Integrative Molecular Profiling of Human Pancreatic Cancer

1)批准并实施一项胰腺癌研究，从PDAC病例中收集临床标本：该研究包括500例原发性PDAC病例，其中150例手术切除，根据NCI-UMD实验室资源合同，在马里兰大学巴尔的摩分校收集新鲜冷冻肿瘤和周围非肿瘤标本。正在收集所有病例的血液样本。收集的样本将用于分子分析研究，具体目的如下：1)PDAC中与生存相关的基因和代谢物的功能作用。2)表征早期PDAC的分子亚群，筛选体液中亚群特异性肿瘤生物标志物。3)鉴别生存期小于6个月和2年以上患者早期肿瘤的关键分子差异。2)建立多个独立验证队列：重要的是要确保我们的结果广泛适用于研究人群以外的PDAC。为了确保来自PDAC独立队列的临床标本的可用性，我们与世界各地的不同研究所建立了合作关系。我们已经成功地接收了来自德国海德堡大学和哥廷根医科大学以及日本东京纪庆大学的合作者的临床样本。我们正在继续努力扩大每个队列的样本量。3)代谢组学和转录组学的整合揭示了脂肪酸网络在人胰腺癌中发挥生长抑制作用(Zhang et al.，临床；Cancer Res., 2013)：为了确定胰腺导管腺癌（PDAC）中紊乱的代谢途径，我们结合代谢组学和转录组学研究了基因代谢物网络。我们对切除的PDAC病例的两个独立队列进行了全球代谢物谱分析，以确定可能导致胰腺癌进展的关键代谢物改变。然后，我们通过整合代谢物和基因表达谱来寻找与关键代谢物显著相关的基因替代品。在一个测试队列（N=33）和一个独立验证队列（N=31）中，与邻近的非肿瘤组织相比，55种代谢物在肿瘤中一致改变。加权网络分析揭示了一组独特的游离脂肪酸（FFAs），它们在PDAC中高度共调节并减少。157个差异表达基因替代物的通路分析显示，脂质代谢网络显著改变，包括关键的脂质分解酶PNLIP、CLPS、PNLIPRP1和PNLIPRP2。与非肿瘤组织相比，这些脂肪酶的基因表达在胰腺肿瘤中显著降低，导致FFAs减少。更重要的是，在两个独立的队列中，肿瘤中较低的PNLIP基因表达与较差的生存率相关。我们进一步证明，棕榈酸酯和硬脂酸酯两种饱和脂肪酸显著诱导胰腺癌细胞TRAIL表达，引发细胞凋亡，抑制细胞增殖。我们的研究结果表明，涉及脂肪酶和一组独特的FFAs的脂溶途径的损伤可能在胰腺癌的发生和进展中发挥重要作用，并为治疗干预提供了潜在的靶点。1)人胰腺导管腺癌（pancreatic ductal adenocaroma， PDAC）中mif -高和mif -低肿瘤的特征胰导管腺癌（pancreatic ductal adenocaroma， PDAC）是一种高度侵袭性和致死性的恶性肿瘤。巨噬细胞迁移抑制因子（MIF）的异常表达与疾病侵袭性相关，PDAC中MIF的升高预示着较差的患者生存。在这项研究中，我们正在研究mif诱导的人类PDAC疾病侵袭性的机制。在这项正在进行的研究中，我们分析了两个PDAC队列中高和低MIF表达的人类肿瘤中编码和非编码基因的表达，以确定高MIF肿瘤中驱动疾病侵袭性的关键信号通路。2)表征早期切除PDAC的分子差异和生存好坏：早期PDAC如果被检测到，是可切除的，并且在切除的患者中提供相对较好的预后，中位生存期约为2年。然而，超过80%的切除病例在两年内复发并死亡，在某些病例中，甚至在手术后6个月内报告。相比之下，少数切除病例可存活5年（12%），甚至10年（5%）。切除后的生存与许多临床预后因素有关，包括肿瘤分期、分级（分化程度）和切除边缘状态，但没有一个因素是一致的预后因素，我们确实发现在具有相似分期、分级或切除边缘状态的病例中有不同的结果。我们假设，具有显著不同预后的早期PDAC的分子特征可能确定与疾病侵袭性和候选治疗干预靶点相关的关键途径。为了验证这一假设，我们正在进行一项试点研究，通过检查和比较两组切除患者肿瘤的蛋白质编码基因和非编码miRNA表达谱：一组PDAC患者切除后生存期短（7个月，N=11），另一组生存期长（2-6年，N=16）。在本研究中，我们提出以下问题：1)与长生存组相比，短生存组肿瘤中有哪些差异表达的蛋白编码基因和mirna ？2)炎症基因表达谱在短生存组和长生存组中是否不同？3)差异表达的炎症基因和相关通路在胰腺癌侵袭性中的功能作用是什么？3)胰腺导管腺癌的分子亚群：肿瘤的分子多样性导致治疗反应的差异。通过对大量PDAC进行多层次、全局的分子变化（包括mRNA、miRNA和代谢物）检测，并对它们进行综合分析，可能有助于表征分子亚群、亚群特异性生物标志物和候选治疗靶点。我们感兴趣的领域之一是炎症在胰腺癌中的作用。一些炎症和免疫相关基因被证明在胰腺癌的侵袭性中具有功能作用。我们假设炎症相关的基因特征定义了可切除PDAC的侵袭性亚型。我们正在对一个大型PDAC队列（N=200）进行全面的转录组学（mRNA和microRNA）和代谢组学分析，具有完整的临床、人口统计学和流行病学概况。我们的目标如下：1)通过整合转录组学和代谢组学特征来表征PDAC的分子亚群。2)在侵袭性亚型中寻找炎症相关基因特征。3)研究亚型特异性基因和代谢物在肿瘤进展中的机制作用。4)评估亚型特异性基因和途径在临床前体外和体内模型中的潜在治疗意义。

Accomplishments: 1) Approval and implementation of a pancreatic cancer study to collect clinical specimens from PDAC cases: The study includes 500 primary PDAC cases, including 150 cases with surgical resection from which fresh-frozen tumor and surrounding nontumor specimens are being collected at the University of Maryland at Baltimore under NCI-UMD resource contract of the laboratory. Blood samples are being collected from all cases. The collected samples are being used for molecular profiling study with the following specific aims: 1) Functional role of genes and metabolites that are associated with survival in PDAC. 2) Characterizing molecular subgroups in early stage PDAC and screening the subgroup- specific tumor biomarkers in body fluid. 3) Identifying the critical molecular differences between early stage tumors from patients surviving less than 6 months and more than 2 years. 2) Establishment of Multiple independent validation Cohorts: It is important to ensure that our results are broadly applicable to PDAC outside the study population. To ensure the availability of clinical specimens from independent cohorts of PDAC, we established collaborations at different institutes around the world. We have been successful in receiving clinical samples from collaborators at University of Heidelberg and University of Medicine, Goettingen in Germany and Jikei University in Tokyo, Japan. We are continuing our efforts to expand the sample size in each of these cohorts. 3) Global-Gene expression and Metabolite Profiling Integration of Metabolomics and Transcriptomics Revealed a fatty Acid Network Exerting Growth Inhibitory Effects in Human Pancreatic cancer (Zhang et. al., Clin. Cancer Res., 2013): To identify metabolic pathways that are perturbed in pancreatic ductal adenocarcinoma (PDAC), we investigated gene-metabolite networks with integration of metabolomics and transcriptomics. We have performed global metabolite profiling analysis on two independent cohorts of resected PDAC cases to identify critical metabolites alteration that may contribute to the progression of pancreatic cancer. We then searched for gene surrogates that were significantly correlated with the key metabolites by integrating metabolite and gene expression profiles. Fifty-five metabolites were consistently altered in tumors as compared with adjacent nontumor tissues in a test cohort (N=33) and an independent validation cohort (N=31). Weighted network analysis revealed a unique set of free fatty acids (FFAs) that were highly co-regulated and decreased in PDAC. Pathway analysis of 157 differentially expressed gene surrogates revealed a significantly altered lipid metabolism network, including key lipolytic enzymes PNLIP, CLPS, PNLIPRP1, and PNLIPRP2. Gene expressions of these lipases were significantly decreased in pancreatic tumors as compared with nontumor tissues, leading to reduced FFAs. More importantly, a lower gene expression of PNLIP in tumors was associated with poorer survival in two independent cohorts. We further demonstrated that two saturated FFAs, palmitate and stearate significantly induced TRAIL expression, triggered apoptosis, and inhibited proliferation in pancreatic cancer cells. Our results suggest that impairment in a lipolytic pathway involving lipases and a unique set of FFAs, may play an important role in the development and progression of pancreatic cancer and provide potential targets for therapeutic intervention. Ongoing Studies: 1) Characterization of MIF-high and MIF-low tumors in human pancreatic ductal adenocarcinoma (PDAC) Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal malignancy. Aberrant expression of macrophage migration inhibitory factor (MIF) is associated with disease aggressiveness and increased MIF in PDAC predicts poor patient survival. In this study, we are investigating the mechanism of MIF-induced disease aggressiveness in human PDAC. In this ongoing study, we analyzed the expression of coding and non-coding genes in high and low MIF-expressing human tumors in two cohorts of PDAC to identify the key signaling pathway in tumors with high MIF that drives disease aggressiveness. 2) Characterizing molecular distinctions in early stage resected PDAC with good and poor survival: Early-stage PDAC, if detected, are resectable and offer relatively better prognosis in resected patients with a median survival of about 2 years. However, more than 80% of the resected cases show recurrence within two years with fatality, in some cases, reported even within 6 months following surgery. In contrast, a small number of the resected cases may survive up to 5 years (12% of the resected cases) and even 10 years (5% of the resected cases). Survival following resection is associated with many clinical prognostic factors including tumor stage, grade (degree of differentiation) and resection margin status but no one factor is consistently prognostic and we do find variable outcomes among cases with similar stage, grade or resection margin status. We hypothesize that molecular characterization of early stage PDAC with markedly different prognoses may identify critical pathways associated with disease aggressiveness and candidate targets for therapeutic intervention. To test this hypothesis we are conducting a pilot study by examining and comparing protein coding genes and non-coding miRNA expression profile of tumors in two groups of resected patients: one with a short survival (7 months, N=11) and the other with a longer survival (2-6 years, N=16) following resection in PDAC cases. In this study, we are asking the following questions: 1) What are the differentially expressed protein coding genes and miRNAs in the tumors from short survival as compared with long survival group? 2) Are inflammatory genes expression profile different in short versus long survival groups? 3) What are the functional roles of the differentially expressed inflammatory genes and associated pathways in the aggressiveness of pancreatic cancer? 3) Molecular Subgroups of Pancreatic Ductal Adenocarcinoma: Molecular diversity in tumors contributes to the discrepancies in therapeutic response. Examining a large cohort of PDAC for multi-level, global molecular changes including mRNA, miRNA and metabolites, and their integrative analyses may help characterize molecular subgroups, subgroup-specific biomarkers, and candidate therapeutic targets. One of our areas of interest is the role of inflammation in pancreatic cancer. Several of the inflammatory and immune-related genes are shown to have a functional role in the aggressiveness of pancreatic cancer. We hypothesize that Inflammation-associated gene signature defines aggressive subtypes in resectable PDAC. We are conducting a comprehensive transcriptomic (mRNA and microRNA) and metabolomic analyses in a large cohort of PDAC (N=200) with complete clinical, demographic and epidemiological profile. Our goals are as follows: 1) Characterize molecular subgroups of PDAC through integration of transcriptomic and metabolomic profiles. 2) Search for inflammation-associated gene signature in aggressive subtypes. 3) Examine the mechanistic role of subtype-specific genes and metabolites in tumor progression. 4) Evaluate the potential therapeutic significance of subtype specific genes and pathways in pre-clinical in vitro and in vivo models.