J Korean Med Sci.  2015 Feb;30(2):155-161. 10.3346/jkms.2015.30.2.155.

Clinical Significance of Aberrant Wnt7a Promoter Methylation in Human Non-Small Cell Lung Cancer in Koreans

• Kim TH
• Moon JY
• Kim SH
• Paik SS
• Yoon HJ
• Shin DH
• Park SS
• Sohn JW

• KMID: 2129645
• DOI: http://doi.org/10.3346/jkms.2015.30.2.155

Abstract

The Wnt signaling pathway has regulatory roles in cell proliferation, differentiation, and polarity. Aberrant Wnt pathway regulation can lead to abnormal cell proliferation and cancer, and loss of Wnt7a expression has been demonstrated in lung cancer cell lines. E-cadherin keeps intercellular integrity and prevents metastasis. Therefore, E-cadherin has been known as a prognostic factor in cancer. In the present study, we investigated the E-cadherin expression status by immunohistochemical stain and the Wnt7a promoter methylation status in human non-small cell lung carcinoma (NSCLC) by methylation-specific PCR. We also analyzed their correlations with clinicopathological factors. Methylation of the Wnt7a gene promoter was detected in the lung tissues of 32 of 121 (26.4%) patients with NSCLC. Wnt7a promoter methylation was correlated with advanced tumor stage (P = 0.036) and distant metastasis (P = 0.037). In addition, Wnt7a promoter methylation showed correlation with loss of E-cadherin expression (P < 0.001). However, Wnt7a promoter methylation was not closely related with gender, age, histological type, or smoking habit. Even though Wnt7a methylation could not show significant correlation with the long term survival of the patients with limited follow up data, these findings suggest that loss of the Wnt7a gene induced by promoter methylation might be another prognostic factor for NSCLC and that restoration of Wnt7a may be a promising treatment for NSCLC.

Keyword

Carcinoma, Non-small Cell Lung; Wnt7a; Biologic Markers; E-cadherin

MeSH Terms

Cadherins/biosynthesis
Carcinoma, Non-Small-Cell Lung/*genetics/mortality
DNA Methylation/*genetics
Female
Humans
Lung Neoplasms/*genetics/mortality
Male
Middle Aged
Neoplasm Metastasis/genetics
Neoplasm Staging
Promoter Regions, Genetic/*genetics
Republic of Korea
Tumor Markers, Biological/genetics
Wnt Proteins/*genetics
Cadherins
Tumor Markers, Biological
Wnt Proteins

Figure

• 

  Fig. 1 Examples of Immunohistochemical analysis of E-cadherin protein expression in lung cancer tissues. (A) trace, (B) low, (C) intermediate, (D) strong expression of E-cadherin (magnification, ×100).

• 

  Fig. 2 Representative results showing Wnt7a gene promoter methylation in non-small cell lung cancer tissues. Wnt7a gene promoter methylation status and corresponding unmethylated non-tumor tissue samples determined by methylation-specific PCR. Sample No. 1 shows promoter methylation. No. 2 is an unmethylated sample. A water blank was used as a negative control, and in vitro methylated genomic DNA was used as a positive control. M, PCR with Wnt7a primer for methylated DNA; U, PCR with primer for unmethylated DNA.

• 

  Fig. 3 Kaplan-Meyer survival curve of the patients according to the Wnt7a methylation status. Black stands for Wnt7a methylation negative, and gray stands for positive. Cox regression analysis including sex, age, smoking status, stage, and E-cadherin expression, showed no significant correlation between Wnt7a methylation and survival.

Reference

1. Dubey S, Powell CA. Update in lung cancer 2008. Am J Respir Crit Care Med. 2009; 179:860–868.

2. Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst. 2001; 93:1062–1074.

3. Graziano SL, Gamble GP, Newman NB, Abbott LZ, Rooney M, Mookherjee S, Lamb ML, Kohman LJ, Poiesz BJ. Prognostic significance of K-ras codon 12 mutations in patients with resected stage I and II non-small-cell lung cancer. J Clin Oncol. 1999; 17:668–675.

4. Hsieh ET, Shepherd FA, Tsao MS. Co-expression of epidermal growth factor receptor and transforming growth factor-α is independent of ras mutations in lung adenocarcinoma. Lung Cancer. 2000; 29:151–157.

5. Ohsaki Y, Tanno S, Fujita Y, Toyoshima E, Fujiuchi S, Nishigaki Y, Ishida S, Nagase A, Miyokawa N, Hirata S, et al. Epidermal growth factor receptor expression correlates with poor prognosis in non-small cell lung cancer patients with p53 overexpression. Oncol Rep. 2000; 7:603–607.

6. Sekido Y, Fong KM, Minna JD. Progress in understanding the molecular pathogenesis of human lung cancer. Biochim Biophys Acta. 1998; 1378:F21–F59.

7. Polakis P. Wnt signaling and cancer. Genes Dev. 2000; 14:1837–1851.

8. Giles RH, van Es JH, Clevers H. Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys Acta. 2003; 1653:1–24.

9. Taniguchi K, Roberts LR, Aderca IN, Dong X, Qian C, Murphy LM, Nagorney DM, Burgart LJ, Roche PC, Smith DI, et al. Mutational spectrum of beta-catenin, AXIN1, and AXIN2 in hepatocellular carcinomas and hepatoblastomas. Oncogene. 2002; 21:4863–4871.

10. Howe LR, Brown AM. Wnt signaling and breast cancer. Cancer Biol Ther. 2004; 3:36–41.

11. Fukuchi T, Sakamoto M, Tsuda H, Maruyama K, Nozawa S, Hirohashi S. Beta-catenin mutation in carcinoma of the uterine endometrium. Cancer Res. 1998; 58:3526–3528.

12. Calvo R, West J, Franklin W, Erickson P, Bemis L, Li E, Helfrich B, Bunn P, Roche J, Brambilla E, et al. Altered HOX and WNT7A expression in human lung cancer. Proc Natl Acad Sci U S A. 2000; 97:12776–12781.

13. Winn RA, Marek L, Han SY, Rodriguez K, Rodriguez N, Hammond M, Van Scoyk M, Acosta H, Mirus J, Barry N, et al. Restoration of Wnt-7a expression reverses non-small cell lung cancer cellular transformation through frizzled-9-mediated growth inhibition and promotion of cell differentiation. J Biol Chem. 2005; 280:19625–19634.

14. Winn RA, Van Scoyk M, Hammond M, Rodriguez K, Crossno JT Jr, Heasley LE, Nemenoff RA. Antitumorigenic effect of Wnt 7a and Fzd 9 in non-small cell lung cancer cells is mediated through ERK-5-dependent activation of peroxisome proliferator-activated receptor gamma. J Biol Chem. 2006; 281:26943–26950.

15. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet. 2002; 3:415–428.

16. Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996; 93:9821–9826.

17. Sato N, Fukushima N, Maitra A, Matsubayashi H, Yeo CJ, Cameron JL, Hruban RH, Goggins M. Discovery of novel targets for aberrant methylation in pancreatic carcinoma using high-throughput microarrays. Cancer Res. 2003; 63:3735–3742.

18. Lindberg D, Akerström G, Westin G. Mutational analyses of WNT7A and HDAC11 as candidate tumour suppressor genes in sporadic malignant pancreatic endocrine tumours. Clin Endocrinol (Oxf). 2007; 66:110–114.

19. Travis WD. Pathology of lung cancer. Clin Chest Med. 2002; 23:65–81. viii

20. Christofori G, Semb H. The role of the cell-adhesion molecule E-cadherin as a tumour-suppressor gene. Trends Biochem Sci. 1999; 24:73–76.

21. Bremnes RM, Veve R, Gabrielson E, Hirsch FR, Baron A, Bemis L, Gemmill RM, Drabkin HA, Franklin WA. High-throughput tissue microarray analysis used to evaluate biology and prognostic significance of the E-cadherin pathway in non-small-cell lung cancer. J Clin Oncol. 2002; 20:2417–2428.

22. Shibanuma H, Hirano T, Tsuji K, Wu Q, Shrestha B, Konaka C, Ebihara Y, Kato H. Influence of E-cadherin dysfunction upon local invasion and metastasis in non-small cell lung cancer. Lung Cancer. 1998; 22:85–95.

23. Sulzer MA, Leers MP, van Noord JA, Bollen EC, Theunissen PH. Reduced E-cadherin expression is associated with increased lymph node metastasis and unfavorable prognosis in non-small cell lung cancer. Am J Respir Crit Care Med. 1998; 157:1319–1323.

24. Retera JM, Leers MP, Sulzer MA, Theunissen PH. The expression of beta-catenin in non-small-cell lung cancer: a clinicopathological study. J Clin Pathol. 1998; 51:891–894.

25. Pantel K, Passlick B, Vogt J, Stosiek P, Angstwurm M, Seen-Hibler R, Häussinger K, Thetter O, Izbicki JR, Riethmüller G. Reduced expression of plakoglobin indicates an unfavorable prognosis in subsets of patients with non-small-cell lung cancer. J Clin Oncol. 1998; 16:1407–1413.

26. Smythe WR, Williams JP, Wheelock MJ, Johnson KR, Kaiser LR, Albelda SM. Cadherin and catenin expression in normal human bronchial epithelium and non-small cell lung cancer. Lung Cancer. 1999; 24:157–168.

27. Pirinen RT, Hirvikoski P, Johansson RT, Hollmén S, Kosma VM. Reduced expression of α-catenin, ß-catenin, and γ-catenin is associated with high cell proliferative activity and poor differentiation in non-small cell lung cancer. J Clin Pathol. 2001; 54:391–395.

28. Catzavelos C, Tsao MS, DeBoer G, Bhattacharya N, Shepherd FA, Slingerland JM. Reduced expression of the cell cycle inhibitor p27Kip1 in non-small cell lung carcinoma: a prognostic factor independent of Ras. Cancer Res. 1999; 59:684–688.

29. Sato G, Saijo Y, Uchiyama B, Kumano N, Sugawara S, Fujimura S, Sato M, Sagawa M, Ohkuda K, Koike K, et al. Prognostic value of nucleolar protein p120 in patients with resected lung adenocarcinoma. J Clin Oncol. 1999; 17:2721–2727.

30. Perl AK, Wilgenbus P, Dahl U, Semb H, Christofori G. A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature. 1998; 392:190–193.

31. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer. 2002; 2:442–454.

32. Cano A, Pérez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, Portillo F, Nieto MA. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol. 2000; 2:76–83.

33. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, Savagner P, Gitelman I, Richardson A, Weinberg RA. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell. 2004; 117:927–939.

34. Yanagawa Si, Lee JS, Haruna T, Oda H, Uemura T, Takeichi M, Ishimoto A. Accumulation of Armadillo induced by Wingless, Dishevelled, and dominant-negative Zeste-White 3 leads to elevated DE-cadherin in Drosophila clone 8 wing disc cells. J Biol Chem. 1997; 272:25243–25251.

35. Huber O, Korn R, McLaughlin J, Ohsugi M, Herrmann BG, Kemler R. Nuclear localization of beta-catenin by interaction with transcription factor LEF-1. Mech Dev. 1996; 59:3–10.

36. Hadeball B, Borchers A, Wedlich D. Xenopus cadherin-11 (Xcadherin-11) expression requires the Wg/Wnt signal. Mech Dev. 1998; 72:101–113.

37. Ohira T, Gemmill RM, Ferguson K, Kusy S, Roche J, Brambilla E, Zeng C, Baron A, Bemis L, Erickson P, et al. WNT7a induces E-cadherin in lung cancer cells. Proc Natl Acad Sci U S A. 2003; 100:10429–10434.