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. 2017 Jan 15;23(2):549-561.
doi: 10.1158/1078-0432.CCR-15-3140. Epub 2016 Aug 3.

ADAM17 is a Tumor Promoter and Therapeutic Target in Western Diet-associated Colon Cancer

Reba Mustafi #  1 , Urszula Dougherty #  1 , Devkumar Mustafi  2 , Fatma Ayaloglu-Butun  1 , Michelle Fletcher  1 , Sarbani Adhikari  1 , Farhana Sadiq  1 , Katherine Meckel  1 , Haider I Haider  1 , Abdurahman Khalil  1 , Joel Pekow  1 , Vani Konda  1 , Loren Joseph  3 , John Hart  3 , Alessandro Fichera  4 , Yan Chun Li  1 , Marc Bissonnette  1
Affiliations

ADAM17 is a Tumor Promoter and Therapeutic Target in Western Diet-associated Colon Cancer

Reba Mustafi et al. Clin Cancer Res. .

Abstract

Purpose: Epidermal growth factor receptors (EGFR) are required for tumor promotion by Western diet. The metalloprotease, ADAM17 activates EGFR by releasing pro-EGFR ligands. ADAM17 is regulated by G-protein-coupled receptors, including CXCR4. Here we investigated CXCR4-ADAM17 crosstalk and examined the role of ADAM17 in tumorigenesis.

Experimental design: We used CXCR4 inhibitor, AMD3100 and ADAM17 inhibitor, BMS566394 to assess CXCR4-ADAM17 crosstalk in colon cancer cells. We compared the expression of CXCR4 ligand, CXCL2, and ADAM17 in mice fed Western diet versus standard diet. Separately, mice were treated with marimastat, a broad-spectrum ADAM17 inhibitor, or AMD3100 to assess EGFR activation by ADAM17 and CXCR4. Using Apc-mutant Min mice, we investigated the effects of ADAM17/10 inhibitor INCB3619 on tumorigenesis. To assess the effects of colonocyte ADAM17, mice with ADAM17 conditional deletion were treated with azoxymethane (AOM). ADAM17 expression was also compared in colonocytes from primary human colon cancers and adjacent mucosa.

Results: CXCL12 treatment activated colon cancer cell EGFR signals, and CXCR4 or ADAM17 blockade reduced this activation. In vivo, Western diet increased CXCL12 in stromal cells and TGFα in colonocytes. Marimastat or AMD3100 caused >50% reduction in EGFR signals (P < 0.05). In Min mice, INCB3619 reduced EGFR signals in adenomas and inhibited intestinal tumor multiplicity (P < 0.05). In the AOM model, colonocyte ADAM17 deletion reduced EGFR signals and colonic tumor development (P < 0.05). Finally, ADAM17 was upregulated >2.5-fold in human malignant colonocytes.

Conclusions: ADAM17 is a Western diet-inducible enzyme activated by CXCL12-CXCR4 signaling, suggesting the pathway: Western diet→CXCL12→CXCR4→ADAM17→TGFα→EGFR. ADAM17 might serve as a druggable target in chemoprevention strategies. Clin Cancer Res; 23(2); 549-61. ©2016 AACR.

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Figures

Fig. 1
Fig. 1. Effects of Western diet on CXCL12 and EGFR signaling components in colonic mucosa
Flash frozen colonic mucosal tissues from control mice fed standard or Western diet (WD) for 40 wks were used from a prior study (9). Tissues were extracted for proteins and RNA, and real time PCR and Western blotting were performed. A. Experimental protocol. B. CXCL12, ADAM17, TGFα and K-Ras transcripts (*p<0.05, †p<0.005, ‡p<0.001, compared to levels in mice fed Std diet; n = 4 mice for each diet). C. Representative Western blots for indicated proteins. D. Expression levels for indicated proteins (*p<0.05, ‡p<0.001 compared to levels in Std diet fed mice; n=4 mice for each diet). E, F in situ TGFα mRNA (white arrows) under Std diet (E) and WD conditions (F), 10x. The relative fluorescence intensity of TGFα mRNA was 2.8±1.4 in WD compared to Std diet (p<0.05). G, H CXCL12 immunostaining (white arrows), 10x. Colon sections in OCT from mice on standard diet (G) or Western diet (H) were stained for CXCL12. Note CXCL12 is predominantly expressed in stromal cells in the pericryptal mucosa and submucosa and increased in WD-fed mice. In situ TGFα mRNA hybridization and CXCL12 immunostained sections are representative of three mice in each dietary group.
Fig. 2
Fig. 2. EGFR activation by CXCL12 requires EGF receptor ligand binding and is mediated by ADAM17 and inhibited by AMD3100
A. anti-EGFR antibody blocks EGF receptor activation by CXCL12. HT29 cells were pretreated for 2 hrs with PBS (vehicle, Ctl) or 20 μg/ml C225, an anti-EGFR antibody. Cells were then stimulated with CXCL12 (50 ng/ml) for the indicated times (1, 5, 10 min) and cell lysates probed for phospho-EGFR (pEGFR), pErbB2, pAKT and pERK, as well as for panEGFR, panErbB2, panAKT and panERK and β-actin as loading control. Separate wells were treated with vehicle or EGF (10 ng/ml) as a positive control. Note that C225 antibodies suppressed EGFR signals, indicating that EGFR activation by CXCL12 involves an EGF receptor ligand-dependent mechanism. B. Quantified phospho-active EGFR signals in cells pretreated with vehicle or C225 normalized to vehicle-treated (control) cells. C. ADAM17 mediates the effect of CXCL12 on EGFR activation. Cells were pretreated for 2 hrs with vehicle (−) or 5 μM BMS566394 (+, BMS), an ADAM17-specific inhibitor, and then stimulated with 50 ng/ml CXCL12 for the indicated times (1, 5, 10 min). Cell lysates were probed for the indicated phospho-active proteins (EGFR cascade). MWS: Lane containing molecular weight standards. Note that BMS566394 inhibits CXCL12-induced EGFR signals, indicating that ADAM17 mediates EGFR transactivation by CXCL12. D. Indicated phospho-protein signals in cells pretreated with BMS566394 normalized to vehicle-treated (control) cells. E. AMD3100 inhibits EGFR activation by CXCL12 as assessed by pErbB2, pERK and pAKT. HT29 cells were pretreated with 10 ng/ml AMD3100 or PBS (vehicle) for 2 hrs, and cells were then stimulated with 100 ng/ml CXCL12 or vehicle. At the indicated times (1, 5, 10 min), cell lysates were probed for pErbB2, pERK and pAKT. F. Phospho-active protein signals in cells pretreated with vehicle or AMD3100 normalized to signals in vehicle-treated cells. Results in Figs 2A-F are representative of two independent platings with comparable findings.
Fig. 3
Fig. 3. CXCR4 inhibitor AMD3100 suppresses EGFR signals and proliferation in colonic mucosa of mice fed Western diet
Mice were implanted with Alzet pumps and received vehicle or AMD3100 (5 mg/kg body weight/day) and were fed a Western diet. After 2 wks, mice were sacrificed and left colons were harvested. Indicated proteins were measured by Western blotting. A. Treatment protocol. B. EGFR signals. Western blots of indicated proteins from scrape-isolated mucosa in left colon from mice on WD alone or WD + AMD3100 (+). C. Quantitative expression levels (*p<0.001, †p<0.005 compared to WD alone, n=4 mice in each group). D. BrdU incorporation in vehicle-treated mouse. E. BrdU incorporation in AMD3100-treated mouse. F. BrdU quantitation (*p<0.05, compared to BrdU in WD alone; n = 4 mice per condition).
Fig. 4
Fig. 4. ADAM17 inhibitor INCB3619 suppresses tumor development in Apc mutant Min mice
A. Effect of INCB3619 on mouse growth. (*

B. Effects of INCB3619 on small intestine and colon tumor incidence. C. INCB3619 suppresses small intestinal tumor multiplicity (n=20 mice on WD and 20 mice on WD+INCB3619; *p<0.01, Mann-Whitney-U test). D. INCB3619 suppresses small intestinal tumor growth (mean±SD, p<0.05).

Fig. 5
Fig. 5. INCB3619 suppresses EGFR signals and proliferation in adenomas from Apc mutant Min mice
Mice were treated as described in the Methods. At 7-mo age, mice were sacrificed, and intestinal adenomas harvested. Diet-matched adjacent normal-appearing intestinal mucosa was scrape-isolated and proteins solubilized in Laemmli buffer. A. Western blots of indicated EGFR signals. B. Densitometry. Values represent 6-8 mice per group [*p<0.05, **p<0.005, phospho-protein levels in adenomas from mice on WD alone (Tumor), compared to adjacent normal-appearing mucosa (Nl); †p<0.05, phospho-protein levels in normal-appearing mucosa in group receiving INCB3619 (Nl + INCB), compared to levels in intestinal mucosa from mice on WD alone (Nl); ‡p<0.05, ‡‡p<0.005, phospho-protein levels in adenomas from mice on INCB3619 (Tumor + INCB), compared to adenomas from mice on WD alone (Tumor)]. C. Western blots of indicated EGFR and Notch1 effectors. D. Densitometry [*p<0.05, **p<0.005, compared to levels in normal-appearing intestinal mucosa; ‡p<0.05, compared to levels in adenomas from mice on WD alone; n= 6-8 mice per group]. E. Ki67 immunostaining in representative adenoma from mouse on WD alone (left panel) and adenoma from mouse on WD + INCB3619 (right panel). F. Quantitation of Ki67 staining (*p<0.001 compared to WD alone, n=10 adenomas from mice on WD alone, n=11 adenomas from mice on WD + INCB3619. Each adenoma is representative of a different mouse).
Fig. 6
Fig. 6. Colonocyte ADAM17 deletion suppresses EGFR signals and reduces colonic tumor development in AOM-treated mice
Villin-Cre and ADAM17 LoxP/+ mice were interbred to an AOM-susceptible A/J background. Villin-Cre-homozygous ADAM17LoxP (ADAM17ΔΔ) and homozygous ADAM17LoxP mice were then treated with AOM or vehicle (saline). Two weeks after the 6th AOM injection, mice were begun on a Western diet (WD, 20%) as described in the Methods. Twenty-eight wks after the first AOM treatment, mice were sacrificed and tumors harvested A. Cell-specific ADAM17 expression. Colonocytes and stromal cells were isolated from scraped mucosa from left colon of villin-Cre+ - homozygous ADAM17LoxP (villin-Cre+) and homozygous ADAM17LoxP mice (villin-Cre −) as described in the Methods. (Mice in Fig 6A received no AOM treatment). The indicated cell fractions were probed by Western blotting for ADAM17, vimentin (VIM, stromal cell marker) and cytokeratin20 (CK20, colonocyte marker). Note that in villin-Cre+ - homozygous ADAM17LoxP mice (ADAM17ΔΔ), ADAM17 was deleted from colonocytes, but not from stromal cells. B. Colon tumor incidence. Tumors were classified histologically as adenomas or carcinomas (*,†p<0.05 compared to control homozygous ADAM17LoxP mice; n=32 homozygous ADAM17LoxP mice and n=29 ADAM17ΔΔ mice). C. Colon tumor multiplicity. The median number of tumors was significantly lower in ADAM17ΔΔ group compared to the homozygous ADAM17LoxP group (2.0 vs. 5.0 tumors per tumor-bearing mouse, respectively; *p<0.05, Mann-Whitney-U test). D. Ki67 staining (*p<0.05, n=14 tumors in each genotype). E. Tumor size (*p<0.05; n=35 tumors from homozygous ADAM17LoxP mice and n=12 tumors from ADAM17ΔΔ mice). F. EGFR and Notch1 signals in AOM tumors (T) and genotype-matched scrape-isolated left colon mucosa from vehicle-treated mice (N). Blots are representative of 6 tumors per group. G. Quantitative densitometry (*p<0.05 compared to homozygous ADAM17LoxP mucosa; †p<0.05 compared to adenomas in the homozygous ADAM17LoxP group).
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