Differentiation of cancer stem cells (CSCs) into cancer cells causes increased 
sensitivity to chemotherapeutic agents. Although inhibition of mammalian target 
of rapamycin (mTOR) leads to CSC survival, the effect of branched chain amino 
acids (BCAAs), an mTOR complex 1 (mTORC1) activator remains unknown. In this 
study, we examined the effects of BCAA on hepatocellular carcinoma (HCC) cells 
expressing a hepatic CSC marker, EpCAM. We examined the effects of BCAA and/or 
5-fluorouracil (FU) on expression of EpCAM and other CSC-related markers, as 
well as cell proliferation in HCC cells and in a xenograft mouse model. We also 
characterized CSC-related and mTOR signal-related molecule expression and 
tumorigenicity in HCC cells with knockdown of Rictor or Raptor, or 
overexpression of constitutively active rheb (caRheb). mTOR signal-related 
molecule expression was also examined in BCAA-treated HCC cells. In-vitro BCAA 
reduced the frequency of EpCAM-positive cells and improved sensitivity to the 
anti-proliferative effect of 5-FU. Combined 5-FU and BCAA provided better 
antitumor efficacy than 5-FU alone in the xenograft model. Stimulation with high 
doses of BCAA activated mTORC1. Knockdown and overexpression experiments 
revealed that inhibition of mTOR complex 2 (mTORC2) or activation of mTORC1 led 
to decreased EpCAM expression and little or no tumorigenicity. BCAA may enhance 
the sensitivity to chemotherapy by reducing the population of cscs via the mTOR 
pathway. This result suggests the utility of BCAA in liver cancer therapy.