Progestin-driven regulatory T cells directly promote an aggressive and metastatic phenotype in triple negative breast cancer

DALOTTO-MORENO T; MENDEZ-HUERGO S; MOSES F; RABINOVICH GA; SALATINO M

Philadelphia

Congreso; ANnual Meeting of the American Association for Cancer Research; 2015

American Association for Cancer Research

   The immune system plays key roles in the recognition and elimination of most tumors. Progesterone (Pg) can shape the immune response favoring a tolerogenic rather than a pro-inflammatory adaptive response. As hormone replacement therapies, supplementation and hormone-based contraceptives have been associated with an increase of malignant breast neoplasia we decided to investigate how progestins can regulate different key immune cell populations in the tumor microenvironment and their influence to tumor progression. To address this issue we used the highly metastatic, triple-negative 4T1 breast tumor. Balb/c mice treated with Pg or its synthetic analog, medroxyprogesterone acetate, showed an increased frequency of Foxp3+ regulatory T cells (Tregs) in draining lymph nodes (DLN) and an impaired antitumor response elicited by a decreased production of IL-17 and IFN-ã by CD4+ T cells (p<0,01). Tregs in DLN of progestin-treated animals showed increased expression of CD44 and CTLA-4. CD44+ Tregs were more suppressive in vitro than their CD44- counterpart. Concordantly, progestin-treated tumor-bearing mice showed a higher frequency of CD44+ Tregs and PD1+TIM3+ ?exhausted? CD8 T cells in the tumor (p<0,05). Both progestins favored in vitro Treg stable differentiation and expansion which resulted in a more robust suppression activity (p<0,01). Progestin-induced Treg differentiation could not be inhibited by the nuclear progesterone receptor (nPR) antagonist, Zk230211. Interestingly, T cells did not express detectable levels of nPR by qPCR but did express membrane progesterone receptor alfa, gamma and progesterone receptor membrane component type 1. In the presence of progestins, in vitro differentiated Tregs expressed higher levels of RANKL, a protein involved in breast stem cell maintenance and malignant transformation of tumors. When cocultured, ex vivo sorted- or in vitro differentiated Tregs increased 4T1 cells invasiveness both in vitro and in vivo following the downregulation of E-cadherin and increased expression of Snail (p=0,05). Concordantly we observed a stable expansion of the CD44+ stem cell-like population when cocultured with progestin-iTregs. The CD44+ 4T1 population formed tumor spheres, exhibited an EMT molecular signature and had augmented tumorigenicity and a metastatic phenotype. Although primary tumor volume was not altered, the number of lung metastases was notably increased in animals treated with progestins (p<0,01). Additonally, overall survival of mice was decreased when injected with 4T1 cells cocultured with progestin-induced Tregs compared with 4T1 cocultured with control Tregs. Our findings highlight the relevance of progestins in modulating immunoregulatory checkpoints and harnessing immunosurveillance in the tumor microenvironment and suggest a mechanism through which Tregs could directly promote a metastatic and aggressive phenotype on tumor cells.