Huaier Induces Immunogenic Cell Death In Triple Negative Breast Cancer

BACKGROUND:Triple-negative breast cancer(TNBC)is the most lethal subtype of breast cancer.Due to the lack of effective treatment strategy,patients often have low objective response rate(ORR)and poor prognosis.With the in-depth understanding of tumor immunology,manipulation of the immune system has become an attractive treatoment option in various malignancies.Although previously held notion suggested that breast cancer was a poorly immunogenic cancer type,recent investigations have demonstrated a markedly higher infiltration of immune cells in TNBC than that in other subtypes.Meanwhile,the tumor infiltration of lymphocytes is tightly associated with the prognosis of TNBC patients.Trametes robiniophila Murr(Huaier)belongs to Class Hymenomycetes,Phylum Basidiomycota.Earliest recorded by "Compendium of Materia Medica"(Ben Cao Gang Mu),Huaier has been used to treat disease by the Chinese folk for approximately 1600 years.According to modern biological studies,as a traditional herbal medicine with immunomodulatory functions,Huaier has significant tumorsurpressing effects in the treatment of TNBC.However,the mechanisms remain to be elucidated.METHODS AND RESULTS:In the present study,we intend to explore the molecular mechanism of Huaier in treatment of TNBC from the unique immune infiltrating pattern of the subtype.The contents include the following three parts:Part Ⅰ Construction of ICD-related prognostic model in TNBCMETHODS:Based on the METABRIC(molecular taxonomy of breast cancer international consortium)dataset,the tumor infiltrating cells of TNBC were quantified by MCP-counter(microenvironment cell populations counter)algorithm and segregated into clusters.Kaplan-Meier method was used to screen the types of tumor infiltrating cells affecting the prognosis of TNBC patients.The METABRIC cohort was equally divided into training cohort and validation cohort by using a random number table.In the training cohort,the immune infiltration-related prediction model of TNBC was established.Subsequently,the prognostic value of the model was evaluated by Kaplan-Meier survival analysis and time dependent ROC curve in training cohort and validation cohort,respectively.RESULTS:In TNBC patients,infiltration of T cells and dendritic cells(DCs)showed positive correlation with the overall survival and played a crucial role in reducing the risk of recurrence.T cells and DC cells are indispensable players to induce immunogenic cell death(ICD).This immune pattern suggests that the prognosis of TNBC could be possibly related to ICD.Therefore,we established an ICD-related risk model(ICDscore)based on the tumor infiltrating T cells and DC cells in the METABRIC training cohort.And the prognostic value of ICDscore was verified in both training cohort and validation cohort.These findings demonstrated the central role of ICD in the progression of TNBC.Part Ⅱ Huaier induces ICD in TNBCMETHODS:Flow cytometry and immunofluorescence analysis were applied to determine the effects of Huaier in the TNBC cell surface exposure of calreticulin(CRT).Adenosine triphosphate(ATP)kit was utilized to detect the ATP concentration in the supernatant of the cells after the treatment by Huaier.The content of CRT in cell membrane protein and the enrichment of high mobility group protein B1(HMGB1)in the cell cultural medium were analyzed by westernblot assays.Mouse bone marrow derived DCs(BMDCs)were isolated to co-culture with Huaier treated TNBC cells.Flow cytometry was used to analyze the surface marker on DCs.The functions of endogenous CD8+T cells in Huaier-induced tumor suppression were further explored by in vivo experiments.The subcutaneous tumor model of TNBC was established in Balb/c mice to compare the tumor inhibition effects of oral administration of Huaier,intraperitoneal injection of programmed cell death protein 1(PD-1)inhibitor,and combined application of two drugs.Tumor growth and weight changes of mice were monitored regularly.The tumor samples were fixed with formalin and made into paraffin-embedded sections.The immunohistochemical staining assay was applied to evaluate the T lymphocyte infiltration in the tumor microenvironment(TME).RESULTS:Huaier could induce ICD in TNBC cell lines via augmenting cell surface CRT exposure and increasing the release of ATP and HMGB1.Co-cultured with Huaier-treated TNBC cells effectively promoted the maturation of DCs.This conclusion was further confirmed by a cell-based in vivo vaccination assay.In the mouse subcutaneous tumor model,oral administration of Huaier could obviously promote the infiltration of lymphocytes and substantially slow the tumor growth.Meanwhile,the depletion of endogenous T cells by antibodies could eliminate the inhibitory effects of Huaier on TNBC.Compared with single-agent therapy groups,the combination of PD-1 inhibitor and Huaier showed significantly stronger tumor suppressing effects.Besides,mice in each group maintained a steady weight.In the immunohistochemical staining assay,the T lymphocyte infiltration in the TME was substantially increased in the combined therapy group.These findings further indicated that Huaier could markedly improve the therapeutic effects of immune checkpoint inhibitors in TNBC patients via inducing ICD.Part Ⅲ The mechanisms of Huaier-induced ICDMETHODS:In the MDA-MB-231 and MDA-MB-468 TNBC cell lines,the differentially expressed messenger RNAs(mRNAs)before and after Huaier treatment were detected by gene expression profiling.Gene Ontology(GO)analysis was utilized to evaluate the associated molecular pathways of Huaier-induced ICD.And the results of bioinformatics analysis were subsequently validated via westernblot assays.To investigate the underlying mechanisms of how Huaier stimulates ER stress,the circRNA expression profiles of MDA-MB-231 and MDA-MB-468 cells before and after Huaier treatment were analyzed with R language "limma" package.The differentially expressed circRNAs were verified using real-time quantitative PCR(qRT-PCR)assays.RNA pulldown and mass spectrometry(MS)assays were employed to determine the downstream binding protein of the circRNA.RNA binding protein immunoprecipitation(RIP),fluorescence in situ hybridization(FISH)and immunofluorescence assays were then utilized to validate the results.RESULTS:In the analysis of cell component(CC),the differentially expressed mRNA was strikingly correlated with the endoplasmic reticulum,while in the analysis of biological process(BP),these mRNA showed noticeably enrichment in"endoplasmic reticulum stress response" and "positive transcriptional regulation of RNA polymerase Ⅱ promoter in endoplasmic reticulum stress response".It suggested that Huaier-stimulated ICD might be related to the activation of endoplasmic reticulum(ER)stress.In the biological experiments,westernblot assays revealed that Huaier could substantially enhance the phosphorylation of eIF2α,thus induces ER stress-related ICD.Bioinformatics tools were applied to analyze the RNA-sequencing results.The expression of circular RNA,circCLASP1,was enormously upregulated in Huaier treated TNBC cells.In vitro assays demonstrated an independent role of circCLASP1 in the Huaier-triggered phosphorylation of eIF2α.PKR/eIF2α/ATF4 was a crucial axis in ER stress.Further studies show that circCLASP1 could directly bind to PKR in the cytoplasm which lead to its up-regulation.Thus,circCLASP1 participates in the stimulation of Huaier-induced ER stress and enhances the activation of ICD.CONCLUSIONS1.ICD-related risk model for TNBC was constructed based on METABRIC.2.Huaier could effectively induce ICD in TNBC.3.Huaier-treated tumor cells showed immune-stimulating effects in the in vivo vaccine assay.4.Combination of PD-1 inhibitor and Huaier could substantially slow TNBC progression.5.Huaier-triggered ICD in TNBC is regulated by ER-stress through circCLASP1/PKR/eIF2α/ATF4 signaling pathway.SIGNIFICANCESTaken together,in this study,bioinformatics methods were employed to analyze the immunological characteristics of TNBC and a prognostic risk model based on dendritic cells and T cells was constructed.The results indicated a vital role of ICD in the progression of TNBC.Huaier,a traditional Chinese medicine,has significant immune regulation function,but the molecular mechanism remains unelucidated.In the study,we demonstrated the crucial effects of circCLASP1/PKR/eIF2α/ATF4 in Huaier-induced ICD via in vivo and in vitro assays.In addition,the study provided theoretical foundations for the combined application of Huaier and immune checkpoint inhibitors.Our findings harboured significant translational potential and proposed a promising research direction for achieving long-term remission in the treatment of TNBC patients.