The Loop Of TGF-β-HAb18G/CD147Promotes Carcinogenesis And Progression Of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC，also called malignant hepatoma) is the mostcommon type of primary liver cancer. HCC is usually hard to be observed at the earlystage. However, when obvious symptoms show up, the prognosis is generally poor due totumor progression. HCC is the fifth most common malignancy and the third leading causeof cancer-related death worldwide. Half the newly diagnosed HCC patients are in China,especially the down-country. Thus exploration of the underlying mechanisms of HCCcarcinogenesis and development is needed to provide theoretical basis for future clinicalapplications.HAb18G/CD147is a novel cancer target that was identified in our lab. As a memberof the immunoglobulin super family, CD147has been found to be highly expressed invarious cancers, while expressed negatively or at a very low level in normal tissues. Preliminary study showed that HAb18G/CD147could induce the secretion ofmatrixmetalloproteinases (MMPs) from HCC cells and fibroblasts, and promote thedegradation of surrounding stroma and enhance the invasion of HCC cells. Previousstudies always focus on the function of HAb18G/CD147, but pathways related to thismolecule remains unclear.Viral hepatitis infection leading to chronic inflammation apparently promotes fibrosisand hepatic carcinogenesis, known as the inflammation-fibrosis-cancer axis. Transforminggrowth factor beta (TGF-β) is one of the important regulators in this axis, and is known toexhibit tumor stage-dependent suppressive and oncogenic properties. During cancerprogression, TGF-β switched from being a tumor suppressor to a tumor promoter in earlycarcinogenic process. However, the mechanism underlying TGF-β-induced HCCprogression has not been completely understood.This study tries to examine the cooperation of HAb18G/CD147and TGF-β in HCCcarcinogenesis and progression. The study is composed of the following three parts.Part1. TGF-β-HAb18G/CD147loop promotes epithelial-mesenchymal transition andhepatocarcinogenesis. TGF-β triggers epithelial-mesenchymal transition (EMT) tomaintain the mesenchymal phenotype of invasive cells, but the role of TGF-β inhepatocarcinogenesis remains unclear. In the first part, we tested the effects of TGF-β1stimulation on HAb18G/CD147expression in a normal hepatic cell line, QZG. Theexpression of HAb18G/CD147and N-cadherin was upregulated and the expression ofE-cadherin was downregulated after stimulation of TGF-β1, implying the occurance ofEMT. To confirm that HAb18G/CD147expression was regulated by TGF-β1signaltransduction, we applied a selective antagonist of the TGF-β type1receptor (TβRI) andfound that SB-431542inhibited TGF-β1-mediated HAb18G/CD147upregulation. Theexpression of HAb18G/CD147was controlled by the cell survival PI3K/Akt-GSK3βsignaling pathway and was directly regulated by the transcription factor Slug. Transfectionof CD147also induced an elevated expression of TGF-β, demonstrating a positivefeedback loop between CD147and TGF-β. CD147-transfected hepatocytes showedmesenchymal phenotypes such as upregulation of N-cadherin and downregulation ofE-cadherin. CD147-transfected hepatocytes also obtained enhanced malignant propertiessuch as migration, MMP-2secretion and colony formation. Tumor formation and tumor metastasis of CD147-transfected QZG cells in vivo were accelerated.Immunohistochemistry analysis displayed a negative correlation betweenHAb18G/CD147and E-cadherin expression (rs=-0.3622, P=0.0105) and a positivecorrelation between HAb18G/CD147and Slug expression (rs=0.3064, P=0.0323) inhuman HCC tissues. Our study revealed a novel role of HAb18G/CD147in mediatingEMT in the process of HCC progression and demonstrated that CD147was a Slug targetgene in the signaling cascade TGF-βâ†'PI3K/Aktâ†'GSK3βâ†'Snailâ†'Slugâ†'CD147.Our results suggest that CD147may be a potential target for the treatment and preventionof HCC.Part2. HAb18G/CD147promotes dedifferentiation progress of hepatocellularcarcinoma via the TGF-β signaling pathway. The degree of dedifferentiation in HCCdetermines the primitiveness of the cells and therefore their rate of growth, which meansthat the malignancy of the HCC is directly related to the degree of de-differentiation. Inthe second part, we found TGF-β and CD147-transfection induced upregulation of CD147and dedifferentiation progress in well differentiated HCC cells, while ATRA andCD147-deletion induced downregulation of CD147and differentiation progress in poorlydifferentiated HCC cells. Overexpression of CD147also enhanced malignant properties ofwell differentiated HCC cells, including invasion and colony formation in vitro, as well astumor formation ability in vivo. These results proved that CD147was involved in thededifferentiation progress of HCC cells and enhances the malignant properties of HCCcells. Further, we tried to investigate the signaling pathway involved in thededifferentiation progress induced by CD147. We found that β-catenin could betranslocated to nuclear and elevated mRNA level and secretion of TGF-β1. MMPsdownstream of CD147could activate pro-TGF-β1. β-catenin and MMPs were bothinvolved in the regulation of TGF-β signaling pathway by CD147. The activated TGF-βsignaling subsequently repressed the expression of HNF4α and enhanced thededifferentiation progress.Part3. Differentiation therapy of hepatocellualr carcinoma by targetingTGF-β-HAb18G/CD147loop. We designed three lentivirus vectors, two were expressionvectors including sequences encoding HNF4α and Smad7, and one was shRNA interferingvector targeting CD147mRNA. We found overexpression of HNF4α and Smad7and deletion of CD147reprogramed the expression profile of characteristics of hepatocytemarkers (G6PT10, CRP, APOCIII, CYP1A2, ALDOB) in hepatoma cells, and attenuatedthe proliferation, colony formation and cell survival of hepatoma cells in vitro. In vivo,poorly differentiated cells transfected with these three lentivirus significantly inhibitedtumor formation. These results confirm the TGF-β-HAb18G/CD147loop as a potentialtarget for differentiation therapy of HCC.All the above results suggest an important role of TGF-β-HAb18G/CD147signalingloop in HCC development. In normal hepatic cells, TGF-β promotes expression of CD147by PI3K-GSK3β-Snail1-Slug signaling pathway, and CD147induces EMT and elevatesexpression of TGF-β. This positive feedback loop enhances the malignant properties ofhepatic cells and induce carcinogenesis. In well differentiated HCC cells, CD147activatesTGF-β signaling via β-catenin and MMPs, and the expression of differentiation markerssuch as HNF4α are repressed, leading to dedifferentiation progress. Differentiation therapytargeting TGF-β-HAb18G/CD147loop promotes the dedifferentiation progression andsuppresses malignancy of poorly differentiated HCC cells. It is the first time to determinean important role of TGF-β-HAb18G/CD147loop in HCC carcinogenesis and progression,and the underlying mechanism is well explored. This study may found a basis for thefurther investigation of the differentiation therapy of HCC targeting HAb18G/CD147.