Abstract
Abstract Background: The understanding of colon cancer stem cell (CSC) biology is essential to developing new treatments, however, little is known about the molecular mechanisms underlying the acquisition of colon CSC properties. Furthermore, the similarity of the predisposition, including the surface markers, between colon CSCs and normal colon stem cells makes it difficult to develop a practical approach that targets colon CSCs. MicroRNA (miRNA) is one of the important factors regulating CSC properties. In addition, doublecortine-like kinase 1 (DCLK1), a microtubule-associated kinase, has been proposed to be a distinctive marker for colon CSCs. In the present study, we aimed to identify miRNAs that potentially target DCLK1 and unveil the role of the miRNA/DCLK1 axis in colon CSC properties. Material and Methods: Human colon cancer and normal colon specimens were dissociated into single cells and sorted by flow cytometry to separate the stem cell enriched population (EpCAM+/CD44+/CD66a-) and stained with anti-DCLK1 antibody. The expression profile of 384 miRNAs and the expression of DCLK1 in the colon CSC and normal colon stem cell populations were determined using a qRT-PCR. A luciferase activity assay and Western blotting were performed to evaluate the relationship between the miRNA and DCLK1. A lentiviral expression system was designed to investigate the miRNA phenotypes. We adopted an organoid assay and in vivo tumorigenicity assay to examine the influence of the miRNA on CSCs. Results: MiRNA-137 was highly expressed in the normal colon stem cell population whereas the DCLK1 mRNA expression was significantly upregulated in the colon CSC population. Actually, DCLK1-positive colon cancer cells were widely distributed in the colon cancer specimens, while DCLK1-positive epithelial cells were rarely detected in normal colon tissues, including the crypt bottoms. We confirmed that the activity of the luciferase gene linked to the 3’UTR of DCLK1 was decreased and that the protein level of DCLK1 was suppressed in the miRNA-137 transduced SW480 cells. The transduction of exogenous miRNA-137 suppressed the organoid development of colon cancer cells as well as shRNAs against DCLK1. The defect in organoid development by the transduction of miR-137 or shRNAs were substantially rescued by co-expression of the exsogenous DCLK1. Although miRNA-137 overexpression did not affect the organoid development of the normal intestine, miRNA-137 knockdown promoted the organoid development of normal colon cells. Xenograft tumor formation and growth were markedly suppressed in the miRNA-137-transduced SW480 cell-injected mice. Conclusion: These results suggest that miRNA-137 has the potential to suppress the tumorigenicity of colon CSCs through the inhibition of DCLK1 and that the dysregulation of the miRNA-137/DCLK1 axis plays an important role in colon CSCs. Citation Format: Masazumi Sakaguchi, Shigeo Hisamori, Nobu Oshima, Yoshiharu Sakai. microRNA-137/DCLK1 axis: A novel mechanism regulating tumorigenicity of colon cancer stem cell. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1898.
Published Version
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