Novel Targets of Indoles in Prostate Cancer

描述(由申请人提供)： 大多数接受激素治疗的前列腺癌(PCa)患者发生激素难治性前列腺癌(HRPC)，并导致主要的骨转移疾病，该疾病仍含有Akt激活的功能性雄激素受体(AR)，目前尚无根治方法。在HRPC(也称为去势抵抗肿瘤)和骨转移疾病中，AR的功能紊乱，至少是由于Akt信号的激活。因此，重要的是要了解Akt是如何激活AR的，最重要的是开发一种无毒的策略，使PCA中的AR完全失活，从而完全破坏通过AR信号的生长反应。先前的研究表明，食用水果和蔬菜提供了吲哚化合物[吲哚-3-甲醇(I3C)及其体内产物3-3‘-二吲哚甲烷(DIM)]，据信这些化合物对人类前列腺癌的发展具有保护作用。我们在上一期的研究表明，从生物反应中获得的DIM和标准化的DIM，命名为BR-DIM(以下简称B-DIM)，下调Akt、NF-B、AR、2-catenin和PSA的表达，并诱导前列腺癌细胞系的细胞死亡，而不是正常的前列腺上皮细胞。此外，我们还发现DIM可以抑制肿瘤的侵袭和血管生成。根据我们的初步结果和对Akt/Wnt/AR信号转导的现有知识，我们假设2-catenin是Akt、Wnt和AR信号网络之间信号转导的关键分子，B-dim的使用将成为一种新的途径，通过调节2-catenin介导的Akt/Wnt/AR信号来抑制PCa的进展、侵袭和转移。我们进一步假设B-DIM将在动物模型中诱导抗肿瘤活性，并且B-DIM可能靶向前列腺癌患者的前列腺癌。为了验证我们的假设，我们设计了四个特定的靶点，雄激素敏感(LNCaP，突变AR+)和雄激素不敏感(LNCaP衍生的C4-2B和CWR22Rv1，突变AR+)细胞将用于回答以下问题：(A)Akt如何通过2-catenin途径调节Wnt信号；(B)2-catenin如何调节AR信号；(C)B-dim在调节细胞增殖和凋亡方面是否对Wnt信号有任何直接影响；(D)B-dim是否对2-catenin介导的信号转导和AR反式激活有任何直接影响，(E)B-dim在体外和体内是否通过调节2-catenin介导的信号转导途径抑制前列腺癌细胞生长和诱导细胞凋亡，以及(F)前列腺癌患者暴露于B-dim是否会在前列腺癌中显示B-dim积聚，这可能导致AR和核因子-B的下调，并导致2-catenin的核排斥。了解Akt激活AR的分子和细胞机制，以及B-DIM如何失活AR信号，将对设计无毒的预防肿瘤进展和/或治疗PCa患者的策略具有重要影响。因此，我们的建议是创新的，对一般公共卫生，特别是对前列腺癌患者来说，具有很高的相关性。 公共卫生相关性： 这一竞争性的更新应用集中于阐明Akt/WNT激活AR的机制，并在动物模型中评估化学预防性药物(B-DIM)如何使AR信号失活，从而抑制前列腺癌(PCA)细胞的生长、侵袭和血管生成，并产生抗肿瘤活性。此外，在我们的I期临床试验的基础上，我们还将进行II期临床研究，以评估B-DIM能否到达前列腺癌并在接受口服B-DIM的患者中产生生物学效应。我们的研究结果将有助于设计基于机制的临床试验，以评估B-DIM在预防和/或治疗前列腺癌中的作用。

DESCRIPTION (provided by applicant): The majority of prostate cancer (PCa) patients treated with hormone ablation therapy develop hormone refractory prostate cancer (HRPC), and leads to predominant bone metastatic disease that still contains Akt- activated functional androgen receptor (AR), and for which there is no curative therapy. In HRPC (also known as castrate resistant tumors) and bone metastatic disease, AR functions promiscuously, at least, due to the activation of Akt signaling. Therefore it is important to understand how Akt activates AR, and most importantly develop a non-toxic strategy by which AR in PCa could be totally inactivated; thereby growth response via AR signaling could be totally destroyed. Previous studies have shown that consumption of fruits and vegetables provides indole compounds [indole-3-carbinol (I3C) and its in vivo product 3-3'-diindolylmethane (DIM)] that are believed to be protective against the development of human PCa. Our studies during the preceding funding period showed that DIM and a well-standardized formulated DIM obtained from BioResponse, named BR-DIM (hereafter referred as B-DIM), down-regulated Akt, NF-:B, AR, 2-catenin and PSA, and induced apoptotic cell death of PCa cell lines but not normal prostate epithelial cells. Moreover, we have shown that DIM could inhibit invasion and angiogenesis. Based on our preliminary results and the existing knowledge on Akt/Wnt/AR signaling, we hypothesize that 2-catenin is a critical molecule for the signal transduction between Akt, Wnt, and AR signaling network and that the use of B-DIM will serve as a novel approach by which the progression, invasion, and metastasis of PCa could be inhibited through the regulation of 2-catenin mediated Akt/Wnt/AR signaling. We further hypothesize that B-DIM will elicit anti-tumor activity in animal model and that B-DIM could be targeted to human prostate gland in PCa patient. To test our hypotheses, we designed four specific aims where androgen sensitive (LNCaP, mutant AR+) and androgen insensitive (LNCaP derived C4- 2B and CWR22Rv1, mutant AR+) cells will be used to answer the following questions: (a) how Akt regulates Wnt signaling via 2-catenin pathway, (b) how 2-catenin regulates AR signaling, (c) whether B-DIM has any direct effect on Wnt signaling in regulating cell proliferation and apoptosis, (d) whether B-DIM has any direct effect on 2-catenin mediated signal transduction and AR trans-activation, (e) whether B-DIM mediated inhibition of PCa cell growth and induction of apoptosis in vitro and in vivo could in part be due to the regulation of 2-catenin mediated signal transduction, and finally (f) does exposure of PCa patients to B-DIM will show B-DIM accumulation in the prostate tumor, which may result in the down-regulation of AR and NF- :B, and nuclear exclusion of 2-catenin. Understanding of the molecular and cellular mechanism by which Akt activates AR and how B-DIM inactivates AR signaling, will have a significant impact on designing non-toxic strategies for the prevention of tumor progression and/or treatment of PCa patients. Thus our proposal is innovative and highly relevant to public health in general and for PCa patients in particular. PUBLIC HEALTH RELEVANCE: This competing renewal application is focused on elucidating the mechanism by which Akt/Wnt activates AR, and assessing how a chemo-preventive agent (B-DIM) could inactivate AR signaling resulting in the inhibition of prostate cancer (PCa) cell growth, invasion and angiogenesis, and causing anti-tumor activity in an animal model. Moreover, based on our phase I clinical trial, we will also conduct a phase II clinical study for assessing whether B-DIM could reach prostate gland and elicit biological effects in PCa patients receiving oral B-DIM. The results of our study will help to design mechanism-based clinical trials for assessing the role of B-DIM in the prevention and/or treatment of PCa.