Human aldo-keto reductases and nuclear receptor action

描述(由申请者提供)：宾夕法尼亚大学为当地经济做出了巨大贡献。2008年，宾夕法尼亚医科大学创造了3.7万个就业机会和54亿美元的区域经济活动，该地区训练有素的劳动力仅为840个宾夕法尼亚大学员工研究职位空缺就产生了超过24600份申请。目前的提案将在两个不同的机构创造或保留5个工作岗位。这是对1R01-CA90744：“醛-酮还原酶和核受体作用”(项目结束日期：06/30/12)的竞争性修订申请，将由2009年美国复苏和再投资法案资助。母基金的重点是AKR1C3(5型17-羟基类固醇脱氢酶(17-HSD)或前列腺素F合成酶)在调节前列腺癌和乳腺癌核受体配体局部产生方面的作用。我们的假设是，前列腺癌是一种老年男性的疾病，它是由腺体局部产生的雄激素驱动的，而不是由睾丸起源的雄激素驱动的。这种雄激素的内分泌形成的前体可能是源于肾上腺的脱氢表雄酮或源于前列腺的孕烯醇酮。AKR1C3在前列腺雄激素的生物合成中起关键作用，因为它将弱雄激素-4-雄烯-3，17-二酮转化为睾酮(一种有效的雄激素)，并直接位于2型5a还原酶的上游。最近，由于雄激素缺乏，耐去势前列腺癌被证明经历了雄激素生物合成的重新编程。这种反应的一部分包括与雄激素生物合成有关的几个基因的上调，包括AKR1C3。此外，CYP17，20裂解酶抑制剂醋酸阿比特龙被发现对晚期前列腺癌有效，这表明局部雄激素信号可能仍在发生。为了验证这一假设，关键是要有可靠的方法来测量不同患者组的前列腺内雄激素水平，并将它们与酶表达水平联系起来。因此，这项补充将超越最初拨款的具体目标：[1]建立稳定的同位素稀释LC/MS分析方法，用于检测和定量孕烯醇酮和脱氢表雄酮(DHEA)下游的所有雄激素代谢物；[2]通过测量LNCaP和LNCaP-AKR1C3稳定转基因细胞中的雄激素代谢谱来验证该方法；[3]应用该方法测量接受根治性前列腺癌切除(阳性对照)的患者和邻近正常组织中的雄激素水平；以及在手术前接受不同雄激素剥夺治疗的患者的前列腺切除样本中，例如LHRH激动剂；LHRH激动剂+比卡鲁胺(雄激素受体拮抗剂)；以及LHRH激动剂+比卡鲁胺+酮康唑(一种CYP17，20-裂解酶抑制剂)；以及[4]使用qPCR检测胆固醇后所有类固醇生成酶在同一前列腺标本中的表达：SCC、CYP17A1、HSD3B2、AKR1C3、SRD5A1、SRD5A2、AKR1C2、AKR1C1、HSD17B2、HSD17B6和UGT2B15，以确定它们的水平是否与观察到的雄激素水平相关。这些研究将带来一种可靠的方法，可以测量接受根治性前列腺切除术的前列腺癌患者的前列腺内雄激素水平，并确定雄激素剥夺疗法的疗效。他们还将确定是否可以通过孕烯醇酮合成前列腺雄激素，以及雄激素水平升高是否与包括AKR1C3在内的类固醇生成酶表达增加有关。 公共卫生相关性：前列腺癌是人类癌症死亡的第二大原因。它最初对雄激素去势治疗有反应，但此后患者复发并发展为耐去势前列腺癌(CRPC)。新出现的数据表明，CRPC经历了对雄激素消融的重新编程或适应性反应，并合成了自己的雄激素。醛酮还原酶(AKR)家族1C成员3(AKR1C3)，也被称为5型17-羟基类固醇脱氢酶，可能在这一反应中发挥关键作用。本研究旨在建立一种稳定的同位素稀释液相色谱质谱检测前列腺癌雄激素代谢组的方法，用于前列腺癌的诊断和治疗。

DESCRIPTION (provided by applicant): Penn Medicine contributes substantially to the local economy. In 2008, Penn Medicine created 37,000 jobs and $5.4 billion in regional economic activity, with the area's highly trained workforce producing more than 24,600 applications for just 840 open Penn staff research positions. The current proposal will create or retain 5 jobs at two different institutions. This is an application for a Competitive Revision to 1R01-CA90744: "Aldo-Keto Reductases and Nuclear Receptor Action" (Project End Date: 06/30/12) to be funded by the American Recovery and Reinvestment Act of 2009. The parent grant is focused on the role of AKR1C3 (type 5 17¿-hydroxysteroid dehydrogenase (17¿-HSD) or prostaglandin F synthase) in regulating the local production of ligands for nuclear receptors in prostate and breast cancer. Our hypothesis is that prostate cancer is a disease of the aging male and that it is driven by the local production of androgens in the gland rather than by androgens of testicular origin. The precursor for this intracrine formation of androgens is likely dehydroepiandrosterone of adrenal origin or pregnenolone of prostatic origin. AKR1C3 plays a key role in prostate androgen biosynthesis since it converts ?4-androstene-3,17-dione (a weak androgen) to testosterone (a potent androgen) and lies immediately upstream from type 2 5a-reductase. Recently, castration resistant prostate cancer has been shown to undergo a re-programming of androgen biosynthesis due to androgen deprivation. Part of this response includes upregulation of several genes involved in androgen biosynthesis including AKR1C3. Moreover, abiraterone acetate a CYP17,20-lyase inhibitor has been found effective in advanced prostate cancer suggesting that local androgen signaling may still be occurring. To test this hypothesis it is critical to have reliable methods to measure intraprostatic androgen levels in different patient groups and relate them to enzyme expression levels. This supplement will therefore go beyond the initial funded specific aims of the grant to: [1] Develop a stable-isotope dilution LC/MS assay for the detection and quantitation of all androgen metabolites downstream from pregnenolone and dehydroepiandrosterone (DHEA); [2] Validate the method by measuring androgen metabolic profiles in LNCaP and LNCaP-AKR1C3 stably transfected cells; [3] Apply the method to measure intrapostatic androgen levels in adenocarcinoma of patients that have undergone radical prostatectomy (positive control) and adjacent normal tissue; and in prostatectomy samples in patients that underwent different androgen deprivation therapies prior to surgery, e.g. LHRH agonist alone; LHRH agonist + bicalutamide (androgen receptor antagonist); and LHRH agonist + bicalutamide + ketoconazole (a CYP17,20- lyase inhibitor); and [4] Use qPCR to measure the expression of all steroidogenic enzymes after cholesterol in the same prostate specimens: Scc, CYP17A1, HSD3B2, AKR1C3, SRD5A1, SRD5A2, AKR1C2, AKR1C1, HSD17B2, HSD17B6, and UGT2B15 to determine if their levels correlate with the androgen levels observed. These studies will lead to a robust method that can measure intraprostatic androgen levels in prostate cancer patients that have undergone radical prostatectomy and determine the efficacy of androgen deprivation therapy. They will also determine whether prostatic androgen synthesis can occur from pregnenolone and whether elevated androgen levels can be correlated to increased expression of steroidogenic enzymes including AKR1C3. PUBLIC HEALTH RELEVANCE: Prostate cancer is the second leading cause of cancer death in man. It is initially responsive to androgen ablative therapy but thereafter patients relapse and develop castration resistant prostate cancer (CRPC). Emerging data suggests that CRPC has undergone a re-programming or adaptive response to androgen ablation and synthesizes its own androgens. Aldo-keto reductase (AKR) family 1C member 3 (AKR1C3) also known as type 5 17¿-hydroxysteroid dehydrogenase may play a key role in this response. This proposal is aimed at developing a stable isotope dilution liquid chromatography mass spectrometry method to measure the intraprostatic androgen metabolome which could be used to better diagnose and treat prostate cancer.