【作者】 郭磊；

【导师】 叶青海；

【作者基本信息】 复旦大学 ， 外科学， 2013， 博士

【摘要】 肝细胞肝癌(Hepatocellular carcinoma, HCC)(简称肝癌)是世界上最常见、恶性程度最高的肿瘤之一,位居全球恶性肿瘤死因第三位、我国恶性肿瘤死因第二位(全世界半数以上病例发生在我国)。在欧洲和美国,由于受丙型肝炎感染的影响,近年来发病率也呈上升趋势。近年来随着肝癌诊断和治疗技术的进步,部分患者通过早期诊断及早期根治切除获得了较好的疗效,根治切除术后的十年生存率已提高到30%左右。当前,手术切除仍是病人获得长期生存最有效的途径,但目前只有10%-20%肝癌病人在诊断时具有根治性治疗的机会；即使是根治性切除,其术后5年复发率仍高达60%-70%。其他治疗方式,诸如经动脉导管化疗栓塞(TACE)、索拉菲尼(Sorafenib)及干扰素(IFNa)治疗可使一部分患者获益,然而,由于无法区分哪些患者适合这类辅助性治疗及患者对此类药物副作用耐受不一,其治疗效果不尽人意。寻找能够区分和预测患者对此类治疗是否敏感的分子指标及提高药物疗效的分子干预对提高肝癌患者生存具有重要意义。IFNα是重要的肿瘤免疫治疗药物,已获美国FDA批准用于多种病毒性疾病和恶性肿瘤的治疗。包括我所在内的多家中心所开展的很多随机临床对照试验表明肝癌术后长疗程(>18个月)IFNα治疗是减少复发、提高生存的有效方法。然而国内肝癌患者绝大多数伴有乙肝肝硬化背景,肝脏代偿功能较差,血小板和白细胞处于一个较低的水平。术后大剂量长疗程IFNα治疗对患者肝功能和白细胞、血小板均构成了较为严重的影响,也增加了急性和慢性神经系统和血液系统损害等毒副作用,患者耐受性和依从性差。另外,患者对IFNα台疗反应不一,部分患者未能取得预期的治疗效果。如何筛选哪些患者适合IFNα治疗及在现有治疗方法基础上联合新的干预手段提高IFNα治疗敏感性是进一步提高IFNα防治效果的关键。课题组前期将激光捕获显微切割(LCM)和基因芯片技术相结合,研究伴和不伴肝外转移肝癌原发瘤之间基因表达谱的差异,发现了一组以高尔基体Ⅱ型膜蛋白GOLPH2(type Ⅱ Golgi membrane protein)(又称GP73)为首、可能在肝癌转移进程中起重要作用并可能成为肝癌转移重要干预靶点的候选基因。已有报道GOLPH2在肝癌组织中表达显著上调,可作为肝癌潜在诊断指标。本研究中,肝癌患者术后应用IFNα治疗临床随机对照试验显示,GOLPH2低表达的患者,术后辅助应用IFNα治疗能显著延长其生存,而GOLPH2高表达患者术后辅助应用IFNα治疗,生存期未得到明显改善；体内外试验中我们发现,抑制GOLPH2表达后明显增强IFNα的抑肿瘤增殖作用,而高表达GOLPH2明显抑制IFNα的抑肿瘤增殖作用；进一步研究表明,作为高尔基体特异性跨膜蛋白,GOLPH2可能通过调节IFNα受体1(IFNαR1)的降解从而抑制干扰素作用的敏感性。当前,肝癌术后应用IFNα缺乏有效的预测指标,IFNα抗肿瘤作用有待提高,IFNα受体在IFNα抗肿瘤中的作用鲜有报道,这些都制约了IFNα在肝癌临床治疗中的广泛应用。因此,GOLPH2作为潜在的肝癌患者应用IFNα治疗预测指标及干预靶点在临床抗肿瘤研究中具有重要意义。第一部分GOLPH2在预测肝癌术后IFNα治疗效果中的价值目的：评估肝癌术后IFNα随机临床对照治疗试验肝癌标本组织中GOLPH2表达水平对肝癌术后IFNα治疗效果的临床预测价值。方法：通过组织芯片免疫组化染色检测142例肝癌术后应用IFNα治疗随机临床对照试验肝癌组织GOLPH2的表达,分析GOLPH2在肝癌术后IFNα(文中要统一用IFNα治疗组(测试组：n=69)和非治疗组(对照组；n=73)中的表达水平及其与肝癌术后IFNa治疗效果的相关性。结果：142例肝癌组织中,55例GOLPH2表达阳性,87例GOLPH2表达阴性。比较GOLPH2不同表达患者临床病理资料,发现GOLPH2阴性肝癌术后接受IFNα治疗(测试组)比不接受IFNα治疗(对照组)可获得更好的总体生存时间(OS,P=0.001)；但GOLPH2阳性肝癌接受IFNα治疗与否其OS没有明显差异。Cox比例风险回归单因素／多因素分析均表明IFNα治疗能显著延长GOLPH2阴性肝癌患者的生存。结论：GOLPH2可作为肝癌术后IFNα治疗效果的有效预测指标。第二部分肝癌细胞GOPH2表达水平及其干预在体内外实验中对IFNα治疗效果的影响目的：通过慢病毒载体介导的RNA干扰和过表达体系观察GOLPH2不同表达水平肝癌细胞系对IFNα治疗的敏感性及其在体内外不同环境中的抑肿瘤作用。方法：运用实时荧光定量PCR技术及Western blot技术比较分析GOLPH2表达水平与IFNα治疗效果的关系。通过慢病毒介导的RNA干扰／过表达技术构建GOLPH2不同表达的肝癌细胞株,经体内外功能实验明确GOLPH2表达水平及其干预对IFNα治疗效果的影响。结果：qRT-PCR及Western blot结果显示,对IFNα干预敏感肝癌细胞系(HepG2、PLC和BEL-7402)其GOLPH2mRNA和蛋白水平均显著低于对IFNα抵抗细胞系(HCC-97H和MHCC-LM3)。体外实验通过慢病毒载体介导的RNA干扰和过表达体系获得稳定的GOLPH2高、低表达肝癌细胞稳转株HCC-97H、 HCC-97H-shGOPH2以及BEL-7402、BEL-7402-GOLPH2,发现对IFNα敏感细胞系BEL-7402过表达GOLPH2后对IFNα治疗抵抗,而IFNα抵抗肝癌细胞系HCC-97H抑制GOLPH2表达后对IFNα治疗敏感。体内实验发现皮下接种BEL-7402、7402-GOLPH2或HCC-97H、HCC-97H-shGOPH2细胞,并于第二周进行IFNα干预,过表达GOLPH2后IFNα抑制肝癌生长的作用明显减弱；而降低GOLPH2后小剂量IFNα就能抑制肝癌细胞的生长。在原位肝癌移植瘤模型中,对HCC-97H-shGOPH2进行回补并IFNα干预,发现回补GOLPH2能恢复对IFNα干预的抵抗,而回补GOLPH2的突变体不能恢复其对IFNα的抵抗。结论：GOLPH2的不同表达水平显著影响IFNα在体内外的抗肿瘤作用,下调GOLPH2明显促进IFNα的抗肿瘤作用,有望成为提高IFNα抗肝癌治疗效果的干预靶点。第三部分GOLPH2影响IFNα肝癌治疗效果的机制目的：探索GOLPH2不同表达影响IFNα肝癌治疗效果的可能机制,为潜在的分子干预提供理论基础。方法：通过慢病毒介导的RNA干扰/过表达技术构建GOLPH2不同表达肝癌细胞株,qRT-PCR和Western-blot等技术检测IFNα经典信号通路中多种分子转录和蛋白水平的变化。通过蛋白质谱分析GOLPH2可能调控的干扰素相关蛋白并进行功能分析。通过双荧光素酶报告基因测试系统分析GOLPH2的不同表达能否影响IFNα信号通路中PISRE启动子的结合及IFNα调控因子(IRFs)如P48的表达,探讨GOLPH2不同表达影响IFNα作用效果的分子机制。结果：不同肝癌细胞系IFNα干预12小时后,对IFNα敏感肝癌细胞BEL-7402、PLC、HepG2其IFNaR1高表达,刺激后STAT1、STAT2及IRF9(P48)显著上调；不敏感肝癌细胞系HCC-97H、MHCCLM3其IFNaR1低表达,干预后STAT1、 STAT2及IRF9(P48)无明显变化。BEL-7402过表达GOLPH2后,IFNaR1转录水平没有差异,但蛋白水平有所降低；HCC-97H降低GOLPH2后,IFNαR1转录水平没变化,蛋白水平有所升高,说明GOLPH2参与了IFNαR1的降解。蛋白质谱分析显示GOLPH2有可能调控IFNαR1,进一步研究发现,GOLPH2通过AP2调控IFNαR1的泛素化降解从而抑制IFNα的相关功能。双荧光素酶报告基因测试显示GOLPH2的不同表达影响了IFNα信号通路中ISRE启动子的结合,荧光共聚焦及RT-PCR和Western-blot显示GOLPH2能影响IFNα调控因子IRF9(P48)的表达及分布。结论：GOLPH2通过影响IFNαR1降解及其下游信号通路影响干扰素抗肝癌作用。更多还原

【Abstract】 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.更多还原