Combined Targeting of AKT and mTOR Inhibits Tumor Formation of EpCAM(+) and CD90(+) Human Hepatocellular Carcinoma Cells in an Orthotopic Mouse Model

SIMPLE SUMMARY: Cancer stem cells are a distinct tumor subpopulation associated with poor outcome. The epithelial cell adhesion molecule (EpCAM) and Thy-1 cell surface antigen (CD90) have been implicated as cancer stem cell (CSC) markers in hepatocellular carcinoma (HCC). Eradication of those CSCs is of high importance with respect to the outcome of treatment. We aimed to verify the impact of targeting HCC cells expressing the CSC marker CD90 and EpCAM with combined therapy of AKT and mTOR inhibitors. Our data demonstrated that combined targeting of AKT and mTOR is highly synergistic in vitro but leads to treatment resistance of xenograft tumors in an orthotopic mouse model in vivo. The development of resistance was observed in large numbers of MK2206/RAD001 treated cells. Restoration of phosphorylated AKT was observed in most tumors during AKT/mTOR therapy underlining the importance of restored AKT signaling as a resistance mechanism. Further work is required to verify the molecular mechanisms of HCC treatment resistance. ABSTRACT: The epithelial cell adhesion molecule (EpCAM) and Thy-1 cell surface antigen (CD90) have been implicated as cancer stem cell (CSC) markers in hepatocellular carcinoma (HCC). Expression of EpCAM and CD90 on HCC cells is associated with increased tumorigenicity, metastasis and poor prognosis. In this study, we demonstrate that combined treatment with AKT and mTOR inhibitors—i.e., MK2206 and RAD001—results in a synergistic reduction in proliferation of EpCAM(+) and CD90(+) HCC cells cultured either as adherent cells or as tumoroids in vitro. In addition, tumor growth was reduced by combined treatment with AKT and mTOR inhibitors in an orthotopic xenograft mouse model of an EpCAM(+) HCC cell line (Huh7) and primary patient-derived EpCAM(+) HCC cells (HCC1) as well as a CD90(+) HCC-related cell line (SK-HEP1) in vivo. However, during AKT/mTOR treatment, outgrowth of therapy-resistant tumors was observed in all mice analyzed within a few weeks. Resistance was associated in most cases with restoration of AKT signaling in the tumors, intrahepatic metastases and distant metastases. In addition, an upregulation of the p38 MAPK pathway was identified in the AKT/mTOR inhibitor-resistant tumor cells by kinome profiling. The development of resistant cells during AKT/mTOR therapy was further analyzed by red-green-blue (RGB) marking of HCC cells, which revealed an outgrowth of a large number of Huh7 cells over a period of 6 months. In summary, our data demonstrate that combined treatment with AKT and mTOR inhibitors exhibits synergistic effects on proliferation of EpCAM(+) as well as CD90(+) HCC cells in vitro. However, the fast development of large numbers of resistant clones under AKT/mTOR therapy observed in vitro and in the orthotopic xenotransplantation mouse model in vivo strongly suggests that this therapy alone will not be sufficient to eliminate EpCAM(+) or CD90(+) cancer stem cells from HCC patients.