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Abstract Background Tumor cells of diffuse-type gastric cancer (DGC) are discohesive and infiltrate into the stroma as single cells or small subgroups, so the stroma significantly impacts DGC progression. Cancer-associated fibroblasts (CAFs) are major components of the tumor stroma. Here, we identified CAF-specific secreted molecules and investigated the mechanism underlying CAF-induced DGC progression. Methods We conducted transcriptome analysis for paired normal fibroblast (NF)-CAF isolated from DGC patient tissues and proteomics for conditioned media (CM) of fibroblasts. The effects of fibroblasts on cancer cells were examined by transwell migration and soft agar assays, western blotting, and in vivo. We confirmed the effect of blocking tubulointerstitial nephritis antigen-like 1 (TINAGL1) in CAFs using siRNA or shRNA. We evaluated the expression of TINAGL1 protein in frozen tissues of DGC and paired normal stomach and mRNA in formalin-fixed, paraffin-embedded (FFPE) tissue using RNA in-situ hybridization (RNA-ISH). Results CAFs more highly expressed TINAGL1 than NFs. The co-culture of CAFs increased migration and tumorigenesis of DGC. Moreover, CAFs enhanced the phosphorylation of focal adhesion kinase (FAK) and mesenchymal marker expression in DGC cells. In an animal study, DGC tumors co-injected with CAFs showed aggressive phenotypes, including lymph node metastasis. However, increased phosphorylation of FAK and migration were reduced by blocking TINAGL1 in CAFs. In the tissues of DGC patients, TINAGL1 was higher in cancer than paired normal tissues and detected with collagen type I alpha 1 chain (COL1A1) in the same spot. Furthermore, high TINAGL1 expression was significantly correlated with poor prognosis in several public databases and our patient cohort diagnosed with DGC. Conclusions These results indicate that TINAGL1 secreted by CAFs induces phosphorylation of FAK in DGC cells and promotes tumor progression. Thus, targeting TINAGL1 in CAFs can be a novel therapeutic strategy for DGC.

Background: Tumor cells of diffuse-type gastric cancer (DGC) are discohesive and infiltrate into the stroma as single cells or small subgroups, so the stroma significantly impacts DGC progression. Cancer-associated fibroblasts (CAFs) are major components of the tumor stroma. Here, we identified CAF-specific secreted molecules and investigated the mechanism underlying CAF-induced DGC progression. Methods: We conducted transcriptome analysis for paired normal fibroblast (NF)-CAF isolated from DGC patient tissues and proteomics for conditioned media (CM) of fibroblasts. The effects of fibroblasts on cancer cells were examined by transwell migration and soft agar assays, western blotting, and in vivo. We confirmed the effect of blocking tubulointerstitial nephritis antigen-like 1 (TINAGL1) in CAFs using siRNA or shRNA. We evaluated the expression of TINAGL1 protein in frozen tissues of DGC and paired normal stomach and mRNA in formalin-fixed, paraffin-embedded (FFPE) tissue using RNA in-situ hybridization (RNA-ISH). Results: CAFs more highly expressed TINAGL1 than NFs. The co-culture of CAFs increased migration and tumorigenesis of DGC. Moreover, CAFs enhanced the phosphorylation of focal adhesion kinase (FAK) and mesenchymal marker expression in DGC cells. In an animal study, DGC tumors co-injected with CAFs showed aggressive phenotypes, including lymph node metastasis. However, increased phosphorylation of FAK and migration were reduced by blocking TINAGL1 in CAFs. In the tissues of DGC patients, TINAGL1 was higher in cancer than paired normal tissues and detected with collagen type I alpha 1 chain (COL1A1) in the same spot. Furthermore, high TINAGL1 expression was significantly correlated with poor prognosis in several public databases and our patient cohort diagnosed with DGC. Conclusions: These results indicate that TINAGL1 secreted by CAFs induces phosphorylation of FAK in DGC cells and promotes tumor progression. Thus, targeting TINAGL1 in CAFs can be a novel therapeutic strategy for DGC. [ABSTRACT FROM AUTHOR]

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin β1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/β1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor β1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin β1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/β1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor β1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications., Competing Interests: Declaration of competing interest None, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)