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NCOA3-mediated upregulation of mucin expression via transcriptional and post-translational changes during the development of pancreatic cancer

Oncogene volume 34, pages 4879–4889 (2015)Cite this article

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Abstract

Pancreatic cancer (PC) is characterized by aberrant overexpression of mucins that contribute to its pathogenesis. Although the inflammatory cytokines contribute to mucin overexpression, the mucin profile of PC is markedly distinct from that of normal or inflamed pancreas. We postulated that de novo expression of various mucins in PC involves chromatin modifications. Analysis of chromatin modifying enzymes by PCR array identified differential expression of NCOA3 in MUC4-expressing PC cell lines. Immunohistochemistry analysis in tumor tissues from patients and spontaneous mouse models, and microarray analysis following the knockdown of NCOA3 were performed to elucidate its role in mucin regulation and overall impact on PC. Silencing of NCOA3 in PC cell lines resulted in significant downregulation of two most differentially expressed mucins in PC, MUC4 and MUC1 (P<0.01). Immunohistochemistry analysis in PC tissues and metastatic lesions established an association between NCOA3 and mucin (MUC1 and MUC4) expression. Spontaneous mouse model of PC (K-rasG12D; Pdx-1cre) showed early expression of Ncoa3 during pre-neoplastic lesions. Mechanistically, NCOA3 knockdown abrogated retinoic acid-mediated MUC4 upregulation by restricting MUC4 promoter accessibility as demonstrated by micrococcus nuclease digestion (P<0.05) and chromatin immuno-precipitation analysis. NCOA3 also created pro-inflammatory conditions by upregulating chemokines like CXCL1, 2, 5 and CCL20 (P<0.001). AKT, ubiquitin C, ERK1/2 and NF-κB occupied dominant nodes in the networks significantly modulated after NCOA3 silencing. In addition, NCOA3 stabilized mucins post translationally through fucosylation by FUT8, as the knockdown of FUT8 resulted in the downregulation of MUC4 and MUC1 at protein levels.

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Acknowledgements

We thank Lynette Smith, Dr Joshua Souchek, Ms Kavita Mallya, Dr Neeru Sharma and Dr Parama Dey for help in statistical analysis and technical support. Microarray analysis was performed using BRB-Array Tools developed by Dr. Richard Simon and BRB-Array Development Team. The work was supported, in parts, by grants from the NIH (TMEN U54 CA163120, RO1 CA183459, EDRN UO1, CA111294, SPORE P50 CA127297 and RO3 CA167342).

Author Contributions

SK, SKB, SD designed the study. SK, SD, SR, S Kaur, SJ performed experiments, analyzed the data and wrote the manuscript. S Kaur performed MUC4 IHC and PMP performed NCOA3 chromatin immuno-precipitation. SR performed the animal experiments and SLJ analyzed the pathological samples. SK, MJ, SKB were involved in manuscript preparation and data analysis.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA,

    S Kumar, S Das, S Rachagani, S Kaur, S Joshi, M P Ponnusamy, M Jain & S K Batra

  2. Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha,, NE, USA

    S L Johansson

  3. Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA,

    M Jain & S K Batra

  4. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA,

    S K Batra

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  1. S Kumar
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Correspondence to S K Batra.

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Kumar, S., Das, S., Rachagani, S. et al. NCOA3-mediated upregulation of mucin expression via transcriptional and post-translational changes during the development of pancreatic cancer. Oncogene 34, 4879–4889 (2015). https://doi.org/10.1038/onc.2014.409

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