedCrossRef

23.

Bryan BB, Schnitt SJ, Collins LC (2006) Ductal carcinoma in situ with basal-like phenotype: a possible precursor to invasive basal-like breast cancer. Mod Pathol 19:617–621PubMedCrossRef

24.

Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93:9821–9826PubMedCrossRef

25.

Waki T, Tamura G, Sato M, Terashima M, Nishizuka S, Motoyama T (2003) Promoter methylation status of DAP-kinase and RUNX3 genes in neoplastic and non-neoplastic gastric epithelia. Cancer Sci 94:360–364PubMedCrossRef

26.

Dhillon VS, Shahid M, Husain SA (2004) CpG methylation of the FHIT, FANCF, cyclin-D2, BRCA2 and RUNX3 genes in Granulosa cell tumors (GCTs) of ovarian origin. Mol Cancer 3:33PubMedCrossRef

27.

Kang GH, Lee S, Lee HJ, Hwang KS (2004) Aberrant CpG island hypermethylation of multiple genes in prostate cancer and prostatic intraepithelial neoplasia. J Pathol 202:233–240PubMedCrossRef

28.

Widschwendter M, Jones PA (2002) DNA methylation and breast carcinogenesis. Oncogene 21:5462–5482PubMedCrossRef

29.

Ito Y, Miyazono K (2003) RUNX transcription factors as key targets of TGF-ß superfamily signaling. Curr Opin Genet Dev 13:43–47PubMedCrossRef

30.

Xie W, Mertens JC, Reiss DJ, Rimm DL, Camp RL, Haffty BG, Reiss M (2002) Alterations of Smad signaling in human breast carcinomas are associated with poor outcome: a tissue microarray study. Cancer Res 62:497–505PubMed

31.

Evron E, Dooley WC, Umbricht CB, Rosenthal D, Sacchi N, Gabrielson E, Soito AB, Hung DT, Ljung B, Davidson NE, Sukumar S (2001) Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR. Lancet 357:1335–1336PubMedCrossRef

32.

Umbricht CB, Evron E, Gabrielson E, Ferguson A, Marks J, Sukumar S (2001) Hypermethylation of 14-3-3 sigma (stratifin) is an early event in breast cancer. Oncogene 20:3348–3353PubMedCrossRef

33.

Futscher BW, O’Meara MM, Kim CJ, Rennels MA, Lu D, Gruman LM, Seftor RE, Hendrix MJ, Domann FE (2004) Aberrant methylation of the maspin promoter is an early event in human breast cancer. Neoplasia 6:380–389PubMedCrossRef

34.

Lo PK, Mehrotra J, D’Costa A, Fackler MJ, Garrett-Mayer E, Argani P, Sukumar S (2006) Epigenetic suppression of secreted frizzled related protein 1 (SFRP1) expression in human breast cancer. Cancer Biol Ther 5:281–286PubMedCrossRef

35.

Zhang Y, Bhat I, Zeng M, Jayal G, Wazer DE, Band H, Band V (2006) Human kallikrein 10, a predictive marker for breast cancer. Biol Chem 387:715–721PubMedCrossRef

36.

Lee JS (2007) GSTP1 promoter hypermethylation is an early event in breast carcinogenesis. Virchows Arch 450:637–642PubMedCrossRef

37.

Yeo W, Wong WL, Wong N, Law BK, Tse GM, Zhong S (2005) High frequency of promoter hypermethylation of RASSF1A in tumorous and non-tumourous tissue of breast cancer. Pathology 37:125–130PubMedCrossRef

38.

Pasquali L, Bedeir A, Ringquist S, Styche A, Bhargava R, Trucco G (2007) Quantification of CpG island methylation in progressive breast lesions from normal to invasive carcinoma. Cancer Lett Aug 14 [Epub ahead of print]

Title: RUNX3 inactivation by frequent promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression
Authors: Manish Mani Subramaniam
Jason Yongsheng Chan
Richie Soong
Kosei Ito
Yoshiaki Ito
Khay Guan Yeoh
Manuel Salto-Tellez
Thomas Choudary Putti
Publication date: 01-01-2009
Publisher: Springer US
Published in: Breast Cancer Research and Treatment / Issue 1/2009
Print ISSN: 0167-6806
Electronic ISSN: 1573-7217
DOI: https://doi.org/10.1007/s10549-008-9917-4

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