BRAF^V595E Mutation Associates CCL17 Expression and Regulatory T Cell Recruitment in Urothelial Carcinoma of Dogs

Abstract

Regulatory T cells may serve as targets in cancer immunotherapy. A previous study showed that the chemokine CCL17 and the receptor CCR4 play roles in regulatory T cell recruitment in canine urothelial carcinoma. In this article, we show that the BRAFV595E mutation is associated with tumor-produced CCL17 and regulatory T cell infiltration in dogs with urothelial carcinoma. In comparison with healthy dogs, dogs with urothelial carcinoma showed increased CCL17 mRNA expression in the bladder and elevated CCL17 protein concentration in urine. Immunohistochemistry showed increased levels of Foxp3+ regulatory T cells in the tumor tissues of urothelial carcinoma. The density of Foxp3+ regulatory T cells was positively correlated with CCL17 concentration in urine, indicating that CCL17 is involved in regulatory T cell recruitment. Moreover, tumor-infiltrating regulatory T cells and urine CCL17 concentration were associated with poor prognosis in dogs with urothelial carcinoma. The number of tumor-infiltrating regulatory T cells, CCL17 mRNA expression, and urine CCL17 concentration in cases with BRAFV595E mutation were higher than those in cases with wild-type BRAF. In vitro, high CCL17 production was detected in a canine urothelial carcinoma cell line with BRAFV595E mutation but not in an urothelial carcinoma cell line with wild-type BRAF. Dabrafenib, a BRAF inhibitor, decreased CCL17 production in the cell line with BRAFV595E mutation. These results suggest that BRAFV595E mutation induced CCL17 production and contributed to regulatory T cell recruitment in canine urothelial carcinoma.

Anti-CCR4 treatment depletes regulatory T cells and leads to clinical activity in a canine model of advanced prostate cancer

“… 38 We have recently shown that BRAF V595E mutation induces CCL17 production and contributes to Treg recruitment in dogs with bladder cancer. 36 Thus, we investigated whether BRAF V595E mutation influences tumor-infiltrating Tregs or the CCL17–CCR4 axis in dogs with prostate cancer. Of the 28 dogs with prostate cancer used in this study, BRAF V595E mutation was detected in 21 (75%) cases ( online supplemental table S1 ).…”

“…BRAF V595E mutations were examined by digital PCR assay using genomic DNA isolated from urine sediments, as previously described. 36 …”

Diseases of the Canine Prostate Gland

“…To differentiate between urothelial and prostate carcinoma, a combination of markers will be necessary. In a recent study [131], the chemokine CCL17 was found to contribute to regulatory T cell (Treg) recruitment in prostate tumors. In dogs with prostate cancer, tumor-infiltrating Tregs were found to be associated with bad prognosis [132].…”

CCR4 blockade leads to clinical activity and prolongs survival in a canine model of advanced prostate cancer

“…A somatic point mutation in the BRAF gene (BRAF V595E ), which is homologous to the human BRAF V600E mutation, is present in over 70% of dogs with bladder and prostate cancers (23). We have recently shown that BRAF V595E mutation induces CCL17 production and contributes to Treg recruitment in dogs with bladder cancer (24). Thus, we investigated whether BRAF V595E mutation influences tumor-infiltrating Tregs or the CCL17/CCR4 axis in dogs with prostate cancer.…”

“…Urinary CCL17 was assessed by ELISA, as described above. BRAF V595E mutations were examined by digital PCR assay using genomic DNA isolated from urine sediments, as previously described (24).…”