BRMS1 transcription is repressed by DNA methylation and functions as both a metastasis and a tumor suppressor gene in non-small cell lung cancer

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Breast cancer metastasis suppressor 1(BRMS1) was originallyidentified as a metastasis suppressor gene in breast cancerand melanoma cell lines. Our prior work and other studies indicated that BRMS1 expression is decreased in cancer including non-small cell lung cancer (NSCLC). However, the molecular mechanisms through which BRMS1 expression is decreased remain unclear. Furthermore, it is unknown whether BRMS1 functions as metastasis suppressor in NSCLC. Utilizing tissue microarrays and immunohistochemistry we examine BRMS1 expression in 80 human NSCLC tumors and matched samples of adjacent non-cancerous lung. BRMS1 proteinlevels were dramatically decreased in 85% of NSCLCtissues compared with matched adjacent tissues. Realizing that hypermethylation of CpG islands in the promoter region repress transcription and are present in many cancers, we analyzed the BRMS1 genomic DNA sequence and identified a large CpG island in the proximal promoter. We then hypothesized that one mechanism through which BRMS1 levels are reduced in NSCLC is through DNA hypermethylation. To experimentally address this, quantitative Methylation-Specific PCR (MSP) for the BRMS1 gene was performed in 9 human NSCLC and matched adjacent non-cancerous tissues. We found a 2 to 7-fold increase in BRMS1 methylation in the NSCLC specimens compared to controls. A similar profile was also found in our NSCLC cell lines (N=5). Moreover, all NSCLC cell lines demonstrated increased basal methylation of the BRMS1 promoter that was reversed following treatment with DNA methylationinhibitor 5-aza-2’-deoxycytidine (5-aza-CdR). Furthermore, the addition of 5-aza-CdR resulted in a robust rescue of endogenous BRMS1 mRNA and protein levels in all detected NSCLC cells. These studies provided strong evidence that decreased BRMS1 levels in human NSCLC and cell lines are secondary to DNA methylation. We next examined whether BRMS1 functions a metastasis suppressor in several NSCLC cell culture metastasis models including matrigel migration and invasion assays and hanging-drop culture following three-dimensional collagen I gel assays. In all the assays stable overexpression of BRMS1 (H1299B) resulted in significant decrements in metastatic movement and invasion compared to controls (H1299V). To determine whether similar results occurred in vivo, we used our NSCLC xenograft metastasis model, and found that H1299B xenografts had markedly decreased lung metastasis and also resulted in 3-fold inhibition of the primary tumor growth compared to H1299V xenografts. In conclusion, these findings support DNA hypermethylation as a mechanism for reduced levels of BRMS1 protein and mRNA in NSCLC. Moreover, in addition to functioning as a metastasis suppressor, BRMS1 appears to function as a tumor suppressor by repressing primary NSCLC tumor growth.