Screening Functional Complex Of CD151 Associated With The Progression Of HCC And Its Role In HCC Invasion And Metastasis

Hepatocellular carcinoma (HCC) is one of the most common malignancies that accounts for approximately 0.6 million deaths each year worldwide. The invasiveness, metastatic potential and recurrence ability of HCC are responsible for majority of mortality. Therefore, the development of effective treatment against metastasis and recurrence, by exploring the mechanism involved in HCC progression in details, could be of great significance to prolong the survival and improve the prognosis of HCC patients.Tetraspanin, TMSF, is a small glycoprotein molecule, as the name implies, with four membrane spanning domains. Tetraspanins interact with other tetraspanins and numerous membrane proteins, such as integrin and growth factor receptors, to form a dynamic network of multimolecular membrane microdomains, often regarded as tetraspanin enriched microdomain (TEM). The main function of tetraspanin is to influence biological activities of its associates in tetraspanin web. Studies have confirmed its modulatory role in regulation of various basic cellular processes such as cell differentiation, cell migration, cell-extracellular matrix adhesion, platelet aggregation, cell lysis and signal transduction. Moreover, the abnormal expression of tetraspanin is significantly related to the incidence and progression of several diseases. Tetraspanin CD151 is an important member of the transmembrane 4 superfamily and its abnormal expression influences the functions of associated proteins. In addition, the structural and functional integrity of tetraspanin web depends on CD151 expression.In our previous study, we found that overexpression of CD151 indicates poor prognosis in HCC, moreover, up-regulates hepatocyte growth factor (HGF)/hepatocyte growth factor receptor (c-Met) signaling. Besides, CD151 forms a functional complex with a6β1 in HCC and its overexpression selectively expandα6β1-PI3K signaling that accumulates and activates nuclear transcription factor Snail, thereby promoting neoangiogenesis and epithelial mesenchymal transition.As tetraspanin CD151, a key molecule in tetraspanin web, regulates invasion and metastasis of HCC, the current study screened its possible functional associates using co-immunoprecipitation and LC MS/MS techniques. Subsequent bioinformatics analysis and western blotting illustrated the characteristic CD151 network. Further, transducted HCC cells to unbalance the CD151 functional network were examined for the motility and invasive properties and validated in HCC tissue specimen. Thus, this study moved a step further to manifest the mechanism involved in HCC progression and to develop new targeted therapy by providing theoretical basis for the treatment of HCC.Chapter IScreening the associates of HCC invasion related CD151 functional complex using co-immunoprecipitation and LC MS/MSObjective:To screen the associates of HCC invasion related CD151 functional complex using co-immnoprecipitation and LC MS/MS.Methods:Freshly extracted proteins from HCCLM3 cell line were immunoprecipitated with CD151 antibody. Liquid chromatography coupled to mass spectrometry (LC-MS/MS) was used to identify the associates of CD151 functional complex. Bioinformatics analysis and protein validation were performed to establish CD151 functional network.Results:Fifty-eight proteins including CD151 were identified using LC-MS/MS. Among them were membrane proteins such as a3, a6,β1, c-Met, LDLR and TNFαIR etc. and signal transduction proteins like 14-3-3, p195/IQGAP1, HSP70, RuvB1 and 2, PKC and IKK etc. The functional complex formed by CD151, integrins a6 andβ1 was examined by Western blot analysis and confirmed the reliability of the LC-MS/MS result. Moreover, a schematic diagram of three-tier CD151 network was depicted.Conclusion:Tetaspanin CD151 and integrins a6 andβ1form a functional complex in HCC cell line.ChapterⅡThe study of interaction between CD151 and integrinβ1 and their influence in HCC invasion Objective:To study the interaction between CD151 and integrin (31 and their influence in HCC invasion and metastasis.Methods:Real-time fluorescence quantitative PCR, Western blot and flow cytometry were used to measure differences in expression level of CD151 and integrinβ1 in various HCC cells with different metastatic potential. RT-PCR and Western blot were used to analysis the interaction of CD151 and integrin (31 by measuring their expression levels before and after CD151-cDNA, shRNA-CD151, vshRNA-Integrinβ1 transduction in HCC cells. Transwell assay was used to measure changes in secretion of MMPs and cell in transfected cells. Nude mice models were analysed for lung metastases to observe difference in metastatic ability of HCC cell before and after transfection.Results:HCC cell lines showed a positive correlation between CD151 expression and their metastatic potential. Increased level of integrinβ1was also observed in those cell lines. In successfully transfected cell line, CD151 and integrinβ1expression positively correlated with MMP 9 secretion, but mildly associated with MMP2 secretion. HCC cells with high expression of both CD151 and integrinβ1 demonstrated enhancement in cell motility and invasiveness, moreover, increased lung metastases. In vivo experiment showed decrease in invasive and metastatic potential of HCC cells expressing high levels of both CD151 and integrinβ1 when transfected with integrinβ1; however, HCC cells expressing high CD151 and low integrinβ1 didnot exibit changes in invasiveness after transfected with integrinβ1.Conclusion:High expression of CD151 promotes integrinβ1-dependent HCC invasion and metastasis.Chapter IIIThe study of CD151and integrinβ1 expression in HCC tissue and their prognostic roleObjective:To study the CD 151 and integrinβ1 expression in primary liver cancer and explore their clinical significance. Methods:The expression of integrinβ1 in 120 HCC tissue specimens and 20 normal liver samples were analysed via qRT-PCR. Immunohistochemical analysis of CD151 and integrinβ1 expression was performed in tissue microarray of 301 cases of HCC. The prognostic significance of clinicopathological features of HCC was analyzed statistically.Results:The quantitative PCR analysis showed high integrinβ1 level in HCC tissue than in corresponding noncancerous tissue and normal liver tissue (p<0.05); moreover, manifested slightly higher expression of integrinβ1 in early recurrence group compared to non-recurrence group. The overexpression of CD151 observed in HCC tissue was related with multiple tumors (number> 1, p=0.46), microvascular invasion (p=0.01), large tumor (diameter> 5cm, p=0.027) and high TNM staging (p=0.001), while and high level of integrinβ1 was associated with microvascular invasion (p=0.028), large tumor (diameter> 5cm, p=0.04) and high TNM staging (p=0.046). The postoperative 3-, 5-, and 7-year overall suvival rates (OS) of HCC patients in the CD151+group was significantly lower than that in CD151- group (p<0.01). The 3-,5-, and 7-year cumulative recurrence rates in the CD151- group was higher than those in CD151+ group. Moreover,3-,5-, and 7-year OS in CD151+/integrinβ1+ group was significantly lower than those in CD151+/integrinβ1-, CD151-/integrinβ1+and CD151-/integrinβ1- groups (p<0.01). Cox multivariate analysis identified that CD151 or CD151/integrinβ1 were independent indicators of poor prognosis in HCC.Conclusion:CD151-integrinβ1 functional complex is associated with invasion, metastasis and poor prognosis in HCC.