An epigenetic switch links stromal pyruvate dehydrogenase activation to extracellular matrix production via proline synthesis

Cancer associated fibroblasts (CAFs) are known to influence tumour progression through the secretion of factors which influence tumour growth and invasion. Collagen production is a major aspect of the secretory CAF phenotype. High collagen content in breast cancer is a marker of poor prognosis and is known to promote tumour growth and metastasis, as well is impeding drug delivery to the tumour through reduced perfusion. Tumour metabolism is also well established as a hallmark of cancer, however, how the metabolism of CAFs influences their pro-tumourigenic phenotype is not yet well understood. We investigated metabolic differences between paired CAFs and normal fibroblasts (NFs) from breast tissue. Using an unbiased phosphoproteomic analysis, we identified pyruvate dehydrogenase kinase 2 (PDK2) as the most downregulated kinase in CAFs. PDK2 phosphorylates and deactivates pyruvate dehydrogenase (PDH), which is a key metabolic protein that converts pyruvate to acetyl-CoA in the mitochondria, providing a key link between the major metabolic pathways of glycolysis and the TCA cycle. However, extensive metabolic profiling of CAFs and NFs did not reveal metabolic differences that could be attributed to PDH activity. Acetyl-CoA is also used for protein acetylation, and, using an MS-proteomic approach, we discovered increased histone acetylation in CAFs, which has epigenetic implications for how CAFs regulate their activated phenotype. Using a combination of proteomics, metabolomics, in vitro assays and imaging analyses we investigated the role of PDH-mediated histone acetylation in CAFs and uncovered the importance of histone acetylation in regulating collagen and ECM production by CAFs. PDH activity also regulated PYCR1 expression in CAFs, and we further discovered that proline production by PYCR1 is further required to maintain PDH-activity induced collagen synthesis in CAFs. Our findings open up new possibilities for targeting the desmoplastic stroma to reduce tumour growth and metastasis, and improve drug delivery