HAb18G/CD147 Regulats Integrin Signal Pathway To Promote Hcc Invasion And Migration

Hepatocellular carcinoma (HCC) is the most common primary hepatic carcinoma. The average live time of HCC patients are only 3 to 6 months. 90% of HCC patients die of complications associated with migration. Migration is the most important lethal factor to HCC patients, and is also the immediate cause of therapeutics failure. Meanwhile, abnormal apotosis pathways help HCC escape from apotosis, thus promoting HCC migration and recurrence. Studies on the regulation factors of HCC invasion and migration will not only broaden our basic knowledge of HCC development, but also provide some promising novel cancer biomarkers and therapeutical targets.HAb18G, a hepatoma-associated antigen, cloned in anti-hepatoma monoclonal antibody HAb18 screening of human hepatocellular carcinoma cDNA library has an identical nucleotide acid and amino acid sequence as CD147. HAb18G/CD147 is a transmembrane glycoprotein of the immunoglobulin superfamily that is broadly expressed in many cell types, especially at high levels in human tumor cells. Our previous studies have demonstrated that HAb18G/CD147 may promote the invasion and metastasis potential of human hepatoma cells by stimulating fibroblast cells to produce elevated levels of MMPs. Although published data support a crucial role for CD147 on migration and metastasis potential of tumor progression, but the detailed investigation on the precise mechanism is required. Now it is becoming increasingly apparent that full understanding of HAb18G /CD147 functional activities may require understanding of its associations with other molecules and its signal transduction pathway. The aim of this study is to identify the exact mechanism of HAb18G/CD147 promoting human hepatoma cells invasion and migration that has eluded investigators for many years. The study is composed of four parts.Part 1. HAb18G/CD147 interacts with integrinα3β1 to promote HCC invasion and migrationIntegrin which is widly expressed in vertebrate cell membrane, may interact with adhesion molecures to promote cell adhesion and movement. Immunofluorescent double staining showed co-localizations of HAb18G/CD147 with integrinα3 andβ1 on the human hepatoma FHCC-98 cells membrane. integrinα3β1 was then found co-immunoprecipitated with endogenous HAb18G/CD147 in 7721 cell lysates. RT-PCR and western-blot analysis showed that there were no significant expression modifications of integrinα3 andβ1 subunits in HAb18G/CD147 upregulated HCC cells and HAb18G/CD147 downregulated HCC cells. In-vitro adhesion, invasion and gelatinase zymography assay showed that upregulation of HAb18G/CD147 in HCC cells could significanty promote cell adhesion,invasion and MMPs secretion (p<0.01), downregulation of HAb18G/CD147 in HCC cells could significanty inhibit cell adhesion, invasion and MMPs secretion (p<0.01). Antibodies toα3 andβ1 integrins completely blocked HAb18G/CD147 enhanced cell adhesion,invasion potential and MMPs secretion (p<0.01). These results demonstrate that HAb18G/CD147 and integrinα3β1 are both required and functionally dependent for HCC adhesion, invasion potential and MMPs secretion.Part 2. Involvement of integrin signal pathways in HAb18G/CD147-mediated HCC invasion and migration processFocal adhesion kinese FAK is a kind of tyrosine kinese, which has protical role in integrin promoting HCC adhesion and movement process. Upregulation of HAb18G/CD147 in HCC cells could significantly increase the expression and phosphorylation of FAK and Paxiilin (p<0.01), and downregulation of HAb18G/CD147 could significantly diminish them (p<0.01). Downregulation of HAb18G/CD147 in HCC cells could decrease focal adhesions and F-actin stress fibers, and also redistribute FAK and paxillin to the periphery. Upregulation of HAb18G/CD147 in HCC cells could increase focal adhesions and filopodial protrusions.The addition of PI3K inhibitor wortmannin or LY294002 could significantly block HAb18G/CD147 enhanced intracellular free calcium ([Ca2+]i) concentration (p<0.01). In-vitro adhesion, invasion and gelatinase zymography assay showed that wortmannin or LY294002 could inhibit HAb18G/CD147-elevated cells adhesion, invasion and MMPs secretion in a dose-dependent manner in both cell lines (p<0.01). These results suggest that HAb18G/CD147 promotes HCC adhesion, invasion and MMPs secretion potential via integrinα3β1 mediated FAK-Paxillin and FAK-PI3K-Ca2+ singnal pathways.Part 3.βig-h3 is involed in HAb18G/CD147-mediated HCC adhesion and metastasis processWestern-blot analysis showed that the expression ofβig-h3 markedly reduced in si-HAb18G transfected 7721 cells (p<0.01). The four homologous recombination sequences and the full length ofβig-h3 were correctly cloned and transiently transfected to 7721 cells. In-vitro adhesion, invasion and gelatinase zymography assay showed that upregulation ofβig-h3 in HCC cells could significanty promote cell adhesion,invasion and MMPs secretion (p<0.01). This finding suggests thatβig-h3 may enhance invasion and metastasis potential of human hepatoma cells.Integrinα3β1 was found to co-immunoprecipitate with endogenousβig-h3 in 7721 cell lysates. The presence of antibodies forα3 andβ1 integrins completely blockedβig-h3-enhanced cell adhesion and adhesion potential (p<0.01). These results suggest that integrinα3β1 is involved inβig-h3 induced invasion and metastasis potential of human hepatoma cells.Downregulation ofβig-h3 in HCC cells could significantly decrease focal adhesions and F-actin stress fibers, and redistribute both FAK and paxillin to the periphery. These results indicate thatβig-h3 may promote HCC invasion and migration by integrinα3β1 and its downstream signal molecules FAK and paxillin.Part 4. HAb18G/CD147 promotes HCC recurrence by ERS induced anti-apotosisThe expression of HAb18G/CD147 increased as time-dependent manner in tunicamycin (Tm) or Thapsigargin (Tg) incubated 7721 cells. The expression levels of ERS markers-CHOP and Bip were also increased in the time-dependent manner. Upregulation of HAb18G/CD147 in HCC cells could markedly increase the Ca2+ level. Western-blot analysis and immunofluorescence analysis showed that in ERS condition, upregulation of HAb18G/CD147 in HCC cells could markedly increase Bip expression, and downregulation of HAb18G/CD147 could significantly decrease Bip expression. These results suggest that the expression of Bip is in positive correlation with HAb18G/CD147.No significant expression and phosphorylation modifications of IRE1, PERK and ATF6 were found in HAb18G/CD147 upregulated HCC cells and HAb18G/CD147 downregulated HCC cells. It is known that TFⅡ-Ⅰand YY1 can form trimer with ATF6 to regulate Bip expression. Western-blot analysis showed that upregulation of HAb18G/CD147 in HCC cells could markedly increase TFⅡ-Ⅰphosphorylation level (p﹤0.01), downregulation of HAb18G/CD147 coule significantly decrease TFⅡ-Ⅰphosphorylation level (p﹤0.01). There were no significant expression modifications of TFⅡ-Ⅰand YY1 in HAb18G/CD147 upregulated HCC cells and HAb18G/CD147 downregulated HCC cells. The results of immunofluorescence staining showed that upregulation of HAb18G/CD147 in HCC cells could obviously increase the fluorenscence indensity representing p-TFⅡ-Ⅰand accumulate p-TFⅡ-Ⅰto the nucleus, downregulation of HAb18G/CD147 in HCC cells could decrease p-TFⅡ-Ⅰand diffuse p-TFⅡ-Ⅰin the cytoplasm. These results indicate that HAb18G/CD147 may promote the expression of Bip by activiting TFⅡ-Ⅰ.Western-blot analysis showed that upregulation of HAb18G/CD147 in HCC cells obviously enhance c-Src and FAK phosphorylation levels (p﹤0.01), and downregulation of HAb18G/CD147 significantly decrease c-Src and FAK phosphorylation levels (p﹤0.01). There were no significant expression modifications of c-Src and FAK in HAb18G/CD147 upregulated HCC cells and HAb18G/CD147 downregulated HCC cells (p﹥0.05). The addition of FAK inhibitor could significantly decrease the phosphorylation levels of FAK, Src, TFⅡ-Ⅰand expression level of Bip in HAb18G/CD147 upregulated HCC cells (p<0.01). There results suggest that by activiting c-Src and TFⅡ-Ⅰ, HAb18G/CD147 may promote Bip expression.AnnexinV/PI double staining showed that upregulation of HAb18G/CD147 in HCC cells could obviously inhibit apoptosis, and downregulation of HAb18G/CD147 could significantly promote HCC apoptosis. The results of caspase4 activity test showed that upregulation of HAb18G/CD147 in HCC cells could obviously dercease caspase4 activity (p<0.01),and downregulation of HAb18G/CD147 could significantly increase caspase4 activity (p<0.01). This result was coincidence with the AnnexinV/PI double staining analysis results. These results demonstrate that in ERS condition, by derceasing caspase4 activity, HAb18G/CD147 may inhibit apoptosis in HCC cells.All the above results suggest that HAb18G/CD147 may enhance invasion and metastatic potential of human hepatoma cells via integrinα3β1 mediated FAK-paxillin and FAK-PI3K-Ca2+ signal pathways.βig-h3 is cooperated with HAb18G/CD147 in this process. In ERS condition, by FAK-Src signal pathways, HAb18G/CD147 may phosphorylate TFⅡ-Ⅰand help p-TFⅡ-Ⅰaccumulate to the nucleus, thus promoting Bip expression. By promoting Bip expression and decreasing the activity of caspase4, HAb18G/CD147 may inhibit HCC apoptosis. This is the first time to determine the signal transduction pathway of HAb18G/CD147 in promoting HCC invasion and migration, and to examine the mechanism of HAb18G/CD147 anti-apoptosis in ERS condition. The present study founds a basis for the further demonstration of the function of HAb18G/CD147 in HCC generation and development, and provides some new evidence for its further application in clinical.