Molecular mechanisms of immune evasion in colorectal carcinoma.

• Applicant: Privatdozentin Dr. Nathalie Britzen-Laurent
• Subject Area: General and Visceral Surgery ; Gastroenterology ; Pathology ; Cell Biology
• Term: from 2016 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 292410854

• Final Report Year: 2019

Final Report Abstract

In colorectal carcinoma (CRC), the presence of an interferon (IFN)-γ immune signature has been associated with an improved clinical outcome. This response is characterized by high expression levels of IFN-γ and interferon-stimulated genes (ISGs), including guanylate-binding protein 1 (GBP-1). We previously reported that GBP-1 expression can be specifically lost in tumor but not stromal cells in CRC, indicating a resistance to IFN-γ. Here, we investigated the IFN-γ response in CRC cell lines and found that ISGs expression and IFN-γ-induced cell death were inhibited in 6 out of 11 CRC lines (non-responders). Analysis of the IFN-γ response pathway revealed no significant driver mutation of the pathway genes IFNGR1, IFNGR2, JAK1, JAK2 and STAT1. At the mRNA level, IFNGR1 expression was downregulated in 4 non-responder cell lines. Treatment with decitabine, a DNA methylation inhibitor, increased IFNGR1 mRNA expression, suggesting an epigenetic regulation of the receptor expression. In CRC, a low mRNA expression of IFNGR1 but not of IFNGR2, STAT1, JAK1 or JAK2 correlated with a reduced disease-free survival in two patients cohorts. At the protein level, IFNγRα (coded by IFNGR1) was undetectable in the 4 non-responder lines showing a reduced mRNA expression and mis-glycosylated in the two remaining resistant cell lines. In a cohort of 363 CRC patients, absence IFNγRα protein expression in tumor cells was significantly associated with a reduced cancer-related survival. In addition, mice with a conditional knockout of the IFN-γ receptor in intestinal epithelial cells developed more tumors than control mice in a carcinogen-induced colon tumorigenesis model, indicating that the absence of IFN-γ receptor expression in intestinal epithelial cells fosters tumor growth. Reconstitution of IFNγRα expression in non-responder cells by transfection failed to reinstate the sensitivity to IFN-γ. In all non-responder cell lines, IFNγRα, either intrinsically or ectopically expressed, was mis-glycosylated. Lectin staining showed an overall reduction of glycosylation in nonresponders compared to IFN-γ-sensitive cell lines, and the expression of N-glycosylation enzymes was deregulated in non-responder cells. Treatment with different N-glycosylation inhibitors abrogated the IFN-γ signalling in responder cells. In addition, both IFNγRα expression and the presence of complex N-glycans were reduced in tumor samples compared to matching normal tissues. Treatment of non-responder cells with MG132 resulted in increased IFNγRα protein expression, indicating that improperly glycosylated IFNγRα is degraded by the proteasome. Altogether, our data showed that the loss of IFN-γ response in CRC is regulated by the glycosylation status of IFNγRα and indicate that dysfunction of the glycosylation pathway, by destabilizing IFNγRα, is involved in immune escape mechanisms of CRC.

Publications

• Pathophysiological role of guanylate-binding proteins in gastrointestinal diseases. World J Gastroenterol. 2016;22(28):6434–6443
  Britzen-Laurent N, Herrmann C, Naschberger E, Croner RS, Stürzl M
  
• Processing and secretion of guanylate binding protein-1 depend on inflammatory caspase activity. J Cell Mol Med. 2017;21(9):1954–1966
  Naschberger E, Geißdörfer W, Bogdan C, Tripal P, Kremmer E, Stürzl M, Britzen-Laurent N
  
• IFN-γ-response mediator GBP-1 represses human cell proliferation by inhibiting the Hippo signaling transcription factor TEAD. Biochem J. 2018;475(18):2955–2967
  Unterer B, Wiesmann V, Gunasekaran M, Sticht H, Tenkerian C, Behrens J, Leone M, Engel FB, Britzen-Laurent N, Naschberger E, Wittenberg T, Stürzl M
  
• IFN-γ drives inflammatory bowel disease pathogenesis through VE-cadherin-directed vascular barrier disruption. J Clin Invest. 2019 Nov 1;129(11):4691-4707
  Langer V, Vivi E, Regensburger D, Winkler TH, Waldner MJ, Rath T, Schmid B, Skottke L, Lee S, Jeon NL, Wohlfahrt T, Kramer V, Tripal P, Schumann M, Kersting S, Handtrack C, Geppert CI, Suchowski K, Adams RH, Becker C, Ramming A, Naschberger E, Britzen-Laurent N, Stürzl M
  
• Permeability analyses and three dimensional imaging of interferon gamma-induced barrier disintegration in intestinal organoids. Stem Cell Res. 2019;35:101383
  Bardenbacher M, Ruder B, Britzen-Laurent N, Schmid B, Waldner M, Naschberger E, Scharl M, Müller W, Günther C, Becker C, Stürzl M, Tripal P