Non-AR mediated DHT-promoted Bladder Cancer initiation and progression

描述(申请人提供)：膀胱癌(BCA)是美国男性/女性中第四/十分之一最常见的癌症。据报道，男性患膀胱癌的风险明显高于女性(约为女性的三倍)。然而，这种发病率性别差异的病因尚不清楚。最近的研究表明雄激素/雄激素受体(AR)信号参与了BCA的进展。利用AR基因敲除(ARKO)小鼠，我们发现雄激素/AR信号可能在化学致癌物N-丁基-N-4-羟丁基亚硝胺(BBN)诱发膀胱癌的发生发展中起关键作用。在BBN治疗下，缺乏AR和检测不到雄激素的ARKO小鼠没有发生膀胱肿瘤，而它们的野生型后代，具有功能AR的小鼠，在50周龄时都出现了膀胱肿瘤。有趣且出乎意料的是，我们发现在补充雄激素双氢睾酮(DHT)后，近25%的ARKO小鼠发生了BBN诱导的膀胱肿瘤，这表明DHT能够通过非AR途径促进膀胱肿瘤的进展。这一发现在AR阴性的人膀胱癌细胞系TCC5637中得到了进一步证实，表明加入DHT可以促进这种AR阴性的膀胱细胞的生长和侵袭。初步数据还表明，DHT可以增强BCA AR阴性细胞从G蛋白信号到ERK/MAPK-MEK信号的通路。基于这些体外人类细胞系和体内小鼠的证据，我们假设DHT可以通过非AR途径促进BCA的启动、生长和侵袭。我们将应用以下4个目标来验证我们的假设并剖析潜在的机制。目的1：利用Uro-SV40T-Arko小鼠模型证实DHT可通过非AR途径促进BCA的进展。目的：用UPII-Ha-ras-Arko小鼠模型证实DHT可通过非AR途径促进BCA的进展。目的：通过细胞转化/成瘤实验证实DHT可通过非AR途径促进BCA的启动和/或进展。目的：利用AR阴性的人膀胱癌细胞株，证实DHT可通过非AR途径促进BCA的生长和侵袭，并探讨其可能的作用机制。影响：目前，BCA在人类中一般不被认为是激素依赖的，因此不认为雄激素剥夺疗法(ADT)结合化学或手术去势可以作为BCA患者的有效治疗选择，以抑制肿瘤进展。本研究的成功证明DHT可以通过非AR途径促进膀胱癌的发生和发展，这不仅为新的雄激素机制提供了体内证据，也可能解决了以往用ADT抑制雄激素与AR结合治疗BCA失败的难题，并可能为抑制BCA提供一种新的预防/治疗方法。 公共卫生相关性：膀胱癌是美国男性/女性中第四/第十常见的癌症。据报道，男性患膀胱癌的风险明显高于女性(约为女性的三倍)。然而，这种发病率性别差异背后的原因尚不清楚。最近的研究表明雄激素/雄激素受体(AR)信号参与了膀胱癌的进展。利用AR基因敲除(ARKO)小鼠，我们发现雄性激素雄激素/AR信号可能在化学致癌物BBN诱导的膀胱癌的发生发展中起关键作用。我们还发现，在BBN和雄激素双氢睾酮(DHT)治疗后，近25%的ARKO小鼠发生膀胱癌，这表明DHT能够通过非AR途径促进膀胱肿瘤的进展。我们将使用这一建议来证明我们的假设，即DHT可以通过非AR途径促进膀胱癌的进展。

DESCRIPTION (provided by applicant): Urinary bladder cancer (BCa) is the fourth/tenth most common cancer among men/women in the United States. It is reported that males have significantly (approximately three times) higher risk of bladder cancer than females. However, the etiology of this sex difference in incidence is unknown. Recent studies have suggested the involvement of androgens/androgen receptor (AR) signaling in BCa progression. Using AR knockout (ARKO) mice, we found that the androgen/AR signaling might play a critical role in the development of the chemical carcinogen, N-butyl-N-4-hydroxybutyl-nitrosamine (BBN), induced bladder carcinogenesis. With BBN treatment, ARKO mice, that lack AR and with undetectable androgen, did not develop bladder tumors while their wild type littermates, with functional AR, all develop bladder tumors at the age of 50 wks. Interestingly and unexpectedly, we found near 25% of ARKO mice develop BBN-induced bladder tumor after supplementation with the androgen, dihydrotestosterone (DHT), suggesting DHT is able to function through non-AR pathways to promote bladder tumor progression. This finding was further confirmed in the human AR-negative bladder cancer cell line, TCC5637, showing that addition of DHT can promote this AR-negative bladder cell growth and invasion. Preliminary data also showed DHT could enhance the pathways from G-proteins signals to ERK/MAPK-MEK signals in BCa AR- negative cells. Based on these in vitro human cell line and in vivo mice evidences, we hypothesize that DHT can function through non-AR pathways to promote BCa initiation, growth, and invasion. We will apply the following 4 aims to prove our hypothesis and dissect potential mechanisms. Aim 1: Using the Uro- SV40T-ARKO mouse model to prove DHT can go through non-AR pathways to promote BCa progression. Aim 2: Using UPII-Ha-ras-ARKO mouse model to prove DHT can go through non-AR pathways to promote BCa progression. Aim 3: Using cell transformation/tumorigenesis assays to prove DHT can go through non-AR mediated pathways to promote BCa initiation and/or progression. Aim 4: Using human AR-negative bladder cell lines to prove DHT can go through non-AR pathways to promote BCa growth and invasion and to dissect potential mechanisms. IMPACT: Currently, BCa in humans is not generally considered hormonally dependent and therefore it is not assumed that androgen deprivation therapy (ADT) with chemical or surgical castration can be an effective treatment option for BCa patients in order to repress tumor progression. The success of this proposal to prove DHT can function through non-AR pathways to promote bladder cancer initiation and progression, not only provides in vivo evidence for a novel androgen mechanism, it may also answer the puzzle for the previous failure of using ADT to suppress androgen binding to AR for the treatment of BCa patients and may provide a new preventive/therapeutic approach to suppress BCa. PUBLIC HEALTH RELEVANCE: Urinary bladder cancer is the fourth/tenth most common cancer among men/women in the United States. It is reported that males have significantly (approximately three times) higher risk of bladder cancer than females. However, the reasons behind this sex difference in incidence are unknown. Recent studies have suggested the involvement of androgens/androgen receptor (AR) signaling in bladder cancer progression. Using AR knockout (ARKO) mice, we found that the male hormone androgen/AR signaling might play a critical role in the development of the chemical carcinogen, BBN, induced bladder carcinogenesis. We also found near 25% of ARKO mice develop bladder tumors after treatment with BBN and the androgen, dihydrotestosterone (DHT), suggesting DHT is able to function through non-AR pathways to promote bladder tumor progression. We will use this proposal to prove our hypothesis that DHT can function through non-AR pathways to promote bladder cancer progression.