Abstract 2513: Convergence of cancer associated fibroblasts (CAFs) and progesterone receptor (PR) signaling in ER+ luminal breast cancer
Authors: Spartz Angela , Diep Caroline , Troness Benjamin , Lange Carol A. , El-Ashry Dorraya 
Published: July 2021

Abstract
Background: Cancer associated fibroblasts (CAFs) in the tumor microenvironment and the circulation secrete a complex array of paracrine factors that contribute to the development of metastasis, stemness and chemotherapy resistance. CAF-derived factors are primary candidates for initiating signaling pathways in ER+/PR+ breast cancer cells even in low/no hormone conditions and particularly at genes that specify stemness and survival in the circulation. Herein, we investigate the convergence of CAFs and PR signaling in ER(+) luminal breast cancer.
Methods: Tumorsphere assay: BC cells were grown in B27-supplemented tumorsphere media in ULA plates for 5-10 days. Soft agar colony formation assay: BC cells were grown in soft agar under serum starvation for 3 weeks. Clustering assay: Cell tracker-labeled CAF or BC cells were cultured together in ULA plates for 24 hrs.
Results: Using the ER(+) T47D cell series that express no PR (T47D-Y), endogenous PR (T47D CO), or either exclusively PR-A or PR-B isoforms, we observed that CAF conditioned media (CM) increased primary and secondary tumorsphere formation relative to controls. In soft agar assays, CAF CM increased adhesion-independent proliferation compared to non-CM. Previously, our group reported that phosphorylation of PR Ser294 is required for PR-induced cancer stem cell phenotypes. Phospho-mutant S294A T47D PR-A or PR-B cell lines attenuated the response to CAF CM in tumorsphere assays. Conversely, in soft agar assays, S294A PR-A+ cells did not differ from WT PR-A+ cells, though S294A PR-B+ cells formed significantly fewer colonies than WT PR-B+ cells. Western blots showed that CAF CM induced rapid phosphorylation of PR S294 and activation of AKT and MAPK/ERK kinases; the MEK inhibitor U0126 blocked CAF-induced PR S294 phosphorylation whereas the AKT inhibitor, LY294006, was without effect. Two candidate cytokines, SDF1 and FGF2, were tested for their ability to induce PR activity, tumorsphere formation, and anchorage independent proliferation. Treatment of cells with FGF2, but not SDF1, mimicked the patterns of phosphorylation and the phenotypes observed for CAF CM. Lastly, physical interaction between CAFs and PR expressing cells as measured by in vitro clustering assays demonstrated that T47D CO, PR-A and PR-B cells preferentially cluster with CAF cells relative to PR null (T47D-Y) cells.
Conclusion: We have shown that CAF-derived paracrine factors, possibly FGF2, activate MAPK/ERK, which in turn phosphorylates PR on Ser294 to initiate tumorsphere formation and adhesion independent proliferation. Our study of the interactions between PR (+) breast cancer cells with neighboring CAFs either in the primary tumor or in the circulation (as CAF/CTC clusters) may shed light on alternative treatment strategies, particularly when endocrine therapies have failed.
Citation Format: Angela Spartz, Caroline Diep, Benjamin Troness, Carol A. Lange, Dorraya El-Ashry. Convergence of cancer associated fibroblasts (CAFs) and progesterone receptor (PR) signaling in ER+ luminal breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2513.