Experimental Study Of Molecular Pathogenesis Of Hepatocyte Carcinoma Metastasis Mediated By P120ctn

Backbround:Hepatocellular carcinoma(HCC) is one of the most common cancers worldwide,with an estimated 500,000 to 1,000,000 newcases annually.The survival of patients is extremely poor by the time they present with symptoms related to the tumor. Invasion and metastasis formation in early-stage HCC is an important feature and a crucial unfavorable prognostic factor.The E(epithelial)-cadherin-mediated cell adhesion plays a critical role in the metastasis of HCC.As the major epithelial adhesion molecule,the loss of E-cadherin is reported to be associated with tumor dedifferentiation and metastasis in many human cancers.E-cadherin function requires a fine interplay with the catenin-cytoskeleton complex in the cytoplasmic space.The catenins,includingα-catenin,β-catenin and P120ctn,may participate signal transduction process in nucleus except their adhesion function.P120ctn was originally identified as a Src sub-strate,and later as a new member of the cadherinsuperfamily of cell-cell adhesion molecules.In contrast to the functions of the classical catenins(α-catenin,β-catenin),which have been studied extensively,its role remains controversial.P120ctn binds to the "Juxtamembrane domain"(JMD) of E-cadherin, and regulates cell-cell adhesion.P120ctn is frequently lost or abnormally expressed in many of human tumors (e.g.Coloncarcinoma,Gastric carcinoma,Breast carcinoma,Lung carcinoma,etc.),suggesting roles for P120ctn as a tumor suppressor and/or a metastasis promoter.Most proteins physically or functionally related to catenins are oncogenes or tumor suppressors(e.g. Src-family kinases,receptor tyrosine kinases,Wnt-1,APC,etc.).But the relationship between P120ctn and human cancer is not yet clear. Recently,P120ctn was found as a binding partner of Kaiso,which is a ubiquitously expressed new member of the POZ zincfinger family of transcription factors.Kaiso was identified as a dual-specificity DNA-binding protein that recognizes the minimal core sequence CTGCNA(where N is any nucleotide) in addition to the methyl-CpG dinucleotides.And Kaiso has a higher affinity for the consensus binding site than for the methyl-CpG sites.These findings suggest that one role of nuclear P120ctn is to indirectly control gene expression by modulating Kaiso-mediated transcriptional regulation of its target genes.And recently,the neuromuscular gene rapsyn and the non-canonical Wnt signaling gene xWnt11,Siamos are identified as the target genes of Kaiso.Worthy of investigation and in-depth study,we found Matrix metalloproteinase-2 (MMP-2),a member of the MMP family which are involved in proteolytic modeling of the extracellular matrix,possesses two conserved copies of the core sequence-specific Kaiso site.Probably,MMP-2 is the target gene of Kaiso.MMP-2,also called 72-kDa typeⅣcollagenase,was expressed by malignant cells and some stroma cells such as fibroblasts.MMP-2 has been shown to play a critical role in invasion/metastasis and correlate with the malignancy grade of the original tumor.However, the regulation of MMP-2 gene expression in both malignant and normal cells is not very clear yet.In this study,we explore the relationship between transcription of MMP-2 and the P120ctn-Kaiso complex.And it will help us to deeply understanding the molecular mechanism in metastasis of HCC.Methods:(1)Immunohistochemical staining for P120ctn,E-cadherin andβ-catenin was performed in 58 hepatocellular carcinoma specimens,17 hepatic cirrhosis specimens,and 8 healthy controls.We evaluate the expression of P120ctn and correlate these results with pathological and clinical parameters.(2)The human MMP-2 promoter-reporter constructs were generated by cloning promoters into KpnI/XhoI sites of pGL3 Basic vector(Promega). To further validate the specificity of the observed Kaiso-mediated transcriptional repression, we also generated the luciferase reporter constructs carrying mutations in the two sites of Kaiso binding by PCR-based point mutagenesis method.All the reporter constructs were amplified by PCR from human genomic DNA.Full-length human Kaiso cDNA were amplified by PCR from human genomic DNA,and were cloned into SalI/XbaI sites of pEGFP-c3 vector(Clontech).Full-length mouse P120ctn in plasmid pRcCMV was kindly supplied by Albert.B.Reynolds,Vanderbilt University,USA.All the constructs were verified by sequencing.Luciferase assays(Promega) and ChIP assays(Santa Cruz) were performed according to the manufacturer's protocol.(3)HA-tag and P120ctn cDNA from plasmid were subcloned into pAdTrack-CMV vector.The resultant plasmid was linearized and subsequently cotransformed into E.coli BJ5183 containing adenovirus backbone plasmid pAdEasy-1.The adenovirus vector was then transferred into 293N cells by liposome to get recombinant adenovirus particles.Finally,P120ctn adenovirus were used to infect HepG-2 cell line.Then the expression of P120ctn and MMP-2 protein in HepG-2 cells were detected by RT-PCR,immunohistochemistry,and Western blot assays.The MMP-2 activity was detected by gelatin zymography.After transfection,the invasive activity of HepG-2 cells was assayed in a transwell cell culture chamber which has been dealed with matrigel first.Results and conclusions:(1)We could only see staining of cytomembrane in cases of healthy controls.But abnormal P120ctn expression was observed in 38/58(65.5%) of HCC, and in 6/17(35.3%) of hepatic cirrhosis specimens.The main change is the loss of membrane expression and/or the increased cytoplasmic accumulation.The staining pattern of P120ctn is associated with tumor cell dedifferentiation,capsule,metastasis(intra- or extra-hepatic metastasis),but does not appear to correlate with gender,age,tumor size and stage of the disease.But E-cadherin,β-catenin expression immunoreactivities were correlated only with capsule and tumor cell dedifferentiation.The proportion of tumors with decreased P120ctn(58.6%) was much greater than that of E-cadherin(44.8%) orβ-catenin (37.9%),indicating that P120ctn loss may in some cases precede loss of other members of the cadherin-catenin complex.There was no correlation between the expression of P120ctn and that of E-cadherin orβ-catenins,suggesting a lack of coordinate regulation of P120ctn and E-cadherin.P120ctn can be an new independent prognostic factor for metastasis of hepatocellular carcinoma.(2)Luciferase assays showed that the MMP-2 promoter can be repressed by Kaiso and P120ctn can relieve this repression.ChIP assays showed that endogenous Kaiso in HepG-2 cells binds the MMP-2 promoter.These findings demonstrate that the MMP-2 promoter is a transcriptional target of Kaiso and P120ctn.(3) P120ctn cDNA was successfully inserted into the adenovirus vector and the recombinant adenoveirus encoding P120ctn cDNA was obtained from this vector.It can infect HepG-2 cells successfully(the infective rate could reach nearly 100%) and estimulate HepG-2 cells to secret MMP-2 via the enhancing effect on MMP-2 promoter activity.Cell migration and invasion assays also demonstrate that transfection with Ad-P120ctn can increase the mobility and invasion of HepG-2 cells.