Metformin Suppresses Cancer Stem Cells through AMPK Activation and Inhibition of Protein Prenylation of the Mevalonate Pathway in Colorectal Cancer

Abstract

Metformin is a well-known AMPK (AMP-activated protein kinase) activator that suppresses cancer stem cells (CSCs) in some cancers. However, the mechanisms of the CSC-suppressing effects of metformin are not yet well understood. In this study, we investigated the CSC-suppressive effect of metformin via the mevalonate (MVA) pathway in colorectal cancer (CRC). Two colorectal cancer cell lines, HT29 and DLD-1 cells, were treated with metformin, mevalonate, or a combination of the two. We measured CSC populations by flow cytometric analysis (CD44+/CD133+) and by tumor spheroid growth. The expression of p-AMPK, mTORC1 (pS6), and key enzymes (HMGCR, FDPS, GGPS1, and SQLE) of the MVA pathway was also analyzed. We investigated the effects of metformin and/or mevalonate in xenograft mice using HT29 cells; immunohistochemical staining for CSC markers and key enzymes of the MVA pathway in tumor xenografts was performed. In both HT29 and DLD-1 cells, the CSC population was significantly decreased following treatment with metformin, AMPK activator (AICAR), HMG-CoA reductase inhibitor (simvastatin), or mTOR inhibitor (rapamycin), and was increased by mevalonate. The CSC-suppressing effect of these drugs was attenuated by mevalonate. The results of tumor spheroid growth matched those of the CSC population experiments. Metformin treatment increased p-AMPK and decreased mTOR (pS6) expression; these effects were reversed by addition of mevalonate. The expression of key MVA pathway enzymes was significantly increased in tumor spheroid culture, and by addition of mevalonate, and decreased upon treatment with metformin, AICAR, or rapamycin. In xenograft experiments, tumor growth and CSC populations were significantly reduced by metformin, and this inhibitory effect of metformin was abrogated by combined treatment with mevalonate. Furthermore, in the MVA pathway, CSC populations were reduced by inhibition of protein prenylation with a farnesyl transferase inhibitor (FTI-277) or a geranylgeranyl transferase inhibitor (GGTI-298), but not by inhibition of cholesterol synthesis with a squalene synthase inhibitor (YM-53601). In conclusion, the CSC-suppressive effect of metformin was associated with AMPK activation and repression of protein prenylation through MVA pathway suppression in colorectal cancer.

Effective Suppression of Hypoxia Induced Angiogenesis by Synergism of Simvastatin and Metformin via Inhibiting ET-1-ETBR-Hif1α Signaling Axis

“…Others were mainly about anti-tumor effect, such as inhibition of prostate cancer, colorectal cancer, hepatocellular carcinoma and endometrial cancer, etc [35][36][37][38][39][40][41][42]. In our study, we also found that combination of simvastatin and metformin had a wide range of anti-cancer effects in breast cancer, hepatocellular carcinoma, lung cancer and cervical cancer.…”

Metformin treatment response is dependent on glucose growth conditions and metabolic phenotype in colorectal cancer cells

“…As an orally ingested drug, its highest concentration is found in the gastrointestinal (GI) tract, and GI derived tumours would most likely be affected 28 , which further indicates that achieved drug concentration play a major role in tumour responses. This is recapitulated in its various mode of action ranging from influence on microbiota 29 , immune modulation 30 , and direct intracellular effect 31 .…”

Tumour Microenvironment Stress Promotes the Development of Drug Resistance

“…Activated AMPK promotes tumour cell survival by increasing mitochondrial fatty acid oxidation, mitophagy-mitochondrial fission and mitochondrial biosynthesis [205]. Indeed, CSCs have been reported to be maintained in their stem-like state by AMPK activation although contradictory results on the role of AMPK in CSCs have also been published [217,218]. Nevertheless, in already developed tumours, AMPK appears to act as a tumour promoter, most likely by enhancing the survival of tumour cells under stress conditions [219].…”