Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17‐dependent ligand release

Abstract High fat diet is implicated in the elevated deoxycholic acid (DCA) in the intestine and correlated with increased colon cancer risk. However, the potential mechanisms of intestinal carcinogenesis by DCA remain unclarified. Here, we investigated the carcinogenic effects and mechanisms of DCA using the intestinal tumour cells and Apc min/+ mice model. We found that DCA could activate epidermal growth factor receptor (EGFR) and promote the release of EGFR ligand amphiregulin (AREG), but not HB‐EGF or TGF‐α in intestinal tumour cells. Moreover, ADAM‐17 was required in DCA‐induced promotion of shedding of AREG and activation of EGFR/Akt signalling pathway. DCA significantly increased the multiplicity of intestinal tumours and accelerated adenoma‐carcinoma sequence in Apc min/+ mice. ADAM‐17/EGFR signalling axis was also activated in intestinal tumours of DCA‐treated Apc min/+ mice, whereas no significant change occurred in tumour adjacent tissues after DCA exposure. Conclusively, DCA activated EGFR and promoted intestinal carcinogenesis by ADAM17‐dependent ligand release.

the activation of numerous of intracellular signals, which are critical to tumour progression, including cell growth, epithelial-mesenchymal transition (EMT), metastasis and angiogenesis. These changes are mediated by the downstream targets of EGFR, including extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt protein kinase. 11,12 The proteolytic processing of EGFR soluble ligands, amphiregulin (AREG), heparin-binding (HB)-EGF or transforming growth factor (TGF)-a requires a disintegrin and metalloprotease (ADAM- 17), which is also known as tumour necrosis factor-a converting enzyme (TACE). As a member of the ADAM family of metalloproteases, ADAM-17 involves in cell adhesion, migration, cellular signalling and proteolysis, 13 recently emerging as a potential therapeutic target in several tumour types. 14,15 Our previous studies have already showed that DCA enhanced the multiplicity of intestinal tumours and accelerated intestinal adenoma-adenocarcinoma sequence in Apc min/+ mice. 7,16 And recent research provided evidence that DCA induced EGFR/STAT3 signalling to promote gastrointestinal cancer progression. 15 In this study, we investigated whether DCA promoted intestinal carcinogenesis through activation of ADAM-17/EGFR signalling axis. Here, we provided the evidence that DCA up-regulated the release of AREG in accordance with EGFR/Akt activation, and ADAM-17 was required in the shedding AREG for activation of EGFR. These data defined a mechanism of DCA in promoting intestinal tumour development through ADAM-17-mediated AREG release, leading to activation of EGFR, and represented a potential target for the bile acidrelated intestinal cancer prevention and therapy.

| Cell culture and treatment
Young adult mouse colonic epithelium cell line (YAMC) cell line was generated using a mouse harbouring thermolabile mutation (tsA58) under the control of an interferon (IFN)-c-inducible H-2Kb promoter and a temperature-sensitive simian virus 40 large T antigen (Immortomouse). Immorto-Min colonic epithelial cell line (IMCE) cell line was derived from the colonic epithelium of F1 Immorto-Apc min/+ mouse hybrid and carried both the mutant Min gene and a temperature-sensitive mutant of the SV40 large T antigen. 17 ADAM17-deficient MCE (ADAM17 À/À MCE) cell line was derived from the colonic epithelium of Adam17 DZn/DZn null mice crossed to the Immortomouse. 18

| Western blot analysis
The cells lysates were solubilized using RIPA buffer with protease The membranes were flowed by horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology). The band density was detected using an Image processor program (ImageJ) and was determined by comparing the density of the indicated band to the internal control band.

| Real-time PCR analysis
Total RNA was extracted using the RNeasy mini kit (Qiagen, Carlsbad, CA, USA), and cDNA reverse transcription was carried out using the TIANScript RT Kit (TIANGEN, Inc. Beijing, China) according to the manufacturer's instructions. The Oligonucleotide primers for target genes (GAPDH and AREG) were shown in Table S1. The ΔΔCt method was used to calculate relative mRNA expression.

| Animal treatment and tissue processing
Apc min/+ mice on a C57BL/6J background were purchased from Animal Model Institution of Nanjing University, China. The mice were provided with either sterile water (n = 10) or 0.2% DCA in drinking water (n = 10) under specific pathogen free (SPF) conditions for 12 weeks as previously described. 7,16 Signs of illness were monitored daily and body weight was recorded weekly. Mice were killed for intestinal tumour burden assessment and tissue collection as previously described. 16,19 Tissue sections were prepared for haematoxylin and eosin (H&E) and immunohistochemical staining. Adenomas of distal small intestinal section were excised, immediately frozen in liquid nitrogen and then stored at À80°C until analysis for protein expression. Animal protocols were approved by the Institutional Animal Care and Use Committee at Tianjin Medical University, Tianjin, China.

| TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay (Roche Applied Science, Mannheim, Germany) was used to detect apoptotic cells. Apoptotic cells were determined by counting percentage of positive-stained cells in five randomly selected fields in each tumour. At least three tumours in each mouse were randomly selected.

| Statistical analysis
Statistical analysis was performed on SPSS 22.0 (SPSS, Chicago, IL, USA). Data were presented as mean AE SD. Differences among groups were tested by one-way ANOVA for multiple comparison and t test for paired samples. P < .05 was considered significant.   F I G U R E 3 Epidermal growth factor receptor (EGFR) kinase activity and ADAM-17 were required for deoxycholic acid-induced intestinal epithelial cell apoptosis resistance. A,. The effects of deoxycholic acid (DCA) on apoptosis associated protein (cleavage of PARP and caspase-3) in YMCE and AG1478-treated cells by Western blot analysis, using internal control protein b-actin for total protein. B, Western blot analysis showed that DCA did not induce apoptosis in YAMC or ADAM-17 À/À MCE + wt ADAM-17; however, EGFR kinase activity blocking and ADAM-17 knock-out induced apoptosis in cells exposed to DCA, using internal control protein b-actin for total protein. AG1478, an EGFR receptor kinase inhibitor; DCA, deoxycholic acid F I G U R E 4 Deoxycholic acid accelerated intestinal carcinogenesis in Apc min/+ mice. A, The numbers of tumours/mouse in the small intestine and colon in DCA-treated Apc min/+ mice were shown compared with untreated Apc min/+ mice (control). The tumour size distribution in the small intestine and the tumour number in each section were also listed. B,C, The representative and histologic appearance of intestinal tumours from DCA-treated or untreated Apc min/+ mice. D, Immunohistochemistry results showed that the cell proliferation (Ki-67) was significantly increased in Apc min/+ mice by DCA. E, Terminal deoxynucleotidyl transferase dUTP nick end labelling staining showed that the apoptotic cells in tumours were significantly reduced in DCA group compared with that in control group DCA, deoxycholic acid; HGD, highgrade dysplasia; LGD, low-grade dysplasia. Scale bar: 50 lm. *P < .05, **P < .01, ***P < .001. n = 10/group

| DISCUSSION
The high fat diet has been linked with increased CRC risk and associated with high DCA production in the intestine. 5 Epidermal growth factor receptor is recognized as a key player in CRC initiation and progression. 31 As a member of the ErbB family, EGFR has an extracellular ligand-binding domain and an intracellular portion that contains a tyrosine kinase domain. EGFR is most frequently activated by binding to its soluble ligands and then triggers a variety of signalling molecules that regulate intracellular signalling networks including Akt and MAPK pathway. 15,32,33 In the Apc Min/+ mouse model, it has been shown that EGFR activity was important in the establishment of intestinal tumours, and Apc deficiency was associated with the increased EGFR activity. 34  F I G U R E 5 ADAM-17/EGFR signalling axis was activated in intestinal tumours of deoxycholic acid-treated Apc min/+ mice. A, Immunohistochemistry showed that DCA treatment increased the percentage of positive cells of ADAM-17 in intestinal tumour in Apc min/+ mice. B, DCA treatment increased the percentage of positive cells of amphiregulin in intestinal tumour in Apc min/+ mice. C,D, Phosphorylation of epidermal growth factor receptor (EGFR) and Akt in intestinal tumours was up-regulated after DCA treatment. E, Western blot analysis showed that DCA increased the phosphorylation of EGFR and Akt in Apc min/+ mice. DCA, deoxycholic acid. Scale bar: 20 lm. **P < .01, ***P < .001. n = 10/group DONG ET AL. Researches had shown that the EGFR pathway was the most important target for CRC therapy. 41 Two FDA-approved monoclonal antibodies against EGFR have become clinically routine, but only a small part of patients had an effective result. 42,43 Thus, the development of combinatory therapeutic target should be introduced into CRC therapy. 44 In this study, we found that DCA accelerated intestinal carcinogenesis through activation of ADAM-17/EGFR signalling axis. Further studies should be arranged to study the relationship between DCA and upstream components of ADAM-17/EGFR signalling axis, as specific nuclear receptors (FXR, PXR and vitamin D receptor) and G-protein-coupled receptors (TGR5, sphingosine-1 phosphate receptor 2 and muscarinic receptors). 45 In conclusion, we investigated the importance of ADAM-17/ EGFR signalling activation in the process of intestinal carcinogenesis by DCA treatment. This report also shows that DCA can stimulate release of AREG mediated by ADAM-17. Consequently, it may provide new insights that DCA promotes adenoma to adenocarcinoma progression. And ADAM-17/EGFR signalling axis will represent a potential target for the bile acid-related CRC therapy.