Identify SNPs and Polymorphisms Involved in the Development of Prostate Cancer

该项目的目的是进行转化研究，以开发新的药物，和/或治疗方法，似乎在前列腺癌中具有抗肿瘤活性，并开发前列腺癌患者的分子图谱，以制定个性化的治疗计划。为了实现这一目标，我们已经广泛参与了解前列腺癌生物学的努力。目前，我们正试图将与前列腺癌和治疗反应相关的生物学变量联系起来。分子药理学科的一个早期成就是首次证实了氟他胺停药的治疗效果，以及同时抑制肾上腺的活性增强。据推测，与氟他胺相关的临床改善是雄激素受体的配体结合区域内存在突变的结果。我们仍然有兴趣在基因组水平上分析可能使个体易患前列腺癌风险增加的遗传变异的候选基因。我们已经完成了与内皮抑素（COL18A1，无统计学差异）自然产生相关的基因分析，一个直接参与从胆固醇合成睾酮的基因（CYP17，结果表明其多态性与雄激素非依赖型前列腺癌患者的总生存率相关），一个参与睾酮毒性代谢分解的基因（CYP1B1，观察到与生存率降低相关），药物代谢（CYP3A4 &5，研究的遗传变异不太可能对癌症患者CYP3A的表型活性有重要的功能意义），以及参与细胞转运和偶联的基因（UGT1A9，功能变异在高加索人中很少见，在伊立替康方案中可能临床上不显著）。由SLCO1B3编码的有机阴离子转运体OATP1B3参与类固醇激素的转运。然而，它在睾酮摄取和前列腺癌进展中的作用尚不清楚。通过测序在NCI-60肿瘤细胞中评估SLCO1B3基因型，而在转染野生型（WT）， 334G和699A SLCO1B3变体的Cos-7细胞中分析睾酮转运。荧光显微镜检测ooatp1b3在前列腺组织中的表达，并检测患者SLCO1B3单倍型与生存的关系。我们最近的研究表明，与正常或良性增生组织相比，前列腺癌过度表达OATP1B3，常见的SLCO1B3 GG/AA单倍型与前列腺癌患者睾酮转运受损和生存率提高有关。我们在68例接受ADT治疗并伴有转移性疾病（D2）或生化失败但无转移性疾病（D0）的高加索晚期前列腺癌患者中，研究了SLCO1B3多态性与ADT到雄激素独立、ADT到前列腺特异性抗原（PSA）最低点和PSA最低点到雄激素独立的时间之间的关系。单独检查时，D0组（P = 0.11）和D2组（P = 0.18）具有T等位基因的患者在各个阶段实现雄激素独立的时间较短。结合这些组并按分期分层，T等位基因的雄激素独立时间较短，具有统计学意义（P = 0.048）。因此，在ADT治疗的前列腺癌患者中，增加睾酮输入的转运蛋白多态性与较短的雄激素独立时间相关。基于这一证据，我们进行了一项研究，以确定携带一种编码更活跃睾酮转运蛋白的多态性的晚期前列腺癌患者是否比不携带这种多态性的患者有更少的持久ADT反应。我们在68例接受ADT治疗并伴有转移性疾病（D2）或生化失败但无转移性疾病（D0）的高加索晚期前列腺癌患者中，研究了SLCO1B3多态性与ADT到雄激素独立、ADT到前列腺特异性抗原（PSA）最低点和PSA最低点到雄激素独立的时间之间的关系。当单独检查时，D0组（p=0.11）和D2组（p=0.18）中T等位基因的患者在各个阶段趋向于较短的雄激素独立时间。结合这些组并按分期分层，T等位基因的雄激素独立时间较短，具有统计学意义（p=0.048）。我们的研究结果表明，在接受ADT治疗的前列腺癌患者中，增加睾酮输入的转运蛋白多态性与较短的雄激素独立时间相关。我们目前正在研究缺氧和雄激素剥夺导致的OATP1B3基因和蛋白表达的变化，并表征雄激素的转运特性。此外，对其他基因（SRD5A1&2， LOX, CYP19， ER）的分析已经显示出初步证据，表明它们可能发挥重要作用，并且处于不同的完成阶段。最近，在筛选前列腺癌患者的DNA与设计用于筛选参与代谢和药物转运的基因的基因芯片后，发现了其他感兴趣的多态性基因。这些重要的基因将被添加到前列腺癌分子指纹的持续目标中。该项目的一些总体目标是：(a)更好地了解重要的雄激素调节基因多态性与前列腺癌风险之间的关系；(b)评估这些多态性和血清激素浓度对非那雄胺作为前列腺癌化学预防剂使用的影响。最近完成的前列腺癌预防试验（PCPT）调查了使用类固醇5 α -还原酶抑制剂非那雄胺在七年治疗期间预防前列腺癌。通过长期合作，我们获得了参与这项研究的18800名男性的组织样本。我们目前正在关注与前列腺癌风险相关的激素相关因素，这可能有助于解释PCPT的发现（即，腺癌的总体发生率降低，但非那雄胺治疗组的高级别疾病患病率增加）。我们假设，与具有野生型等位基因的男性相比，具有积极影响雄激素水平的基因多态性的男性患前列腺癌和高级别疾病的风险更高。此外，长期暴露于非那雄胺可能导致躯体改变，增加血清睾酮水平和潜在有害的睾酮分解产物。目前正在评估AR、SRD5A2和HSD3B2基因的多态性变异是否与前列腺癌活检检测的风险相关。我们正在通过激光捕获显微解剖和直接核苷酸测序来确定AR和HSD3B2的体细胞改变，这些改变可能是由于长期暴露于非那雄胺而选择的。此外，我们正在确定这些基因的前列腺癌体细胞突变在安慰剂组和非那雄胺组、PIA组、HGPIN组、前列腺癌组和正常上皮组之间的患病率是否存在差异。这些发现将有助于确定药物基因组学概况，以确定最有可能从5 -还原酶抑制剂治疗中获益的男性。

The purpose of this project is to perform translational research to develop new agents, and/or therapeutic maneuvers, that appear to have antitumor activity in prostate cancer, and to develop molecular profiles of patients with prostate cancer to tailor an individualized treatment plan. To achieve this goal, we have become extensively involved in the efforts to understand the biology of prostate cancer. Currently, we are attempting to correlate biological variables associated with prostate cancer and response to therapy. One early achievement by the Molecular Pharmacology Section was to report the first confirmation of the therapeutic efficacy of flutamide withdrawal, as well as the enhanced activity of simultaneous adrenal suppression. It has been hypothesized that the clinical improvement associated with flutamide is a result of the presence of a mutation within the ligand-binding domain of the androgen receptor. We remain interested in analyzing candidate genes at the genomic level for genetic variations that may predispose individuals to increased risk of prostate cancer. We have completed the analysis of genes involved in the natural production of endostatin (COL18A1, no statistical difference), a gene directly involved in the synthesis of testosterone from cholesterol (CYP17, the results suggest that the polymorphism is associated with overall survival in patients with androgen independent prostate cancer), a gene involved in the toxic metabolic breakdown of testosterone (CYP1B1, an association with decreased survival was observed), drug metabolism (CYP3A4 &5, the studied genetic variants are unlikely to have an important functional significance to phenotypic CYP3A activity in patients with cancer), and a gene involved in cellular transport and conjugation (UGT1A9, functional variants are rare in Caucasians and likely to be clinically insignificant in irinotecan regimens). The organic anion transporter OATP1B3, encoded by SLCO1B3, is involved in the transport of steroid hormones. However, its role in testosterone uptake and progression of prostate cancer is unknown. SLCO1B3 genotype was assessed in the NCI-60 panel of tumor cells by sequencing, while testosterone transport was analyzed in Cos-7 cells transfected with wild-type (WT), 334G and 699A SLCO1B3 variants. OATP1B3 expression in prostatic tissues was examined by fluorescence microscopy and the relationship between SLCO1B3 haplotypes and survival was examined in patients. Our recent study showed that prostate cancer over-expresses OATP1B3 compared to normal or benign hyperplastic tissue, and the common SLCO1B3 GG/AA haplotype is associated with impaired testosterone transport and improved survival in patients with prostate cancer. We examined the association between this SLCO1B3 polymorphism and time from ADT to androgen independence, ADT to prostate-specific antigen (PSA) nadir and PSA nadir to androgen independence in 68 Caucasian patients with advanced prostate cancer who were treated with ADT with metastatic disease (D2) or biochemical failure with no metastatic disease (D0). When examined separately, patients in the individual stages tended to have a shorter time to androgen independence with the T allele in the D0 (P = 0.11) and D2 (P = 0.18) groups. Combining these groups and stratifying by stage yielded a statistically significant shorter time to androgen independence with the T allele (P = 0.048). Thus, a polymorphism in a transporter that increases testosterone import is associated with a shorter time to androgen independence in patients with prostate cancer who are treated with ADT. Based on this evidence, we conducted a study to determine whether patients with advanced prostate cancer carrying a polymorphism that codes for a more active testosterone transporter have less durable responses ADT than patients not carrying this polymorphism. We examined the association between this SLCO1B3 polymorphism and time from ADT to androgen independence, ADT to prostate-specific antigen (PSA) nadir and PSA nadir to androgen independence in 68 Caucasian patients with advanced prostate cancer who were treated with ADT with metastatic disease (D2) or biochemical failure with no metastatic disease (D0). When examined separately, patients in the individual stages tended to have a shorter time to androgen independence with the T allele in the D0 (p=0.11) and D2 (p=0.18) groups. Combining these groups and stratifying by stage yielded a statistically significant shorter time to androgen independence with the T allele (p=0.048). Our findings demonstrate that a polymorphism in a transporter that increases testosterone import is associated with a shorter time to androgen independence in patients with prostate cancer who are treated with ADT. We are currently examining the changes in gene and protein expression of OATP1B3 due to hypoxia and androgen deprivation and characterizing the androgen transport properties. Furthermore, the analysis of other genes (SRD5A1&2, LOX, CYP19, ER) which have shown preliminary evidence that suggests that they may play important roles are ongoing and at various stages of completion. Other polymorphic containing genes of interest have been recently identified after screening the DNA from patients with prostate cancer against a gene chip designed to screen genes involved in metabolism and drug transport. These important genes will be added to the ongoing goal of a molecular fingerprint of prostate cancer. Some of the overall goals of this project are: (a) to better understand associations between important androgen regulatory gene polymorphisms and prostate cancer risk and (b) to evaluate the effects of these polymorphisms and serum hormone concentrations on the use of finasteride as a chemopreventive agent for prostate cancer. The recently completed Prostate Cancer Prevention Trial (PCPT) investigated the prevention of prostate cancer using the steroid 5 alpha-reductase inhibitor finasteride over a seven year treatment period. Through a longstanding collaboration we have access to tissue samples of the 18,800 men enrolled in this study. We are currently focusing on hormone-related factors that are associated with prostate cancer risk, which may help explain the findings of the PCPT (i.e., decreased overall occurrence of adenocarcinoma, but increased prevalence of high-grade disease in the finasteride treatment arm). We hypothesize that men with polymorphisms within genes that positively impact androgen levels will have a higher risk of developing prostate cancer and high grade disease than those with the wild-type alleles. In addition, long-term exposure to finasteride may select for somatic alterations and increase serum levels of testosterone and potentially harmful testosterone breakdown products. The evaluation of whether the polymorphic variations in the AR, SRD5A2 and HSD3B2 genes are associated with the risk of biopsy-detected prostate cancer in the PCPT are underway. We are identifying by laser-capture microdissection and direct nucleotide sequencing somatic alterations in the AR and HSD3B2 that may have been selected for by long-term exposure to finasteride. Furthermore, we are determining whether prostate cancer somatic mutations of these genes differ with regard to their prevalence between the placebo and finasteride arms, and among PIA, HGPIN, prostate cancer and normal epithelium. These findings will help define a pharmacogenomic profile to identify men that are most likely to benefit from treatment with 5 alpha-reductase inhibitors.