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

Neidler, Sarah (2015) Identification of tumour progression genes in a mouse model for non-small cell lung cancer. PhD thesis, University of Glasgow.

Abstract

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.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: NSCLC, lung cancer, lung ADC, mouse models, GEMM, Myc, Ras, p53, cancer, tumour progression, p-Erk, AdenoCre, conditional alleles, RNA-Seq, Next-Generation Sequencing, RNA, transcriptome analysis, identification of early diagnostic markers, Wnt pathway, ErbB signalling, therapeutic targets, FFPE, Illumina sequencing, Laser Capture, LCM, PCR, IHC, tumourigenesis, Incucyte, quantitative Real Time PCR, Intranasal installation, Neratinib, LGK974, siRNA, oncogenes, Rosa26, Gene expression analysis
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QH Natural history > QH426 Genetics; R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences > Beatson Institute of Cancer Research
• Supervisor's Name: Murphy, Dr. Daniel
• Date of Award: 2015
• Depositing User: Ms Sarah Neidler
• Unique ID: glathesis:2015-7111
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 02 Mar 2016 14:57
• Last Modified: 20 Feb 2019 09:51
• URI: https://theses.gla.ac.uk/id/eprint/7111