GOLPH2(GP73) Expression, Response To Interferon α In Liver Cancer And The Underlying Mechanism

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignancies worldwide. It has ranked the sixth most important cancer in terms of numbers of cases and the third in terms of cancer mortality in the world and become the second cancer killer in China since1990s. Due to the advance of diagnostic techniques and its therapeutic methods in the past several decades, the HCC outcome is improved in a proportion of patients who were diagnosed at an early stage and received curative treatments. To date, surgery with curative potential remains the most effective treatment. However, only about10to20%of patients with HCC are currently eligible for surgical intervention at the time of diagnosis. In addition, patients who undergo curative resection often have a high rate of recurrence with the rate still up to60-70%in5years. Therapies such as transcatheter arterial chemoembolization (TACE), interferon alfa (IFNa) and sorafenib may prolong survival in some patients. However, the response is often not satisfactory because of the unpredictable tolerance to the side effects of these treatments among patients and the limited ability to identify patients who are most likely to benefit from such targeted adjuvant therapies. There is a need for molecular tools that can be used to stratify patients with respect to prognosis and response to therapy and improve the effects of these treatments.IFNa is an important tumor immunotherapy drug, as first-line drugs are widely used in cancer treatment. It has been approved by FDA for the treatment of various viral diseases and cancer. Randomized controlled study conducted by our institute to evaluate the effectiveness of IFNa in the prevention of recurrence after curative resection has indicated that that18months of IFNa treatment delayed recurrence and improved overall survival (OS). And the result has been confirmed by other Randomized controlled studies in other medical centers. However, IFNa in the clinical treatment of long-term administration of large doses required, as so often lead to acute and chronic toxicity, damage to the nervous system and blood system and other significant side effects, has seriously hampered its wide application in clinical practice.On the other hand, many patients failed to achieve the desired benefits when received IFNa treatment. So it is imporntant to stratify patients with respect to prognosis and response to IFNa and improve the effect of IFNa by combining other interventions in clinical treatment of HCC patients.In our previous studies, by comparing the gene expression profiles between40HCCs with and without intrahepatic metastases using cDNA micorarray, we proposed for the first time a new hypothesis that genes favoring HCC metastasis and progression were likely initiated from the primary tumors (Ye QH, et al. Nat Med2003). Based on the previous study, recently, the laser capture microdissection (LCM), gene expression array were introduced to explore the genomic aberration of paired HCCs with and without extra-hepatic metastasis. The new hypothesis that genes favoring HCC metastasis are initiated in primary tumors got further validated and some important candidate genes associated with HCC extrahepatic metastasis were disclosed. Of them, type II Golgimembrane protein (GOLPH2) was found to be one of the leading gene favoring HCC extra-hepatic metastasis. It has been reported that GOLPH2is an accurate serum marker for the detection of HCC and its recurrence after surgery, with comfortable sensitivity and specificity (Mao YL, et al. Gut2010). Moreover, we found that the GOLPH2expression status is associated with survival and response to adjuvant therapy with IFNa. High expression of GOLPH2could inhibit cellular resposes to IFNa via activating the ligand-independent pathway for the phosphorylation and subsequent ubiquitination and accelerated degradation of the IFNaRl chain of Type I IFN receptor. Thus, GOLPH2can be used as a predictive serum marker for outcome of postoperative IFNa treatment in patients with hepatocellular carcinoma and intervention of GOLPH2may reduce the clinical dose of IFNa and improve the beneficial effects of IFNa treatment in the future.PART ONEGOLPH2Is a Predictive Marker for Outcome of Postoperative Interferon-a Treatment in Patients with Hepatocellular CarcinomaPurpose:Postoperative interferon-a (IFNa) therapy improved survival in patients with hepatocellular carcinoma (HCC). The aim of this study was to evaluate the importance of the type II Golgimembrane protein (GOLPH2) levels as a predictive marker of outcome that will help to select patients who are most likely to benefit from treatment.Patients and Methods:An immunohistochemical study of GOLPH2was performed on specimens that were collected from patients in a randomized trial who received postoperative IFNa therapy (Group1) and who did not receive postoperative IFNa therapy (Group2).Results:Fifty-five patients were positive for GOLPH2, and eighty-seven patients were negative for GOLPH2. The clinicopathologic data were comparable between patients with GOLPH2-negative and GOLPH2-positive staining. Overall survival (OS) in GOLPH2-negative patients were better than that in GOLPH2-positive patients (OS, P=.030). OS in GOLPH2-negative patients from Group1was better than that in patients with GOLPH2-negative patients from Group2(OS, P=.001) but did not differ when GOLPH2was positive. Cox proportional-hazards regression to evaluate the effect of treatment on survival in patients who had low GOLPH2expression showed that in both univariate and multivariate analyses, IFNa was associated with significant improvement in survival.GOLPH2expression emerged as an independent predictor of the response to IFNa.Conclusions:GOLPH2was useful as a predictive marker of outcome after postoperative IFNa treatment in patients with HCC.PART TWOThe Imortance of GOLPH2In Regulating The Efficiency of IFNa In Vivo And Vitro StudiesBackground and Purpose:GOLPH2was useful as a predictive marker of outcome after postoperative IFNa treatment in patients with HCC. The aim of this study was to evaluate the importance of it in regulating the efficiency of IFNa in vivo and vitro.Methods:The expression levels of GOLPH2mRNA in different HCC cell lines with different response to treatment of IFNa were assessed by qRT-PCR.Lentivirus-mediated RNAi or over-expression was used to obtain a stable HCC cell line with low or high expression of GOLPH2. Both in vitro cell proliferation assay, and in vivo tumor growth were investigated in the absence or presence of IFNa treatment to validate the functional role of GOLPH2in regulating the efficiency of IFNa.Results:Both the mRNA and protein expression levels of GOLPH2in two IFNa-resistant HCC cell lines (HCC-97H and MHCCLM3) were found to be significantly increased in comparison to three IFNa-sensitive HCC cell lines(HepG2, PLC and BEL-7402). Gain-and loss-of-function studies both in vitro and in vivo validated GOLPH2as a potent regulater of the efficiency of IFNa treatments. Overpression of GOLPH2in BEL-7402resulted in reduced antiproliferative effects of IFNa, while the knock-down of GOLPH2in HCC-97H enhenced the cytostasis mediated by IFNa. When GOLPH2-cDNA but not mutant-GOLPH2-cDNA was reintroduced into the GOLPH2knock down HCC-97H cells, it could make a rescue in vitro proliferation agaist IFNa treatments. In vivo, the similar resuilt was seen in growth of the tumor treated by IFNa, when stable HCC cell lines with low or high expression of GOLPH2mediated by lentivirus RNAi or over-expression were separately transplanted into nude mice subcutaneously, and orthotopic implantation of intact subcutaneous tumor tissue derived form indicated cell lines respectively, before conduting the IFNa treatment a week later.Conclusions:Down regulation of GOLPH2could enhence the efficiency of IFNa, while up regulation of GOLPH2reverse this.PART THREE Proliferative Resistance Mechanisms of GOLPH2on IFNa TreatmentBackground and Purpose:IFNa is a multiple functional cytokine whose biological fuctions are often mediated by many different signaling pathways. The Jak/Stats signaling pathway is thought to be a pivotal one in mediating its antiproliferative effects on many other tumor cell lines. The aim of this part was to explore the mechanisms underlying proliferative resistance on IFNa treatment regulated by GOLPH2.Methods:Lentivirus-mediated RNAi or over-expression was used to obtain a stable HCC cell line with low or high expression of GOLPH2. Further study was conducted to detect the transcriptional and post-translational expression of the key moleculars in the Jak/Stats signaling pathway. Luciferase activity of ISRE activated by IFNa and the expressions of level of P48(ISGFy, IRF9) protein were also estimated by Luciferase Reporter Assay and by confocal microscopy separately.Results:RT-PCR and western blotting analysis showed that there was no difference about key moleculars in the Jak/Stats signaling pathway at the transcriptional level, such as IFNa receptor, STAT1and STAT2, while significant difference of IFNaRl was seen at the protein leval. Further studies showed that HCC cells with high expression of GOLPH2consistently maintained a lower level of IFNaRl expression and displayed no proper response to IFNa, while GOLPH2-lacking cells exhibited a proper response to IFNa treatment. GOLPH2could inhibit IFNa signaling and promote phosphorylation of IFNαR1degron (S535), and ensuing degradation of IFNaRl.Conclusions:GOLPH2downregulates IFNaRl leading to the avoidance of extracellular IFNa signal transduction.