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Inhibition of autophagy and tumor growth in colon cancer by miR-502.

ABSTRACT: Autophagy is a catabolic process that allows cellular macromolecules to be broken down and recycled as metabolic precursors. The influence of non-coding microRNAs in autophagy has not been explored in colon cancer. In this study, we discover a novel mechanism of autophagy regulated by hsa-miR-502-5p (miR-502) by suppression of Rab1B, a critical mediator of autophagy. A number of other miR-502 suppressed mRNA targets (for example, dihydroorotate dehydrogenase) are also identified by microarray analysis. Ectopic expression of miR-502 inhibited autophagy, colon cancer cell growth and cell-cycle progression of colon cancer cells in vitro. miR-502 also inhibited in-vivo colon cancer growth in a mouse tumor xenografts model. In addition, the expression of miR-502 was regulated by p53 via a negative feedback regulatory mechanism. The expression of miR-502 was downregulated in colon cancer patient specimens compared with the paired normal control samples. These results suggest that miR-502 may function as a potential tumor suppressor and therefore be a novel candidate for developing miR-502-based therapeutic strategies.

SUBMITTER: Zhai H

PROVIDER: S-EPMC3422411 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Inhibition of autophagy and tumor growth in colon cancer by miR-502.

Zhai H H Song B B Xu X X Zhu W W Ju J J

Oncogene 20120514 12

Autophagy is a catabolic process that allows cellular macromolecules to be broken down and recycled as metabolic precursors. The influence of non-coding microRNAs in autophagy has not been explored in colon cancer. In this study, we discover a novel mechanism of autophagy regulated by hsa-miR-502-5p (miR-502) by suppression of Rab1B, a critical mediator of autophagy. A number of other miR-502 suppressed mRNA targets (for example, dihydroorotate dehydrogenase) are also identified by microarray an ...[more]

PMID: 22580605

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Project description:BackgroundChemoresistance is a major obstacle that limits the benefits of 5-Fluorouracil (5-Fu)-based chemotherapy for colon cancer patients. Autophagy is an important cellular mechanism underlying chemoresistance. Recent research advances have given new insights into the use of natural bioactive compounds to overcome chemoresistance in colon cancer chemotherapy. As one of the multitargeted and safer phytomedicines, curcumin has been reported to work as cancer-specific chemosensitizer, presumably via induction of autophagic signaling pathways. The precise therapeutic effect of curcumin on autophagy in determining tumorous cells' fate, however, remains unclear. This study was conducted to investigate the differential modulations of the treatments either with 5-Fu alone or 5-Fu combined with curcumin on cellular autophagic responses and viabilities in the human colon cancer cells HCT116 and HT29, and explore molecular signaling transductions underlying the curcumin-mediated autophagic changes and potentiation of 5-Fu's cytotoxicity in vitro and in vivo.MethodsCell proliferation assay and morphology observation were used to identify the cytotoxicity of different combinations of curcumin and 5-Fu in HCT116 and HT29 cells. Cell immunofluorescence assay, Flow cytometry and Western blot were employed to detect changes of autophagy and the autophagy-related signaling pathways in the colon cancer cells and/or xenograft mice.ResultsCurcumin could significantly augment the cytotoxicity of 5-Fu to the tumorous cells, and the pre-treatment with curcumin followed by 5-Fu (pre-Cur) proved to be the most effective one compared to other two combinations. The chemosensitizing role of curcumin might attribute to the autophagy turnover from being activated in 5-Fu mono-treatment to being inhibited in the pre-Cur treatment as indicated by the changes in expression of beclin-1, p62 and LC3II/LC3I and the intensity of Cyto-ID Green staining. The autophagic alterations appeared to be contributed by down-regulation of not only the phospho-Akt and phospho-mTOR expressions but the phospho-AMPK and phospho-ULK1 levels as well. The cellular activation of AMPK by addition of A-769662 to the pre-Cur combination resulted in reversed changes in expressions of the autophagy protein markers and apoptotic status compared to those of the pre-Cur combination treatment. The findings were validated in the xenograft mice, in which the tumor growth was significantly suppressed in the mice with 25-day combination treatment, and meanwhile expressions of the autophagy markers, P-AMPK and P-ULK1 were all reversely altered in line with those observed in HCT116 cells.ConclusionPre-treatment with curcumin followed by 5-Fu may mediate autophagy turnover both in vitro and in vivo via AMPK/ULK1-dependent autophagy inhibition and AKT modulation, which may account for the increased susceptibility of the colon cancer cells/xenograft to the cytotoxicity of 5-Fu.

Project description:BACKGROUND:Achaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controls the fate of intestinal stem cells. However, the role of Ascl2 in colon cancer progenitor cells remains unknown. The cell line HT-29 (47.5-95% of CD133(+) population) and LS174T (0.45% of CD133(+) population) were chosen for functional evaluation of Ascl2 in colon cancer progenitor cells after gene knockdown by RNA interference. METHODOLOGY/PRINCIPAL FINDINGS:Immunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas. Downregulation of Ascl2 using RNA interference in cultured colonic adenocarcinoma HT-29 and LS174T cells reduced cellular proliferation, colony-forming ability, invasion and migration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2 protein level in CD133(+) HT-29 cells was significantly higher than in CD133(-) HT-29 cells. Ascl2 blockade via shRNA interference in HT-29 cells (shRNA-Ascl2/HT-29 cells) resulted in 26.2% of cells staining CD133(+) compared with 54.7% in control shRNA-Ctr/HT-29 cells. The levels of 'stemness' associated genes, such as CD133, Sox2, Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft (CD133 was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/HT-29 cells had inhibited abilities to form tumorspheres compared with control. The microRNA (miRNAs) microarrays, identified 26 up-regulated miRNAs and 58 down-regulated miRNAs in shRNA-Ascl2/HT-29 cells. Expression levels of let-7b, miRNA-124, miRNA-125b, miRNA-17, miRNA-20a and miRNA-302b, involved in the regulation of 'stemness', were quantified with qPCR, which confirmed their identities. Restoration of miRNA-302b, via its mimic, led to the restoration of shRNA-Ascl2/HT-29 'stemness' characteristics, including tumorsphere formation and 'stemness' associated genes levels, and the recovery of cellular behaviors, including colony-forming ability, invasion and migration in vitro. CONCLUSIONS/SIGNIFICANCE:Ascl2 may be a potential target for the inhibition of colon cancer progenitor cells, and functions through a miR-302b-related mechanism.

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Inhibition of autophagy and tumor growth in colon cancer by miR-502.

Publications

Inhibition of autophagy and tumor growth in colon cancer by miR-502.

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