Integrative Molecular Profiling of Human Pancreatic Cancer

1)批准和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PDAC病例，包括150例手术切除的病例，根据NCI-UMD实验室的资源合同，正在巴尔的摩的马里兰大学收集新鲜冰冻肿瘤和周围非肿瘤标本。正在从所有病例中采集血液样本。收集的样本用于我们的研究，具体目的如下：1)炎症介质的功能作用，与PDAC的生存相关，并评估它们作为治疗靶点。2)鉴别早期PDAC炎症基因信号所定义的分子亚群，筛选体液中亚群特异的肿瘤标志物。3)确定生存期<6个月和>2年的早期肿瘤的关键分子差异。2)建立多个独立验证队列：确保我们的结果广泛适用于研究人群以外的PDAC，这一点很重要。为了确保来自PDAC独立队列的临床样本的可用性，我们在世界各地的不同研究所建立了合作关系。我们已经成功地从德国戈廷根的海德堡大学和医科大学、东京的日本经济大学和日本千叶的日本医学院的合作者那里收到了临床样本。我们正在继续努力扩大每个群体的样本规模。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.艾尔，克莱恩。癌症研究，2013)：为了确定胰腺导管腺癌(PDAC)中受到干扰的代谢途径，我们结合代谢组学和转录组学研究了基因-代谢物网络。我们对两组独立的PDAC切除病例进行了全球代谢物图谱分析，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物。在测试队列(N=33)和独立验证队列(N=31)中，与邻近非肿瘤组织相比，肿瘤中有55种代谢物持续改变。加权网络分析揭示了一组独特的自由脂肪酸(FFA)，它们在PDAC中高度协同调节并减少。对157个差异表达基因替代物的通径分析显示，脂质代谢网络发生了显著变化，包括关键的脂解酶PNLIP、CLPs、PNLIPRP1和PNLIPRP2。与非肿瘤组织相比，胰腺肿瘤组织中这些脂肪酶的基因表达显著降低，导致游离脂肪酸减少。更重要的是，PNLIP基因在肿瘤中的低表达与两个独立队列中较差的生存有关。我们进一步证明了两种饱和脂肪酸棕榈酸和硬脂酸显著诱导胰腺癌细胞TRAIL的表达，触发细胞凋亡，并抑制细胞增殖。我们的结果表明，脂肪酶和一组独特的游离脂肪酸参与的脂解途径的损伤，可能在胰腺癌的发生和发展中发挥重要作用，并为治疗干预提供潜在的靶点。4)早期PDAC切除的分子特征：早期PDAC如果被发现是可以切除的，并且在中位生存期约2年的切除患者中预后相对较好。然而，超过80%的切除病例在两年内复发，有些病例甚至在手术后6个月内报告死亡。相比之下，少数切除病例可存活长达5年(占切除病例的12%)，甚至10年(占切除病例的5%)。术后生存率与许多临床预后因素有关，包括肿瘤分期、分级(分化程度)和切除切缘状况，但没有一个因素是始终如一的预后因素，我们确实发现在具有相似分期、分级或切除切缘状况的病例中，预后存在差异。我们假设，预后明显不同的早期PDAC的分子特征可以确定与疾病侵袭性相关的关键途径和治疗干预的候选靶点。为了验证这一假设，我们正在进行一项先导性研究，通过检测和比较两组切除患者的肿瘤蛋白编码基因和非编码miRNA表达谱：一组患者在PDAC病例切除后生存时间较短(7个月，N=11)，另一组患者生存时间较长(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，短生存组肿瘤中差异表达的蛋白编码基因和miRNAs是什么？2)炎性基因在短生存组和长生存组中的表达谱有何不同？3)差异表达的炎性基因和相关通路在胰腺癌侵袭性中的作用是什么？使用这一策略，我们最近报道了内皮型一氧化氮合酶交通诱导剂(NOSTRIN)是胰腺癌疾病侵袭性的负面调节因子(Wang等，临床研究，2016)

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 our studies with the following specific aims: 1) Functional role of inflammatory mediators, associated with survival in PDAC, and their assessment as therapeutic targets. 2) Characterizing molecular subgroups, defined by inflammatory gene signature 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, Jikei University, Tokyo, and Nippon Medical School, Chiba, 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. 4) 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? Using this strategy, we have recently reported that Endothelial Nitric Oxide Synthase Traffic Inducer (NOSTRIN) is a negative regulator of disease aggressiveness in pancreatic cancer (Wang et.al., Clinical Can Res., 2016)