Abstract
Metformin inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, which is frequently upregulated in hepatocellular carcinoma (HCC). Metformin has also been shown to induce apoptosis in this cancer. Here, we investigate whether metformin-induced apoptosis in HCC is mediated by the downstream mTORC1 effectors eukaryotic initiation factor 4E and (eIF4E)-binding proteins (4E-BPs). Further, we ask whether changes in 4E-BPs activity during metformin treatment negatively regulate translation of the anti-apoptotic myeloid cell leukemia 1 (Mcl-1) mRNA. A genetic HCC mouse model was employed to assess the ability of metformin to reduce tumor formation, induce apoptosis, and control 4E-BP1 activation and Mcl-1 protein expression. In parallel, the HCC cell line Huh7 was transduced with scrambled shRNA (control) or shRNAs targeting 4E-BP1 and 4E-BP2 (4E-BP knock-down (KD)) to measure differences in mRNA translation, apoptosis, and Mcl-1 protein expression after metformin treatment. In addition, immunohistochemical staining of eIF4E and 4E-BP1 protein levels was addressed in a HCC patient tissue microarray. We found that metformin decreased HCC tumor burden, and tumor tissues showed elevated apoptosis with reduced Mcl-1 and phosphorylated 4E-BP1 protein levels. In control but not 4E-BP KD Huh7 cells, metformin induced apoptosis and repressed Mcl-1 mRNA translation and protein levels. Immunostaining of HCC patient tumor tissues revealed a varying ratio of eIF4E/4E-BP1 expression. Our results propose that metformin induces apoptosis in mouse and cellular models of HCC through activation of 4E-BPs, thus tumors with elevated expression of 4E-BPs may display improved clinical chemopreventive benefit of metformin.
Highlights
Hepatocellular carcinoma (HCC) is the most rapidly rising cause of cancer-related deaths among men in the United States [1]
Staining for the anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1), whose expression was shown to be regulated by mammalian target of rapamycin complex 1 (mTORC1) [35], revealed that there was a significant decrease in Mcl-1 positive cells in nodules of metformin-treated mice as compared to those from mice injected with vehicle alone (p
These results show that metformin treatment in a hepatocellular carcinoma (HCC) mouse model limits HCC progression, increases tumor apoptosis, and reduces Mcl-1 and phosphorylated 4E-BP1 protein expression
Summary
Hepatocellular carcinoma (HCC) is the most rapidly rising cause of cancer-related deaths among men in the United States [1]. A biguanide drug commonly used to treat type 2 diabetes, has been shown to inhibit the mTORC1 pathway through two mechanisms: it induces AMPK activation, and decreases Insulin Growth Factor (IGF-1)mediated stimulation of the PI3K/Akt/mTOR pathway [11,12,13]. Retrospective studies have suggested that metformin prevents development of HCC among diabetic patients with chronic liver disease [21,22,23,24,25,26,27,28,29,30]. Chemoprevention with metformin could be most effective where insulin resistance is present and the mTORC1 pathway is upregulated, such as in diabetic and obese patients with chronic liver disease
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