The Effects And Mechanisms Of PEBP4 On The Biological Behavior Of Hepatocellular Carcinoma

Background and Objective:Primary liver cancer is a common human malignant tumor,among which 75% to85% are hepatocellular carcinoma.The major risk factor of hepatocellular carcinoma is hepatitis virus infection.Owing to the control of hepatitis virus infection in the world,the incidence of liver cancer remains stable.However,the mortality of liver cancer has been increasing year by year.The main reason is the diversity of incompletely understood molecular mechanisms,which makes it difficult to implement targeted therapy.Akt,an important molecule of tumor growth,is regulated by growth factors and oncogenes,and its biological function engages mTORC1 and mTORC2.On the one hand,many biological functions of Akt are mediated by mTORC1,which phosphorylates S6K1 and thus regulates protein synthesis.On the other hand,Akt together with serum glucokinase(SGK)and protein kinase C(PKC)serve as substrates that are phosphorylated and activated by mTORC2.Studies have found that PEBP4 is up-regulated in many cancers and plays an important role in cancer progression;In addition,it has been reported that PEBP4 binds to Akt and participates in the regulation of its activity.However,the role of PEBP4 and the functional interaction between PEBP4 and Akt-mTOR has not been documented in hepatocellular carcinoma.Thus,it is the focus of this study,and outcomes of the present study would provide molecular targets for the treatment of hepatocellular carcinoma.Method:1.To assess the effect of PEBP4 on biological behavior of hepatocellular carcinoma cells(1)Immunohistochemistry was employed to examine PEBP4 expression in cancer and adjacent normal tissues of patients with hepatocellular carcinoma.(2)Western blot(WB)and qPCR were carried out to examine the expression of PEBP4 in normal hepatocytes(LO2)and hepatocellular carcinoma cell lines with different degrees of malignancy,MHCC97L(low degree),He PG2 and SMMC-7721(middle degree),and MHCC97 H and HCCLM3(high degree).(3)Construction of stable cell lines: knockdown of PEBP4 in MHCC97 H cells containing a relatively high level of PEBP4 and overexpression of PEBP4 in MHCC97 L cells containing a relatively low level of PEBP4 with sh RNA-PEBP4 and PCMV6-PEBP4,respectively.WB and qPCR were used to test success of stable transfection.(4)The effects of knockdown and overexpression of PEBP4 on cell growth of hepatoma cells were assessed by CCK8 and clone formation assay.(5)The effects of knockdown and overexpression of PEBP4 on the migration and invasion of hepatoma cells were assessed by scratch test and Transwell assay.(6)Subcutaneous transplantation experiment: hepatoma cells were inoculated subcutaneously(axillary)in nude mice,and the effect of knockdown and overexpression of PEBP4 on tumor growth was examined.(7)Construction of lung metastasis model: the effect of knockdown and overexpression of PEBP4 on the metastatic ability of hepatoma cells was examined by injection of hepatoma cells into the tail vein of nude mice and metastatic nodes observed.2.To examine the effects of knockdown and overexpression of PEBP4 on Akt/mTOR signal pathway(1)Changes in the Akt/mTOR signal pathway in knockdown and overexpression of PEBP4 in cell lines were measured by WB.(2)WB was used to examine Akt/mTOR signaling pathway after stimulation with IGF-1 of the MHCC97 H cells where PEBP4 was knocked down.(3)The MHCC97 H cells with PEBP4 knockdown were infected by Akt adenovirus(Akt-S473D)and changes in Akt/mTOR signal pathway were examined by WB.(4)MHCC97L cells with overexpressed PEBP4 were treated with the Akt inhibitor MK-2206 and changes in Akt/mTOR signal pathway were measured by WB.3.To explore the role of PEBP4 and Akt/mTOR signaling pathways in biological behavior of hepatocellular carcinoma cells(1)CCK8 and Transwell were used to assess the cell viability,migration and invasion after the MHCC97 H cells with PEBP4 knockdown were infected by Akt-S473 D.(2)MHCC97L cells with overexpressed PEBP4 were treated with MK-2206 and the cell viability,migration and invasion were examined by CCK8 and Transwell,respectively.4.To assess the effect of PEBP4 on EMT(1)EMT markers were examined in cancer cells with PEBP4 knockdown or overexpression by WB.(2)Immunofluorescence was used to assessed expression of E-cadherin and N-cadherin in cells with PEBP4 knockdown or overexpression.(3)MHCC97H cells with PEBP4 knockdown were treated with IGF-1,followed by WB to examine changes in EMT.(4)MHCC97H cells with PEBP4 knockdown were infected by Akt-S473 D and changes in EMT were examined by WB and immunofluorescence.(5)MHCC97L cells overexpressed with PEBP4 were treated with MK-2206,and changes in EMT were examined by WB and immunofluorescence.5.To investigate interaction between PEBP4 and Akt/mTOR(1)Co-immunoprecipitation was performed to detect the interaction between PEBP4 and Akt/mTOR in MHCC97 H cells.(2)HEK293T cells were transfected with N-myc-PEBP4 and then the interaction between PEBP4 and Akt/mTOR was examined by coimmunoprecipitation.(3)In the MHCC97 H cells with PEBP4 knocked down,interaction between PEBP4 and Akt/mTOR was examined by co-immunoprecipitation.(4)After siRNA silencing mTOR in HEK293 T cells,interaction between PEBP4,Akt was examined by co-immunoprecipitation.Results:1.Effect of PEBP4 on Biological behavior of Hepatocellular carcinoma cells(1)Immunohistochemistry study showed that PEBP4 expression was greater in cancer tissues than that in adjacent normal tissues.(2)The expression of PEBP4 in highly malignant hepatoma cells(MHCC97H and HCCLM3)was higher than that in normal hepatocytes(LO2)and low malignant hepatoma cells(MHCC97L).(3)Knockdown of PEBP4 impaired the cell viability,migration and invasion of hepatoma cells,whereas overexpression of PEBP4 exhibited the opposite changes.(4)Xenograft tumor model showed that tumor growth was suppressed by PEBP4 knockdown in hepatoma cells whereas it was accelerated by its overexpression.(5)Data with injection of hepatoma cells into tail veins of mice revealed that PEPB4 knockdown reduced metastatic nodules in the lung,whereas the nodules were increased by the overexpression of PEBP4.2.The impact of PEBP4 on Akt/mTOR signal pathway(1)Knockdown of PEBP4 diminished the following phosphorylation events:(a)mTORC2 Ser2481 and other two substrates of mTORC2,SGK1 and PKC?,(b)Akt Ser473 and Thr308,(c)RSK2 and mTORC1 Ser2448,both of which are the phosphorylated by Akt,(d)S6K1 at the site mediated by mTORC1.All of these events were enhanced by overexpression of PEBP4.(2)IGF-1 could not affect the expression of PEBP4,nor reverse the effect of knockdown of PEBP4.(3)Introduction of Akt-S473 D into MHCC97 H cells where PEBP4 was knocked down had no effect on the expression of PEBP4,nor restore phosphorylation of mTORC2 Ser2481;however,it restored phosphorylation of Akt Thr308,RSK2,and mTOR Ser2448 phosphorylation as well as p-S6K1.All these events are related to Akt or mTORC1.(4)Treatment of MHCC97 L cells overexpressing PEBP4 with the Akt inhibitor MK-2206 had no effect on phosphorylation of mTOR Ser2481,SGK1 and PKC?,events related to mTORC2 activity,while it suppressed phosphorylation of Akt Ser473 and Thr308,mTORC Ser2448 and p-S6K1.3.Role of PEBP4 and Akt/mTOR signaling pathways on biological behavior of hepatocellular carcinoma cells Akt-S473 D partially reversed the effects of PEBP4 knockdown on the cell viability,migration and invasion of hepatoma cells,although these behaviors in the cells overexpressing PEBP4 was completely suppressed by MK-2206.4.The effects of PEBP4 on EMT(1)Results obtained with immunofluorescence and WB showed that knockdown of PEBP4 up-regulated of E-cadherin and down-regulated N-cadherin,vimentin and integrin,while opposite results were observed in the cells overexpressing PEBP4.(2)IGF-1 stimulation of cells with PEBP4 knockdown did not change EMT.(3)Akt-S473 D reversed the effects of PEBP4 knockdown on EMT,while the opposite effects of PEBP4 overexpression were counteracted by MK-2206.5.The interaction between PEBP4 and Akt /mTOR(1)Co-immunoprecipitation study using endogenous proteins in MHCC97 H or HEK293 T cells expressing amminoterminally tagged myc-PEBP4 revealed a complex among PEBP4,Akt and mTOR.(2)In the cells with PEBP4 knockdown,Akt associated with mTOR.(3)Silencing mTOR did not prevent Akt from binding to PEBP4.Conclusion:We conclude that(1)PEBP4 promotes malignant behaviors of hepatocellular carcinoma cells;(2)PEBP4 facilitates activation of the Akt/mTOR signal pathway,thereby regulating the malignant behaviors of hepatocellular carcinoma cells;(3)some of PEBP4 functions are not completely mediated by Akt/mTORC1,and other signal molecules may also be engaged,which are possibly regulated by mTORC2;(4)Physical association between PEBP4 and Akt/mTOR exists and contributes to biological activities of these molecules.