ARKADIA AND CANCER · CHARACTERISATION OF ARKADIA FUNCTION IN THE REGULATION OF SNON STABILITY AND ITS ROLE AS A POTENTIAL TUMOUR SUPPRESSOR

Characterisation of Arkadia Function in the Regulation of SNON Stability and its Role as a Potential Tumour Suppressor

Project objectives Among cancer patients, it has been estimated that around 90 % of deaths are due to complications related to the growth of metastases. In recent years, the study of the metastatic process and the development of new therapeutic strategies to stop it have become a critical part of the battle against cancer. Cancer cells use a wide variety of gene expression programmes to achieve dissemination, allowing them to colonise distant tissues with distinct microenvironments, compared to those present in the primary tumour. Smad proteins are the main effectors of the TGF-beta signalling pathway and they are tightly regulated in order to modulate their transcriptional activity. Ski and SnoN are transcriptional co-repressors that bind the Smads and their overexpression has been linked to several cancer types. Our group discovered that ARKADIA is the ubiquitin ligase that targets Ski and SnoN for proteasomal degradation upon TGF-beta stimulation. Ski and SnoN degradation is required for TGF-beta-induced transcriptional activation of genes that are dependent on activated Smad3-Smad4 complexes, a transcriptional signature often lost in tumour cells. We therefore hypothesised that ARKADIA would be involved in regulating tumourigenesis. The main objective of the project was to dissect the role of ARKADIA in tumourigenesis, investigating both potential tumour suppressive and tumour promoting roles. Results For the study, I selected two cell lines where ARKADIA has two different functional profiles. The cell line NCI-H460 is a lung carcinoma cell line that lacks functional ARKADIA due to a mutation that generates a truncated version of the protein lacking the C-terminus, where the catalytic RING domain is located. Upon ARKADIA replacement in this cell line, we restored TGF-beta-induced Ski and SnoN degradation, and also rescued TGF-beta/Smad3-dependent transcriptional responses. I showed that ARKADIA replacement affects the tumourigenic phenotype in vitro by inhibiting the ability of these cells to form colonies in soft-agar assays. However, restoration of ARKADIA activity does not have any effect on the ability of NCI-H460 cells to develop primary tumours in xenograft assays or to colonise lungs in tail vein assays in nude mice. This suggests that if the loss of ARKADIA was a tumour-driving event in these cells, i.e. that ARKADIA is a tumour suppressor, then additional cancer driver mutations must have been acquired subsequently. In addition, I was unable to identify any other cancer cell lines with functionally inactivating mutations in ARKADIA, suggesting that loss of ARKADIA function in human cancer is very rare. We found that even cell lines exhibiting loss of heterozygosity (LOH) at the ARKADIA locus did not acquire mutations in the other allele. The other cellular model used in this study was chosen to determine what happens to the behaviour of a tumour cell if ARKADIA is functionally inactivated. I selected the breast cancer cell line, MDA-MB-231 where tumour-promoting activity is driven by TGF-beta signalling. I observed that ARKADIA inactivation does not affect primary tumour formation using xenograft assays in nude mice. However ARKADIA inactivation either by knocking down with siRNAs or by stable expression of a dominant negative ARKADIA resulted in a dramatic decrease in the ability of the tumour cells to colonise the lungs when injected into the tail vein of nude mice. I was able to observe this effect just 48 hours after injection of the cells, suggesting that ARKADIA is required for extravasation rather than for survival at the site of metastasis. In support of this view, I found no evidence that ARKADIA is required to regulate cell proliferation in vitro or in vivo. However cells lacking ARKADIA function adhere more tightly to endothelial cells, but spread less efficiently, suggesting that ARKADIA may be required for cells to exit blood vessels and colonise the lungs. Taken together, our data do not support a prominent tumour suppressive role for ARKADIA. Instead, we show that ARKADIA is required for metastasis in a breast cancer model, probably at the level of extravasation. These findings are very important. ARKADIA is an activating component of the TGF-beta signalling pathway, being absolutely required for a subset of downstream responses. Targeting the TGF-beta signalling pathway for cancer therapy has long been an aim of the field, but has been very difficult because the pathway has both tumour suppressive and tumour-promoting roles. Since ARKADIA is an E3 ubiquitin ligase and moreover, is only required for a subset of TGF-beta responses, it might be amenable to inhibition by small molecules, and thus represent a possible therapeutic target for cancer.

Data: CORDIS, © European Union