Characterization of endogenous BRMS1, and splice variants, in the MCF10 breast cancer progression model

484 Breast cancer metastasis suppressor 1 ( BRMS1 ) suppresses metastasis without blocking tumorigenesis in multiple human and murine cancer cells of diverse origins. The molecular mechanisms of BRMS1 are not well understood and thus far, mechanistic studies of BRMS1 have been limited to systems in which BRMS1 was re-expressed in metastatic cell lines that normally have no detectable levels of BRMS1 protein. The purpose of this study was to determine the functional role of endogenous BRMS1 in the progression of breast cancer using the isogenic MCF10 cell lines. These genetically related cell lines represent normal breast epithelial cells (MCF10A), pre-malignant breast disease (MCF10AT and MCF10DCIS.com), and metastatic carcinoma (MCF10CA a.1 and MCF10CA d.1α). Real-time RT-PCR showed higher levels of BRMS1 mRNA in the metastatic cell lines, although BRMS1 protein expression and nuclear localization were not significantly different. Alternatively spliced variants were detected by RT-PCR and found to be differentially expressed; specifically, BRMS1.v2 and BRMS1.v4 were only detected in the pre-malignant MCF10A and AT cell lines, while BRMS1 and BRMS1.v3 were detected in all lines. However, translated protein for these splice variants was not detected. Since BRMS1 protein, but not splice variants, was expressed in both the metastatic and non-metastatic cell lines, BRMS1 exons were sequenced and scanned for possible mutations. All sequences were found to be wild-type with the exception of a synonymous T/C transition in exon 7. Together, these results demonstrate that BRMS1 expression alone is not sufficient to prevent metastasis and further suggests that the expression and/or activation of BRMS1 protein interactors are necessary for metastasis suppression. Support: CA87728, F32CA113037, and National Foundation for Cancer Research