Abstract
De novo and acquired resistance, which are largely attributed to genetic alterations, are barriers to effective anti-epidermal-growth-factor-receptor (EGFR) therapy. To generate cetuximab-resistant cells, we exposed cetuximab-sensitive colorectal cancer cells to cetuximab in three-dimensional culture. Using whole-exome sequencing and transcriptional profiling, we found that the long non-coding RNA MIR100HG and two embedded microRNAs, miR-100 and miR-125b, were overexpressed in the absence of known genetic events linked to cetuximab resistance. MIR100HG, miR-100 and miR-125b overexpression was also observed in cetuximab-resistant colorectal cancer and head and neck squamous cell cancer cell lines and in tumors from colorectal cancer patients that progressed on cetuximab. miR-100 and miR-125b coordinately repressed five Wnt/β-catenin negative regulators, resulting in increased Wnt signaling, and Wnt inhibition in cetuximab-resistant cells restored cetuximab responsiveness. Our results describe a double-negative feedback loop between MIR100HG and the transcription factor GATA6, whereby GATA6 represses MIR100HG, but this repression is relieved by miR-125b targeting of GATA6. These findings identify a clinically actionable, epigenetic cause of cetuximab resistance.
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Acknowledgements
We acknowledge the support of the Vanderbilt University Cell Imaging, Chemical Biology Synthesis, and Flow Cytometry Shared Resources. We thank J. Higginbotham for help with flow cytometry, W. Fry for help with plasmid construction, and E. Poulin and N. Markham for critical editing of the manuscript. We thank X. Wang and Y. Nie (Xijing Hospital of Digestive Diseases) for providing clinically annotated samples. This work was supported by National Cancer Institute (NCI) R01 CA046413, R35 CA197570 and P50 CA095103 GI Specialized Programs of Research Excellence to R.J.C., Natural Science Foundation of China (NSFC) 81430072 and 81421003 and National Key R&D Program 2016YFC1303200 to D.F., and Emmy Noether-Programme of the German Research Foundation KL 2374/2-1 to J.H.K.
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Y.L., X.Z., C.L., D.F. and R.J.C. designed the research. Y.L., X.Z., Q.L., C.L., R.G.-D., Z.C., B.S., J.W., H.H., T.W., M.Y. and S.H. performed experiments, analyzed data, and prepared figures and tables. T.Y., E.L., K.S.-D., C.H.C., S.E., J.-H.K. and D.F. contributed new reagents and/or analytical tools. Y.L., X.Z., Q.L., C.L., J.L.F., T.J.Y., E.L., J.G.P., C.H.C., D.F. and R.J.C. analyzed the data and provided critical input. Y.L., X.Z. and R.J.C. wrote the paper. R.J.C. and D.F. conceived the project, and supervised and coordinated all aspects of the work.
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Supplementary Tables 2–3,5–11 and Supplementary Figures 1–11 (PDF 9231 kb)
Supplementary Table 1
Genetic mutations found in CC-CR compared to CC by whole-exome sequencing (XLSX 21 kb)
Supplementary Table 4
Functional enrichment analysis of miR-100 and miR-125b putative targets (XLSX 39 kb)
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Lu, Y., Zhao, X., Liu, Q. et al. lncRNA MIR100HG-derived miR-100 and miR-125b mediate cetuximab resistance via Wnt/β-catenin signaling. Nat Med 23, 1331–1341 (2017). https://doi.org/10.1038/nm.4424
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DOI: https://doi.org/10.1038/nm.4424
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