Final Report Summary - SYSTUMS (Systems biology approaches to novel Tumour Suppressors)
Treatment of non-small cell lung cancer is based on histological analysis and identification of targetable driver oncogenes. Patient-specific therapeutic responses are further critically defined by the landscape of passenger mutations, or loss of tumor suppressor genes, the validation of which requires appropriate in vivo model systems. We applied a cell-based senescence screen to search for putative novel tumor suppressors (Lahtela et al. 2013), and identified the EPHA3 receptor tyrosine kinase, one of the most frequently mutated genes in human lung cancers. EPHA receptors constitute the largest family of transmembrane RTKs and critically regulate cell-cell interactions and cell migration. Their role in epithelial cancers is pleiotropic, with dual roles in tumor suppression and progression. This project specifically addressed the putative EPHA3 tumor suppressor functions, through studying cell system responses to manipulation of EPHA3 receptor signalling. Importantly, since EPH receptors critically regulate cell positioning and migration in the native microenvironment, an additional objective was the study of EPHA3’s in vivo function, using genetically-engineered mouse models (GEMMs) of lung cancer.
In support of a tumor suppressor function, we showed that selected EPHA3 lung cancer-associated point mutations decrease the receptor expression level and/or kinase activity. Furthermore, and consistent with public data reporting frequent loss of its 3p11 genomic locus, we found that around 15-20% of lung cancer samples lack EPHA3 protein expression. We further showed that selected intracellular EPHA3 lung cancer-associated point mutations caused a decrease in receptor expression level and/or RTK activity. Importantly, loss of EPHA3 promoted proliferation when key senescence-inducing signals, such as the p53 pathway, were inactivated. These results provided compelling evidence in support of the hypothesis that EPHA3 may act as a bona fide lung tumor suppressor.
To assess this hypothesis in vivo, we studied lung tumor formation in EphA3 null and conditional oncogenic KrasG12D and p53 compound mice. We showed that constitutive loss of EphA3 did however not alter the latency or severity of mutant KRAS- or p53-driven lung adenocarcinomas. We cannot rule out the possibility that sustained expression of oncogenic KRAS is functionally linked to decreased EPHA3 activity. However, this most likely implies functional redundancy of lung-expressed EPH receptors, or lack of EPHA3 function in adult murine lungs. Hence, we undertook a detailed comparative expression analysis, and demonstrated expression of EPHA3 uniquely in the embryonic lung distal mesenchyme. We are currently addressing how loss of EPHA3 affects lung branching morphogenesis. Importantly, our data suggest that the in vivo function of murine EPHA3 may differ from its role in human lung tissue, warranting ethical use of the constitutive EphA3 null GEMM. Our data further provide an incentive for the design of more versatile knock-in or conditional mouse models, to unequivocally assign how EPHA3 mutations control lung development, and tumorigenesis.
In support of a tumor suppressor function, we showed that selected EPHA3 lung cancer-associated point mutations decrease the receptor expression level and/or kinase activity. Furthermore, and consistent with public data reporting frequent loss of its 3p11 genomic locus, we found that around 15-20% of lung cancer samples lack EPHA3 protein expression. We further showed that selected intracellular EPHA3 lung cancer-associated point mutations caused a decrease in receptor expression level and/or RTK activity. Importantly, loss of EPHA3 promoted proliferation when key senescence-inducing signals, such as the p53 pathway, were inactivated. These results provided compelling evidence in support of the hypothesis that EPHA3 may act as a bona fide lung tumor suppressor.
To assess this hypothesis in vivo, we studied lung tumor formation in EphA3 null and conditional oncogenic KrasG12D and p53 compound mice. We showed that constitutive loss of EphA3 did however not alter the latency or severity of mutant KRAS- or p53-driven lung adenocarcinomas. We cannot rule out the possibility that sustained expression of oncogenic KRAS is functionally linked to decreased EPHA3 activity. However, this most likely implies functional redundancy of lung-expressed EPH receptors, or lack of EPHA3 function in adult murine lungs. Hence, we undertook a detailed comparative expression analysis, and demonstrated expression of EPHA3 uniquely in the embryonic lung distal mesenchyme. We are currently addressing how loss of EPHA3 affects lung branching morphogenesis. Importantly, our data suggest that the in vivo function of murine EPHA3 may differ from its role in human lung tissue, warranting ethical use of the constitutive EphA3 null GEMM. Our data further provide an incentive for the design of more versatile knock-in or conditional mouse models, to unequivocally assign how EPHA3 mutations control lung development, and tumorigenesis.