Rac guanine nucleotide exchange factors in lung cancer

点击翻译按钮获取中文摘要

ABSTRACT KRAS and EGFR mutations are the most prevalent genetic alterations detected in human lung adenocarcinomas, and play essential roles in malignant transformation and disease progression. The small GTPase Rac1, a member of the Rho family, is a key signaling effector of KRAS and EGFR oncogenic pathways. Rac1 has been widely implicated in the formation of actin-rich protrusive structures required for cancer cell motility and invasion, as well as in the activation of oncogenic and metastatic gene expression networks. Activation of Rac1 (i.e. GTP loading) is mediated by Rac-GEFs, a large family of Guanine nucleotide Exchange Factors largely associated with tumorigenesis and invasiveness. Strikingly, there is limited information on the contribution of Rac-GEFs to lung cancer progression. Moreover, their relationship to specific lung cancer oncogenic mutations remains unknown. We carried out a systematic and unbiased screening for Rac-GEFs responsible for driving pro-motile phenotypes in KRAS mutant NSCLC cell lines. This analysis unambiguously identified three Rac-GEFs (ARHGEF39, FARP1 and TIAM2) as mediators of ruffle formation and motility in NSCLC cells. To our surprise, well-studied GEFs, such as TIAM1, TRIO, VAV isoforms and P-REX isoforms, were either poorly expressed or dispensable in our model. We therefore hypothesize that these Rac-GEFs are major players in lung cancer progression. In Aim 1, we will generate KRAS mutant cell lines deficient in RacGEFs using a CRISPR/Cas9 approach, and determine their contribution to invasion, ECM protease production and metastasis in mouse models. In addition, the requirement of selected Rac-GEFs to the development of autochthonously-arising metastatic lung cancer will be determined using a lentiviral CRISPR-based gene editing approach in Kras G12D/WT; p53 flox/flox mice (KP mice). In Aim 2 the goal is to identify and characterize Rac-GEFs as EGFR effectors in NSCLC. To unequivocally elucidate their permissive roles in mutant EGFR lung cancer progression phenotypes, in vivo lentiviral CRISPR-based Rac-GEF gene editing in an EGFR L858R-driven, p53 deficient lung adenocarcinoma mouse model will be performed. Mechanistically, we aim to disentangle the basis of Rac-GEF activation by pursuing a comprehensive signaling analysis of proximal EGFR adaptors and effectors. In Aim 3, we will first elucidate Rac-GEF-dependent gene transcriptomes and network signatures driven by mutant KRAS and mutant EGFR. Finally, we will determine Rac-GEF expression in single tumor cells isolated from malignant pleural effusions (a site of lung metastatic dissemination), as well as in single and clustered circulating tumor cells (CTCs) from peripheral blood of mutant KRAS and mutant EGFR lung adenocarcinoma patients. The identification of novel Rac-GEFs provides unprecedented information to predict metastatic disease outcome in lung cancer patients and increase the likelihood of identifying metastasis biomarkers, ultimately aiding in refining patient prognosis and decision-making in a clinical setting.