Huaier Polysaccharides Inhibit The Development Of Triple Negative Breast Cancer By Inducing Autophagy

Triple-negative breast cancer(TNBC)is a subtype of breast cancer that is negative for the expression of estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor receptor-2(Her-2).It has characteristics of high malignancy,high metastasis and recurrence rate,and poor prognosis.Currently,surgery and chemotherapy are still the main strategy for treating TNBC due to the lack of specific therapeutic targets[1,2].However,only about 31%of TNBC patients achieve pathological complete response after chemotherapy,and all metastatic TNBC patients develop chemotherapy resistance[3-5].Therefore,finding new strategies to treat TNBC is currently an urgent problem to be solved.Autophagy is a highly conserved lysosome-dependent intracellular degradation pathway that maintain cellular homeostasis by eliminating abnormal proteins and organelles[6-8].On the one hand,autophagy prevents the accumulation of denatured proteins and damaged organelles in cells,maintains the stability of intracellular substance circulation,and thereby inhibits the occurrence and development of tumors.On the other hand,autophagy can provide metabolic substrates by recycling intracellular components to meet the energy needs of highly metabolic tumors,thus promoting the survival of tumor cells[9,10].In breast cancer,autophagy inhibits cell invasion and migration,prevents tumor immune escape,and enhances the sensitivity of tumor cells to treatment[11-13].In summary,autophagy plays an important role in breast cancer and may have an impact on the occurrence,development,and treatment response of breast cancer.Therefore,further research on autophagy may help to develop new treatment strategies and improve the treatment efficacy of TNBC.Traditional Chinese medicine(TCM)has unique advantages in cancer treatment[14,15].Among them,Huaier granules are widely used in the adjuvant treatment of various malignant tumors,with the characteristics of good therapeutic effect,few side effects,and high safety[16,17].The previous clinical research of our research group showed that Huaier granules can effectively limit the progression of TNBC,providing a possibility for the treatment of TNBC[18].To further investigate,we purified its main active ingredient,polysaccharides of Trametes robiniophila Murr(PS-T)[19].Numerous studies have confirmed that PS-T could promote tumor cell apoptosis,inhibit cell stemness,and block invasion and metastasis,thus exerting anti-tumor effects[20-22].In addition,PS-T can induce autophagy in tumor cells by inhibiting the AKT/m TOR/S6K signaling pathway,thereby inhibiting tumor progression[23-25].However,the role and specific mechanism of PS-T–induced autophagy in the occurrence and development of TNBC are still to be studied.This study aims to explore the effects and specific mechanisms of PS-T on the cell motility and tumor immune microenvironment in TNBC,and to investigate the role of PS-T–induced autophagy in tumor invasion,migration,and immune response.Further,we demonstrated the potential of combining immune checkpoint inhibitors with PS-T for the treatment of TNBC.Experimental Methods and Results1.PS-T inhibits the invasion and metastasis of triple-negative breast cancer by inducing autophagy(1)Transwell invasion,migration and scratch experiments showed that PS-T inhibits the invasion and migration abilities of MDA-MB-231 and 4T1 cells.(2)The expression of EMT-related markers were detected using Western Blot.The results showed that PS-T blocks TMT transformation in TNBC cells.(3)The PS-T–induced autophagy in TNBC cells was confirmed by Western Blot and immunofluorescence experiments in vitro.(4)Additionally,the suppressive effects on cell invasion and migration were reversed in TNBC cells interfering with ATG5.(5)We then performed Western Blot to detect the expression of Snail,a key transcription factor of EMT.The result showed that PS-T significantly downregulated Snail expression,while Snail expression level was restored after ATG5 interference.(6)The results of the acute toxicity experiment showed that PS-T did not influence the body weight and food intake of mice,nor did cause obvious damage to major organs,including the heart,liver,spleen,lung,and kidney.(7)We constructed 4T1 orthotopic mouse breast cancer models and verified the inhibitory effect of PS-T on tumor growth and induction of autophagy in breast tumor.Conclusion:PS-T inhibited the invasion and metastasis by inducing autophagy-dependent degradation of Snail,thereby inhibiting the tumor growth in TNBC.2.PS-T promotes antitumor immunity against triple-negative breast cancer by inducing autophagy(1)Flow cytometry and immunohistochemical staining revealed that the number of CD45~+,dendritic cells(DCs),CD3~+T,CD4~+T,CD8~+T,and cytotoxic CD4~+and CD8~+T cells in the tumor and spleen of PS-T treated mice was significantly increased,whereas that of immunosuppressive regulatory T cells(Tregs),myeloid-derived suppressor cell(MDSCs),and M2-macrophages was decreased.(2)in vitro and in vivo experiments,PS-T downregulates PD-L1 expression in a dose-dependent manner.(3)The m RNA level of PD-L1(CD274)in MDA-MB-231 treated with or without PS-T was detected by transcriptome sequencing and found no significantly difference.(4)The immunohistochemistry results showed that the expression of LC3 was significantly increased,while that of PD-L1 was decreased after PS-T treatment.There was a correlation between the expression levels of LC3 and PD-L1 protein.(5)By autophagy inhibition by pharmacological inhibitors and ATG5 knockdown,we found autophagy inhibition reverses the degradation of PD-L1 protein and activation of T-cell–mediated immune responses in TNBC.(6)We then confirmed that the autophagy status is associated with the PD-L1 protein level as well as T cell infiltration in TNBC samples.(7)Furthermore,the synergisms of anti-PD-L1 antibodies and PS-T were analyzed in vivo.Comparing to single administration group,the combination of atezolizumab and PS-T exhibited enhanced inhibition of tumor growth,Ki67 expression,and Fox P3~+Tregs count in TNBC,and promotion of CD4~+,and CD8~+T cells.Conclusion:PS-T significantly activated antitumor immune responses in TNBC.PS-T significantly downregulates PD-L1 expression by inducing autophagy,thus activating the T cell antitumor immune responses in TNBC.PS-T enhances the inhibitory effect of anti-PD-L1 antibodies on tumor growth and cell proliferation,and the activation of T-cell antitumor immune response in TNBC.