Mutationally-activated PI3'-kinase-a promotes de-differentiation of lung tumors initiated by the BRAFV600E oncoprotein kinase

Human lung adenocarcinoma exhibits a propensity for de-differentiation, which complicates diagnosis and treatment, and predicts for poor overall patient survival. In genetically engineered mouse (GEM) models of lung cancer, expression of the BRAFV600E oncoprotein kinase initiates the growth of benign tumors that retain characteristics of their cell of origin, alveolar type II (ATII) pneumocytes. Cooperating genetic alterations such as silencing of the PTEN tumor suppressor or expression of mutationally-activated PI3-kinase-a (PIK3CAH1047R) promote malignant progression of such benign tumors to malignant adenocarcinoma, though their effects on differentiation status are unknown. To address this in vivo, we generated a new conditional BrafCAT allele in which Cre-mediated recombination leads to expression of a bi-cistronic mRNA encoding both BRAFV600E and the tdTomato fluorescent protein. Using this model, we demonstrate that coincident expression of BRAFV600E and PIK3CAH1047R in ATII pneumocytes leads to rapid and widespread cell de-differentiation. Surprisingly, the combined effects of BRAFV600E and PIK3CAH1047R on ATII pneumocyte identity occurred without loss of expression of the lung lineage transcription factors NKX2.1, FOXA1, or FOXA2. Instead, we demonstrate a novel role of PGC1a in maintaining pneumocyte identity, which is lost upon PIK3CAH1047R expression. These findings provide additional insight into how two of the most commonly mutated growth factor signaling pathways contribute to the pathogenesis of lung adenocarcinoma. Overall design: BRAFV600E mutant mouse lung adenocarcinoma (n=6) vs BRAFV600E;PIK3CAH1047R mutant lung adenocarcinoma (n= 8), and BRAFV600E;PGC1aHET (n=5) vs BRAFV600E;PGC1aNULL tumors (n=4)

人类肺腺癌具有去分化倾向，该特性不仅加剧了临床诊断与治疗的难度，还提示患者整体生存期不佳。在肺癌基因工程小鼠（genetically engineered mouse, GEM）模型中，致癌蛋白激酶BRAFV600E的表达可启动良性肿瘤的生长，此类肿瘤保留其起源细胞——II型肺泡上皮细胞（alveolar type II pneumocytes, ATII）的特征。诸如肿瘤抑制基因PTEN沉默或突变激活的PI3-激酶α（PIK3CAH1047R）表达等协同遗传改变，可促进此类良性肿瘤向恶性腺癌进展，不过其对细胞分化状态的影响尚不明确。为在体内探究这一科学问题，我们构建了一种新型条件性BrafCAT等位基因，经Cre介导的重组后，该等位基因可表达编码BRAFV600E与tdTomato荧光蛋白的双顺反子mRNA。利用该模型，我们证实，在II型肺泡上皮细胞中同时表达BRAFV600E与PIK3CAH1047R可引发快速且广泛的细胞去分化。令人意外的是，BRAFV600E与PIK3CAH1047R对II型肺泡上皮细胞身份的联合影响，并未伴随肺谱系转录因子NKX2.1、FOXA1或FOXA2表达的缺失。与之相反，我们证实PGC1α具有维持肺泡上皮细胞身份的全新功能，而该功能在PIK3CAH1047R表达时会丧失。上述发现进一步阐明了两种最常见突变的生长因子信号通路如何参与肺腺癌的发病机制。实验整体设计：BRAFV600E突变型小鼠肺腺癌（n=6）与BRAFV600E;PIK3CAH1047R突变型肺腺癌（n=8）的对比分析，以及BRAFV600E;PGC1aHET（n=5）与BRAFV600E;PGC1aNULL肿瘤（n=4）的对比分析。