| The"Cancer Stem Cell"theory provides a new theoretical basis for malignancy recurrence and metastasis,and also points out new directions and strategies for cancer treatment.According to the cancer stem cells theory,there is a small group of cells with strong self-renewal ability in malignancy tissues.They have both the characteristics of tumor cells and the characteristics of stem cells called cancer stem cells(CSCs).The current view is that cancer stem cells have strong resistance for chemotherapy,and are the real source of malignancy recurrence and metastasis.Therefore,targeted treatment of cancer stem cells has become the focus of antineoplastic therapy field.c-MYC is one of the most amplified oncogenes in various types of cancer.About 75%of all types of cancer overexpress c-MYC.It plays an important role in the development of malignant tumors and the maintenance of the cancer stem cells related capability,it is also an important target for the treatment of cancer stem cells.However,similar to RAS and other well-known oncogenes,it is considered to be a"undruggable"cancer target protein,because its protein structure lacks suitable sites for drug binding,so far there is still lack of effective c-MYC targeting drugs in clinic.Targeting c-MYC has become one of the key challenges in the field of anti-cancer research.China has many patients with hepatocellular carcinomas.The number of patients is huge and growing rapidly.It is often difficult to treat advanced liver cancer surgically.However,the long-term therapeutic effect of sorafenib,a first-line drug for advanced liver cancer,is also unsatisfactory.There is an urgent need for more effective therapeutic drugs for advanced liver cancer patients.In view of the significant demand for treatment of advanced liver cancer patients,the special performance of cancer stem cells,and the characteristics of the above related oncogenes.Our objective is to find candidate small molecule drugs that can effectively inhibit liver cancer stem cells capability,and to study the molecular mechanism and therapeutic targets of these drugs,which can help to inhibit the recurrence and metastasis of advanced hepatocellular carcinoma and providing new ways for targeted therapy of advanced liver cancer.Selenium compounds have excellent therapeutic effects in the anti-cancer stem cells research.It has been reported that some selenium compounds can change the redox metabolism of cancer stem cells,and some selenium compounds can target Wnt/beta-catenin signaling pathway to downregulate the stemness of cancer stem cells.In our study,we found a new organic selenium compound CU27 through screening comparison,which can effectively downregulate the stemness of liver cancer stem cell by inhibiting the transcriptional function of c-MYC,and also significantly inhibit the tumorigenesis and metastasis ability of hepatocellular carcinoma cells in vivo.This study also revealed the important role of c-MYC-hn RNPU axis in the capability of liver cancer stem cells and provided a new target for the treatment of liver cancer stem cells.This study mainly includes four parts:?.Screening of Small Molecular Compounds with the Function of Inhibiting Liver Cancer Stem CellsFirstly,we selected 16 candidate molecules from the organic selenium small molecule compound library for functional verification in the three-dimensional cultivation system.The liver cancer stem cell marker CD13,CD133 and tumor-sphere forming ability were used as preli minary screening criteria to evaluate the effect of small molecules compounds on stemness.Among them,small molecule CU27 could not only down-regulate the expression level of liver cancer stem cell markers,but also inhibit the Tumor-Sphere forming ability,and has less cytotoxicity,which has a good potential to inhibit stemness and capability of liver cancer stem cells,and beco ming a candidate target molecule for further research.?.In Vivo and In Vitro Validation of CU27 Inhibiting the Capability of Liver Cancer Stem CellsThrough primary screening,we obtained the ideal candidate molecule CU27.In order to further clarify the role of CU27 on liver cancer stem cells,we designed in vitro and in vivo experiments to test the therapeutic effect of CU27 according to the specific biological capability of liver cancer stem cells.In vitro,we investigated the effects of CU27 on the Tumor-Sphere forming ability,drug resistance,invasiveness and stemness markers expression of hepatocellular carcinoma cells.The results showed that CU27 could effectively reduce the self-renewal ability of Huh7.5.1,PLC/PRF/5 and Hep3b cells by68.8%,78.5%and 86.1%,respectively;and also inhibite drug resistance,invasiveness of hepatocellular carcinoma cells as well as the expression of stemness markers of liver cancer cells.It also inhibited the capability of stemness of enriched liver cancer stem cells.These results suggest that CU27can inhibit the capability of liver cancer stem cells in vitro.In vivo,we investigated the effects of CU27 on tumorigenicity,metastasis and drug resistance of hepatocellular carcinoma cells in mice model.The results showed that CU27 could effectively inhibit the tumorigenicity of hepatocellular carcinoma cells in mice,reducing the tumor formation rate of tumor-bearing mice in groups 10~6,10~5 and 10~4 by 28.6%,87.5%and 66.7%,respectively;and also could inhibit lung metastases of liver cancer cells,compared with the control group,the total number of lung metastasis nodules in the CU27 treatment group decreased by 67.9%,and the total volume of metastases nodules decreased by43.6%;and also could improve the survival rate and survival status of mice bearing hepatocellular carcinoma when combined with doxorubicin.Moreover,the combination of CU27 and sorafenib could significantly enhance the therapeutic effect on sorafenib-resistant hepatocellular carcinoma.In conclusion,CU27 can reduce the stemness and capability of liver cancer stem cells in vitro and in vivo,showing valuable therapeutic potential and application value.?.CU27 binds c-MYC protein and inhibits transcription of its downstream target genesTo study the molecular mechanism of CU27,we detected the gene microarray of hepatocellular carcinoma cells treated by CU27,and screened out the common differentially genes in two hepatocellular carcinoma cell lines.Bioinformatics analysis and IPA upstream regulatory targets prediction of these common differentially genes indicated that c-MYC might be the key biological target molecule of CU27.CU27 inhibited the transcriptional function of c-MYC to reduce the expression of downstream target genes,especially RNA Splicing regulators.In order to clarify the interaction between CU27 and c-MYC,we designed Double Luciferase Report assay,chromatin immunoprecipitation assay(Ch IP),Co-Immunoprecipitation assay(Co-IP)and protein-small molecule binding assay(SPR)to investigate the effect of CU27 on c-MYC.The results showed that CU27 could directly bind the b HLH domain of c-MYC protein,hinder the effective binding of c-MYC to DNA promoters,and decreased the expression of c-MYC target genes.While,overexpression of c-MYC in hepatocellular carcinoma cells can greatly weaken the therapeutic effect of CU27,which also shows that c-MYC is a key biological target molecule of CU27.?.CU27 Decreases the Capability of Liver Cancer Stem Cells by Inhibiting the c-MYC-hn RNPU AxisOn the basis of previous experiments,we further explored which of the down-regulated c-MYC target genes are the key factors for CU27 effects.IPA analysis and experimental verification showed that hn RNPU may be the key target gene for CU27.In order to verify the biological role of c-Myc and hn RNPU in hepatocellular carcinoma cells,we knocked down or overexpressed the expression of hn RNPU in hepatocellular carcinoma cells.The results showed that hn RNPU and c-Myc regulates each other,and have a synergistic effect on the biological properties(proliferation,self-renewal and so on)of HCC cells,knockdown or overexpress one of them,and the other one would have similar changes.c-MYC and hn RNPU might be both important for the proliferation and self-renewal of advanced c-MYC-driven HCC cells.In addition,the expression of hn RNPU was co-localized and positively correlated with c-Myc in HCC samples.This is inspiring that c-MYC-hn RNPU feedback loop in HCC which might be partially responsible for HCC progression.In this study,we demonstrated that CU27 can reduce the expression of hn RNPU by inhibiting the transcriptional function of c-MYC,and hn RNPU overexpression could significantly weaken the therapeutic effect of CU27suggesting that hn RNPU is a key downstream target gene of CU27.CU27 could effectively block the c-MYC-hn RNPU regulatory loop and then inhibit the stemness and capability of liver cancer stem cells as well as reducing self-renewal ability and hepatocellular carcinoma cells metastasis.It is the first time to reveal the important role of c-MYC-hn RNPU regulatory loop in the maintenance of liver cancer stem cells,which also provides a new therapeutic strategy for anti-cancer stem cells. |
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