【作者】 李云峰；

【导师】 杨连粤； 袁志民；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 肝细胞癌(HCC)是全球最为常见的恶性肿瘤之一,2002年全球HCC新发病例数为62.6万,而年死亡人数达59.8万,死亡人数与发病人数比接近1：1,预后相当恶劣。中国每年HCC死亡病例数占全球总数的55%,HCC死亡率已达20.37／10万,居国内恶性肿瘤死亡率第二位。综合分析究其原因,其中HCC的高侵袭转移能力是导致其不良预后的关键原因之一。深入探讨HCC侵袭转移的分子机制,阐明在侵袭转移中起关键作用的基因,有助于发现新的HCC预后标志物和研制新的有效的抗HCC药物。我们在先前的临床实践中发现孤立性大肝癌(SLHCC)较结节性肝癌(NHCC)有着明显较低的侵袭转移潜能和较好的预后。在这个非常有意义的临床发现的基础上,我们以SLHCC为切入点对HCC侵袭转移潜能的内在分子机制进行了一系列研究。我们采用了基因芯片的技术分析了在SLHCC和NHCC存在显著差异表达的基因,发现Ackl (Activated Cdc42-associated tyrosine kinase 1)在NHCC中的表达水平较SLHCC显著升高,提示其可能为一个与HCC侵袭转移相关的基因。Ack1基因定位于人染色体3q29,其编码蛋白大小约120KD,主要定位于细胞胞浆,其分子结构包括一个激酶区、一个SH3区、一个Cdc42/Rac结合区和一个富含脯氨酸的羧基端。Ack1蛋白属于酪氨酸激酶家族,最早作为Cdc42下游的作用蛋白被发现。已有的研究表明,Ack1作为Cdc42下游的效应器参与了多条信号通路,在细胞增殖、运动和侵袭中发挥重要作用。尽管如此,Ack1在恶性肿瘤中作用还远未阐明,其与肿瘤患者的预后关系及肿瘤的临床病理特征的关系尚不清楚,且其在HCC中的作用尚未有研究涉及。基于此,我们在本研究中通过体内和体外两个水平,运用质粒转染、小RNA干扰和裸鼠成瘤等实验方法,对Ack1在HCC侵袭转移中的作用,以及其可能的分子机制进行了一系列研究,主要结果如下：1.我们运用real-time RT-PCR及Western blot方法在检测了38例手术切除的新鲜HCC和相应的邻近非瘤肝组织(ANLT)以及5例正常肝组织(NL)中Ack1 mRNA和蛋白的表达水平。结果显示Ack1 mRNA和蛋白均在HCC中明显高表达(P<0.05)。免疫组化法检测了131例HCC石蜡切片中Ack1蛋白的表达强度。结果显示Ack1在85.5%(112／131)的HCC中表达,根据免疫组化的结果将此131例HCC分为Ack1高表达组和Ack1低表达组并比较两组患者的预后,结果显示Ack1高表达组患者的术后无瘤生存率(P<0.05)和总生存率(P<0.01)皆明显短于Ack1低表达组。进一步将Ack1蛋白表达水平纳入单因素和多因素Cox比例风险回归模型分析。结果显示Ack1的高表达是HCC患者预后的独立危险因素(RR,1.758；P=0.029)。继而我们又进一步分析了Ack1表达水平与HCC不同临床病理特征之间的关系,结果显示Ack1的表达水平与肿瘤结节数目、细胞分化程度以及有无静脉浸润等与HCC侵袭性生长相关的临床病理特征密切相关(P<0.05),并且在NHCC中Ack1的表达明显高于SLHCC。提示Ack1的高表达与HCC的侵袭转移密切相关,并可能因此而导致了HCC患者的不良预后。2. RT-PCR及Western blot方法检测Ackl mRNA和蛋白在HepG2、MHCC97-L和HCCLM3这3种侵袭转移潜能依次递增的HCC细胞系中的表达水平,并以常氏肝细胞系CCL13作为对照。结果显示Ack1 mRNA和蛋白在3种HCC细胞系中的表达皆显著高于CCL13 (P<0.01),且其表达水平在HepG2、MHCC97-L和HCCLM3细胞中依次升高(任意两者比较,P<0.05),提示Ack1的高表达与HCC细胞的侵袭转移潜能密切相关。3.为了寻找Ack1参与HCC的生长和侵袭转移的直接证据,我们接下来采用体内实验进行了验证。我们选取了侵袭转移潜能相对较低的MHCC97-L细胞,利用质粒转染的方法获得了过表达Ack1的MHCC97-L细胞系MHCC97-LAck1,以转染空载体的MHCC97-L细胞系MHCC97-LVector作为对照,并建立了MHCC97-L裸鼠原位HCC生长转移模型,以此观察过表达Ack1对HCC生长和转移的作用。结果显示MHCC97-LAck1组裸鼠所形成的肝脏原位种植瘤的平均体积较MHCC97-LVector组裸鼠明显增大(P<0.05),且有着更高肝内转移率(P<0.05)和肺转移率(P<0.01),提示上调Ack1的表达促进了HCC在体内的生长和转移。4.为进一步深入研究Ack1在HCC中的作用,解释我们在体内实验观察到的Ack1对HCC生长和转移的作用。我们随后进行了一系列体外细胞学实验,从细胞的层面来观察上调Ack1对HCC细胞恶性生物学行为的作用。生长曲线和平板克隆形成实验的结果显示MHCC97-LAckl细胞较MHCC97-LVector细胞的增殖能力显著增强,生长速度显著加快(P<0.05)；划痕愈合实验的结果显示MHCC97-LAck1细胞较MHCC97-LVector细胞迁移能力显著增强(P<0.01)；基质胶侵袭实验显示MHCC97-LAck1细胞较MHCC97-LVector细胞的侵袭能力亦显著增强(P<0.01)。进一步行细胞骨架(F-actin)荧光染色发现显示过表达Ack1以后,MHCC97-L细胞的肌动蛋白细胞骨架发生明显形变。这些结果提示上调Ack1能够增强HCC细胞的恶性表型,促进HCC细胞生长增殖、运动和侵袭能力,从而促进HCC的生长和转移。5.接下来我们进一步研究试图弄清楚Ack1通过何种分子机制促进HCC细胞的恶性生物学行为。鉴于已有的研究证实Crk在肿瘤运动侵袭中发挥着重要作用,并且Crk参与了Ack1的同源蛋白Ack2诱导的细胞运动。因此我们推测Crk亦可能参与了Ack1在HCC中的信号通路。我们首先检测了MHCC97-L细胞中Crk的表达水平,以常肝细胞系CCL13作为对照,结果显示Crk在MHCC97-L细胞中存在明显的表达上调,提示Crk可能在HCC的发生和／或发展发挥了一定的作用。接下来我们用免疫共沉淀的方法证明了在MHCC97-L中Ack1与Crk存在相互作用。随后我们利用小RNA干扰的方法阻断Crk的表达,再重复前面所进行的一系列体内和体外实验,以观察阻断Crk对Ack1诱导的HCC细胞增殖、运动、侵袭和转移的影响。体内和体外的实验显示阻断Crk对Ack1诱导增强的HCC生长和增殖并无显著影响(P>0.05)；而对Ack1诱导增强的HCC运动、侵袭和转移能力皆有明显抑制作用(P<0.05),且进一步研究发现在Crk被阻断的情况下,过表达Ack1不能再对HCC的运动、侵袭和转移产生显著的促进作用(P>0.05),提示Crk参与了Ack1诱导的HCC运动、侵袭和转移的信号通路。通过以上的研究,我们发现了Ack1在HCC中表达上调并且在HCC的侵袭转移中发挥了重要作用。我们还首次证实了Ack1的高表达与肿瘤患者的不良预后以及侵袭转移相关的临床病理特征密切相关；并且首次证实了过表达Ack1通过Crk信号通路实现促进肝细胞癌的侵袭转移。我们的研究提示,Ack1可作为一个新的HCC预后标志物和潜在的侵袭转移干预靶点。更多还原

【Abstract】 Hepatocellular carcinoma (HCC), one of the most common malignancies in China and worldwide, has a very high mortality and poor prognosis. The poor prognosis of HCC is mainly due to its high potential of invasion and metastasis. Clarifying the molecular mechanisms underlying the invasion and metastasis of HCC and find "key genes" in this process will help to better understand the progression of HCC and therefore provide potent prognostic biomarkers and molecular targets for developing effective drugs against HCC. But until now, the invasion and metastasis process of HCC is still not fully understood and need more investigation to give us more detailed information. During our long-term clinical practice, we have previously identified a specific subtype of hepatocellular carcinoma, which we categorized as solitary large hepatocellular carcinoma (SLHCC, one nodule, and diameter>5 cm), had a lower tumor recurrence and metastasis rate and a better outcome compared with nodular hepatocellular carcinoma (NHCC, nodule number≥2, and diameter>5 cm). Based on this interesting clinical finding, we have previously carried out a series of studies on the mechanisms underlying the invasiveness and metastasis of HCC. By using cDNA microarray analysis to identify differentially expressed genes between SLHCC and NHCC, we found a number of genes which were significantly up-regulated in NHCC as compared with SLHCC, including Ackl.Ackl (activated Cdc42-associated tyrosine kinase 1), a downstream tyrosine kinase of Cdc42, have being found to be a key transducer in several cancer-related signaling pathway such as integrins, EGF, PDGF pathways and so on, and played an important role in cell proliferation, migration and invasion. Although previous studies have provided us some information about the role of Ack1 in malignant tumors, its role and detailed mechanism in cancers are still far from clear, its correlations with prognosis and clinicopathological characteristics of cancer patients have never been revealed, and its role in HCC has not been investigated. Therefore, we carried out the present study to investigate the expression of Ack1 in HCC and its correlations with prognosis and clinicopathological characteristics of HCC, as well as the role of Ack1 in the growth and metastasis of HCC and the possible underlying molecular mechanism. By using plasmid transfection and RNA interference, as well as a series of in vitro and in vivo assays, we got the following results.1. Real-time RT-PCR and Western blot were employed to examined Ack1 mRNA and protein expressions in 38 HCCs and their corresponding adjacent non-tumorous liver tissues (ANLTs), as well as in 5 normal liver tissues (NLs). The results showed that both mRNA and protein levels of Ack1 were significantly increased in HCCs than in ANLTs and NLs (P<0.05). Next, we used immunohistochemistry (IHC) to detect Ack1 expression in the paraffin sections of 131 cases of HCC and observed Ack1 protein was expressed in 85.5%(112/131) of all HCCs. Based on the results of IHC, we divided these 131 HCC patients into two subgroups:Ack1 high expression group and Ack1 low expression group, and then studied the prognosis of the two groups of patients. Our results showed HCC patients of the Ack1 high expression group had both a shorter disease-free survival (P<0.05) and a shorter overall survival (P<0.01) than those of the Ack1 low expression group. By univariate and multivariate Cox regression analysis, high Ack1 expression was found to be an independent prognostic factor for overall survival (RR,1.758; P<0.05). In further analysis of correlations of Ack1 with clinicopathological characteristics, we found overexpression of Ack1 in HCCs was significantly related with several aggressive clinicopathological characteristics, including multiple nodules, high Edmondson-Stainer grade, and with vein invasion (P<0.05).2. We further examined Ack1 mRNA and protein expressions in three HCC cell lines:HepG2, MHCC97-L and HCCLM3, using the Chang’s liver cell line CCL13 as a control. By RT-PCR and Western blot, we found all three HCC cell lines exhibited elevated Ackl mRNA and protein levels (P<0.01), and the expression levels in the three cell lines increased with the rising of metastatic potentiality of the cell lines, which were the highest in HCCLM3, followed by MHCC97-L (P<0.001) and HepG2 (P<0.05), indicating Ackl expression may be correlated with metastatic potential of HCC.3. To further verify the role of Ackl in HCC, we forcibly overexpressed Ackl into MHCC97-L cell line, which has a relative low metastatic potential, by transfecting with Ack1-expressing plasmids pCMV-Tag2B-Ack1, and used the empty vector pCMV-Tag2B as a control. After G418 selection, we got the Ackl overexpressed cell line MHCC97-LAckl and the control cell line MHCC97-LVector. By establishing mice orthotopic hepatocellular carcinoma growth and metastasis models, we observed that compared with mice implanted with MHCC97-LVector cells, those implanted with MHCC97-LAck1 cells have a significantly larger average volume of in situ liver tumors (P<0.05), as well as significantly higher incidences of intrahepatic metastasis (P<0.05) and pulmonary metastasis (P<0.01). The results based on the mice models indicated Ackl promotes tumor growth and metastasis.4. To explain our findings from in vivo nude mice models and further elucidate how Ackl promotes the tumor growth and metastasis of HCC, we then observed the role of Ackl in malignant biological behaviors of HCC cells by employing a series of in vitro assays. Results of growth assay and plate colony formation assay showed overexpression Ackl in MHCC97-L cells significantly enhanced cell proliferation (P<0.05) and speeded up cell growth (P<0.05). Wound-healing assay and matrigel-invasion assay demonstrated MHCC97-LAck1 cells exhibited a significantly higher motility and invasive ability compared with MHCC97-LVector cells (P<0.01). By immunofluorescence stain of cytoskeleton (F-actin), we observed overexpression of Ackl induced significant cytoskeleton reorganization and morphological change of MHCC97-L cells.5. To further investigate the underlying molecular mechanism in Ack1-enhanced HCC growth and metastasis, we investigated the possible downstream protein of Ackl in MHCC97-L. We focused on Crk, an adaptor protein which has crucial functions in the signaling pathways regulating cell proliferation and migration by acting as an adaptor to link tyrosine kinases and its effectors. More importantly, Crk has been previously shown to be involved in the signaling pathway in cell motility enhanced by Ack2, which is a homolog of Ack1. By RT-PCR and Western blot, we firstly observed Crk was overexpressed in HCC cell line MHCC97-L compared with Chang’s liver cell line CCL13. We then confirmed the interaction between Ackl and Crk in MHCC97-L cells by using co-immunoprecipitation assay. Next, we silenced the expression of Crk in MHCC97-L cells by RNA interference, and then investigated the influence of Crk-silencing on Ack1-induced HCC growth, invasion and metastasis, by repeating the in vitro and in vivo assays as we had employed earlier. The results showed silencing of Crk did not cause significant alteration in Ack1-promoted HCC cell proliferation or growth in vitro (P>0.05), or HCC tumor formation in vivo (P>0.05). But more interestingly, we observed that silencing of Crk significantly reversed Ack1-enhanced HCC cell migration and invasion in vitro (P<0.05), as well as reduced incidence of intrahepatic and pulmonary metastasis of HCC in vivo (P<0.05). And when Crk being silenced, overexpression of Ack1 failed to induce significant enhancement neither in cell migration or invasion in vitro (P>0.05), nor in incidence of intrahepatic or pulmonary metastasis in vivo (P>0.05), as compared with control cells. These results indicated Crk was required in Ack1-induced HCC invasion and metastasis but not growth and proliferation.In conclusion, our results have demonstrated Ack1 was overexpressed in HCC and played an important role in invasion and metastasis of HCC. We also have shown for the first time that high Ack1 expression closely correlated with poor prognosis and aggressive clinicopathological characteristics of HCC. Furthermore, we have identified for the first time that Crk was involved in Ack1-driven invasion and metastasis of HCC. Taken together, our findings suggest Ack1 as a novel prognostic marker and a potential therapeutic target for treatment of HCC.更多还原