Gankyriji-Mediated Dedifferentiatipn Facilitates Hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is one of the most common cancers and the thirdmost frequent cause of cancer death in the world. The high heterogeneity of HCC makes itdifficult to eliminate the cancer cells with chemotherapy alone. Meanwhile, surgicalresection and liver transplantation are limited to early stage of HCC. Late diagnosis,recurrence and metastasis result in the poor prognosis of HCC worldwide. Five-yearsurvival rate of patients undergone surgical resection is disappointedly low. It is necessaryto elucidate the underlying mechanisms of hepatocarcinogenesis and identify novel targetsfor HCC prevention and therapy.Hepatocyte dedifferentiation is a key cellular event during hepatocarcinogenesis,which is featured by alteration of morphology and loss of hepatic function. Hepatoma hasbeen characterized as a heterogeneous complex that includes undifferentiated cells withinfinite proliferative capacity. Induction of hepatoma cell differentiation has beenconsidered as a promising strategy for HCC treatment. Clinicopathological studies havedemonstrated that poorly differentiated HCCs are associated with poorer convalescence,and that a higher differentiation grade of HCCs is related to a lower recurrence rate.Assessment of differentiation grades of HCCs is not only important for the evaluation ofpathological diagnosis and prognosis, but also critical for the optimization of therapeuticstrategies.The maintenance of liver architecture and hepatic function is cross-regulated by a setof liver enriched nuclear factors termed hepatocyte nuclear factors, which includes HNF1α,HNF3β and HNF4α, etc. Among the members of HNF family, HNF4α is most closelyassociated with the differentiation status of HCC. Our previous study has indicated thatforced re-expression of HNF4α was, to some extent, sufficient to induce the differentiationof hepatoma cells into hepatocytes.Gankyrin, also named as26S proteasome non-ATPase regulatory subunit10(PSMD10), was reported to be an oncoprotein principally overexpressed in human HCC by our and other studies. It has been documented that the interaction of gankyrin and CDK4facilitates the degradation of Rb. Gankyrin also binds MDM2directly and acceleratesMDM2-dependent ubiquitination and degradation of p53. Fujita at el. showed thatIGFBP-5levels were up-regulated by gankyrin in hepatoma cells and their data suggestedthat gankyrin may play an oncogenic role at the early stages of humanhepatocarcinogenesis. We also reported that gankyrin binds RelA in the nucleus of HCCcells and transports NF-kB back to cytoplasm, thus inhibiting the activation of NF-kB. Ourmost recent data also clarified p28GANKover-expression accelerates hepatocelluarcarcinoma invasiveness and metastasis via PI-3K/Akt/HIF-1α pathways and HCC patientswith high gankyrin expression in general had worse prognosis than those with lowgankyrin expression, which indicates gankyrin could be a candidate gene for riskprognostication of HCC.However, the role of gankyrin in the differentiation of hepatocytes or hepatoma cellshas not been reported so far. In this study, we clarified, for the first time, that gankyrinexpression was inversely correlated with the differentiation of HCC and over-expression ofgankyrin significantly enhanced the degradation of HNF4α in hepatoma cells, whichindicates that gankyrin-mediated dedifferentiation of hepatocytes and hepatoma cells wasat least partially via an HNF4α-dependent mechanism. Moreover, we showed thatdifferentiation induced by gankyrin interference dramatically reduced the proportion ofcancer stem cells (CSCs) in hepatoma cells and induced the differentiation of hepatomacells, implying a novel strategy for HCC prevention and differentiation therapy. Liver cancer is a malignant tumor with dismal prognosis, and half patients withHCC reside in China. Up to now, surgical resection remains as the primary treatment forpatients with HCC. For those with severe liver cirrhosis, however, alternative therapy isdesirable due to poor physical condition. Recent findings suggested that aberrantexpression of oncogenes and growth factors were closely related with malignanttranformation and metastatic invasion of hepatoma cells, but effective therapeutic targetsof HCC remain obscure. Therefore, it is urgent to illustrate the mechanism underlyinghepatocarcinogenesis and elicit target for prevention therapies as well as prognosticmarkers.Previous studies preferentially focused on the regulation of particular gene expressionand protein synthesis while little is known about the protein degradation process. It isrecently acknowledged that degradation plays an essential role in protein turnover to helpmaintain cellular balance. Strictly regulated, protein degradation system is involved inmany diseases, including tumor development. Key mediators in the process are believed tobe important targets for treatment and crosstalk with other signaling pathways arecommonly observed.There are two different degradation pathways in cells: the lysosome pathway and theubiquitin-proteasome pathway. While lysosome is responsible for the degradation ofmembrane proteins and autophagy, the ubiquitin-proteasome system (UPS) is capable ofmaintaining cellular function by selective degradation of cytoplasmic proteins.Malfunction of UPS is frequently observed in tumorigenesis. Dysregulation of proteinubiquitination leads to elevated expression of oncoproteins and down-regulation oftumor suppressors. Notably, the UPS not only functions in protein degradation, but alsocross-talks with other signaling pathways. For instance, our laboratory previouslydemonstrated that p28GANK(also known as Gankyrin, encoded by26S proteasomenon-ATPase regulatory subunit10) was up-regulated in HCC tissues, releasingtranscription factor E2F1via suppressing phosphorylation of Rb.Our previous studies on mRNA expression microarray of70cases of para-tumortissues and tumor samples of HCC patients demonstrated that PSMD4(Proteasome26S non-ATPase regulatory subunit4) was aberrantly over-expressed in HCC tissues. In ourcurrent study, PSMD4was found to be closely related with malignant transformation viaRas/Raf/MAPKs and PI-3K/Akt signaling cascades in hepatoma cells. This project willfocus on the role of PSMD4in the malignancy of HCC and its molecular mechanismsusing lentivirus-delivered microRNA targeting PSMD4. The results not only shed newlight on the mechanism of hepatocarcinogenesis but also provide new target for diagnosisand treatment of HCC.