Study On Biological Functions Of Breast Cancer Metastasis Suppressor 1 (BRMSI)

Objective To investigate the anti-cancer activities of breast cancer metastasis suppressor1 (BRMS1) in two invasive breast cancer cell lines. Method1.Based on the BRMS1 gene sequence from the Genebank, a full-length BRMS1 cDNA was amplified by a pair of specifically designed primers and constructed into a pcDNA3 eukaryotic expression vector.2. Cell lines with high expression of BRMS1 were established by stable transfection of pcDNA3-BRMS1 expression under the help of cationic liposome (Lipofactamin2000) and selection in medium containing G418.3.The effect of BRMS1 overexpression on cell growth and proliferation were observed using MTT method.4.Cell migration and invasion were determined by in vitro scratch and modified Boyden chamber assays.5.Alteration of cell cycle progression and apoptosis induction were studied by flow cytometry.6.BRMS1 role on the tumor growth and metastasis in vivo were studied in nude mice with orthotopic injection of breast cancer cells.7.The effects of BRMS1 on lung metastasis was observed via tail intravenous injection of breast cancer cells.8.Radiosensitivity was analyzed by colony forming assays.9.Analysis of chromosomal aberrations and PCC fragments were performed to determine the effect of BRMS1 overexpression on genomic instability.10.Western blot assay was used to measure protein expression.11.In vitro GST pull-down and in vivo immunoprecipitation-WB assays were employed to determine protein-protein interaction.12.Luciferase assay was used to determine transcriptional activityResults1.Construction of the BRMS1 eukaryotic expression vector was confirmed by sequencing and digestion with restriction endonucleases BamH I and Xho I.2.Characterization of G418 resistance clone by RT-PCR and Western blot indicates that BRMS1 over-expressing cell lines have been established.3.Ectopic expression of BRMS1 did not produce any effects on cell growth in both MDA-MB-231 and MDA-MB-435.4.Ectopic expression of BRMS1 did not alter migration ability of MDA-MB-231 and MDA-MB-435 cells, but significantly reduced cell invasion.6.BRMS1 overexpression did not cause any effects on cell cycle progression in both MDA-MB-231 and MDA-MB-435.7.Enhanced expression of BRMS1 did not impact the body weight and tumor growth rate and tumor size. However, tumor with BRMS1 overexpression had well differentiation and low malignant degree compared to control tumors.8.BRMS1 significantly reduced lung and liver metastases and angiogenesis (formation of new blood vessels).10.BRMS1 overexpression did not influence sensitivity of breast cancer to radiotherapy, accompanying with no alteration of DNA damage repair proteins.11.Less chromosomal aberrations and PCC fragments were found in the cells with BRMS1 overexpression.12.BRMS1 associated with nuclear hormone receptors, including AR, ER(ER-αand ER-β), PR(PRA and PR-B), GR, RXRαand VDR and inhibited AR and ER-αtranscriptional activations.ConclusionIn in vitro cell models, enhanced expression of BRMS1 by stable transfection of a full-length of BRMS1 cDNA significantly reduces or inhibits potentials of cell invasion, without alteration of cell growth and migration in two highly invasive breast cancer cell lines MDA-MB-231 and MDA-MB-435. Also, no alteration of cell cycle progression and apoptosis induction were observed in the cells with BRMS1 overexpression. In in vivo animal models, tumor formation and growth in nude mouse by orthotopic injection of breast cancer cells with BRMS1 overexpression exhibited no difference from those in the control cells. However, a significant decrease in malignant degree, remote metastasis and angiogenesis was observed in BRMS1 overexpression tumors. Increased expression of BRMS1 did not affect radiosensitivity, but indeed decreased the genomic instability caused by stresses. In summary, BRMS1 plays an important role in breast cancer development and progression as a metastatic and angiogenesis suppressor, and is a potential target to improve therapy of breast cancer. Furthermore, the BRMS1 association with different nuclear hormone receptors as a co-repressor will provide a new avenue to explore other biological functions of BRMS1, besides metastasis and angiogenesis suppression.