The Role of Ring domain AP-1 Coactivator (RACO)-1 in Lung cancer
Final Report Abstract
Lung cancer is responsible for the most cancer-related deaths worldwide, with total mortality estimated at 1.37 million in 2008 [WHO Globocan 2008]. Adenocarcinoma, a form of non small cell lung cancer, is the most common type of lung cancer in humans, but its initiation and progression are poorly understood. K-Ras mutations are found in 35% of non small cell lung cancers and are believed to play a key role in this malignancy. Oncogenic K-Ras mutations constitutively activate the Ras-Raf-MEK-ERK cellular signalling cascade, and this hyperactivation is commonly found in many cancer types, highlighting its role in proliferation control. Inducible expression of an active mutant K-RasG12D in mice has been shown to provide a valuable model to study the function of oncogenic Ras, one example being lung carcinogenesis. Mutant K-RasG12D expression in this tissue accurately models the process of lung tumourigenesis, from hyperplasia to adenocarcinoma. It has been known for over a decade that c-Jun is an important component of Ras-mediated tumourigenesis, since fibroblasts lacking c-jun are resistant to transformation by oncogenic Ras. The AP-1 transcription factor is a heterodimeric complex of various Jun, Fos and CREB/ATF-2 family members and mediates diverse cellular responses ranging from cell proliferation, migration and differentiation to tumourigenesis and cellular apoptosis. c-Jun is essential for cellular proliferation and transformation, by controlling cell cycle regulator genes including cyclinD1 and cdc2. Consequently, c-jun null fibroblasts display severe proliferation defects and deficiency in cell cycle re-entry after serum withdrawal. We have recently described a novel c-Jun coactivator, RING domain AP-1 co-activator 1 (RACO-1), that links growth factor/oncogenic Ras signalling to AP-1 activation. Ras activation stimulates RACO-1 function by increasing protein stability. Mechanistically, RACO-1 stability is controlled by the competition of degradative K48- and non-degradative K63-linked ubiquitination. Upon activation of the Ras-Raf-MEK-ERK pathway, K63-linked ubiquitin chains are attached to the same residues targeted for degradative K48-linked ubiquitination, thereby resulting in enhanced protein levels and consequent upregulation of c-Jun target genes. RACO-1 depletion reduced cellular proliferation and downregulated several growth-associated AP-1 target genes, such as cdc2, cyclinD1 and hb-egf. On the other hand, transgenic overexpression of RACO-1 augmented intestinal tumour formation triggered by aberrant Wnt signalling and cooperated with oncogenic Ras in colon epithelial hyperproliferation. This work identified RACO-1 as the much sought-after missing link between Ras signalling and AP-1 activation. Given that RACO-1 is downstream of the Ras- Raf-MEK-ERK pathway and regulates the transcription of AP-1 target genes associated with cell proliferation, we have hypothesised that RACO-1 is an important component of the Rasc-Jun pro-tumourigenic pathway. In this study we wanted to address the role of RACO-1 in non-small cell lung cancer and investigate if RACO-1 is required for and cooperates with mutant K-Ras to drive tumour formation and progression. Using a conditional mutant K-Ras-driven mouse model of non small cell lung cancer, we were able to show that RACO-1 overexpression cooperates with K- RasG12D in promoting tumourigenesis, increasing tumour burden, tumour grade and tumour cell proliferation. Using stable knockdown of RACO-1 in human lung cancer cell lines, we demonstrated that cells with reduced RACO-1 levels proliferate more slowly and were less able to initiate tumours in xenograft experiments. Surprisingly, however, loss of RACO-1 did not correlate with lower expression of c-Jun, a finding that is now being investigated in more detail. We have now also generated an inducible knockout mouse model for RACO-1 and demonstrated that the targeted allele can be successfully recombined in vitro using adenoviral infection with Cre recombinase. In combination with intratracheal administration of adenovirus, a technique that we have newly adopted and optimised for use in this institute, this is an ideal tool with which to investigate the requirement for RACO-1 in lung cancer in vivo.
