Biomarkers of Human Lung Cancer

miRNA在人类疾病和癌症中起着重要作用。我们试图研究miRNA在非小细胞肺癌中的表达状态、临床相关性和功能作用。 与Jun Jen等人合作，我们使用56对新鲜冷冻（FF）和47对福尔马林固定、石蜡包埋（FFPE）的从不吸烟者的样本，在匹配的肺腺癌和未受累的肺中进行了miRNA表达谱分析。采用考克斯分析和log-rank检验对差异表达最高的miRNA基因进行评价。在最佳候选者中，进一步检查miR-708在两个独立群组中的差异表达。通过鉴定其候选mRNA靶标并通过操纵其在培养细胞中的表达水平来检查miR-708在肺癌中表达的功能意义。 在测试的肿瘤和正常样品之间最差异表达的20种miRNAs中，在校正所有临床显著因素（包括年龄、性别和肿瘤分期）后，肿瘤中miR-708的高表达水平与死亡风险增加最强相关。（FF队列：HR，1.90; 95%CI，1.08 - 3.35; P = 0.025; FFPE队列：HR，1.93; 95%CI，1.02 - 3.63; P = 0.042）。TMEM88基因的转录物在其3'UTR中具有miR-708结合位点，并且在miR-708高的肿瘤中显著减少。强制miR-708表达降低了TMEM 88转录水平，并增加了培养物中细胞增殖、侵袭和迁移的速率。 miRNA-708作为癌基因，通过直接下调TMEM 88（肺癌中Wnt信号通路的负调节因子）而促进肿瘤生长和疾病进展。与Tahaski Kohno等人合作，我们使用全转录组测序鉴定了KIF5B（驱动蛋白家族5B基因）和RET癌基因的框内融合转录物，它们存在于来自日本和美国的1 - 2%的肺腺癌（LADC）中。KIF5B-RET融合导致RET激酶的异常激活，并且被认为是LADC的新驱动突变，因为它与EGFR、KRAS、HER2和ALK中的突变或融合分离，并且RET酪氨酸激酶抑制剂凡德他尼抑制NIH3T3细胞的融合诱导的锚定非依赖性生长活性。肺癌的令人沮丧的致死率是由于诊断时的晚期和固有的治疗抗性。靶向治疗的结合适度改善了临床结局，但新靶点的识别可以通过指导低风险早期患者的分层和个性化治疗选择来进一步改善临床结局。与Phil Dennis等人合作，我们假设一种顺序的、组合的微阵列方法对于鉴定和验证肺癌中的新靶点是有价值的。我们分析了肺上皮细胞永生化和转化过程中的基因表达特征，发现参与有丝分裂的基因在癌变过程中逐渐增强。28个基因通过免疫印迹法验证，4个基因在非小细胞肺癌组织芯片中进一步评估。虽然CDK1在肿瘤组织中高度表达，但其从细胞质中的丢失意外地预测了较差的存活率，并在多种细胞系中赋予了对化疗的抗性，特别是微管导向剂。NCI60细胞系数据库中CDK1和CDK1相关基因表达的分析证实了这些基因与化疗反应性的广泛关联。这些结果对个性化肺癌治疗具有意义，并突出了生物标志物发现的联合方法的潜力。

miRNA plays an important role in human disease and cancer. We seek to investigate the expression status, clinical relevance, and functional role of miRNA in non-small cell lung cancer. In collaboration with Jun Jen et al., we conducted miRNA expression profiling in matched lung adenocarcinoma and uninvolved lung using 56 pairs of fresh-frozen (FF) and 47 pairs of formalin-fixed, paraffin-embedded (FFPE) samples from never smokers. The most differentially expressed miRNA genes were evaluated by Cox analysis and log-rank test. Among the best candidate, miR-708 was further examined for differential expression in two independent cohorts. Functional significance of miR-708 expression in lung cancer was examined by identifying its candidate mRNA target and through manipulating its expression levels in cultured cells. Among the 20 miRNAs most differentially expressed between tested tumor and normal samples, high expression level of miR-708 in the tumors was most strongly associated with an increased risk of death after adjustments for all clinically significant factors including age, sex, and tumor stage (FF cohort: HR, 1.90; 95% CI, 1.08-3.35; P = 0.025 and FFPE cohort: HR, 1.93; 95% CI, 1.02-3.63; P = 0.042). The transcript for TMEM88 gene has a miR-708 binding site in its 3' UTR and was significantly reduced in tumors high of miR-708. Forced miR-708 expression reduced TMEM88 transcript levels and increased the rate of cell proliferation, invasion, and migration in culture. miRNA-708 acts as an oncogene contributing to tumor growth and disease progression by directly downregulating TMEM88, a negative regulator of the Wnt signaling pathway in lung cancer. In collaboration with Tahaski Kohno et al., we identified in-frame fusion transcripts of KIF5B (the kinesin family 5B gene) and the RET oncogene, which are present in 1-2% of lung adenocarcinomas (LADCs) from people from Japan and the United States, using whole-transcriptome sequencing. The KIF5B-RET fusion leads to aberrant activation of RET kinase and is considered to be a new driver mutation of LADC because it segregates from mutations or fusions in EGFR, KRAS, HER2 and ALK, and a RET tyrosine kinase inhibitor, vandetanib, suppresses the fusion-induced anchorage-independent growth activity of NIH3T3 cells.The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early stage patients and individualizing therapeutic choices. In collaboration with Phil Dennis et al., we hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery.