Abstract PR01: CLIC3 is secreted by CAFs and enhances angiogenesis and tumor cell invasion by cooperating with TGM2
Authors: Hernández-Fernaud Juan Ramon , Ruengeler Elena , Casazza Andrea , Neilson Lisa , Pulleine Ellie , MacPherson Iain , Blyth Karen , Yin Huabing , Mazzone Massimiliano , Norman Jim , Zanivan Sara 
Published: December 2015

Abstract
Cancer-associated fibroblasts (CAFs) play a central role in tumor progression through the mechanical remodeling of the stroma. Indeed CAFs secrete a plethora of extracellular matrix (ECM) components and ECM modifiers which contribute to generate stiff and dense tumors. Increased tumor stiffness induces endothelial cell (EC) sprouting and tumor cell invasion. Moreover, excessive stiffness represents a critical barrier to therapy because it blocks perfusion thus preventing diffusion of drugs and favoring hypoxia. Tuning tumor stiffness has therefore the potential to contribute to improve the efficacy of conventional anti-cancer therapies. Our study aims at using unbiased proteomics approach to identify CAF proteins which alter the tumor stroma, and investigating their functional role.
We have established mass spectrometry-based proteomics approach to accurately and in-depth analyze secretomes of cells in culture, and used it to compare cell lines of human mammary normal (iNF) and myofibroblasts-like cancer-associated (iCAF) fibroblasts. Unexpectedly, we detected the chloride intracellular channel protein 3 (CLIC3), which so far was known as an intracellular regulator of receptor trafficking in tumor cells, amongst the most up-regulated proteins in the ECM generated by iCAF. Highlighting the relevance of CLIC3 in a clinical context, high levels of CLIC3 were also measured in CAFs isolated from patient samples. Notably, CLIC3 silencing reduces the pro-angiogenic and pro-invasive activity of CAFs in vitro. To investigate the role of secreted CLIC3, we have generated the recombinant protein (rCLIC3) and the mutant for the active site (rCLIC3mut) and show that rCLIC3 induces angiogenesis in vitro and in vivo, and tumor cell invasion in vitro and that its active site is required for this activity. Furthermore, we show that rCLIC3 is used by iCAF to increase the stiffness of the ECM that they produce. Using gene silencing and specific inhibitors in combination with functional assays in vitro and in vivo, we show that rCLIC3 exerts its functions through the cooperation with the ECM remodeling enzyme transglutaminase 2 (TGM2). Importantly, TGM2 is highly abundant in many tumors. In a clinical context, CLIC3 staining of various TMAs shows that this protein is expressed in the stroma of some tumors and that it is particularly abundant in the stroma of ovarian cancers. Preliminary analysis of ovarian TMAs shows that the stroma of aggressive and highly vascularized tumors strongly stains for CLIC3.
In conclusion, our work has discovered a completely unexpected function for CLIC3, which we have now characterized as a stromal protein which is secreted and alters the ECM stiffness cooperating with TGM2, thus enhancing the pro-angiogenic and pro-invasive properties of the CAFs. CLIC3 is therefore a promising candidate to be further investigated for its role in tumor progression and as a target to improve current anti-cancer therapy.
Citation Format: Juan Ramon Hernández-Fernaud, Elena Ruengeler, Andrea Casazza, Lisa Neilson, Ellie Pulleine, Iain MacPherson, Karen Blyth, Huabing Yin, Massimiliano Mazzone, Jim Norman, Sara Zanivan. CLIC3 is secreted by CAFs and enhances angiogenesis and tumor cell invasion by cooperating with TGM2. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr PR01.