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。 公共卫生相关性： 项目简介：前列腺癌是老年男性的主要健康问题，转移性前列腺癌是美国男性癌症死亡的主要原因，仅次于肺癌死亡。患这种疾病的风险从生命的第五个十年开始上升。退伍军人管理局的人口结构正在稳步向老年人口转移，预计到2020年，约46%的退伍军人人口将达到65岁或更高年龄。维生素D缺乏是老年人普遍存在的问题。基于人群的研究表明，男性血清维生素D水平低与前列腺癌风险增加相关。在实验室中，涉及细胞和动物的研究表明，维生素D的活性形式(即维生素D3)在抑制前列腺肿瘤细胞的生长和增殖方面非常有效。事实上，近年来的研究表明，高水平的维生素D可以保护人体免受多种癌症的侵袭，例如结肠癌、乳腺癌和前列腺癌。然而，大剂量临床有效的D3和类似D3的合成化合物(称为D3类似物)毒性很高。因此，它们不能用于前列腺癌的治疗。一种名为E2F1的蛋白质在促进正常细胞的生长和增殖方面发挥着关键作用。然而，E2F1的高活性和这种蛋白的高水平促进了前列腺和其他组织中细胞的过度和不受控制的增殖，从而导致癌症。大量耐药前列腺癌组织中的E2F1含量远高于正常前列腺癌组织。我们设计了一项研究，以调查为什么前列腺癌细胞中E2F1活性和E2F1蛋白水平增加，以及D3如何抑制这种增加。我们将利用尖端研究技术进行各种分析，以找出D3如何影响特定的调节蛋白质分子，使其沉默或减少E2F1的活性和产生。我们的研究有望找到降低E2F1活性和蛋白质水平的替代方法，以避免使用高剂量D3。从我们的研究中收集的信息可能会为前列腺癌的预防和临床治疗开辟新的策略。

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. PUBLIC HEALTH RELEVANCE: Project Narrative: Prostate cancer is a major health concern among elderly men, and metastatic prostate cancer is a leading cause of cancer death in men in the USA, second only to death from lung cancer. Risk for the disease rises from the 5th decade of life. The VA demography is steadily shifting to a late-life population, with a projection that by the year 2020 about 46% of the veteran population will reach age 65 or more. Vitamin D deficiency is a common problem among the elderly. Population based research has shown that low serum vitamin D levels in men correlate with increased prostate cancer risk. In the laboratory, research involving cells and animals has shown that the active form of vitamin D (known as vitamin D3) is very effective in reducing growth and proliferation of prostate tumor cells. Indeed, research in recent years revealed that high vitamin D levels protect human body from many cancers, such as cancer of the colon, breast and prostate. However, high-dose clinically effective D3 and synthetic compounds that act like D3 (known as D3 analogs) are highly toxic. Thus they cannot be used in prostate cancer treatment. A protein known as E2F1 plays a key role in promoting the growth and multiplication of normal cells. However, high activity of E2F1 and high levels of this protein promote excessive and uncontrolled multiplication of cells in the prostate and other tissues, causing cancer. Treatment-resistant prostate cancer tissues in a large number of patients are found to contain much higher levels of E2F1 than normal prostate tissues. We have designed a study to investigate why E2F1 activity and E2F1 protein levels increase in prostate cancer cells, and how D3 can inhibit this increase. We will undertake various analyses using cutting edge research technology to find out how specific regulatory protein molecules are influenced by D3 in such a way that they silence or reduce the activity and production of E2F1. Our study is expected to find alternative ways to reduce E2F1 activity and protein levels to circumvent use of high-dose D3. Information gleaned from our research may open up new strategies for prevention and clinical management of prostate cancer.