| Breast cancer is the most common malignancy in women, the risk of which is higher year by year in our country these twenty years, threatening the feminine seriously. The occurrence of breast cancer is a multi-stages and multi-factors process. Many evidences show that estrogen exposure is con- sidered a significant risk factor for breast cancer development. Estrogen action is mediated by estrogen receptors (ERs), members of the nuclear receptor family and ligand-activated transcription factors that control the expression of estrogen- responsive genes. ERαcontains three members: ERα-66, ERα-46 and ERα-36. Estrogen- stimulated cell proliferation by 17β-estradiol (E2) is believed to be largely mediated by activation of ERα-66 localized in the nucleus. Furthermore, ERα-66 and ERα-46 proteins are found to stimulate the synthesis of NO. ERα-66 is expressed at low levels in the normal epithelia, and its expression is dramatically up-regulated as transformation progresses during mammary adeno- carcinoma development. The mechanism(s) driving ERα-66 up-regulation during mammary transformation remains unclear. ERα-36 is a novel variant of ERα, which is also a memberane-receptor. It is found that estrogens and antiestrogens can both stimulate cell proliferation through membrane-associated ERα-36. It will be interesting to study the mechanisms by which ERα-36 mediates membrane-initiated estrogen and antiestrogen signaling.Caveolin-1 (Cav-1) is the structural protein of plasmalemmal invaginations, termed caveolae, which functions as a tumor suppressor gene. Interestingly, Cav-1 dominant-negative mutations are exclusively found in ERα-positive breast cancer samples. In support of these clinical findings, ERαexpression is increased in Cav-1 (-/-) null mammary epithelia. Cav-1 is tightly associated with the expression of ERα-66. There is also emerging evidence that ERαis resided in plasma membrane, colocalization with the caveolae. In MCF-7 cells, over-expression of Cav-1 stimulates ER translocation from the cytoplast to the plasma membrane. However, earlier studies also reported that E2 binds to a cell surface receptor and stimulates a rapid generation of cAMP. Subsequently, other reports of a plasma membrane-localized ER that transduces membrane-initiated estrogen signaling appeared. This membrane-initiated pathway was found to activate different cytoplasmic signaling proteins and later pathways initiated through membrane estrogen signaling included the adenylate cyclase, the phospholipase C, G protein coupled receptor-activated, the mitogen-activated protein kinase (MAPK) pathways, and phosphatidylinositol 3-kinase (PI3K) /AKT pathway. As a suppressor, the mechanisms of Cav-1 down-regulation in the early transformation and signaling transduction of mammary epithelial cells associated with ERαare still being elucidated.Through gene trapping, we have obtained two cell lines, named MCF 10A-ST1 and -ST3, which express 30% and 50% of Cav-1 compared with parental cells MCF 10A ( human mammary epithelial cell line). The interaction between Cav-1 and ERαis confirmed by immunoprecipitation. RNAi technology is performed to silence the expression of Cav-1 in mammary epithelia cells, then the expression of ERαand the proteins of ER-induced signaling pathway are examined by Western Blot. We discuss the relationship and the possible mechanism of Cav-1 and ERα-activated signalings, in order to clarify the role of Cav-1 in the tumor early transformation and oncogenesis, and provide the further evidence of pathogenesis.Our results showed that: (1) Through siRNA technology, we establish a cell model of Cav-1-silence successfully; (2) Cav-1 is interacted with ERαboth in MCF 10A and ST1 cell lines; Cav-1 down-regulation could effect on the binding level between them; (3) With the decrease of Cav-1, the expression of ERα-66 is markedly increased by transient transfection of MCF 10A and ST1, but the ERα-36 expression is changed variously; (4) The expression of Cav-1 is less and less by transient transfection of ST1; (5) With the down-regulation of Cav-1, the pERK1/2 is up-regulation at 48h and 72h by siRNA transfection, and comes back to be normal level at 96h, but the ERK1/2 expression has no obviously change, the same result is to be found in 7SD8 cell line; (6) The expression of pAKT is increased at 72h, and the AKT expression is also increased at 48h by siRNA transfection, the upstream protein—PI3K expression was up-regulation at 48h and 72h, the downstream protein P21is down-regulation at 48h and 96h; (7) With the Cav-1 transient down-regulation, the expression of Cyclin D1 has no obviously change.Conclusion:1. Cav-1 down-regulation could not only increase the binding level with ERα, but also induce the up-regulation of ERα, with the interaction of Cav-1, which lead the early transformation of mammary epithelium.2. Cav-1 down-regulation could induce the activation of ERα-associated signaling pathway, such as the activation of MAP kinase and Akt signaling pathway, including the phosphorylation of ERK1/2 and AKT, in order to adjust the development and proliferation. |
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