The Molecular Profile of Prostate Tumors in African-American Men

先前，我们获得了33名非裔美国人和36名欧洲裔美国人切除的原发性前列腺肿瘤的基因表达谱。分析结果数据集，我们确定了基因表达差异，描绘了这两组患者不同肿瘤微环境的存在。许多不同表达的基因都是免疫调节基因。也许最重要的是在许多非裔美国人肿瘤中存在明显的干扰素特征。正如我们现在发现的那样，这个特征几乎与干扰素相关的DNA损伤抵抗特征（IRDS）相同，IRDS可以预测对化疗和放疗的耐药性。非裔美国人肿瘤中IDRS的存在不仅可能影响他们对免疫治疗的反应，而且可能使他们对包括放射治疗在内的标准治疗更具抵抗力。此外，IDRS与癌细胞的前转移性上皮向间质转化有关，并由成纤维细胞和肿瘤细胞之间的相互作用诱导。因此，IDRS也可能促进转移过程。我们最近完成了对非洲裔美国人和欧美人肿瘤基因表达谱的两个独立数据集的分析，结果表明，在两个数据集中，IDRS在非洲裔美国患者的肿瘤中比在欧美患者的肿瘤中更常见。为了探索IDRS的起源，我们对14名非裔美国人和13名欧美患者分离的原代人前列腺上皮细胞的表达谱进行了研究，以确定IDRS的存在。该分析发现，IDRS存在于这些培养细胞中，因此在从肿瘤微环境中移除后仍存在于癌细胞中，并且在14个非裔美国患者分离株中有5个（36%）观察到IDRS，而在13个欧美患者分离株中有2个（15%）观察到IDRS。我们认为，IDRS在非裔美国患者癌细胞中的高患病率在临床上可能非常重要，值得进一步研究这种特征的起源，以及如何靶向这种特征。为了进一步了解非洲裔美国人和欧洲裔美国人前列腺癌患者肿瘤中检测到的免疫生物学差异的可能起源，我们开始评估非洲裔美国人和欧洲裔美国人前列腺癌患者的血液免疫细胞谱和年龄匹配的基于人群的对照，重点关注在癌症生物学中具有免疫调节功能的亚群。本项目的假设是，在癌症生物学中具有免疫调节功能的免疫细胞亚群在这两个人群中丰度不同。前列腺肿瘤中干扰素基因标记的存在提示可能涉及疾病病理中的病毒感染或肿瘤微环境中内源性逆转录病毒的再激活。我们发现前列腺肿瘤中的干扰素信号与逆转录病毒激活的基因信号一致，这进一步支持了这一假设。因此，我们开始了一个项目，探索非裔美国人和欧美患者肿瘤中病毒感染和内源性逆转录病毒再激活的存在。在一项初步研究中，我们用免疫组织化学方法研究了肿瘤样本，用定量PCR方法研究了外周血单个核细胞分离物对内源性逆转录病毒HERV-K家族的表达。这个项目是与MD Anderson的Feng Wang-Johanning博士合作的。研究结果表明，通过免疫组织化学可以在前列腺肿瘤中检测到HERV-K包膜蛋白，并且在非裔美国男性的肿瘤中更常见，这表明HERV-K编码蛋白在这些肿瘤中表达，并且与非癌症对照人群相比，前列腺癌患者的血液单个核细胞中HERV-K gag表达升高。下一步，我们将分析被纳入PLCO研究的男性的血液样本。一项试点研究已获批准，并将开始分析30份不受病例对照状态（15/15）影响的血液样本。这些血液样本取自患前列腺癌和未患前列腺癌的男性，并将与位于戈尔韦的爱尔兰国立大学的Sharon Glynn博士的实验室合作进行分析，以检查是否可以在后来患前列腺癌的男性中检测到HERV-K的增加。如果试点研究成功，将进行一项更大规模的PLCO样本研究，以评估HERV-K作为前列腺癌的早期检测标志物。对非裔美国人和欧裔美国人前列腺肿瘤的微阵列分析发现了一种双基因肿瘤特征，可以准确区分非裔美国人和欧裔美国人的癌症患者。PSPHL包含在这个双基因标记中。PSPHL基因在非裔美国患者的前列腺肿瘤中上调幅度最大。目前对PSPHL的功能知之甚少。有趣的是，PSPHL位于染色体7q11.2上，已知该染色体区域具有与前列腺癌晚期肿瘤相关的功能增益。PSPHL编码一个富含半胱氨酸的小蛋白，有72个氨基酸（预计大小7.6 kDa）。尽管PSPHL位于染色体上，但没有研究将其表达与癌症进展联系起来。在与博士合作。约翰霍普金斯大学的罗军和威廉艾萨克斯，我们正在研究PSPHL的基因组区域。这些研究表明，PSPHL位点在欧裔美国人中经常被删除，而在非洲血统的受试者中则存在。PSPHL基因编码两种转录本，这两种转录本都在前列腺癌细胞中表达。siRNA实验数据显示PSPHL可能影响内质网功能、蛋白质泛素化、O-linked n -乙酰氨基葡萄糖代谢和tgf - β信号通路。以PSPHL为诱饵的双杂交筛选结果表明，COPS5是PSPHL的候选结合伴侣。这种蛋白质，也被称为CSN5或JAB1，是COP9信号体的一个亚基，COP9信号体是一种高度保守的蛋白质复合物，在多种信号通路中起调节作用。未来的研究需要明确PSPHL蛋白是否确实与COPS5特异性相互作用。目前，我们正在测试PSPHL表达诱导系统，以观察PSPHL的诱导是否影响肿瘤细胞的基因表达谱和异种移植物生长，以及PSPHL是否是肿瘤细胞应激信号通路的修饰剂。最后，我们开始研究前列腺肿瘤的代谢组，比较非裔美国人和欧美人的肿瘤。在一项试验中，研究人员分析了来自非裔美国患者的12个肿瘤组织和12个非肿瘤组织，以及来自欧裔美国患者的相同数量的组织。使用该公司的专有技术Metabolon，在组织中鉴定了总共600种代谢物。发现113种代谢物的相对丰度在所有肿瘤合并与所有正常周围组织合并之间存在差异。非裔美国人和欧裔美国人肿瘤之间的差异相对较少（n = 16），这表明这两个患者组之间的肿瘤代谢组差异很小，或者试点的样本量太大。在后续研究中，我们将每组患者的肿瘤数量增加到n = 24。该项目正在与贝勒医学院的Arun Sreekumar博士合作继续进行，导致了新的发现，即肿瘤代谢组学表明非裔美国人和欧裔美国人之间肿瘤生物学的某些差异。

Previously, we obtained gene expression profiles from primary prostate tumors resected from 33 African-American and 36 European-American men. Analyzing the resulting datasets, we identified gene expression differences that portray the existence of a distinct tumor microenvironment for these two patient groups. Many of the differently expressed genes were immune-regulatory. Perhaps most significant was the presence of a distinct interferon signature in many of the African-American tumors. As now discovered by us, this signature is almost identical with an interferon-related DNA damage resistance signature (IRDS) that predicts resistance to chemotherapy and radiation. The presence of IDRS in African-American tumors may not only affect the response of them to immune-based therapies but may also make them more resistant to standard therapy including radiation therapy. In addition, IDRS has been linked to the pro-metastatic epithelial to mesenchymal transition of cancer cells and is induced by interactions between fibroblasts and tumor cells. Thus, IDRS may also promote the metastatic process. We completed very recently the analysis of two independent datasets with gene expression profiles from African-American and European-American tumors, which showed that IDRS is significantly more common in tumors from the African-American patients than in tumors from European-American patients in both datasets. To explore the origin of IDRS, we interrogated the expression profiles of isolated primary human prostate epithelial cells from 14 African-American patients and 13 European-American patients for the presence of IDRS. This analysis led to the finding that IDRS is present in these cultured cells, thus persisting in cancer cells after being removed from the tumor microenvironment, and was observed in 5 out of 14 (36%) isolates from African-American patients versus 2 out of 13 (15%) isolates from European-American patients. We believe that the heightened prevalence of IDRS in cancer cells from African-American patients could be clinically very significant and warrants further investigations into the origin of this signature, and also how this signature can be targeted. To further understand the possible origin of the detected immunobiological differences in tumors of African-American and European-American prostate cancer patients, we have started with the evaluation of blood-based immune cell profiles of African-American and European-American prostate cancer patients and age-matched population-based controls with a focus of subpopulation that have immune-regulatory functions in cancer biology. It is the hypothesis of this project that immune cell subpopulation that have immune-regulatory functions in cancer biology are different in abundance in these two population groups.The presence of an interferon gene signature in prostate tumors suggests a possible involvement of either a viral infection in disease pathology or the reactivation of endogenous retroviruses in the tumor microenvironment. This hypothesis was further supported by our finding that the interferon signature in prostate tumors coincides with a gene signature of retroviral activation. Thus, we started a project exploring the presence of viral infections and the reactivation of endogenous retroviruses in tumors from African-American and European-American patients. In a pilot study, we investigated tumor samples by immunohistochemistry and peripheral blood mononuclear cell isolates by quantitative PCR for expression of a family of endogenous retroviruses, HERV-K. This project is a collaboration with Dr. Feng Wang-Johanning at MD Anderson. The findings showed that the HERV-K envelope protein can be detected in prostate tumors by immunohistochemistry and is more commonly detected in tumors from African-American men, indicating that HERV-K encoded proteins are expressed in these tumors, and that HERV-K gag expression is elevated blood mononuclear cells from prostate cancer patients when compared with a non-cancer control population. As a next step, we will analyze blood samples from men prospectively recruited into the PLCO study. A pilot study has been approved and will start to analyze 30 blood samples blinded to the case-control status (15/15). These blood samples have been drawn from men that developed prostate cancer and those who did not develop prostate cancer and will be analyzed in collaboration with laboratory of Dr. Sharon Glynn at the National University of Ireland, Galway, to examine whether increased HERV-K can be detected in men who later developed prostate cancer. If the pilot study is successful, a larger study with PLCO samples will be conducted to assess HERV-K as an early detection marker for prostate cancer.The microarray analysis of African-American and European-American prostate tumors identified a two-gene tumor signature that accurately differentiated between African-American and European-American cancer patients. PSPHL is included in this two-gene signature. PSPHL was the most highly up-regulated gene in prostate tumors from African-American patients. Little is known about the function of PSPHL. Interestingly, PSPHL is located on chromosome 7q11.2, a chromosomal region known to have gain of function related to advanced tumor stage in prostate cancer. PSPHL encodes a cysteine-rich small protein of 72 amino acids (predicted size 7.6 kDa). Despite its chromosomal location, there are no studies linking PSPHL expression to cancer progression. In collaboration with Drs. Jun Luo and William Isaacs at Johns Hopkins University, we were examining the genomic region of PSPHL. These studies showed that the PSPHL locus is frequently deleted in European-Americans while present in subjects of African ancestry. The PSPHL gene encodes for two transcripts that are both expressed in prostate cancer cells. Data from siRNA experiments showed that PSPHL may influence endoplasmic reticulum function, protein ubiquitination, O-linked N-acetylglucosamine metabolism and the TGF-beta signaling pathway. A two hybrid screen with PSPHL as bait identified, COPS5, as a candidate binding partner of PSPHL. This protein, also known as CSN5 or JAB1, is a subunit of the COP9 signalosome, a highly conserved protein complex that functions as a regulator in multiple signaling pathways. Future research is needed to clarify whether PSPHL protein indeed specifically interacts with COPS5. Currently, we are testing inducible systems of PSPHL expression to see if induction of PSPHL affects gene expression profiles and xenograft growth of cancer cells, and whether PSPHL is modifier of the stress signaling pathway in tumor cells.Lastly, we started studying the metabolome of prostate tumors comparing African-American and European-American tumors. In a pilot, 12 tumors and 12 non-tumor tissues from African-American patients and the same number of tissues from European-American patients were analyzed. A total of 600 metabolites were identified in the tissues using a proprietary technology of the company, Metabolon. The relative abundance of 113 metabolites was found to be different between all tumors combined and all normal surrounding tissues combined. A relative small number of those (n = 16) were found to be different between African-American and European-American tumors, suggesting that there are either only few differences in the tumor metabolome between these two patient groups or that the sample size of the pilot was too sample. In a follow up study, we increased the number of tumors to n = 24 for each patient group. This project is being continued in collaboration with Dr. Arun Sreekumar from Baylor College of Medicine, leading to the new finding that the tumor metabolome is indicative of certain differences in tumor biology between African-American and European-American patients.