Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin

<div><p>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. <i>In-vitro</i> 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. </p> </div

Changes in the percentage of EpCAM-positive cells by using the target activation array scan protocol (A), and CYP3A4 and Bmi mRNA levels (B) in HAK1-B cells cultured in RPMI1640 containing 10% FBS only, or with 1, 2, or 4 mM BCAA added for 72 h.

<div><p>Dunnett's test, *p < 0.05, **p < 0.01 n = 6, mean ± SE.</p> <p>The percentage of Annexin V-positive cells (C) and relative viable cell number (D) by array scan in HAK-1B cells cultured in RPMI1640 containing 10% FBS with or without 2 mM BCAA in the presence (1 or 2 µg/mL) or absence of 5-FU for 72 h by using target activation protocol of array scan. Student <i>t</i>-test, *p < 0.05, **p < 0.01, n = 7, mean ± SE.</p></div

The rate change of EpCAM positive cell in 5000 cells in the presence of 100 nM rapamycin treatment for 1 h or 72 h (A).

<div><p>Dunnett's test, *p < 0.05, ***p <0.001 vs. control, n = 6, mean ± SE.</p> <p>The relative expressions of EpCAM, c-myc, and FOXO3a mRNA upon Raptor and Rictor knockdown (B-D).</p> <p>Dunnett's test, **p < 0.01, ***p < 0.001 vs. control n = 8, mean ± SE.</p></div

Changes in the percentage of EpCAM-positive cells upon control medium (DMEM containing 10% FBS) with 4 mM BCAA stimulation or 100 nM rapamycin pretreatment and 4 mM BCAA stimulation (A) or liver cirrhosis modified medium (LC) containing 10% FBS stimulation (B) for 72 h in Huh7 by using the target activation protocol of array scan.

<div><p>The rate change of EpCAM-positive cells in 5000 cells with overexpression of caRheb or control plasmid cDNA (pc DNA) in control medium (DMEM containing 10% FBS) with and without 4 mM BCAA stimulation for 24 h in Huh7 (C).</p> <p>The detection of P70S6 kinase phosphorylation, a member of downstream mTOR signaling, in the presence of DMEM, BCAA treatment, pretreatment with rapamycin and BCAA treatment, or LC stimulation for 72 h in Huh7 (A,B). </p> <p>Tukey’s test **p < 0.01 vs. control, $$$p < 0.001 vs. BCAA, n = 8, mean ± SE (A).</p> <p>Student t-test *p < 0.05, ****p < 0.0001, n = 8, mean ± SE (B,C).</p></div

Tumor volume change over 14 days (A) and tumor weight (B) in the HAK-1B xenograft mouse model on the 14^th day after administration of BCAA and 5-FU injection.

<p>The relative expression of mRNA of various molecules of each tumor was associated with CSC properties (C, D). Control: 10% DMSO/saline/tumor injection + 3% casein containing diet, 5-FU: 250 µg/tumor injection + 3% casein containing diet, BCAA diet: 10% DMSO/saline/tumor injection + 3% BCAA containing, 5-FU+BCAA diet: 250 µg/tumor injection + 3% BCAA containing diet for 14 days. Tukey’s test: *p < 0.05, **p < 0.01, ***p < 0.001 vs. control, #p < 0.05 vs. 5-FU, $$p < 0.01 vs. BCAA, n = 6, mean ± SE.</p

Protein expression and phosphorylation in Huh7 cells under Knockdown conditions for 5 days and overexpression conditions for 1 day, and detection of phosphorylation after 4 mM BCAA treatment for 30 min.

<div><p>Rictor or Raptor Knockdown compared to negative control (NC), caRheb compared to control plasmid cDNA (pc DNA), BCAA treatment compared to DMEM (FBS 10%) only (Ctrl) (A). The average tumor volumes and tumorigenesis ratio at the 4^th week in a xenograft model with transplanted cells with negative control, knockdown of Raptor, Rictor for 5 days, or overexpression of control plasmid DNA, caRheb for 1 day (C), and tumorigenesis rate (B).</p> <p>Dunnett's test, *p<0.05, ***p < 0.001 vs. N.C. n = 5, mean ± SE.</p></div