Estrogen receptor-alpha (ERα) is a clinically important therapeutic target for breast cancer. However, tumors that lose ERα are less responsive to anti-estrogens such as tamoxifen. MicroRNAs (miRNAs) are small RNAs that regulate expression of their target gene and dysregulations of miRNA has been identified in many diseases including human cancer. However, only a few miRNAs associated with tamoxifen resistance has been reported. In this study, we found that lymphocyte antigen 6 complex (LY6K), which is a member of the Ly-6/μPAR superfamily and related to breast cancer progression and metastasis, is inversely correlated with ERα expression. We, for the first time, found miRNAs involved in the regulatory molecular mechanism between ERα and LY6K and related to tamoxifen susceptibility in breast cancer. miR-192-5p, induced by LY6K, downregulates ERα directly and induced tamoxifen resistance in ERα-positive breast cancer cells. In addition, re-expression of ERα in ERα-negative breast cancer cells increased miR-500a-3p expression and directly inhibits LY6K expression. Ectopic expression of miR-500a-3p sensitized ERα-negative cells to tamoxifen by increasing apoptosis. Finally, we observed an inverse correlation between LY6K and ERα in primary breast cancer samples. We found that patients with recurrence showed high expression of miR-192-5p after tamoxifen treatments. In addition, expression of miR-500a-3p was significantly correlated to survival outcome. As miRNAs involved in the regulatory mechanism between LY6K and ERα can affect tamoxifen resistance, downregulating miR-192-5p or re-expressing miR-500a-3p could be a potential therapeutic approach for treating tamoxifen resistant patients.

The role of lymphocyte antigen 6 complex, locus K (LY6K) in breast cancer has been studied, whereas the epigenetic control of LY6K transcription is not fully understood. Here, we report that breast cancer patients with increased LY6K expression had shorter disease-free and overall survival than the patients with low levels of LY6K by multivariate analysis. LY6K also was upregulated in breast cancer patients with distant metastases than those without distant metastases, downregulating E-cadherin expression. Furthermore, xenograft tumor volumes from LY6K knockdown nude mice were reduced than those of mice treated with control lentivirus. Interestingly, LY6K has a CpG island (CGI) around the transcription start site and non-CGI in its promoter, called a CGI shore. LY6K expression was inversely correlated with methylation in not only CGI but CGI shore, which are associated with histone modifications. Additionally, LY6K methylation was increased by the PAX3 transcription factor due to the SNP242 mutation in LY6K CGI shore. Taken together, breast cancer risk and metastasis were significantly associated with not only LY6K expression, but also methylation of CGI shore which induced by SNP242 mutation. Our results suggest that an understanding epigenetic mechanism of the LY6K gene may be useful to diagnose carcinogenic risk and predict outcomes of patients with metastatic breast cancer.

Lymphocyte antigen 6 complex locus K (LY6K) is associated with development of the immune response and carcinogenesis. Elevated LY6K by AP-1 activation induces cell invasion and metastasis through activating the Raf-1/MEK/ERK signaling pathway. However, the exact epigenetic mechanism of LY6K gene expression has yet to be clarified. To elucidate the epigenetic mechanism of LY6K gene regulation and expression, CpG island and 5′CGI shore in the region around transcription start site of the LY6K were predicted using UCSC genome browser and Methprimer software. We performed MSP, bisulfite pyro-sequnecing, and bisulfite sequencing to investigate the DNA methylation status of CpG sites from -400 to +500 on the LY6K CGI and 5′CGI shore. Breast cancer cells with low LY6K expression were highly methylated in the both CGI and 5′CGI shore region, whereas high LY6K expression cell lines had low methylation levels. Moreover, 5-Aza-dC, demethylaing agent, treatment of cells with low LY6K expression caused elevation of LY6K expression and finally leads wound healing. To further validate the inverse correlation between LY6K 5′CGI shore methylation and LY6K expression in vivo seen in breast cancer cell lines, we performed bisulfite pyro-sequencing and tissue microarray using breast carcinoma samples. LY6K expression inversely correlates with methylation status and histone modification of 5′CGI shore in the LY6K promoter in human breast cancers. An understanding of epigenetic changes in LY6K may contribute to the diagnosis of carcinogenic risk and to prediction of outcome in patients with breast cancer. Citation Format: Hyun Kyung Kong, Sae Jeong Park, Ye Sol Kim, Jong Hoon Park. Epigenetic modification of LY6K in CGI shore and CGI regulates LY6K gene activation and metastatic function in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1365. doi:10.1158/1538-7445.AM2014-1365