Abstract
Breast cancer metastasis suppressor 1 (BRMS1) is a predominantly nuclear protein that suppresses metastasis in multiple human and murine carcinoma cell lines. BRMS1 interacts with several nuclear proteins including SIN3:HDAC chromatin remodeling complexes that are involved in repressing transcription. However, recent reports suggest BRMS1 may function in the cytoplasm. BRMS1 has two predicted nuclear localization sequences (NLS) that are located near the C-terminus (amino acids 198–205 and 238–244, NLS1 and NLS2 respectively). We hypothesized that nuclear localization sequences of BRMS1 were essential for BRMS1 mediated metastasis suppression. Replacement of NLS2 with NLS1 (BRMS1NLS1,1), truncation at 238 (BRMS1ΔNLS2), or switching the location of NLS1 and NLS2 (BRMS1NLS2,1) did not affect nuclear localization; but, replacement of NLS1 with NLS2 (BRMS1NLS2,2) or truncation at 197 (BRMS1ΔNLS which removes both NLS) promoted cytoplasmic localization. MDA-MB-231 human metastatic breast cancer cells transduced with BRMS1NLS1,1, BRMS1NLS2,2 or BRMS1NLS2,1 were evaluated for metastasis suppression in an experimental xenograft mouse model. Interestingly, while NLS2 was not necessary for nuclear localization, it was found to be important for metastasis suppression since BRMS1NLS2,2 suppressed metastasis by 85%. In contrast, BRMS1NLS2,1 and BRMS1NLS1,1 did not significantly suppress metastasis. Both BRMS1 and BRMS1NLS2,2 co-immunoprecipitated with SIN3A in the nucleus and cytoplasm; however, BRMS1NLS1,1 and BRMS1NLS2,1 were associated with SIN3A in the nucleus only. Moreover, BRMS1 and BRMS1NLS2,2, but not BRMS1NLS1,1 and BRMS1NLS2,1, down-regulated the pro-metastatic microRNA, miR-10b. Together, these data demonstrate an important role for NLS2 in the cytoplasm that is critical for metastasis suppression and is distinct from nuclear localization.
Highlights
Molecules regulating gene transcription either directly or indirectly have the potential to dramatically impact the metastatic process
We identified potential cytoplasmic functions of Breast cancer metastasis suppressor 1 (BRMS1) through interaction with SIN3A that correlates with the ability of BRMS1 to suppress metastasis
We hypothesized that NLS1 would be required for BRMS1 function as a metastasis suppressor and generated several mutations with alterations of NLS1 and NLS2 (Fig. 1A)
Summary
Molecules regulating gene transcription either directly or indirectly have the potential to dramatically impact the metastatic process. It has been suggested that BRMS1 robustly blocks the overall process of metastasis through small, albeit significant, inhibition of each step in the metastatic cascade [10] This has complicated the studies regarding molecular mechanisms, BRMS1 has been demonstrated to alter specific cellular pathways associated with metastasis including gap junctional intercellular communication [21,22,23], phosphoinositide signaling [24,25], nuclear factor kappa B signaling [26,27,28,29], cell motility and invasion [30,31,32], apoptosis [28,33], and tumor cell dissemination [33]. This study adds to our understanding of the BRMS1 metastasis suppressor protein that will expand our knowledge of metastatic disease
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