Role of Lysyl oxidase in TGF-beta-induced Fibroblast Activation and Cancer-Associated Fibroblast-mediated Breast Cancer Progression

Abstract

Cancer-associated Fibroblasts (CAFs) are a dominant cell type of the breast cancer microenvironment and are proposed to be involved in metastatic progression. TGF-β has been reported to influence fibroblast to myofibroblast activation, and the myofibroblast phenotype is similar to cancer-associated fibroblast phenotype. To understand the mechanism of TGF-β mediated fibroblast activation and CAF phenotype, we performed a comparative proteomic analysis of conditioned media from CAFs (isolated from breast cancer biopsy tissues) and normal mammary fibroblasts engineered to over-express TGF-β. Liquid chromatography/ tandem mass spectrometry (LC ESI Q-TOF-MS/MS) assay of conditioned media and Venn analysis of the data revealed approximately 185 common proteins secreted by the three fibroblast types (CAFs, normal mammary fibroblasts and TGF-β over-expressing mammary fibroblasts). Among these, 12 proteins exclusively overlap between normal fibroblasts and CAFs and 20 proteins exclusively overlap between CAFs and TGF-β over-expressing fibroblasts. Proteome data are available via ProteomeXchange with identifier PXD022374. Further experiments revealed lysyl oxidase (LOX), one of the common proteins between TGF-β over-expressing fibroblasts and CAFs, is critical for TGF-β induced α-smooth muscle actin (α-SMA) induction, migration and contractility of human mammary fibroblasts. Studies using shRNA and specific small molecule inhibitors revealed that TGF-β activates human mammary fibroblasts through LOX/FAK/GSK3β/β-catenin pathway. We also show that LOX is essential for maintaining α-SMA expression and β-catenin stabilization in CAFs. In addition, we explored the role of TGF-β activated fibroblasts and CAFs in breast cancer progression, in vivo. For this, we assessed tumour progression of breast cancer cell lines implanted into immunocompromised mice with or without CAFs, normal mammary fibroblasts and TGF-β over-expressing mammary fibroblasts. We observed that cancer cells showed increased tumour growth and metastatic colonies on the liver in the presence of TGF-β over-expressing fibroblasts and CAFs. We also observed that CAF-derived LOX has a critical role for breast cancer progression, in vivo. Our results highlight a novel mechanism involving LOX during TGF-β mediated activation of mammary fibroblasts and in the regulation of CAF phenotype. Further, we also propose LOX as a critical mediator of TGF-β induced breast cancer progression. Our study highlights the importance of communication between cancer cell and CAF mediated by TGF-β in the tumour microenvironment that may help in devising newer strategies for the management of breast cancer patients.