The Role And Mechanism Study Of Arsenic Trioxide Inhibiting Metastasis And Cancer Stem Cells In Liver Cancer

Hepatocellular carcinoma(HCC)is one of the most common solid tumors with 840,000 new cases diagnosed and 780,000 cases of deaths worldwide each year.Its tumorrelated mortality ranks top three among malignant tumors.This high mortality rate not only due to the high metastatic potential,the lack of effective treatments and drugs are also the main causes.Thus,exploring safe and effective treatment strategies for advanced HCC is always a main focus in liver cancer research.While investigating novel targeted medicine and immunotherapy for advanced HCC,the empirical use of the traditional Chinese medicine Arsenic Trioxide(ATO)in advanced HCC has proved to be a successful case of drug repositioning or repurposing.ATO,which has been recommended by both the U.S.Food and Drug Administration(FDA)and China Food and Drug Administration(CFDA)as the first-line therapy for acute promyelocytic leukemia(APL),also shows therapeutic potential in advanced HCC in several clinical trials.However,unlike in APL,its potential targets and anticancer mechanisms in HCC remain unclear,which limited the safety and efficacy of its application and still need further investigation.Advanced HCC is often associated with metastasis,including portal vein tumor thrombosis,regional lymph nodes invasion,or distal organs metastasis.The true driver of metastasis is a small group of cells defined as cancer stem cells(CSCs),which lying at the apex of the hierarchically organized tumor.CSCs may reconstitute tumors in the process of tumor metastasis or relapse,through the stem cell processes of self-renewal and differentiation.Based on the molecular characteristics and clinical features of ATO,we hypothesized that the therapeutic effect of ATO in advanced HCC may be related to the inhibition of liver cancer stem cells.Our goal is to elucidate whether and how arsenic trioxide inhibits liver cancer stem cells.Here,we demonstrated that ATO inhibits tumorigenesis,relapse and distant metastasis in multiple mouse models,including a subcutaneous tumor formation model,a secondary transplantation model and a metastatic HCC xenograft model.The median survival time of orthotopic tumor-bearing mice was extended from 32 days to 41 days after ATO treatment.Also,ATO was found to significantly inhibit the CSC-associated traits and functions in HCC cells and CSC markers/tumorspheres enriched CSC-like HCC cells,through flow cytometry detecting CSC markers,real-time quantitative PCR(q PCR)detecting stemness related genes,evaluating drug resistance,tumorspheres formation ability and transwell invasion ability.Next,to elucidate the underlying mechanism of ATO in HCC,the target molecule alterations of HCC cells treated by ATO were investigated by a c DNA microarray.Minichromosome maintenance protein 7(MCM7)was identified to be inhibited significantly both in vitro and in vivo after ATO treatment.Knocking down MCM7 expression in HCC cells lead to a decreased rate of lung metastasis from 64.7% to 30% in HCC xenograft model and inhibitory effects on CSCs,implying that MCM7 is a key regulator of CSC and HCC metastasis.While MCM7 ectopic expression abolishes the inhibitory effect of ATO on tumorspheres formation,we confirmed that MCM7 is a key functional target of ATO.Moreover,we investigated the expression profile of MCM7 in HCC and normal tissues derived from 80 liver cancer patients,we found an upregulated expression of nuclear MCM7 in all of the HCC tissues and its correlation with disease progression and prognosis of patients.This finding may provide a new theoretical basis for ATO’s therapeutic effects in advanced HCC and a precision guidance of ATO’s effective application.To explore the underlying mechanism,we performed the immunoprecipitation(IP)and elucidated the role of serum response factor(SRF)/MCM7 complex in transcriptional regulation of MCM7 in HCC cells.The direct binding between ATO and MCM7 was confirmed by an organic FIAs H probe.The dual luciferase reporter system further clarified that ATO suppresses MCM7 transcription by targeting serum response factor(SRF)/MCM7 complex.Taken together,our results suggest that ATO inhibits liver CSCs through downregulating MCM7.Mechanistically,ATO binds to MCM7 and suppresses the transcription activity of SRF/MCM7 complex in modulating MCM7 expression.Our findings reveal the underlying mechanism of ATO in advanced HCC,which may benefit the appropriate and effective use of this agent in the treatment of HCC,and also provide important clues for the screening of new drugs targeting MCM7 pathway.