Tumour suppressor function for the AP-1 transcription factor c-Jun in Ras-induced lung cancer
General Genetics and Functional Genome Biology
Biochemistry
Final Report Abstract
Lung cancer is the leading cause of cancer-related death in the world, claiming an estimated 1.8 million lives in 2018 (The Cancer Atlas). The lack of marked improvement in survival rates over the past decade places lung cancer as a major area of unmet need. Lung adenocarcinoma is the most common type of lung cancer and research into the molecular mechanisms driving this disease will enable promising drug targets and potential treatments to be identified. Abnormal activation of the Ras signalling pathway commonly occurs in human lung adenocarcinomas, with 33% of cases harbouring mutations in K-Ras. K-Ras mutations, such as K-RasG12D, constitutively activate growth factor signalling pathways and drive uncontrolled cell growth, proliferation and invasiveness, and are believed to play a key role in lung carcinogenesis. However, since direct targeting of oncogenic Ras has proved extremely challenging, a detailed understanding of Ras downstream pathways is critical to enable targeted therapy for Ras-driven tumours. Among its many outputs, oncogenic Ras signalling stimulates the AP-1 transcriptional activator family, which in turn controls a vast suite of genes involved in proliferation, migration and apoptosis. AP-1 family transcription factors are dimeric complexes, composed of various Fos and Jun proteins. The three Jun proteins, c-Jun, JunD and JunB, can either homodimerise, or heterodimerise with the Fos family of AP-1 transcription factors to form a dimeric AP-1 complex, and each shows subtle but important differences in regulation and output. Since its discovery as the cellular counterpart of a viral oncoprotein, c-Jun has had a wellestablished role in tumourigenesis and is considered as a classical proto-oncogene. Studies in cultured cells demonstrated that c-Jun is required for Ras-mediated oncogenic transformation. c-Jun cooperates with oncogenic Ras, whereas JunD partially suppresses transformation by activated Ras in MEFs. A mutant allele of c-Jun, which has the JNK phosphoacceptor serines 63 and 73 changed to alanines (c-JunAA), reduced oncogenic transformation caused by constitutive activation of the Ras pathway in immortalized fibroblasts and inhibited skin tumour development, indicating a pro-tumourigenic role of c-Jun N-terminal phosphorylation in Ras-induced transformation. The importance of AP-1 transcription as an output of Ras signalling suggested that c-Jun could be that be crucial for other tumour types, particularly those driven by oncogenic Ras, such as lung adenocarcinoma. In this study, we aimed to investigate the requirement for c-Jun in a well-established mouse model of K-RasG12D-induced lung adenocarcinoma. We simultaneously activated the oncogenic K-RasG12D and inactivated c-Jun in the adult lung. Surprisingly, we found that inactivation of c-Jun, or mutation of its JNK phosphorylation sites, actually increased lung tumour burden. We observed a similar effect in K-RasG12D/p53∆/∆-induced lung adenocarcinoma. Moreover, a partially inactive c-Jun S63A, S73A N-terminal phosphorylation mutant allele, c-JunAA, phenocopied c-Jun deletion, suggesting an essential role for JNK signalling. Mechanistically, we found that protein levels of the Jun family member JunD were increased in the absence of c-Jun. In c-Jun-deficient cells, JunD phosphorylation was increased, and expression of a dominant-active JNKK2-JNK1 transgene further increased lung tumour formation. Strikingly, deletion of JunD completely abolished Ras-driven lung tumourigenesis. This work identifies JunD, not c-Jun, as the crucial substrate of JNK signalling and oncogene required for Ras-induced lung cancer.
