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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References

  1. 1.

    , & Cancer statistics, 2016. CA Cancer J. Clin. 66, 7–30 (2016).

  2. 2.

    et al. The genomic landscape of response to EGFR blockade in colorectal cancer. Nature 526, 263–267 (2015).

  3. 3.

    et al. FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab for metastatic colorectal cancer (FIRE-3): a post-hoc analysis of tumour dynamics in the final RAS wild-type subgroup of this randomised open-label phase 3 trial. Lancet Oncol. 17, 1426–1434 (2016).

  4. 4.

    , , , & Resistance to anti-EGFR therapy in colorectal cancer: from heterogeneity to convergent evolution. Cancer Discov. 4, 1269–1280 (2014).

  5. 5.

    et al. Emergence of multiple EGFR extracellular mutations during cetuximab treatment in colorectal cancer. Clin. Cancer Res. 21, 2157–2166 (2015).

  6. 6.

    & The noncoding RNA revolution—trashing old rules to forge new ones. Cell 157, 77–94 (2014).

  7. 7.

    , , , & Targeting noncoding RNAs in disease. J. Clin. Invest. 127, 761–771 (2017).

  8. 8.

    , , & Identification of mammalian microRNA host genes and transcription units. Genome Res. 14 10A 1902–1910 (2004).

  9. 9.

    , , & Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs. Nat. Struct. Mol. Biol. 22, 319–327 (2015).

  10. 10.

    et al. The H19 lincRNA is a developmental reservoir of miR-675 that suppresses growth and Igf1r. Nat. Cell Biol. 14, 659–665 (2012).

  11. 11.

    et al. The Myc–miR-1792 axis blunts TGFβ signaling and production of multiple TGFβ-dependent antiangiogenic factors. Cancer Res. 70, 8233–8246 (2010).

  12. 12.

    et al. miR-99a/100125b tricistrons regulate hematopoietic stem and progenitor cell homeostasis by shifting the balance between TGFβ and Wnt signaling. Genes Dev. 28, 858–874 (2014).

  13. 13.

    et al. LincRNAs MONC and MIR100HG act as oncogenes in acute megakaryoblastic leukemia. Mol. Cancer 13, 171 (2014).

  14. 14.

    et al. Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer. J. Clin. Invest. 124, 2172–2187 (2014).

  15. 15.

    et al. Three-dimensional culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer. Proc. Natl. Acad. Sci. USA 114, E2852–E2861 (2017).

  16. 16.

    et al. PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to the epidermal growth factor receptor inhibitor cetuximab. Cancer Res. 68, 1953–1961 (2008).

  17. 17.

    et al. The molecular landscape of colorectal cancer cell lines unveils clinically actionable kinase targets. Nat. Commun. 6, 7002 (2015).

  18. 18.

    , , & miR-31 and its host gene lncRNA LOC554202 are regulated by promoter hypermethylation in triple-negative breast cancer. Mol. Cancer 11, 5 (2012).

  19. 19.

    et al. A multigene mutation classification of 468 colorectal cancers reveals a prognostic role for APC. Nat. Commun. 7, 11743 (2016).

  20. 20.

    et al. Transcriptional pathway signatures predict MEK addiction and response to selumetinib (AZD6244). Cancer Res. 70, 2264–2273 (2010).

  21. 21.

    & Identification and consequences of miRNA–target interactions—beyond repression of gene expression. Nat. Rev. Genet. 15, 599–612 (2014).

  22. 22.

    et al. The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo. Proc. Natl. Acad. Sci. USA 108, 3665–3670 (2011).

  23. 23.

    et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614–620 (2009).

  24. 24.

    et al. A small molecule inhibitor of β-catenin/CREB-binding protein transcription. Proc. Natl. Acad. Sci. USA 101, 12682–12687 (2004).

  25. 25.

    et al. MATCH: a tool for searching transcription factor binding sites in DNA sequences. Nucleic Acids Res. 31, 3576–3579 (2003).

  26. 26.

    et al. Transcription factors GATA-4 and GATA-6 in normal and neoplastic human gastrointestinal mucosa. BMC Gastroenterol. 8, 9 (2008).

  27. 27.

    et al. The transcription factor GATA6 enables self-renewal of colon adenoma stem cells by repressing BMP gene expression. Nat. Cell Biol. 16, 695–707 (2014).

  28. 28.

    et al. GATA6 regulates EMT and tumour dissemination, and is a marker of response to adjuvant chemotherapy in pancreatic cancer. Gut 66, 1665–1676 (2017).

  29. 29.

    , & Role of GATA factors in development, differentiation, and homeostasis of the small intestinal epithelium. Am. J. Physiol. Gastrointest. Liver Physiol. 306, G474–G490 (2014).

  30. 30.

    et al. Transcripts of unknown function in multiple-signaling pathways involved in human stem cell differentiation. Nucleic Acids Res. 37, 4987–5000 (2009).

  31. 31.

    et al. Characterization of long non-coding RNA expression profiles in lymph node metastasis of early-stage cervical cancer. Oncol. Rep. 35, 3185–3197 (2016).

  32. 32.

    et al. miR-100 induces epithelial–mesenchymal transition but suppresses tumorigenesis, migration and invasion. PLoS Genet. 10, e1004177 (2014).

  33. 33.

    et al. Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis. Lancet Oncol. 11, 136–146 (2010).

  34. 34.

    & Secreted and transmembrane wnt inhibitors and activators. Cold Spring Harb. Perspect. Biol. 5, a015081 (2013).

  35. 35.

    , & Wnt/β-catenin signaling: components, mechanisms, and diseases. Dev. Cell 17, 9–26 (2009).

  36. 36.

    , , , & Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow. Nat. Cell Biol. 3, 683–686 (2001).

  37. 37.

    & Analysis of Dickkopf3 interactions with Wnt signaling receptors. Growth Factors 28, 232–242 (2010).

  38. 38.

    , , & The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength. Genes Dev. 28, 305–316 (2014).

  39. 39.

    , , , & Dishevelled promotes Wnt receptor degradation through recruitment of ZNRF3/RNF43 E3 ubiquitin ligases. Mol. Cell 58, 522–533 (2015).

  40. 40.

    et al. RNF43 and ZNRF3 are commonly altered in serrated pathway colorectal tumorigenesis. Oncotarget 7, 70589–70600 (2016).

  41. 41.

    et al. Functional comparison of human adenomatous polyposis coli (APC) and APC-like in targeting β-catenin for degradation. PLoS One 8, e68072 (2013).

  42. 42.

    et al. The poly(ADP-ribose) polymerase enzyme tankyrase antagonizes activity of the β-catenin destruction complex through ADP-ribosylation of axin and APC2. J. Biol. Chem. 291, 12747–12760 (2016).

  43. 43.

    et al. CXCL12/CXCR4 axis induced miR-125b promotes invasion and confers 5-fluorouracil resistance through enhancing autophagy in colorectal cancer. Sci. Rep. 7, 42226 (2017).

  44. 44.

    , & miR-125b can enhance skin tumor initiation and promote malignant progression by repressing differentiation and prolonging cell survival. Genes Dev. 28, 2532–2546 (2014).

  45. 45.

    , , & Current understanding on EGFR and Wnt/β-catenin signaling in glioma and their possible crosstalk. Genes Cancer 4, 427–446 (2013).

  46. 46.

    & Convergence between Wnt–β-catenin and EGFR signaling in cancer. Mol. Cancer 9, 236 (2010).

  47. 47.

    et al. Differential WNT activity in colorectal cancer confers limited tumorigenic potential and is regulated by MAPK signaling. Cancer Res. 72, 1547–1556 (2012).

  48. 48.

    , & Wnt1 and Wnt5a induce cyclin D1 expression through ErbB1 transactivation in HC11 mammary epithelial cells. EMBO Rep. 4, 166–171 (2003).

  49. 49.

    et al. Tankyrase and the canonical Wnt pathway protect lung cancer cells from EGFR inhibition. Cancer Res. 72, 4154–4164 (2012).

  50. 50.

    β-catenin contributes to lung tumor development induced by EGFR mutations. Cancer Res. 74, 5891–5902 (2014).

  51. 51.

    et al. Epidermal growth factor receptor: a novel target of the Wnt/β-catenin pathway in liver. Gastroenterology 129, 285–302 (2005).

  52. 52.

    et al. GATA6 activates Wnt signaling in pancreatic cancer by negatively regulating the Wnt antagonist Dickkopf-1. PLoS One 6, e22129 (2011).

  53. 53.

    et al. The acinar regulator Gata6 suppresses KrasG12V-driven pancreatic tumorigenesis in mice. Gut 65, 476–486 (2016).

  54. 54.

    et al. The miR-363–GATA6–Lgr5 pathway is critical for colorectal tumourigenesis. Nat. Commun. 5, 3150 (2014).

  55. 55.

    et al. REG4 is a transcriptional target of GATA6 and is essential for colorectal tumorigenesis. Sci. Rep. 5, 14291 (2015).

  56. 56.

    & Wnt/β-catenin signaling in cancer stemness and malignant behavior. Curr. Opin. Cell Biol. 19, 150–158 (2007).

  57. 57.

    , & Truncated APC regulates the transcriptional activity of β-catenin in a cell cycle dependent manner. Hum. Mol. Genet. 16, 199–209 (2007).

  58. 58.

    et al. Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells. Nat. Commun. 4, 2610 (2013).

  59. 59.

    et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

  60. 60.

    , & edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

  61. 61.

    et al. ncPRO-seq: a tool for annotation and profiling of ncRNAs in sRNA-seq data. Bioinformatics 28, 3147–3149 (2012).

  62. 62.

    et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6, 80–92 (2012).

  63. 63.

    et al. Strelka: accurate somatic small-variant calling from sequenced tumor–normal sample pairs. Bioinformatics 28, 1811–1817 (2012).

  64. 64.

    & MicroRNA sponges: progress and possibilities. RNA 16, 2043–2050 (2010).

  65. 65.

    , & MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat. Methods 4, 721–726 (2007).

  66. 66.

    et al. MicroRNA-7/NF-κB signaling regulatory feedback circuit regulates gastric carcinogenesis. J. Cell Biol. 210, 613–627 (2015).

  67. 67.

    , & Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment. Nat. Protoc. 5, 1061–1073 (2010).

  68. 68.

    et al. A composite gene expression signature optimizes prediction of colorectal cancer metastasis and outcome. Clin. Cancer Res. 22, 734–745 (2016).

  69. 69.

    et al. Comprehensive analysis of β-catenin target genes in colorectal carcinoma cell lines with deregulated Wnt/β-catenin signaling. BMC Genomics 15, 74 (2014).

  70. 70.

    et al. Metastasis-associated gene expression changes predict poor outcomes in patients with Dukes stage B and C colorectal cancer. Clin. Cancer Res. 15, 7642–7651 (2009).

  71. 71.

    et al. Gene expression classification of colon cancer into molecular subtypes: characterization, validation, and prognostic value. PLoS Med. 10, e1001453 (2013).

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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.

Author information

Author notes

    • Yuanyuan Lu
    •  & Xiaodi Zhao

    These authors contributed equally to this work.

Affiliations

  1. Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

    • Yuanyuan Lu
    • , Xiaodi Zhao
    • , Ramona Graves-Deal
    • , Zheng Cao
    • , Bhuminder Singh
    • , Jeffrey L Franklin
    •  & Robert J Coffey
  2. State Key Laboratory of Cancer Biology, National Clinical Research Center for Digestive Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, China.

    • Yuanyuan Lu
    • , Xiaodi Zhao
    •  & Daiming Fan
  3. Department of Biomedical Informatics and Center for Quantitative Sciences, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

    • Qi Liu
    •  & Jing Wang
  4. Jiaen Genetics Laboratory, Beijing Jiaen Hospital, Beijing, China, and Molecular Pathology, Cancer Research Center, Medical College of Xiamen University, Xiamen, China.

    • Cunxi Li
    • , Huaying Hu
    •  & Tianying Wei
  5. Gibbs Cancer Center & Research Institute, Spartanburg, South Carolina, USA.

    • Mingli Yang
    •  & Timothy J Yeatman
  6. Department of Cell and Developmental Biology and Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

    • Ethan Lee
    •  & Kenyi Saito-Diaz
  7. Department of Biological Sciences, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

    • Scott Hinger
    •  & James G Patton
  8. Moffitt Cancer Center & Research Institute, Tampa, Florida, USA.

    • Christine H Chung
  9. Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.

    • Stephan Emmrich
    •  & Jan-Henning Klusmann
  10. Department of Veterans Affairs Medical Center, Nashville, Tennessee, USA.

    • Robert J Coffey

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Daiming Fan or Robert J Coffey.

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    Supplementary Tables 2–3,5–11 and Supplementary Figures 1–11

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    Supplementary Table 1

    Genetic mutations found in CC-CR compared to CC by whole-exome sequencing

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    Supplementary Table 4

    Functional enrichment analysis of miR-100 and miR-125b putative targets

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https://doi.org/10.1038/nm.4424