Adenosine/TGF[beta] axis in regulation of mammary fibroblast functions

Cancer associated fibroblasts (CAF) play a key role in cancer progression and metastasis. Diminished TGF[beta] response on CAF correlates with poor outcome and recurrence in cancer patients. Mechanisms behind lost TGF[beta] signaling on CAF are poorly understood, but, utilizing MMTV-PyMT mouse model, we have previously demonstrated that in tumor microenvironment myeloid cells, producing adenosine, contribute to downregulated TGF[beta] signaling on CAFs. In the current work, we performed serial in vitro studies to investigate the role of adenosine/TGF[beta] axis in mouse mammary fibroblast functions, i.e., proliferation, protein expression, migration, and contractility. We found that adenosine analog NECA diminished TGF[beta]-induced CCL5 and MMP9 expression. Additionally, we discovered that NECA completely inhibited effect of TGF[beta] to upregulate [alpha]SMA, key protein of cytoskeletal rearrangements, necessary for migration and contractility of fibroblasts. Our results show that TGF[beta] increases contractility of mouse mammary fibroblasts and human fibroblast cell lines, and NECA attenuates theses effects. Using pharmacological approach and genetically modified animals, we determined that NECA effects on TGF[beta] pathway occur via A.sub.2A /A.sub.2B adenosine receptor-AC-PKA dependent manner. Using isolated CD11b.sup.+ cells from tumor tissue of CD73-KO and CD39-KO animals in co-culture experiments with ATP and AMP, we confirmed that myeloid cells can affect functions of mammary fibroblasts through adenosine signaling. Our data suggest a novel mechanism of interaction between adenosine and TGF[beta] signaling pathways that can impact phenotype of fibroblasts in a tumor microenvironment.