Abstract A34: Hypoxic cancer-associated fibroblasts secrete regulators of angiogenesis: Novel potential players revealed by MS

Abstract

Abstract Hypoxia correlates with increased risk of metastasis and poor prognosis in breast cancer patients. Cancer-associated fibroblasts (CAFs) aid cancer spread by inducing the formation of an aberrant tumor stroma through secretion of growth factors, cytokines, ECM proteins, and ECM modifiers; this altered stroma can foster a pro-angiogenic environment in tumors. However, the role of CAFs under hypoxic conditions is still controversial. Here we aim to understand how hypoxia modulates the repertoire of intracellular and secreted proteins of mammary CAFs and how CAFs affect the angiogenic response of endothelial cells (ECs) in the hypoxic environment. To tackle this question, we combined a 3D co-culture model of ECs and CAFs which enabled to evaluate the impact of CAF-secreted factors on EC sprouting with SILAC-based MS secretomics approach. Using the 3D angiogenesis assay we found that ECs co-cultured with CAFs under hypoxia or exposed to the conditioned medium (CM) of hypoxic CAFs sprouted significantly more than the normoxic counterpart. The increased sprouting was the result of both autocrine and paracrine signals, although the pro-angiogenic capacity of CAFs was more pronounced than the one of the ECs. Furthermore, we found that the CM of hypoxic CAFs was able to significantly enhance EC sprouting even upon VEGFA blockage with bevacizumab, thus indicating the presence of other hypoxia-induced pro-angiogenic factors. These findings prompted us to profile the proteome and secretome of hypoxic mammary CAFs in culture. The proteome analysis revealed regulation of metabolic proteins, particularly up-regulation of glycolysis-related proteins and down-regulation of proteins involved in mitochondrial metabolism, mirroring the known metabolic switch induced by hypoxia. The secretomics analysis unveiled increased levels of known HIF1α targets, including VEGFA and STC1, and, interestingly, additionally identified a subset of proteins which have not been previously related to hypoxia nor functionally characterized. Integrated bioinformatic analysis revealed that the chromosomic region flanking these genes contains hypoxia responsive elements (HRE), suggesting that their transcription is driven by HIFs. We are currently investigating the pro-angiogenic capacity of these newly identified secreted proteins. Our findings points out that there are hypoxia-induced players still uncharacterized, whose investigation may lead to a better understanding of the CAFs response to hypoxia and how these cells affect tumor progression. Citation Format: Fernanda Grande Kugeratski, Juan Ramon Hernandez, Gabriela Kalna, Sara Zanivan. Hypoxic cancer-associated fibroblasts secrete regulators of angiogenesis: Novel potential players revealed by MS. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A34.