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症的限制。参与D3介导的前列腺癌细胞增殖抑制的调节分子和途径可以靶向抑制肿瘤生长，为治疗开发和开发针对前列腺癌的新防线提供了机会。由于转移性前列腺癌通常与E2 F1水平升高相关，E2 F1是一种关键的细胞周期调节转录因子，因此我们设计了该项目以鉴定通过与雄激素受体（AR），维生素D3受体（VDR）和E2 F1的串扰与D3的抗增殖作用整体相关的调节因子。 在新的研究结果中，我们表明，雄激素调节的E2 F1诱导在人类前列腺癌细胞中的干扰D3由于转录抑制。辅阻遏物Alien和抗增殖蛋白Prohibitin似乎在阻遏中起作用。在E2 F1的诱导和去诱导过程中，AR和VDR都存在于受调节的启动子处。转录因子WSTF和E47占据调节染色质响应雄激素。Prohibitin在D3和雄激素+D3处理的细胞中占据了该区域，但外来占据只发生在雄激素+D3共处理的细胞中。临床上重要的抗雄激素Casodex诱导的外来招募。我们假设a）WSTF和E47是AR相关的辅激活因子复合物的一部分，而D3结合的VDR促进辅调节因子转换为包含抑制素和外源蛋白的辅阻遏物组装，其介导E2 F1基因阻遏并降低细胞增殖; B）E2 F1阻遏通过E2 F1启动子中的自调节位点处的自抑制来辅助，所述自抑制由增强的E2 F1与外源基因的相互作用产生，prohibitin或众所周知的E2 F1伴侣Rb（视网膜母细胞瘤蛋白），并与外来/prohibitin/Rb募集的辅阻遏物。 本研究的目的有四个：1）外源蛋白、抑制素、Rb及相关辅阻遏物在雄激素、D3或雄激素+D3处理的前列腺癌细胞E2 F1活性中的作用; 2）外源蛋白、抑制素与WSTF和E47在调节E2 F1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中的作用以及在免疫缺陷小鼠中异种移植前列腺肿瘤的生长中的作用; 4），在亲和富集后，全息辅阻遏物复合物的另外组分在E2 F1基因沉默和抑制前列腺癌细胞增殖中的作用。研究方法：我们将使用Co-IP、染色质IP、转染、ShRNA、诱导蛋白表达、慢病毒感染、细胞增殖、SCID小鼠中的异种移植肿瘤生长、蛋白富集和质谱。重要性：对雄激素介导的增殖刺激和D3介导的抗增殖信号传导的响应因素的了解将定义可以靶向开发新的前列腺癌治疗剂的机制。该项目与退伍军人的保健问题密切相关。 公共卫生相关性： 项目叙述：前列腺癌是老年男性的主要健康问题，并且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。这种疾病的风险从生命的第五个十年开始上升。退伍军人事务部的人口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岁或以上。 维生素D缺乏是老年人的常见问题。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（称为维生素D3）在减少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，高维生素D水平可以保护人体免受许多癌症的侵害，如结肠癌，乳腺癌和前列腺癌。然而，高剂量的临床有效的D3和合成的化合物，像D3（称为D3类似物）是高毒性的。因此，它们不能用于前列腺癌治疗。 一种被称为E2 F1的蛋白质在促进正常细胞的生长和增殖中起着关键作用。然而，E2 F1的高活性和高水平的这种蛋白质促进前列腺和其他组织中细胞的过度和不受控制的增殖，导致癌症。在大量患者中发现治疗抗性前列腺癌组织含有比正常前列腺组织高得多的E2 F1水平。 我们设计了一项研究来研究为什么E2 F1活性和E2 F1蛋白水平在前列腺癌细胞中增加，以及D3如何抑制这种增加。我们将使用尖端的研究技术进行各种分析，以找出特定的调节蛋白分子如何受到D3的影响，从而使它们沉默或减少E2 F1的活性和产生。我们的研究有望找到降低E2 F1活性和蛋白水平的替代方法，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管理开辟新的策略。

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.