Mechanism Of Gankyrin Promoting Colorectal Cancer Hepatic Metastasis

Colorectal cancer (CRC) remains to be one of the most lethal forms of cancersworldwide. Synchronous hepatic metastases are detected in25%of all CRC patientsand additional25%CRC patients without distant metastasis at initial diagnosissubsequently develop hepatic metastasis. Attempted chemotherapy for hepaticmetastatic CRC has been showing a disappointing patient responsiveness rate. theefforts for screening for cellular molecules that are responsible for CRC progressionin recent years have added several proteins that could be targeted pharmaceutically.Gankyrin (also known as PSMD10or p28gank or nas6) was initially identified asthe p28component, a regulatory subunit of the26S proteasome complex. Gankyrinconsists of seven ankyrin repeats, which are required for protein-protein interactions.Multiple mechanisms by which Gankyrin acts as an oncoprotein have been proposed:i) Gankyrin enhances Rb phosphorylation and inactivation, and dis-engages E2Fsfrom Rb repression complex. E2F, as an oncoprotein, initiates the expression of geneswhich encode proteins required for cell cycle progression. ii) Overexpression ofGankyrin promotes oncogenic transformation in NIH3T3cells. iii)Gankyrin targetsp53for degradation through ubiquitination-proteasome pathways.Gankyrin interactswith several key molecules of significant signaling pathways such as p53, Rb,and etc,and plays critical roles in cell transformation, apoptosis and cell cycle control.The expression of Gankyrin has been investigated in a variety of cancers withnearly universal findings that showed elevated protein and/or mRNA levels in cancerscomparing to normal counterpart. Our immunohistochemistry (IHC) results of clinicaltissue samples of colon cancers showed that Gankyrin is obviously overexpressed incolon cancer tissues compared to adjacent normal tissues.Although oncoprotein Gankyrin has been implicated in cancer metastasis, itsexact role and underlying mechanisms of CRC hepatic metastasis remain largelyunknown. Herein, we demonstrated that the expression of Gankyrin was significantlyhigher in the primary tumors from48cases with hepatic metastasis comparing to cancer tissues from33cases without hepatic metastasis. The expression levels ofGankyrin were significantly upregulated in the nucleus of CRCs with hepaticmetastasis compared to those without metastasis.Our observation that Gankyrin expression correlates with hepatic metastasispromoted us to further investigate the role of Gankyrin in cellular migration andinvasion. To do so, we performed Transwell migration assay using SW480cellsstably expressing Gankyrin shRNA (shGankyrin) or control shRNA (shControl). Wefound that Gankyrin knockdown reduced the number of migrating cells，suggestingthat Gankyrin is required for cell movement in cancer cells, a key aspect of cancermetastasis.In order to evaluate the effect of Gankyrin in tumor metastasis in vivo, weadopted a well-established spleen-hepatic metastasis model. We transduced Gankyrinknockdown and control SW480cells with a retroviral plasmid DNA encodingluciferase in order to visualize the metastasis. We found that the “primary” tumorswere detected in spleen of both Gankyrin knockdown and control groups and hepaticmetastasis was only present in mice implanted with SW480control group. Theimmune-deficient mouse models are ideal for evaluating tumor growth using humancancer cells, however, these models do not reflect the nature of colon cancer in hostsdeveloping antitumor immune response. Two murine colon carcinoma cells weretested in immune-competent allograft models. We found that knockdown of Gankyrinin Two murine colon carcinoma cells also significantly reduced hepatic metastasis.These results suggest that Gankyrin expression is required for hepatic metastasis ofCRC in vivo.To clarify exact role and underlying mechanisms of CRC hepatic metastasis,we performed genome-wide screening using SW480cells with Gankyrin knockdownon an Affymetrix gene expression array. Ingenuity Pathway analysis revealed that theMolecular and Cellular Functions were affected by Gankyrin knockdown. The genesregulated by Gankyrin also populated canonical pathways including IL-8Signaling,AMPK Signaling, PI3K/AKT Signaling, and etal. It is worth noting that IL-8Signaling, including IL-8and Cyclin D1, has been linked to colorectal cancermetastasis. The protein levels of IL-8and cyclin D1, the two important molecules inthe IL-8pathway, were positively correlated to Gankyrin expression in human CRCspecimens. Furthermore, genetic deletion of Ccnd1gene demonstrated therequirement of cyclin D1in Gankyrin-mediated cell migration and recombinant IL-8 protein rescued the migratory defect of CRC cells with knockdown of Gankyrin.Our findings highlight the importance of Gankyrin in hepatic metastasis ofCRC and suggest that Gankyrin is a potential prognostic marker and therapeutic targetin clinical management of metastatic CRC.