Tumor infiltration by T regulatory cells (Tregs) occurs in a wide variety of cancers. Although there is evidence that tumor Tregs decrease immune responses to tumor cells and increase metastasis, there is much to be learned about the mechanisms involved. Tregs constitutively express the prolactin receptor (PRLR), suggesting that PRL is important to function. Essentially all Treg PRLR are the long isoform. To investigate the role that PRL might play in tumor Treg recruitment and function, we used the syngeneic 4T1 orthotopic breast cancer model. Mice were treated systemically with a splice modulating oligomer (SMO) that specifically knocks down expression of only the long isoform (LF) of the PRLR. Treatment with this SMO resulted in decreased tumor Tregs (0.65% of gated tumor cells in PRLRLF knockdown compared to 3.31% in control SMO-treated mice), demonstrating that prolactin (PRL) signaling through the PRLRLF directly or indirectly recruited Tregs to this kind of tumor. Further investigation in vitro and in vivo revealed two different ways that PRL assists in recruitment: 1) PRL directly stimulated tumor cell production of CCL17, a chemokine that attracts Tregs. This was inhibited when PRLRLF was knocked down. Furthermore, the PRLRLF antagonist, S179DPRL, inhibited basal CCL17, demonstrating that tumor cell autocrine PRL could stimulate CCL17 output. 2) PRL signaling is required for CCR4 expression on tumor Tregs. CCR4 is normally constitutively expressed on Tregs and is the receptor that responds to CCL17. Analysis of tumor cell chemokine production in response to estrogen or progesterone in vitro showed estrogen induced CCL22, another ligand for Treg CCR4, while progesterone induced CCL17. All three hormones therefore could promote Treg recruitment, but the ability of PRLRLF knockdown, which has little effect on levels of estrogen and progesterone, to inhibit recruitment suggests the effects of the other two hormones are mediated through PRL. PRL treatment of tumor cells in vitro showed a small decrease in mesenchymal markers consistent with reports in other breast cancer cells. Importantly however, in the presence of Tregs, PRL markedly increased expression of twist-1, snail-2, vimentin and fibronectin. Furthermore, if PRLRLF was knocked down in Tregs, there was no increase in mesenchymal markers in the tumor cells in response to PRL. Cancer stem cells are considered important players in metastatic spread. Analysis of isolated stem cells showed the same PRL and Treg interactions as for bulk tumor cells in terms of mesenchymal markers. Tregs with PRLRLF knockdown showed no lack of suppressive ability in a splenocyte response to lipopolysaccharide test. Taken together, results show that PRLRLF signaling in Tregs is essential for Treg-mediated EMT, but is dispensable for general immune suppressive ability. These effects contributed to an 80% inhibition of metastatic spread coincident with a marked reduction in inflammatory damage from responses to metastatic cells after 40days of PRLRLF knockdown.

Citation Format: KuanHui E. Chen, Mrinal K. Ghosh, Tomohiro Yonezawa, Ameae M. Walker. Prolactin drives the recruitment of tumor Tregs and Treg-mediated metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-255. doi:10.1158/1538-7445.AM2014-LB-255