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Investigation of target molecule alterations by a cDNA microarray in ATO-treated HCC cells

ABSTRACT: BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) has a high mortality rate due to the lack of effective treatments and drugs. Arsenic trioxide (As2O3, ATO, arsenious acid), which has been proved to successfully treat acute promyelocytic leukemia (APL), was recently reported to show therapeutic potential in solid tumors including liver cancer. Although the mechanistic effects of ATO in APL are established, its anticancer mechanism of action in HCC is still unclear. METHODS: We established an HCC subcutaneous xenograft and intrahepatic metastasis mouse model. In CSC models, tumorspheres and the flow cytometry analysis of CSC markers together with limiting dilution and serial transplantation were used. We compared mRNA expression profiles of the ATO-treated and control cells with mRNA microarray. The expression of target molecule or a clinical correlation was analyzed by immunohistochemistry staining in tissues from ATO-treated mouse and 76 HCC patients. We generated five stable minichromosome maintenance protein 7 (MCM7)-knockdown and two stable MCM7-overexpression HCC cell lines. The chromatin immunoprecipitation (ChIP) assays, immunoprecipitation (IP) assays, the dual luciferase reporter assays, a green biarsenical labeling reagent (FlAsH-EDT2) and a “competing endogenous RNA” (ceRNA) analysis were used. RESULTS: ATO could inhibit the liver tumor-initiating capacity and distant metastasis and prolongs survival in mice. We then screened and found that ATO downregulates the overexpression of MCM7 which is correlated with the progression and prognosis in HCC patients. Knockdown of MCM7 expression recapitulates the inhibition function of ATO on self-renewal of cancer stem cells (CSCs), while overexpression of MCM7 abolishes the inhibition function of ATO on tumorsphere formation. Most importantly, we revealed that ATO directly binds to the MCM7 protein and disturbs the interaction between serum response factor (SRF) and MCM7, resulting in downregulation of MCM7 transcription. A ceRNA analysis also indicated the alterations of endogenous MCM7-associated gene networks involved in stemness-related signaling pathways and cell differentiation. CONCLUSIONS: Here, for the first time, we report that ATO inhibits liver CSCs through blocking the interaction between SRF/MCM7 and suppressing MCM7 autoregulation activity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE111935 | GEO | 2019/07/02

REPOSITORIES: GEO

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Project description:BACKGROUND & AIMS: Growing lines of evidence suggests that a subpopulation of cancer cells with self-renewal and tumor-initiating capacity, termed as cancer stem cells (CSCs) or tumor initiation cells (TICs), play pivotal roles in tumor relapse and tumor distant organ metastasis. Therefore, to prevent and treat metastasis based on targeting essential molecules regulating CSCs emerges a promising strategy. Here we describe CU-27, a novel organic selenium compound, significantly diminishes hepatocarcinoma cancer cell (HCC) stemness and inhibits cacner cell metastasis in vitro and in vivo. METHODS: we performed a functional screen to identify compounds with anti-CSC and metastasis effects, In vitro CSC models, tumorspheres, drug resistance and the flow cytometry analysis of CSC markers together were used. In vivo CSC models, limiting dilution and metastatic mouse model were used. We compared mRNA expression profiles of the CU27-treated and control cells with mRNA microarray. The key biological target of CU27 was analyzed by Ingenuity pathway analysis (IPA). The surface plasmon resonance (SPR), chromatin immunoprecipition (ChIP) assays and the dual luciferase reporter assays were used to explore mechanisms of CU27. RESULTS: A novel selenium-containing small molecule-CU-27, was demonstrated to significantly inhibit CSCs self-renewal and function as a metastasis suppressor of HCC cells in vitro and in vivo. Through differentially expressed gene screening with cDNA microarray and IPA analysis, c-MYC, which is mostly recognized to play very important roles in tumor initiation and maintenance in many kinds of tumors, was identified as the major biological target of CU-27. CU-27 was found to significantly suppress c-MYC transcription function and downregulate expression levels of its target genes, thereby inhibit CSC self-renewal and distant metastasis of HCC cells. Ectopic expression of c-MYC rescues the inhibition effects of CU-27 on HCC cells. More important, CU-27 was further demonstrated to interact directly with the bHLH domain of c-MYC protein and decrease its occupancy of the promoter region of target genes, which, in turn, downregulates c-MYC target gene expressions. CONCLUSIONS: CU-27 which can inhibit HCC CSCs and distant metastasis through suppressing c-MYC transcriptional function. Our study provides a vital strategy for targeting CSC and metastasis by inhibiting c-MYC function with novel selenium-containing small molecules.

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Investigation of target molecule alterations by a cDNA microarray in ATO-treated HCC cells

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