| Object:Sorafenib,as an inhibitor of multiple oncogenic kinases,is the first-line treatment of advanced hepatocellular carcinoma(HCC).However,due to the complex pathogenesis and tumor microenvironment of HCC,single-agent chemotherapy is difficult to alleviate the disease process,and the survival benefit of sorafenib on HCC patients is unsatisfactory.Therefore,the combination strategy based on sorafenib has become a research hotspot in the systemic therapy of HCC.In the updated NCCN guidelines for liver cancer in 2022,the first-line systemic treatment options for liver cancer have been increased to six.Among them,a breakthrough in the combination therapy of atezolizumab(anti-PD-L1 mAb)and bevacizumab(anti-VEGF mAb)confirms the theoretical advantage of simultaneously targeting tumor angiogenesis and immunosuppression in the treatment of HCC.Therefore,it is expected to improve the clinical treatment dilemma of sorafenib by combining it with means of reversing tumor immunosuppression.Tumor microenvironment(TME)is both crucial to cancer progression and metastasis,as well as mediating tumor resistance.STAT3 in the tumor microenvironment plays a central role in regulating antitumor immune responses,excessive activation of STAT3 can reduce the expression of immunostimulatory factors(IFN,TNFα,etc.)and increase the expression of IL-6,IL-10 and TGF-β to drive the immunosuppressive state of the tumor.In HCC,STAT3 overexpression and constitutive activation are closely related to pathogenesis and survival,regulating HCC development,chemotherapy resistance and immune escape.Targeting STAT3 in HCC can not only inhibit the intrinsic proliferation and stem cell properties of tumor cells,but also enhance the antitumor effect of tumorinfiltrating immune cells,which makes STAT3 one of the potential targets for HCC therapy.In this study,we aimed to explore the anti-tumor effect of sorafenib combined with STAT3 knockdown in the treatment of HCC,to clarify the mechanism of inducing tumor cell death,and to further reveal the effect of the combination therapy on tumorinfiltrating immune cells.In order to provide experimental and theoretical basis for establishing new and effective therapeutic strategies for clinical treatment of HCC.Methods:1.The GEO database was used to analyze the relationship between the expression of STAT3 in tumor tissue and the response to sorafenib,and the correlation between STAT3 and ERS-induced apoptosis-related genes.2.The effect of sorafenib combined with sh-STAT3 on HCC proliferation was detected by CCK-8 assay.3.Annexin V/7-AAD double staining was used to detect the ability of sorafenib combined with sh-STAT3 to induce HCC apoptosis.4.Transwell evaluated the effect of sorafenib combined with sh-STAT3 treatment on the invasive ability of HCC.5.The effect of sorafenib combined with sh-STAT3 on the proliferation and tumor stemness of HCC was detected by clonogenic assay and spheroidization assay.6.Western blotting was used to detect the effect of sorafenib combined with shSTAT3 on the regulation of ERS-related protein expression.7.Confocal microscopy and flow cytometry to detect the effects of sorafenib combined with sh-STAT3 on protein accumulation in the endoplasmic reticulum of HCC cells.8.RNA-seq,qPCR and western blotting detected the effects of sorafenib combined with sh-STAT3 on ERS-induced apoptosis-related gene expression and protein expression.9.Immunofluorescence and flow cytometry were used to detect the effect of sorafenib combined with sh-STAT3 on ROS production in HCC.10.Flow cytometry to detect the effect of sorafenib combined with sh-STAT3 on mitochondrial membrane potential in HCC cells.11.The interaction between STAT3 and PKR protein was detected by coimmunoprecipitation(IP)and confocal microscopy.12.The effects of PKR and STAT3 expression on eIF2a phosphorylation were detected by Western blotting.13.The Hepa1-6 cell tumor-bearing mouse model was constructed,and the antitumor effect of sorafenib combined with sh-STAT3 was evaluated by observing the tumor volume,tumor weight and survival of the mice,and the safety of the combined treatment strategy was assessed by HE staining and monitoring the body weight of mice before and after treatment.14.The expression of STAT3,CHOP and Cleaved-Caspase3 in mouse tumor tissue was detected by immunofluorescence.15.The infiltration ratio and number of CD4+T cells,CD8+T cells,NK cells,DCs and macrophages in mouse tumor tissues were detected by flow cytometry.16.The effect of Hepa1-6 cells after sorafenib combined with sh-STAT3 treatment on the phenotype and activation of CD11b+DCs,CD8+DCs and CD103+DCs subsets was detected by flow cytometry.17.BMDCs were observed to take up damaged DNA from tumor cells using confocal microscopy.18.The effect of Hepa1-6 cells after sorafenib combined with sh-STAT3 treatment on the phosphorylation of TBK1 and IRF3 in BMDCs was detected by flow cytometry.19.The effect of sbrafenib combined with sh-STAT3 treatment of Hepa1-6 cells on the expression levels of IFNA1 and IFNB1 in BMDCs was detected by qPCR.Result:1.The expression of STAT3 in tumor tissues of HCC patients with "nonresponders" to sorafenib was significantly higher than that of "responders"GEO data analysis showed that the mRNA level of STAT3 in tumor tissues of HCC patients(46 cases)that did not respond to sorafenib was significantly higher than that of patients who responded(21 cases),suggesting that the high expression of STAT3 in HCC patients may be the cause of sorafenib resistance.2.Knockdown of STAT3 promotes the antitumor effect of sorafenib in vitroCompared with sorafenib and or sh-STAT3 treatment alone,combined treatment of sorafenib with sh-STAT3 significantly inhibited HCC cell proliferation and invasion,and decreased the number of clone formation and spheroidization,and promoted HCC cells apoptosis,indicating that STAT3 knockdown can promote the antitumor effect of sorafenib in vitro.3.Sorafenib combined with sh-STAT3 treatment triggers endoplasmic reticulum stress(ERS)-induced apoptosisSorafenib combined with sh-STAT3 up-regulated the ERS signaling pathway in HCC cells,and the expressions of PERK,ATF6,IRE1α and downstream pathway-related molecules were up-regulated.Sorafenib combined with sh-STAT3 triggered the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum of HCC cells as observed by confocal microscopy.These results suggest that sorafenib combined with sh-STAT3 treatment can promote unfolded protein response(UPR)in HCC cells.Through RNA-seq,qPCR,and Western blotting,it was found that the sorafenib combined with sh-STAT3 treatment up-regulated the expression of ATF4CHOP signaling axis-related molecules and induced apoptosis.The ERS inhibitor 4PBA reversed the apoptosis induced by the combination therapy,indicating that the combination therapy triggered extreme ERS-induced apoptosis.4.Sorafenib combined with sh-STAT3 impairs mitochondrial function and accelerates apoptosisImmunofluorescence and flow cytometry showed that sorafenib combined with shSTAT3 treatment significantly increased ROS levels in HCC cells,and a JC-1 staining showed that the combination treatment significantly decreased mitochondrial membrane potential in HCC cells,suggesting that combined therapy can damage the mitochondrial function of HCC cells,thereby accelerating the apoptosis of HCC cells.5.STAT3 knockdown aggravates ERS-induced apoptosis by stimulating the PKR/eIF2α/ATF4/CHOP pathwayIn sorafenib-responsive HCC patients,the DDIT3(CHOP)was significantly upregulated in tumor tissues after sorafenib treatment,and there was a significant negative correlation between STAT3 and ERS-induced apoptosis-related genes DDIT3,PPP1R15A,and TRIB3.STAT3 knockdown promoted PKR dissociation and eIF2αphosphorylation,leading to increased apoptosis.Overexpression of PKR promoted the phosphorylation of eIF2α and significantly increased apoptosis.Elevated eIF2αphosphorylation increases ATF4 translation,which in turn upregulates CHOP expression and induces apoptosis,the effect of combination therapy was significantly suppressed when siRNA reduced CHOP expression.These results suggest that STAT3 knockdown expression can activate apoptosis mediated by the PKR/peIF2α/ATF4/CHOP axis.6.Sorafenib combined with sh-STAT3 exerts significant antitumor effect in Hepa1-6 cells subcutaneous tumor modelUsing low-burden and high-burden Hepa1-6 cells subcutaneous tumor models to demonstrate that the combination of sorafenib and sh-STAT3 has a strong antitumor effect,manifested by slowing tumor growth,significantly reducing tumor volume and weight,and significantly prolonging survival of mice.The toxicity of the combined treatment in vivo was evaluated by HE staining,and no tissue damage was observed,indicating that the combined treatment of sorafenib and sh-STAT3 not only has excellent anti-tumor effects,but also has good safety.7.Sorafenib combined with sh-STAT3 activates antitumor immune responsesSorafenib combined with sh-STAT3 treatment promoted the infiltration and activation of CD8+T and NK cells in tumor tissue,behave as the proportion of CD8+ T cells of IFN-γ+,Perforin+,and TNF-α+increased,and the proportion of exhausted Tim3+ CD8+ T cells decreased;The proportion of NK cells of perforin+,and TNFα+increased,and the proportion of Tim-3+NK cells decreased;At the same time,it promotes the infiltration and maturation of DCs,which is manifested as the proportions of IL-12+,CD86+,MHC Ⅱ+DCs increased,but there was no significant difference in the proportions of PD-L1+DCs.Changes in CD4+T cells and macrophages were not significant.8.Sorafenib and sh-STAT3 combined treatment of HCC cells can activate the cGAS-STING signaling pathway in DCsHepa1-6 cells treated with sorafenib and sh-STAT3 were co-incubated with BMDCs,which could promote the proportion of CD103+DCs and increase the expressions of CD86,MHC Ⅰ and MHC Ⅱ.Sorafenib combined with sh-STAT3 treatment induced the death of Hepal-6 cells,released damaged DNA to be phagocytosed by BMDCs,and promoted the phosphorylation of TBK1 and IRF3 as well as the expression of IFNA1 and IFNB1.However,STING antagonist and the ERS inhibitor 4-PBA reversed this phenomenon,indicating that the combined treatment of sorafenib and sh-STAT3 activates the cGAS-STING signaling pathway in BMDCs through ERS-induced apoptosis9.The cGAS-STING-Type Ⅰ IFNs signaling pathway mediates the antitumor immune response induced by the combination therapy of sorafenib and sh-STAT3Blockade of type Ⅰ interferon signaling pathway by anti-IFNAR antibody weakened the effect of combination therapy,while the infiltration and activation of CD8+T and NK cells in tumor tissue and the infiltration and maturation of CD 103+DCs were significantly reduced.It shows that the anti-tumor effect of combined therapy depends on the type Ⅰ interferon signaling pathway.It was further found that the sorafenib combined with sh-STAT3 treatment reduced the proportion of CD103+DCs in the spleen of mice,suggesting that the CD103+DCs infiltrated by tumor tissue might migrate from the spleen of mice.Conclusions:Our study showed that the combination of sorafenib with STAT3 knockdown showed significant anti-HCC effects in vivo and in vitro.STAT3 knockdown promoted PKR dissociation,increased the phosphorylation of eIF2α,which in turn increased the expression of ATF4 and CHOP,which ultimately caused ERS-induced apoptosis.Subsequently,the apoptotic HCC cells further activated the cGAS-STING signaling pathway in CD103+DCs by releasing damaged DNA,induced the expression of IFNA1 and IFNB1,and finally promoted the antitumor activity of CD8+T and NK cells.Therefore,the multi-kinase inhibitory effect of sorafenib combined with sh-STAT3,which reverses the immunosuppressive microenvironment,can directly exert an antitumor effect on HCC,and at the same time can improve the tumor microenvironment and stimulate anti-tumor immune responses,which is expected to be a novel therapy for HCC. |
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