Abstract
Simple SummaryTumor suppressing effect of metformin has been reported, and one of mechanism of this effect is suppression of cancer stem cells (CSCs). However, detailed mechanism of metformin-induced CSC-inhibitory effect has not been known. We demonstrated that the CSC-suppressive effect of metformin was associated with AMPK activation/mTOR inhibition and repression of protein prenylation through suppression of mevalonate pathway in colorectal cancer. Further studies would be needed to investigate cross-reactions with other mechanisms of the antitumor effect of metformin, and clinical impact of metformin should be considered as chemopreventive or adjunctive treatment for colorectal tumor.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.
Highlights
Colorectal cancer (CRC) is the third most common cause of cancer-related deaths worldwide [1].Approximately 25% of CRC patients are already stage IV at diagnosis, and a significant proportion of advanced CRC patients develop resistance to treatment and experience recurrence and metastasis [2].to develop more effective drugs for advanced CRC, we need to target the critical cells and signaling pathways involved in tumor progression, recurrence, and metastasis.Cancer stem cells (CSCs) are a main target for decreasing cancer risk, recurrence, and metastasis and for overcoming resistance to chemotherapy
Tumor growth and CSC populations were significantly reduced by metformin, and this inhibitory effect of metformin was abrogated by combined treatment with mevalonate
Spheres and found significantly elevated protein and mRNA levels of key enzymes of the MVA. These findings suggest that the MVA pathway may have an important role in CRC tumorigenesis by pathway, including HMGCR, farnesyl diphosphate synthase (FDPS), GGPS1, and squalene epoxidase (SQLE), in tumor spheroids compared to 2D adherent promoting CSCs of CRC
Summary
Colorectal cancer (CRC) is the third most common cause of cancer-related deaths worldwide [1]. To develop more effective drugs for advanced CRC, we need to target the critical cells and signaling pathways involved in tumor progression, recurrence, and metastasis. Metformin decreases colon polyp development, as well as the risk, recurrence, and mortality of CRC [5] These beneficial effects of metformin are related to AMPK activation, mammalian target of rapamycin (mTOR) inhibition, cell cycle regulation, and CSC suppression [6,7,8,9]. The mevalonate (MVA) pathway leads to the synthesis of sterols and protein prenylation that have important roles in tumor growth Multiple enzymes of this pathway are essential for proliferation and survival of various types of cancer cells, and are upregulated in many cancers [15,17]. We showed that CSC suppression by metformin is associated with inhibition of protein prenylation, rather than cholesterol synthesis, through the MVA pathway
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