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Role of the E3 Ubiquitin-ligase Hakai in intestinal inflammation and cancer bowel disease

ABSTRACT: E3 ubiquitin-ligases are important for the cellular protein homeostasis and their deregulation is implicated in cancer. The E3 ubiquitin-ligase Hakai is involved in tumour progression and metastasis, through the regulation of the tumour suppressor E-cadherin. Hakai is overexpressed in colon cancer, however, the implication in colitis-associated cancer is unknown. Here, we investigated the potential role of Hakai in intestinal inflammation and cancer bowel disease.Several mouse models of colitis and associated cancer were used including AOM-DSS, acute colitis, and genetically modified mice deficient for the IL-10 gene, to analyse Hakai expression by immunohistochemistry. Interactome analysis of Hakai was performed and effect on selected protein was determined by plasmid and siRNA transfection, western-blotting, immunoprecipitation, immunofluorescence and ubiquitination assays. Lipid accumulation was assayed by oil red staining. Immunohistochemistry was also performed in inflamed colon biopsies from ulcerative colitis, Crohn's disease and colorectal cancer patients. Our results show that Hakai was downregulated in inflammatory tissues in different mouse models. Fatty Acid Synthase (FASN) protein was identified as a novel Hakai-interacting protein. Hakai induces FASN ubiquitination and degradation via lysosome, thus regulating FASN-mediated lipid accumulation. An inverse expression of FASN with Hakai expression was detected in inflammatory AOM/DSS mouse model. In conclusion, Hakai regulates FASN ubiquitination and degradation, resulting in the regulation of FASN-mediated lipid accumulation, which is associated to the development of inflammatory bowel disease. The interaction between Hakai and FASN may be an important mechanism for the homeostasis of intestinal barrier function and in the pathogenesis of this disease.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Colon

DISEASE(S): Intestinal Disease

SUBMITTER: Susana Bravo

LAB HEAD: Susana B Bravo Lopez

PROVIDER: PXD029136 | Pride | 2022-10-25

REPOSITORIES: Pride

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Project description:MUC1 is a tumor-associated antigen that is aberrantly expressed in cancer and inflammatory bowel disease (IBD). Even though immune cells express low MUC1 levels, their modulations of MUC1 are important in tumor progression. Consistent with previous clinical data that show increased myeloid-derived suppressor cells (MDSCs) in IBD, we now show that down-regulation of MUC1 on hematopoietic cells increases MDSCs in IBD, similar to our data in tumor-bearing mice. We hypothesize that MDSC expansion in IBD is critical for tumor progression. In order to mechanistically confirm the linkage between Muc1 down-regulation and MDSC expansion, we generated chimeric mice that did not express Muc1 in the hematopoietic compartment (KOM-bM-^FM-^RWT). These mice were used in 2 models of colitis and colitis-associated cancer (CAC) and their responses were compared to wildtype chimeras (WTM-bM-^FM-^RWT). KOM-bM-^FM-^RWT mice show increased levels of MDSCs during colitis that was responsible for the larger colon tumors that eventually developed in these mice. Using microarray and qRT-PCR analysis, we observed increased pro-tumorigenic signaling in the colons of KOM-bM-^FM-^RWT mice during colitis as compared to WTM-bM-^FM-^RWT mice. Our RNA (microarray and qRT-PCR analysis) and protein analysis demonstrate increased up-regulation of metalloproteinases, collagenases, defensins, complements, growth factors, cytokines and chemokines in KOM-bM-^FM-^RWT mice as compared to WTM-bM-^FM-^RWT mice. Antibody-mediated depletion of MDSCs in mice during colitis reduced colon tumor formation during CAC. Development of CAC is a serious complication of colitis and our data highlight MDSCs as a targetable link between inflammation and cancer. A total of 12 samples were analysed. RNA was made from the mucosa of the colon of WTM-bM-^FM-^RWT and KOM-bM-^FM-^RWT mice that were either untreated or treated with azoxymethane (AOM) and dextran sodium sulfate (DSS). There are 4 groups, n=3 for each group: untreated WTM-bM-^FM-^RWT mice, untreated KOM-bM-^FM-^RWT mice, AOM+DSS treated WTM-bM-^FM-^RWT mice, AOM+DSS treated KOM-bM-^FM-^RWT mice = 12 samples in total.

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Role of the E3 Ubiquitin-ligase Hakai in intestinal inflammation and cancer bowel disease

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