Arsenic trioxide inhibits liver cancer stem cells and metastasis by targeting SRF/MCM7 complex

Hepatocellular carcinoma (HCC) has a high mortality rate due to the lack of effective treatments and drugs. Arsenic trioxide (ATO), which has been proved to successfully treat acute promyelocytic leukemia (APL), was recently reported to show therapeutic potential in solid tumors including HCC. However, its anticancer mechanisms in HCC still need further investigation. In this study, we demonstrated that ATO inhibits tumorigenesis and distant metastasis in mouse models, corresponding with a prolonged mice survival time. Also, ATO was found to significantly decrease the cancer stem cell (CSC)-associated traits. Minichromosome maintenance protein (MCM) 7 was further identified to be a potential target suppressed dramatically by ATO, of which protein expression is increased in patients and significantly correlated with tumor size, cellular differentiation, portal venous emboli, and poor patient survival. Moreover, MCM7 knockdown recapitulates the effects of ATO on CSCs and metastasis, while ectopic expression of MCM7 abolishes them. Mechanistically, our results suggested that ATO suppresses MCM7 transcription by targeting serum response factor (SRF)/MCM7 complex, which functions as an important transcriptional regulator modulating MCM7 expression. Taken together, our findings highlight the importance of ATO in the treatment of solid tumors. The identification of SRF/MCM7 complex as a target of ATO provides new insights into ATO’s mechanism, which may benefit the appropriate use of this agent in the treatment of HCC.


Supplementary Figure 5 Establishment of MCM7-knockdown HCC cells. (A)
Validation the knockdown effects of two constructed psicoR-shMCM7-GFP lentivirus vector (shRNA-1 and shRNA-2) by western blot analysis in Huh7.5.1 cells with the scramble as controls (psicoR-scramble-GFP, scram). (B) Western blot analysis of MCM7 expression in HCC cells with stable knockdown of MCM7 by the psicoR lentivirus vector (psicoR-shMCM7) with the scramble as a control (psicoR-scramble). Figure 6 Immunofluorescence analysis of CK18 expression in MCM7-knockdown HCC cells. CK18 expression of psicoR-shMCM7-GFP stable cells and control cells were evaluated under the same exposure conditions. Bars, 50 µm.

Microarray
The microarray experiments were performed in the laboratory of the OE Biotech

Vector constructs and cell transfections
The expression construct for human full-length MCM7 was generated by cloning PCR-amplified MCM7 cDNA fragments into the pcDNA3.

Dual-luciferase reporter assay
The luciferase reporter assay was performed in triplicate. The 293T cells were co-

In vitro arsenic-protein binding assay
The microarray slides were provided by CapitalBio Technology (Beijing, China). We

Immunoprecipitation (IP)
The HCC cells were treated by 3.6 µM ATO with PBS as the control. After

Cell viability assay
Cell viability was determined using the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) as previously described (3). A total of 2×10 3 -3×10 3 L02 cells or HCC cells per well were seeded in 96-well plates with DMEM (Sigma) containing 10% fetal bovine serum (FBS, Defined) (ExCell Biology, Shanghai, China). The cells were treated with ATO at the indicated concentration. The medium was exchanged for 100 µL DMEM plus 10 µL CCK-8 reagents and incubated at 37°C for 2 hours. Absorbance was measured at 450nm at indicated time. For doxorubicin and sorafenib chemoresistance assay, a total of 2×10 3 -3×10 3 CD13 + CD133 + EpCAM + cells per well were seeded in 96-well plates. Twenty-four hours later, the medium was replaced with doxorubicin (or sorafenib)-containing medium at the indicated concentration with or without 3.6 µM ATO and incubated for 48 h.

Cell apoptosis assay
The cell apoptosis was assayed with Annexin V, FITC Apoptosis Detection Kit

Flow cytometry analysis and fluorescence-activated cell sorting (FACS)
The constructed stable cells and CD13 + and CD13 + /CD133 + EpCAM + cells were isolated via sorting after transfection as previously described (3). The expression of CD13, EpCAM and CD133 was examined via flow cytometry in the HCC cells.
After incubation, the cells were washed three times and then sorted on a FACS Aria instrument (BD Bioscience, Franklin Lakes, NJ). The expression of CD13, EpCAM and CD133 was examined via flow cytometry after ATO treatment or genetic manipulation in the HCC cells.

Immunofluorescence (IF) and immunohistochemistry (IHC) staining
Immunofluorescent staining and IHC staining were performed as previously described The staining intensity of IHC was analysed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD).

Western blots
Western blots were performed and analysed as previously described (3)(4)(5). Briefly, cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (Roche) overnight at 4°C with rotation. The whole-cell lysates were fractionated into supernatants and pellets by centrifugation. The cell lysates were separated on SDS-PAGE and transferred to PVDF membranes (Millipore). The blots were blocked with TBST containing 5 % skimmed milk and incubated with primary antibodies overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies (Zhongshan Bio-tech Co., Ltd.) for 1 h. Immunoreactivity was detected and the signals were analysed were detected via Immobilon Western Chemiluminescent HRP Substrate (Millipore). All the antibodies used in this study are summarized in the supplementary Table 2.

Supplemental tables
Supplementary Table 1 The primers used in the study.

Gene
Forward primer (