Identification of tumour progression genes in a mouse model for non-small cell lung cancer

The 5-year survival rate of lung cancer patients is only 16%. As most patients are diagnosed at an advanced stage, little is known about early stages and mechanisms underlying the progression to metastatic disease. There are few targeted therapies available and targeting KRas driven lung cancer is especially challenging. KRAS is one of the most frequently mutated oncogenes in lung adenocarcinomas at ~33% of cases and is notably associated with resistance to EGFR inhibitors. In order to study tumour progression in vivo we chose a Cre/loxP inducible system in which Cre recombinase expressing Adenovirus is delivered to the lung by intranasal installation. In this model, Cre-mediated induction of a conditional KRasG12D allele gives rise to benign papillary adenomas (BPAs) that rarely progress to adenocarcinoma. Combined activation with conditional modest MYC overexpression however increases both the growth rate of the BPAs and their frequency of progression to adenocarcinoma. Deregulated MYC expression alone however gives rise to focal proliferation in the bronchioles but does not lead to tumours. Loss of functional Tp53 does not increase MYC’s tumour initiating potential in this model. Importantly, the KRasG12D/MYC model faithfully recapitulates the morphology of a subset of the human disease. I used Erk phophorylation status to distinguish between benign (p-Erk negative) and more advanced (p-Erk positive) tumour regions, and laser capture microdissection to harvest regions of interest. RNA was isolated from those regions and analyzed by RNA-Sequencing. GeneGo pathway analysis revealed that the ErbB and Wnt pathways are significantly upregulated in the p-Erk positive dataset. In order to validate the importance of these pathways, we treated cells derived from the same KRasG12D- and MYC-driven mouse tumours with the pan-ErbB-family inhibitor Neratinib and the WNT-inhibitor LGK974. Single treatment with either inhibitor suppressed cell propagation, migration and invasion into Matrigel, whereas combined treatment had a stronger effect on both characteristics. A panel of KRas mutant human lung adenocarcinoma cell lines were similarly sensitive to at least one inhibitor or to the combination of both. With KRas being downstream of ErbB family receptors and EGFR- and KRAS-mutations being mutually exclusive in NSCLC, the reliance on ErbB family signalling in KRas mutant cells was not expected. These results suggest that broad-specificity inhibitors of these proteins may be effective against a broader spectrum of NSCLC than hitherto anticipated. These results moreover indicate significant cooperation between the Ras and Wnt pathways that likewise may be exploited for therapy. Individual p-Erk associated genes that are also amplified or overexpressed in human NSCLC were selected for an in vitro siRNA screen. A significant number of these genes also correlate with decreased overall survival of NSCLC and in particular lung ADC patients. Screening of 3 KRas mutant human lung adenocarcinoma cell lines revealed that a considerable number of genes is important for cell viability of all tested cell lines. Also, knockdown of certain genes considerably suppressed cell migration in two efficiently migrating cell lines. These results suggest, that I have identified a list of genes that play an important role in KRas mutant lung adenocarcinoma

The ERBB network facilitates KRAS-driven lung tumorigenesis

KRAS is the most frequently mutated driver oncogene in human adenocarcinoma of the lung. There are presently no clinically proven strategies for treatment of KRAS-driven lung cancer. Activating mutations in KRAS are thought to confer independence from upstream signaling; however, recent data suggest that this independence may not be absolute. We show that initiation and progression of KRAS-driven lung tumors require input from ERBB family receptor tyrosine kinases (RTKs): Multiple ERBB RTKs are expressed and active from the earliest stages of KRAS-driven lung tumor development, and treatment with a multi-ERBB inhibitor suppresses formation of KRASG12D-driven lung tumors. We present evidence that ERBB activity amplifies signaling through the core RAS pathway, supporting proliferation of KRAS-mutant tumor cells in culture and progression to invasive disease in vivo. Brief pharmacological inhibition of the ERBB network enhances the therapeutic benefit of MEK (mitogen-activated protein kinase kinase) inhibition in an autochthonous tumor setting. Our data suggest that lung cancer patients with KRAS-driven disease may benefit from inclusion of multi-ERBB inhibitors in rationally designed treatment strategies

Repression of the type I interferon pathway underlies MYC & KRAS-dependent evasion of NK & B cells in pancreatic ductal adenocarcinoma

MYC is implicated in the development and progression of Pancreatic cancer, yet the precise level of MYC deregulation required to contribute to tumour development has been difficult to define. We used modestly elevated expression of human MYC, driven from the Rosa26 locus, to investigate the pancreatic phenotypes arising in mice from an approximation of MYC trisomy. We show that this level of MYC alone suffices to drive pancreatic neuroendocrine tumours, and to accelerate progression of KRAS-initiated precursor lesions to metastatic pancreatic ductal adenocarcinoma. Our phenotype exposed suppression of the Type I Interferon pathway by the combined actions of MYC and KRAS and we present evidence of repressive MYC/MIZ1 complexes binding directly to the promoters of type I Interferon regulators IRF5, IRF7, STAT1 and STAT2. De-repression of Interferon regulators allows pancreatic tumour infiltration of B and NK cells, resulting in increased survival

Studying lung cancer progression: insights from genetically engineered mouse models of cancer

No abstract available

Progression of KRas^G12D;Rosa26-MYC tumours to invasive disease

The aim of the study was to investigate gene expression tumour progression of KRas*/MYC driven lung tumours from adenocarcinoma in situ to invasive disease

Studying lung cancer progression: insights from genetically engineered mouse models of cancer

No abstract available

Identification of a Clinically Relevant Signature for Early Progression in KRAS-Driven Lung Adenocarcinoma

Inducible genetically defined mouse models of cancer uniquely facilitate the investigation of early events in cancer progression, however, there are valid concerns about the ability of such models to faithfully recapitulate human disease. We developed an inducible mouse model of progressive lung adenocarcinoma (LuAd) that combines sporadic activation of oncogenic KRasG12D with modest overexpression of c-MYC (KM model). Histological examination revealed a highly reproducible spontaneous transition from low-grade adenocarcinoma to locally invasive adenocarcinoma within 6 weeks of oncogene activation. Laser-capture microdissection coupled with RNA-SEQ (ribonucleic acid sequencing) was employed to determine transcriptional changes associated with tumour progression. Upregulated genes were triaged for relevance to human LuAd using datasets from Oncomine and cBioportal. Selected genes were validated by RNAi screening in human lung cancer cell lines and examined for association with lung cancer patient overall survival using KMplot.com. Depletion of progression-associated genes resulted in pronounced viability and/or cell migration defects in human lung cancer cells. Progression-associated genes moreover exhibited strong associations with overall survival, specifically in human lung adenocarcinoma, but not in squamous cell carcinoma. The KM mouse model faithfully recapitulates key molecular events in human adenocarcinoma of the lung and is a useful tool for mechanistic interrogation of KRAS-driven LuAd progression