Transcription profiling by array of human bronchial epithelial cells overexpressing TTF1, NKX2-8, or PAX9

We investigated the clinical implications of lung developmental transcription factors (TTF-1, NKX2-8, and PAX9) which we recently discovered as cooperating oncogenes activated by way of gene amplification at chromosome 14q13 in lung cancer. Using stable transfectants of human bronchial epithelial cells, RNA expression profiles (signatures) representing activation of the biological pathways defined by each of the three genes were determined and used to risk stratify a non-small cell lung cancer (NSCLC) clinical dataset consisting of ninety-one early stage tumors. Co-activation of the TTF-1 and NKX2-8 pathways identified a cluster of patients with poor survival, representing approximately 20% of patients with early stage NSCLC, whereas activation of individual pathways did not reveal significant prognostic power. Importantly, the poor prognosis associated with co-activation of TTF-1 and NKX2-8 was validated in two other independent clinical datasets. Further, lung cancer cell lines showing co-activation of the TTF-1 and NKX2-8 pathways were shown to exhibit resistance to cisplatin, the standard of care for the treatment of NSCLC. Since TTF-1 and NKX2-8 lack specific inhibitors at the current time, we explored an alternative therapeutic strategy. Using signatures of signaling pathway activation, we identified deregulation of specific oncogenic pathways (Ras and Myc) in the TTF-1/NKX2-8 co-activated cohort. In vitro experiments demonstrated the ability of a Ras pathway-specific therapy to inhibit tumor cell growth in TTF-1/NKX-2 activated cells, thus, suggesting that modulation of the Ras pathway is a rational strategy to targeted therapy in high risk NSCLC patients with co-activation of specific lung developmental pathways. Experiment Overall Design: Six controls and six replicates of each transcription factor (TTF1, NKX2-8, PAX9) were prepared and analyzed.

本研究针对肺发育转录因子（lung developmental transcription factors）的临床意义展开探究，我们近期发现TTF-1、NKX2-8及PAX9可作为协同致癌基因，通过染色体14q13区域的基因扩增在肺癌中激活。本研究借助人支气管上皮细胞的稳定转染株，测定了代表三个基因各自调控的生物学通路激活状态的RNA表达谱特征签名（signatures），并利用该特征对包含91例早期肿瘤的非小细胞肺癌（non-small cell lung cancer, NSCLC）临床数据集开展风险分层。TTF-1与NKX2-8通路的共激活可鉴定出一类预后不良的患者亚群，该亚群约占早期非小细胞肺癌患者的20%；而单一通路激活则未展现出显著的预后预测价值。尤为关键的是，TTF-1与NKX2-8共激活所关联的不良预后，在另外两项独立临床数据集中均得到了验证。此外，呈现TTF-1与NKX2-8通路共激活的肺癌细胞系，被证实对顺铂——非小细胞肺癌的标准治疗药物——产生耐药性。鉴于目前尚无针对TTF-1与NKX2-8的特异性抑制剂，本研究探索了替代性治疗策略。借助信号通路激活特征签名，本研究在TTF-1/NKX2-8共激活队列中鉴定出特定致癌通路（Ras与Myc）的失调现象。体外实验证实，靶向Ras通路的治疗手段可抑制TTF-1/NKX2-8共激活细胞的肿瘤细胞增殖，由此表明，针对Ras通路进行调控，可为伴随特定肺发育通路共激活的高危非小细胞肺癌患者提供合理的靶向治疗策略。实验总体设计：针对每个转录因子（TTF1、NKX2-8与PAX9）均设置6个对照样本与6个生物学重复样本，并完成制备与分析。