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. 2018 Mar 7;26(3):744-754.
doi: 10.1016/j.ymthe.2017.12.023. Epub 2018 Jan 4.

miR-145 Antagonizes SNAI1-Mediated Stemness and Radiation Resistance in Colorectal Cancer

Yun Zhu  1 , Cindy Wang  2 , Scott A Becker  3 , Katie Hurst  2 , Lourdes M Nogueira  4 , Victoria J Findlay  5 , E Ramsay Camp  6
Affiliations

miR-145 Antagonizes SNAI1-Mediated Stemness and Radiation Resistance in Colorectal Cancer

Yun Zhu et al. Mol Ther. .

Abstract

Epithelial-to-mesenchymal transition (EMT) has been closely linked with therapy resistance and cancer stem cells (CSCs). However, EMT pathways have proven challenging to therapeutically target. MicroRNA 145 (miR-145) targets multiple stem cell transcription factors and its expression is inversely correlated with EMT. Therefore, we hypothesized that miR-145 represents a therapeutic target to reverse snail family transcriptional repressor 1 (SNAI1)-mediated stemness and radiation resistance (RT). Stable expression of SNAI1 in DLD1 and HCT116 cells (DLD1-SNAI1; HCT116-SNAI1) increased expression of Nanog and decreased miR-145 expression compared to control cells. Using a miR-145 luciferase reporter assay, we determined that ectopic SNAI1 expression significantly repressed the miR-145 promoter. DLD1-SNAI1 and HCT116-SNAI1 cells demonstrated decreased RT sensitivity and, conversely, miR-145 replacement significantly enhanced RT sensitivity. Of the five parental colon cancer cell lines, SW620 cells demonstrated relatively high endogenous SNAI1 and low miR-145 levels. In the SW620 cells, miR-145 replacement decreased CSC-related transcription factor expression, spheroid formation, and radiation resistance. In rectal cancer patient-derived xenografts, CSC identified by EpCAM+/aldehyde dehydrogenase (ALDH)+ demonstrated high expression of SNAI1, c-Myc, and Nanog compared with non-CSCs (EpCAM+/ALDH-). Conversely, patient-derived CSCs demonstrated low miR-145 expression levels relative to non-CSCs. These results suggest that the SNAI1:miR-145 pathway represents a novel therapeutic target in colorectal cancer to overcome RT resistance.

Keywords: SNAI1; colorectal cancer; microRNA-145; radiation; resistance; stem cell.

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Figures

Figure 1
Figure 1
Elevated SNAI1 Expression in Human Colorectal Cancer Datasets Whole exome and RNA Seq data of colorectal adenocarcinoma from TCGA was mined for the frequency of SNAI1, SNAI2, ZEB1, and ZEB2 using bioportal.
Figure 2
Figure 2
Ectopic Expression of SNAI1 Induces CSC Phenotype (A) Western blot analysis for expression of critical EMT mediators and CSC transcription factors in parental colon cancer cell lines. (B) Western blot analysis and relative density quantitation of EMT biomarker and stem-cell-related transcription factors in ectopic-expressing SNAI1 cell lines compared with empty vector control cells. (C and D) Limited dilution spheroid assay comparing DLD1-SNAI1 (C) and HCT116-SNAI1 (D) cells with empty vector (Vec) control cells (data are shown as mean ± SD; n = 12; * indicated p < 0.05).
Figure 3
Figure 3
Ectopic Expression of SNAI1 Increases Resistance to Radiation Therapy (A) Clonogenic assay on DLD1-SNAI1 cells and vector control cells treated with increasing doses of radiation. (B) Ectopic SNAI1 expression enhanced cancer cell viability after radiation treatment (data are shown as mean ± SD; n = 3; * indicated as p < 0.05).
Figure 4
Figure 4
SNAI1 Represses miR-145 Promoter Activity and Expression in Colorectal Cancer Cells (A) Relative miR-145 expression level assessed by real-time PCR in parental colorectal cancer cell lines: HCT116 cells versus other four colorectal cancer cell lines (data are shown as mean ± SD; n = 3; * indicated p < 0.05). (B and C) Relative miR-145 expression level of (B) DLD1-SNAI1 cells and (C) HCT116-SNAI1 cells compared to vector control cells (data are shown as mean ± SD; n = 3; * indicated p < 0.05). (D and E) Luciferase reporter activity of (D) DLD1-SNAI1 and (E) HCT116-SNAI1 cells compared to vector control cells (data are shown as mean ± SD; n = 3; *p < 0.05).
Figure 5
Figure 5
miR-145 Sensitizes SNAI1-Overexpressing Colorectal Cancer Cells to Radiation Therapy (A and B) Clonogenic assay (A) and quantitation (B) following radiation of DLD1-SNAI1 cells after transfection of miR-145 compared with scr vector (data are shown as mean ± SD; n = 3; * indicated p < 0.05, DLD1-SNAI1/scr versus DLD1-Vec/scr; ** indicated p < 0.05, DLD1-SNAI1/miR-145 versus DLD1-SNAI1/scr). (C) Clonogenic assay following radiation of SW620 cells after transfection of miR-145 compared with scr control for 48 hr (data are shown as mean ± SD; n = 3; * indicated p < 0.05). (D) Limited dilution in vitro spheroid assay of SW620 cells after transfection of miR-145 compared with scr control for 48 hr (data are shown as mean ± SD; n = 6; * indicated p < 0.05). (E) Western blot analysis of EMT mediators and miR-145 targets in SW620 cells after transfection with miR-145 and scr control for 48 hr.
Figure 6
Figure 6
SNAI1 and miR-145 Expression in CSC and Non-CSC Population in Patient-Derived Rectal Cancer Xenografts (A) Droplet digital PCR analysis of genes involved in the SNAI1:miR-145 axis. (B) miR-145 in FACS-sorted non-CSC (EpCAM+/ALDH−; black bars) and CSC (EpCAM+/ALDH+; gray bars) cells from two patient-derived rectal cancer xenografts (* indicated p < 0.05).
Figure 7
Figure 7
Therapeutic Mechanism of miR-145 Delivery to Overcome SNAI1-Mediated Colorectal Cancer Radiation Resistance SNAI1 can activate Nanog and repress miR-145. Thus, SNAI1 mediates the CSC program, resulting in a radiation-resistant colorectal cancer cell population. Delivery of miR-145 to target multiple stemness genes, including Nanog, KLF4, and c-Myc, can impair CSC self-renewal capacity and increase sensitivity of radiation therapy.
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References

    1. Wu J., Hirata I., Zhao X., Gao B., Okazaki M., Kato K. Influence of alkyl chain length on calcium phosphate deposition onto titanium surfaces modified with alkylphosphonic acid monolayers. J. Biomed. Mater. Res. A. 2013;101:2267–2272. - PubMed
    1. Sauer R., Becker H., Hohenberger W., Rödel C., Wittekind C., Fietkau R., Martus P., Tschmelitsch J., Hager E., Hess C.F., German Rectal Cancer Study Group Preoperative versus postoperative chemoradiotherapy for rectal cancer. N. Engl. J. Med. 2004;351:1731–1740. - PubMed
    1. Willett C.G., Duda D.G., di Tomaso E., Boucher Y., Ancukiewicz M., Sahani D.V., Lahdenranta J., Chung D.C., Fischman A.J., Lauwers G.Y. Efficacy, safety, and biomarkers of neoadjuvant bevacizumab, radiation therapy, and fluorouracil in rectal cancer: a multidisciplinary phase II study. J. Clin. Oncol. 2009;27:3020–3026. - PMC - PubMed
    1. Velenik V., Ocvirk J., Music M., Bracko M., Anderluh F., Oblak I., Edhemovic I., Brecelj E., Kropivnik M., Omejc M. Neoadjuvant capecitabine, radiotherapy, and bevacizumab (CRAB) in locally advanced rectal cancer: results of an open-label phase II study. Radiat. Oncol. 2011;6:105. - PMC - PubMed
    1. Dewdney A., Cunningham D., Tabernero J., Capdevila J., Glimelius B., Cervantes A., Tait D., Brown G., Wotherspoon A., Gonzalez de Castro D. Multicenter randomized phase II clinical trial comparing neoadjuvant oxaliplatin, capecitabine, and preoperative radiotherapy with or without cetuximab followed by total mesorectal excision in patients with high-risk rectal cancer (EXPERT-C) J. Clin. Oncol. 2012;30:1620–1627. - PubMed

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