Vitamin D3, Interference with Androgen Signal and Inhibition of Prostate Cancer

描述(由申请人提供)： 1，25-(OH)2维生素D3(D3)在前列腺癌细胞中的抗增殖活性突出了D3/D3类似物的治疗潜力。然而，这种潜力受到D3/D3类似物诱导的高钙血症和高钙尿素的限制。参与D3介导的前列腺癌细胞增殖抑制的调节分子和调控通路可以靶向抑制肿瘤生长，为前列腺癌的治疗开发和新防线的开发提供了机会。由于转移性前列腺癌通常与E2F1水平升高有关，E2F1是一种关键的细胞周期调控转录因子，我们设计这个项目来识别通过与雄激素受体(AR)、维生素D3受体(VDR)和E2F1的相互作用而与D3的抗增殖作用整合在一起的调节因子。在新的发现中，我们发现雄激素调节的人前列腺癌细胞E2F1的诱导由于转录抑制而受到D3的干扰。辅抑制子Alien和抗增殖蛋白Prohibitin似乎在抑制中发挥了作用。在E2F1的诱导和去诱导过程中，受调控的启动子上都存在AR和VDR。转录因子WSTF和E47占据了雄激素调节的染色质。Prohibitin在D3和雄激素+D3处理的细胞中占据这一区域；而外源占据仅在雄激素+D3联合处理的细胞中发生。临床上重要的抗雄激素药Casodex诱导外星人招募。我们假设a)WSTF和E47是AR相关的辅活化子复合体的一部分，而D3结合的VDR促进共调节因子切换到禁止素和外星人包括的辅阻遏子组件，从而介导E2F1基因抑制和细胞增殖减少；b)E2F1抑制是通过E2F1启动子上的自我调节位点的自我抑制而实现的，这是由于增强了E2F1与Alien、Prohibitin或众所周知的E2F1合作伙伴Rb(视网膜母细胞瘤蛋白)以及与Alien/Prohibitin/RB招募的共抑制子的相互作用。将有四个特异性目标研究：1)Alien、Prohibitin、Rb及相关辅抑制子在雄激素、D3或雄激素+D3处理的前列腺癌E2F1活性中的作用；2)Alien和Prohibitin与WSTF和E47在调节E2F1基因表达中的相互作用；3)内源性WSTF/E47/Alien/Prohibitin在雄激素刺激和D3抑制的前列腺癌细胞增殖和免疫缺陷小鼠异种前列腺癌移植瘤生长中的作用；4)亲和力增强后的完整共抑制物复合体的附加成分，在E2F1基因沉默和抑制前列腺癌细胞增殖中的作用。方法：应用Co-IP、染色质IP、转染法、shRNAs、诱导性蛋白表达、慢病毒感染、细胞增殖、SCID小鼠移植瘤生长、蛋白质浓缩和质谱学等方法。意义：了解雄激素介导的增殖刺激和D3介导的抗增殖信号的反应因素将确定可能针对开发新的前列腺癌治疗方法的机制。该项目与退伍军人群体的医疗保健问题具有很高的相关性。

DESCRIPTION (provided by applicant): Anti-proliferation activity of 1,25-(OH)2vitamin D3 (D3) in prostate cancer cells highlights the therapeutic potential of D3/D3 analogs. This potential is, however, limited by D3/D3 analog-induced hypercalcemia and hypercalciurea. Regulatory molecules and pathways, which are involved in D3-mediated proliferation inhibition of prostate cancer cells, can be targeted to inhibit tumor growth, presenting opportunities for therapeutic exploitation and development of new lines of defense against prostate cancer. Since metastatic prostate cancer is often associated with elevated levels of E2F1, which is a key cell cycle regulatory transcription factor, we designed this project to identify regulatory factors which, through cross-talk with androgen receptor (AR), vitamin D3 receptor (VDR) and E2F1, are integrally linked to the anti-proliferation action of D3. In novel findings we show that androgen-regulated E2F1 induction in human prostate cancer cells is interfered by D3 due to transcriptional repression. The corepressor Alien and anti-proliferative protein prohibitin appear to play roles in the repression. AR and VDR were present at the regulated promoter during both induction and de-induction of E2F1. Transcription factors WSTF and E47 occupied regulated chromatin in response to androgen. Prohibitin occupied this region in D3- and androgen+D3-treated cells; but Alien occupancy occurred only in androgen+D3 co-treated cells. Clinically important anti-androgen Casodex induced Alien recruitment. We hypothesize a) WSTF and E47 are part of an AR-associated coactivator complex, while D3-bound VDR promotes co-regulator switch to a prohibitin- and Alien-included corepressor assembly, which mediates E2F1 gene repression and reduced cell proliferation; b) E2F1 repression is aided by self inhibition at an auto-regulated site in the E2F1 promoter that results from enhanced E2F1 interaction with Alien, prohibitin or well-known E2F1 partner Rb (retinoblastoma protein), and with Alien/prohibitin/Rb-recruited corepressors. Four Specific Aims will examine: 1), roles of Alien, prohibitin, Rb, and associated corepressors in E2F1 activity in prostate cancer cells treated with androgen or D3 or androgen+D3; 2), interplay of Alien and prohibitin with WSTF and E47 in regulating E2F1 gene expression; 3), roles of endogenous WSTF/E47/Alien/prohibitin in androgen-stimulated and D3-inhibited prostate cancer cell proliferation and in the growth of xenograft prostate tumors in immune-deficient mice; 4), additional components of the holo corepressor complex, following affinity enrichment, for roles in E2F1 gene silencing and in the inhibition of prostate cancer cell proliferation. Methods: We will use Co-IP, chromatin IP, transfection, ShRNAs, inducible protein expression, lentivirus infection, cell proliferation, xenograft tumor growth in SCID mice, protein enrichment and mass spectrometry. Significance: Knowledge of the factors responding to the androgen-mediated proliferation stimulation, and to the D3-mediated anti-proliferation signaling will define mechanisms that may be targeted to develop novel prostate cancer therapeutics. The project has high relevance to health care issues of the Veterans population.