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Sestrin 2 suppresses cells proliferation through AMPK/mTORC1 pathway activation in colorectal cancer.

ABSTRACT: Sestrin 2 is a conserved antioxidant protein that reduces reactive oxygen species (ROS) and inhibits mammalian target of rapamycin complex 1 (mTORC1). We previously showed that sestrin 2 is abnormally decreased in colorectal cancer (CRC). To elucidate the molecular mechanism behind the potential contribution of sestrin 2 to CRC, we used a lentiviral expression vector system to determine the effects of sestrin 2 overexpression on human CRC cells. We found that sestrin 2 overexpression decreased ROS production, inhibited cell growth, and stimulated apoptosis in two CRC cell lines. In parallel, expression of the proliferation marker PCNA was decreased, proapoptotic caspase 3, 7, and 9 levels were increased, and expression of the anti-apoptotic protein survivin was reduced. Sestrin 2 overexpression also activated the adenosine monophosphate-activated protein kinase (AMPK) pathway, and suppressed mTORC1 signaling. Treating CRC cells with compound C, an AMPK inhibitor, reversed or attenuated changes in proliferation, apoptosis, and signaling proteins of the AMPK/mTORC1 axis. In a xenograft mouse model, CRC growth was attenuated by sestrin 2 overexpression. These results suggest that sestrin 2 suppresses CRC cell growth through activation of the AMPK/mTORC1 pathway and induction of apoptosis, and could be a novel pharmacological target for the treatment of CRC.

SUBMITTER: Wei JL

PROVIDER: S-EPMC5564770 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

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Project description: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.

Project description:Yes-associated protein (YAP), as a major downstream effector in the Hippo signaling pathway, is considered as an oncogene in cancer. The present study aimed to investigate the potential role of YAP in the development and progression of colorectal cancer (CRC). The mRNA and protein expression levels of YAP in human CRC tissue samples and adjacent normal tissue were analyzed using public databases, as well as clinical samples. The potential roles of YAP and the underlying mechanism regulating the proliferation and migration of CRC cells were examined using genetic manipulation in vitro. The correlation between the expression of the YAP gene and epithelial-to-mesenchymal transition (EMT) markers was investigated in order to determine the mechanism underlying the observed effects of YAP. YAP mRNA expression levels were significantly upregulated in CRC tissue compared with in normal tissue, as determined using datasets obtained from Oncomine. Similarly, in clinical samples, the protein expression levels of YAP were significantly upregulated in CRC tissue samples compared with in normal tissue samples. YAP knockdown inhibited the proliferation and migration of CRC cells in vitro, whereas its overexpression resulted in the opposite effect. The expression levels of the YAP gene were positively correlated with those of EMT markers (such as vimentin and N-cadherin) and EMT-inducing transcription factors (such as Snail1, Slug and zinc finger E-box binding homeobox 1 and 2) in CRC samples from Gene Expression Profiling Interactive Analysis. Furthermore, YAP silencing increased the protein expression of E-cadherin and decreased that of vimentin in CRC cells. By contrast, the overexpression of YAP had the opposite effect. YAP promoted the glucose transporter 3 (Glut3)/AMP-activated protein kinase (AMPK) signaling pathway in CRC cells. In conclusion, YAP promoted the proliferation and migration of CRC cells, as well as the expression of EMT markers, possibly by regulating the Glut3/AMPK signaling pathway.

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Sestrin 2 suppresses cells proliferation through AMPK/mTORC1 pathway activation in colorectal cancer.

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