| AimsHepatocellular carcinoma(HCC)is the most common form of primary liver cancer and has high morbidity and mortality.The annual mortality rate of liver cancer is 800,000 worldwide,and the number of newly diagnosed HCC is estimated to increase further in the coming decades.Surgery,chemotherapy,and radiotherapy are widely used for the treatment of HCC,but cannot effectively control recurrence and metastasis.Since it is asymptomatic in the early stages,HCC is usually diagnosed in its advanced stage,which precludes the possibility of surgical resection.At present,sorafenib is the only chemotherapeutic agent approved for treating advanced HCC,although it provides a survival benefit of only a few months.Therefore,early diagnosis and treatment of HCC is currently a major research focus.Apoptosis can be divided into physiological cell death and immunogenic cell death(ICD)according to the expression of immunogenic molecules on the apoptotic cell surface,which cause immune attack responses.Cells undergoing ICD upregulate the expression of "eat me" signalling molecules on the cell surface,such as calreticulin(CRT)and endoplasmic reticulum resident protein 57(ERp57),Meanwhile,heat shock proteins(HSPs)and high mobility group box 1(HMGB1)are also released during ICD induction.All these ICD-related molecules can stimulate immune cells to recognise and attack tumour cells.Anthracyclines can induce the translocation of calreticulin from cytoplasm to cell surface in tumour cells,which leads to their recognition by dendritic cells(DCs)and tumour antigen processing and presentation to CD4+T and CD8+T cells,thereby stimulating the anti-tumour immune response.Calreticulin and ERp57 are recognised as "eat me" signals,promoting the phagocytosis of cancer cells by the immune system.And extracellular ATP also as a"find me" signal acts on purinergic receptors of metabotropic P2Y2 which could recruit the ATP-mediated chemotaxis of DCs into the tumor site.By contrast,the glycoprotein CD47 on the tumour cell membrane,which inhibits macrophage-mediated phagocytosis,is considered a "don't eat me" signal.At the early stage of ICD,numerous molecules of calreticulin and ERp57 translocate to the cell surface,while the expression of CD47 is significantly decreased.The alteration of"don't eat me" and "eat me" signals results in the effective recognition and phagocytosis of tumour cells by DCs and macrophages.Therefore,ICD induction is one of the anti-tumour strategies that can elicit more effective anti-tumour immune responses.Signal transducer and activator of transcript ion 3(STAT3)is a latent cytoplasmic transcription factor that translocates to the nucleus following tyrosine phosphorylation at position 705 and dimerization.STAT3 activation is tightly controlled in healthy cells,and is constitutively activated during tumorigenesis,where in it upregulates genes involved in tumor cell proliferation,invasion,migration,and angiogenesis.STAT3 is overexpressed in approximately 60%of the HCC tissues,and is associated with poor prognosis.Consistent with this,blocking the STAT3 signaling pathway in human HCC cells using a decoy oligonucleotide induced apoptosis.Our previous studies have confirmed that blocking STAT3 signalling in HCC with STAT3 decoy-oligodeoxynucleotide(-ODN)could effectively inhibit the proliferation of HCC cells in vitro and in vivo.Significantly,blocking STAT3 improved the anti-tumour immune responses against HCC and tumour immune microenvironment,and induced anti-tumour immune memory.However,whether this process is associated with ICD has not been thoroughly investigated.Napabucasin is a neoteric small molecule that targets the STAT3 pathway,and has shown encouraging results in phase III clinical trials on metastatic colorectal carcinoma,pancreatic cancer,gastric cancer and non-small cell lung cancer.In addition,napabucasin prevented relapse and metastasis of human tumor xenografts by inhibiting CSCs.However,the efficacy of napabucasin in HCC and the underlying molecular mechanisms have not been elucidated.In this study,we tried to investigate the influences of STAT3 on ICD of HCC cells in vitro and in vivo,and clarify how STAT3 inhibition though shRNA and napabucasin improves tumour immune microenvironment by directly disturbing the glycolysis pathway and balance between "eat me" and "don't eat me" molecules in HCC,thus providing a solid experimental basis for treatment strategies and clinical trials of HCC.Methods1.Lentivirus carrying STAT3-shRNA was used to interfere with the expression of STAT3 in Huh7 and HepG2.2.15 cells,and the non-phosphorylation mutant of STAT3-Y705F were used for rescue experiments.After 48h,STAT3 mRNA levels in Huh7 and HepG2.2.15 cells were analysed by qRT-PCR;The cell ability and apoptosis rate were detected by CCK-8 and Annexin-V/PI;The ICD-related molecules on HCC cell surface were analysed by flow cytometry;The levels of ATP in the cultural supernatant of HCC cells were assayed by Enhanced ATP Detection Kit.2.The hDC precursor monocytes were purified from PBMCs by adherence,elutriation,and differentiation,and analysed by hCD11c+flow cytometry.3.HCC cells labelled with CFSE were added to THP-1-derived macrophages labelled with CM-Dil,placed in an incubator for 2h at 37? and 5%CO2,and subsequently resuspended in PBS and analysed by flow cytometry.4.Western blotting analysed STAT3/p-STAT3Tyr705,PKR/p-PKR,eIF2?/p-eIF2? levels in Huh7 and HepG2.2.15 cells;IP and GST pull-down experiments confirmed the formation of STAT3-PKR complex.5.TCGA database analysis showed the correlation of the expression of CD47 with STAT3 in HCC;The promoter sequence of CD47 were collected from JASPAR database;ChIP and sequencing analyses demonstrated that STAT3 could directly bind to the promoter regions in CD47 or not.6.CCK-8 analysed the proliferation of Huh7 and HepG2.2.15 cells after 2-DG treatment;The ICD-related molecules on HCC cell surface were analysed by flow cytometry.7.Seahorse XF24 Extracellular Flux Analyzer analysed the extracellular acidification rate(ECAR)of Huh7 and HepG2.2.15 cells.8.TCGA database analysis showed the correlation of the expression of HIF-1?,GLUT1,HK2,LDHA with overall survival of HCC patients,and the correlation of the expression of HIF-1?,GLUT1,HK2,LDHA with STAT3 in HCC.9.Key glycolysis-related molecules such as HIF-1?,GLUT1,HK2 and LDHA mRNA levels in Huh7 and HepG2.2.15 cells were analysed by qRT-PCR.10.TCGA database analysis showed the correlation of the expression of GLUT1 with CD47 in HCC;The promoter sequence of GLUT1 were collected from JASPAR database;ChIP and sequencing analyses demonstrated that STAT3 could or not directly bind to the promoter regions in GLUT1.11.CCK-8 analysed the cell viability of Huh7,HepG2,HepG2.2.15 and Hepa1-6 cells after treatment of napabucasin,cryptotanshinone or oxaliplatin for 48h.12.Western blotting analysed STAT3 and p-STAT3Tyr705 levels in Huh7 and HepG2.2.15 and Hepa1-6 cells after treatment of napabucasin for 12h;The cell cycle,apoptosis rate and pyknosis were detected by PI,Annexin-V/PI and hoechst staining respectively;The expression levels of Nanog,SOX2,Klf4 and Oct4 mRNA were analysed by qRT-PCR;The self-renewal ability were assessed by colony formation assay and spheroid formation culture.13.Cell viability of Huh7?HepG2.2.15 and Hepa1-6 spheroids were detected by CCK-8;The expression levels of Nanog,SOX2,Klf4 and Oct4 mRNA were analysed by qRT-PCR;JuLITM Stage Real-Time Cell History Recorder captured the images of Huh7?HepG2.2.15 and Hepa1-6 spheroids under napabucasin treatment at real-time for 48h.14.HBx,HBc and HBs/p mRNA levels in HepG2.2.15 cells were analysed by qRT-PCR.15.After treatment of napabucasin,the extracellular acidification rate(ECAR)of Huh7 and HepG2.2.15 cells were analysed by Seahorse XF24 Extracellular Flux Analyzer;Flow cytometry analysed the ICD-related molecules on HCC cell surface;After co-incubated with hDC for 48h at 37? and 5%CO2,flow cytometry analysed the expression of CD80?CD86?CD40 and MHC II on hDC.16.Plasmid carrying STAT3-shRNA was used to interfere with the expression of STAT3 in Hepal-6 cells for 48h.STAT3 mRNA levels in Hepa1-6 cells were analysed by qRT-PCR;The ICD-related molecules on Hepa1-6 cell surface were analysed by flow cytometry.17.The mDCs generated from mouse bone marrow cells of C57BL/6J male mice was determined by flow cytometry analysis of mCD11c+cells.18.The liver orthotopic transplantation mouse model with Hepa1-6 cells were intraperitoneally injected with 20 mg/kg napabucasin or placebo every 2 days for 8 consecutive times after 2 weeks of tumour inoculation;Flow cytometry analysed the expression of CD95 and PDL1 on tumor cells;The number of infiltrated DCs,macrophages and CD8+T cells in the liver were assessed by flow cytometry.19.The Hepa1-6 homograft mouse model with Hepa1-6 cells were intraperitoneally injected with 20 mg/kg napabucasin or placebo every 2 days for 8 consecutive times at 5th days of tumour inoculation,the overall survival and tumour growth of HCC-bearing mice were assessed;The tumour recurrence rate and tumour growth were recorded in mice underwent a second Hepa1-6 cell inoculation 7 days after tumour resection.20.Immunofluorescence analysed the expression of calreticulin,ERp57,CD47 and GLUT1 in tumor tissues;Flow cytometry analysed the levels of Ki67,CD95 and PDL1 in tumor cells;The expression levels of Nanog,SOX2,Klf4 and Oct4 mRNA were analysed by qRT-PCR.21.The number of infiltrated DCs,macrophages,CD4+T and CD8+T cells in the tumors were assessed by flow cytometry.22.The Hepa1-6 homograft mouse model bearing large tumour load were intraperitoneally injected with 20 mg/kg napabucasin or placebo every 2 days for 8 consecutive times.The proportion of apoptotic tumor cells and the necrotic area in the tumor tissues were detected by HE and Tunel staining;The expression levels of Nanog,SOX2,Klf4 and Oct4 mRNA were analysed by qRT-PCR;The number of infiltrated CD8+T and NK cells in the tumors were assessed by flow cytometry.Results1.Knockdown of STAT3 induces immunogenic cell death of HCC cells expressionThe expression of STAT3 in Huh7,HepG2.2,15 and Hepa1-6 cells were interfered by lentivirus carrying STAT3-shRNA,along with the increase in apoptosis and inhibition of proliferation in HCC cells.The expression of calreticulin and ERp57 on the surface of Huh7 and HepG2.2.15 cells were also significantly increased accompanied with augmented ATP release by STAT3 knockdown.Meanwhile,compared to DCs co-cultured with control HCC cells,CD80 and CD86 expression was higher on DCs co-incubated with STAT3-silenced Huh7 or HepG2.2.15 cells.TCGA database analysis showed that the expression of CD47 is increased and positively correlated with STAT3 in HCC.And ChIP assay verified that STAT3 could directly bound to the promoter of CD47 and regulated its gene transcription.In addition,STAT3 knockdown could promote macrophage-mediated phagocytosis of HCC cells.2.STAT3 regulates the phosphorylation of ICD marker eIF2?through interaction with PKRKnockdown of STAT3 significantly upregulated the expression and phosphorylation of eIF2? kinase PKR in Huh7 and HepG2.2.15 cells,accompanied by decreased formation of STAT3-PKR complex,which confirms STAT3 regulate eIF2? activation via the formation of STAT3-PKR complexes in HCC cells.3.STAT3 directly regulates GLUT1 in HCC cellsThe TCGA database showed an elevated expression of key glycolysis-related molecules such as HIF-1?,GLUT1,and HK2 in HCC,associated with short survival of HCC patients.Although we found no significant difference in LDHA expression between normal and tumour tissues,HCC patients with high LDHA levels showed a decreased survival time.Moreover,STAT3 was positively correlated with HIF-1?,GLUT1,HK2,and LDHA.Additionally,STAT3 knockdown significantly inhibited glycolysis and maximum glycolysis capacity of Huh7 and HepG2.2.15 cells,as well as downregulated HIF-1?,GLUT1,HK2,and LDHA.The glucose analogue 2-DG could inhibit the proliferation of Huh7 and HepG2.2.15 cells in a time-and concentration-dependent manner.Furthermore,2-DG treatment resulted in calreticulin increase but CD47 decrease on HepG2.2.15 cells glucose-starved for 6 h and then allowed(or not)to recover.TCGA database analysis showed that expression of GLUT1 is increased and positively correlated with CD47 in HCC.Furthermore,the selective GLUT1 inhibitor STF-31 significantly increased the expression of calreticulin but decreased that of CD47 on HepG2.2.15 cells.And ChIP assays proved that STAT3 could directly bind to the promoter of GLUT1 and regulated its gene transcription.4.Napabucasin triggers ICD of HCC cells and improves tumor immune environment in vivoNapabucasin exhibited similar effects to STAT3-shRNA and doxorubicin in upregulating calreticulin,ERp57,HSP70 and HSP90 on the surface of Huh7 and HepG2.2.15 cells and DC activation in vitro.In Hepa1-6 homograft mouse model,napabucasin significantly suppressed tumour growth,along with the upregulated calreticulin,ERp57 and downregulated CD47,GLUT1 in the tumour cells.Although the number of infiltrated DCs in the liver did not noticeably change,the expression of CD80 and MHCII was significantly increased by napabucasin treatment.In addition,napabucasin enhanced the infiltration of CD8+T cells and downregulated immunosuppressive molecules such as LAG-3 and PD-1.Infiltration and CD86 expression of CD11b+F4/80+macrophages were also significantly increased in the livers of napabucasin-treated animals.Significantly,mice underwent a second Hepa1-6 cell inoculation 7 days after tumour resection had a 100%tumour recurrence rate in placebo group,but only 25%in the napabucasin-treated group,in addition to slower tumour growth.In mice bearing large tumour load,athough tumour growth was not significantly inhibited by napabucasin,LAG-3+and Tim-3+CD8+T cells were significantly reduced,while the expression of IFNy was significantly increased.Similar results were seen in infiltrated NK cells.5.STAT3 inhibition induces anti-tumour immunity in liver orthotopic transplantation mouse model with Hepa1-6 cellsIn liver orthotopic transplantation mouse model with Hepa1-6 cells,napabucasin significantly suppressed tumour growth,along with the downregulated PD-L1 and upregulated CD95 in the tumours.Although the number of infiltrated DCs in the liver did not noticeably change,the expression of CD80 and MHCII was significantly increased by napabucasin treatment.In addition,napabucasin enhanced the infiltration of CD8+ T cells and downregulated immunosuppressive molecules such as LAG-3 and PD-1.Infiltration and CD86 expression of CD11b+F4/80+ macrophages were also significantly increased in the livers of napabucasin-treated animals.6.STAT3 inhibitor napabucasin inhibits stemness characteristics in HCC cellsCompared to that of oxaliplatin and the plant-derived STAT3 inhibitor cryptotanshinone,napabucasin exhibited a stronger suppressive effect compared to either cryptotanshinone or oxaliplatin on the viability of HCC cells,and had a significantly lower IC50 value.And napabucasin significantly decreased the colonies formation and spherogenesis of Huh7,HepG2.2.15 and Hepa1-6 cells.Meanwhile,the viability of the multicellular spheroids derived from Huh7 cells was significantly reduced by napabucasin,and the cell masses were obliterated at the high dose of 5?M.The expression levels of Nanog,Sox-2,Klf4 and Oct4 mRNA were significantly decreased in the napabucasin-treated HCC cells and spheroids in a dose-dependent manner.7.STAT3 inhibitor napabucasin inhibits tumor growth in Hepa1-6 homograft mouse modelIn Hepa1-6 homograft mouse model,napabucasin prolonged the overall survival of HCC-bearing mice,and markedly delayed the tumour growth compared to that in the placebo group.Furthermore,napabucasin down-regulated Nanog,Sox2,Klf4 and Oct4 in the tumor tissues,and also decreased the proportion of Ki67+tumor cells.In mice bearing large tumour load,napabucasin treatment increased the proportion of apoptotic tumor cells as well as the necrotic area in the tumor tissues.Furthermore,napabucasin downregulated Nanog,Sox2,Klf4 and Oct4,and the percentage of Ki67+cells in the tumors.ConclusionsIn this study,we found that STAT3 inhibition could induce ICD of HCC cells,which manifested as translocation of "eat me" molecules such as calreticulin and ERp57 to the cell surface while exposure of "don't eat me" molecule CD47 significantly decreased.And extracellular ATP as a "find me" signal were also increased.STAT3 inhibition enhanced the recognition and phagocytosis of HCC cells by macrophages,and diminished HCC-induced disturbance of DC maturation and activation.Furthermore,STAT3 knockdown inhibited the glycolysis via GLUT1,facilitating the induction of ICD in HCC.In HCC mouse models,STAT3 inhibitor napabucasin inhibited tumour growth along with tumour-infiltrated DCs and macrophages increased.More CD4+T and CD8+T cells accumulated in tumour tissues,and CD8+T displayed lower expression of LAG-3 and PD-1.Significantly,the anti-tumour immune memory induced by napabucasin enabled resistance against tumour re-challenge and recurrence in vivo.Further,napabucasin inhibited sternness characteristics in HCC cells,and markedly prolonged the overall survival of HCC-bearing mice.SignificanceWe confirmed that STAT3 inhibition induced immunogenic cell death of HCC cells,improved tumour immune microenvironment,and reversed the immune tolerance in tumors,which is important for evoking anti-tumour immune memory in vivo.The present study proves that STAT3 is a potential target for the treatment of HCC,and provides a solid experimental basis for treatment strategies and clinical trials of HCC. |
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