ABSTRACT
The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion.
Subject(s)
Chloride Channels/metabolism , Disease Progression , Glutathione/metabolism , Animals , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Cell Line, Tumor , Extracellular Matrix/metabolism , Female , GTP-Binding Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Mice, Inbred C57BL , Mice, Nude , Models, Biological , Neoplasm Invasiveness , Neoplasms/blood supply , Neoplasms/pathology , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Oxidoreductases/metabolism , Protein Binding , Protein Glutamine gamma Glutamyltransferase 2 , Proteome/metabolism , Proteomics , Survival Analysis , Transglutaminases/metabolism , Treatment OutcomeABSTRACT
Expression of the initiator methionine tRNA (tRNAi(Met)) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi(Met) expression levels influence tumor progression. We have found that tRNAi(Met) expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi(Met) in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi(Met) contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi(Met) gene (2+tRNAi(Met) mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi(Met) mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi(Met) mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi(Met) significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi(Met)-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi(Met)-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi(Met) mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi(Met) levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis.
Subject(s)
Collagen Type II/metabolism , Fibroblasts/metabolism , Neovascularization, Pathologic/genetics , RNA, Transfer, Met/genetics , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Collagen Type II/genetics , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Mice, Inbred C57BL , Mice, Transgenic , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Neovascularization, Pathologic/pathology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/mortality , Ovarian Neoplasms/pathology , RNA, Transfer, Met/metabolism , Stromal Cells/pathologyABSTRACT
Creating patterns of biomolecules and cells has been applied widely in many fields associated with the life sciences, including diagnostics. In these applications it has become increasingly apparent that the spatiotemporal arrangement of biological molecules in vitro is important for the investigation of the cellular functions found in vivo. However, the cell patterning techniques often used are limited to creating 2D functional surfaces on glass and silicon. In addition, in general, these procedures are not easy to implement in conventional biological laboratories. Here, we show the formation of a living poly(ethylene glycol) (PEG) layer that can be patterned with visible light on plastic surfaces. This new and simple method can be expanded to pattern multiple types of biomolecule on either a previously formed PEG layer or a plastic substrate. Using common plastic wares (i.e., polyethylene films and polystyrene cell culture Petri-dishes), we demonstrate that these PEG-modified surfaces have a high resistance to protein adsorption and cell adhesion, while at the same time, being capable of undergoing further molecular grafting with bioactive motifs. With a photomask and a fluid delivery system, we illustrate a flexible way to immobilize biological functions with a high degree of 2D and 3D spatial control. We anticipate that our method can be easily implemented in a typical life science laboratory (without the need for specialized lithography equipment) offering the prospect of imparting desirable properties to plastic products, for example, the creation of functional microenvironments in biological studies or reducing biological adhesion to surfaces.
