Overexpression Of Ack1 Promotes Invasion And Metastasis Of Hepatocellular Carcinoma Through Crk Signaling

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.