ACSS2 Mediating ATG5-dependent Autophagy In Cd-induced Breast Cancer Progression

Background:Cadmium(Cd)is a heavy metal which is widely used in the chemical,electroplating and nuclear industries,and is an environmental pollutant which is harmful to humans and animals.In addition to the occupational population,general population was also exposed to Cd through several ways,which might lead to various health risks.The IARC classifies Cd as a group 1 carcinogen for its increasing risk of triggering various cancers,such as prostate cancer,lung cancer and renal cancer.Besides,further researches revealed that autophagy plays a critical role in Cd-induced prostate cancer progression.Recent studies have suggested that Cd is associated with an increased risk of breast cancer(BC),nevertheless,the function of autophagy in Cd-induced BC have not yet been elucidated.Therefore,we proposed a hypoesis that Cd-induced BC proliferation and metastasis through regulating autophagy.Rapamycin(Rap)possesses a wide array of pharmacological activities,including anti-BC activity.However,the effects of Rap on Cd-increased BC progression and the essential mechanisms are not available yet.In the present study,we evaluated the function and mechanism of autophagy in the Cd-induced BC cell proliferation,migration and invasion,and investigated the effects and mechanisms of rapamycin antagonized Cd-induced BC progression.Furthermore,we verified the clinical significance of ACSS2 and ATG5 in BC tissues.Contents:1.Cd promotes BC progression through inhibiting autophagy.We treated MCF-7 and T47-D cells with 0,0.1,1 and 10 μM Cd Cl2 for 72 h to establish the Cd exposure model.To evaluate the capacity of cell proliferation,migration and invasion,the CCK-8 assay,transwell-migration and invasion assays were applied in this study,and we found that Cd exposure promotes BC cell proliferation,migration and invasion.Monitoring the stages of autophagy revealed that the number of GFP-LC3 puncta in Cd-treated cells significantly decreased;however,the colocalization of GFP-LC3 with Lysotracker or lysosomal function did not exhibit significant changes under Cd exposure,these data suggesting that Cd inhibits autophagosome formation in BC cells but does not affect autophagosome-lysosome fusion and lysosomal functions.We further assessed the expression of autophagosome formationrelated genes and found the critical role of ATG5 in Cd-inhibited autophagosome formation,in addition,ATG5 overexpression counteracted Cdinduced suppression of GFP-LC3 puncta formation,as well as prevented Cdmediated BC cell proliferation,migration and invasion.2.ACSS2 deficiency is essential for Cd inhibiting autophagy and promoting BC progression.We performed GSEA based on the BC cohorts in the GO database and showed that higher expression of ACSS2 was positively related to the expression of certain components of the autophagy pathway,including ATG5,moreover,the interaction between ATG5 and ACSS2 was also predicted with Gene MANIA,suggesting that ACSS2 may be a potential target for regulating Cd-suppressed autophagy.ACSS2 overexpression clearly upgraded the autophagosome content levels and ATG5 expression under Cd exposure,additionally,Cd-increased BC cell proliferation,migration and invasion were suppressed by ACSS2 overexpression.More importantly,a Cistrome Data Browser analysis confirmed that the epigenetic regulation of the ATG5 promoter was mainly dependent on H3K27 acetylation.H3K27 acetylation was markedly decreased after Cd treatment,while ACSS2 overexpression significantly rescued the Cd-mediated inhibition of H3K27 acetylation in MCF-7 cells.3.Rap antagonizes Cd-induced BC cell proliferation,migration and invasion through directly modulating ACSS2.CCK-8,transwell-migration and invasion assay were preformed to verify that Cd-induced BC cell proliferation,migration and invasion was antagonized by Rap.Moreover,a SPR assay confirmed that rapamycin directly binding to the ACSS2 protein and molecular docking showed that Rap binds at the COA binding site,indicating that ACSS2 is a direct binding protein for Rap.While the effects of rapamycin antagonized Cd-mediated BC progression were abolished by an ACSS2 inhibitor.Together,these results confirmed that Rap antagonized Cd-promoted BC progression through directly modulating ACSS2.4.Downregulation of ACSS2 and ATG5 in BC tissues is associated with poor prognosis.Based on the GEPIA and TCGA databases,the expression levels of ACSS2 in BC tissues and normal tissues were obtained;and the association between ACSS2 expression level in BC patients and prognosis was analyzed by KaplanMeierplotter database.The expression levels of ACSS2 and ATG5 in tissues were measured by q-PCR and IHC staining.We found that the expression of both ACSS2 and ATG5 was lower in BC tissues than in mammary fibroma tissues,and there is a positive correlation between ACSS2 and ATG5 expression.Low expression of ACSS2 and ATG5 showed poor prognosis,exhibiting that ACSS2 and ATG5 expression was inversely correlated with tumor size,and the DFS of patients with BC and high ACSS2 expression was significantly longer than those with low ACSS2 expression.Conclusion:Our present study first demonstrated that Cd promoted BC progression via suppressing ACSS2 mediated ATG5-dependent autophagy,and Rap antagonized Cd-induced BC cell proliferation and metastasis through directly modulating ACSS2.This study revealed the molecular mechanism of the relationship between heavy metal exposure and BC.We found that autophagy played a vital role in Cd exposure promoted BC progression,and ACSS2 was an essential therapeutic target of rapamycin.