Characterization of Distinct Populations of Carcinoma-Associated Fibroblasts from Non-Small Cell Lung Carcinoma Reveals a Role for ST8SIA2 in Cancer Cell Invasion.

Carcinoma-associated fibroblasts (CAFs) are abundant stromal cells in tumor microenvironment that are critically involved in cancer progression. Contrasting reports have shown that CAFs can have either pro- or antitumorigenic roles, indicating that CAFs are functionally heterogeneous. Therefore, to precisely target the cancer-promoting CAF subsets, it is necessary to identify specific markers to define these subpopulations and understand their functions. We characterized two CAFs subsets from 28 non-small cell lung cancer (NSCLC) patient tumors that were scored and classified based on desmoplasia [mainly characterized by proliferating CAFs; high desmoplastic CAFs (HD-CAF; n = 15) and low desmoplastic CAFs (LD-CAF; n = 13)], which is an independent prognostic factor. Here, for the first time, we demonstrate that HD-CAFs and LD-CAFs show different tumor-promoting abilities. HD-CAFs showed higher rate of collagen matrix remodeling, invasion, and tumor growth compared to LD-CAFs. Transcriptomic analysis identified 13 genes that were differentially significant (fold ≥1.5; adjusted P value < .1) between HD-CAFs and LD-CAFs. The top upregulated differentially expressed gene, ST8SIA2 (11.3 fold; adjusted P value = .02), enhanced NSCLC tumor cell invasion in 3D culture compared to control when it was overexpressed in CAFs, suggesting an important role of ST8SIA2 in cancer cell invasion. We confirmed the protumorigenic role of ST8SIA2, showing that ST8SIA2 was significantly associated with the risk of relapse in three independent NSCLC clinical datasets. In summary, our studies show that functional heterogeneity in CAF plays key role in promoting cancer cell invasion in NSCLC

Keratan sulphate in the tumour environment

Keratan sulphate (KS) is a bioactive glycosaminoglycan (GAG) of some complexity composed of the repeat disaccharide D-galactose β1→4 glycosidically linked to N-acetyl glucosamine. During the biosynthesis of KS, a family of glycosyltransferase and sulphotransferase enzymes act sequentially and in a coordinated fashion to add D-galactose (D-Gal) then N-acetyl glucosamine (GlcNAc) to a GlcNAc acceptor residue at the reducing terminus of a nascent KS chain to effect chain elongation. D-Gal and GlcNAc can both undergo sulphation at C6 but this occurs more frequently on GlcNAc than D-Gal. Sulphation along the developing KS chain is not uniform and contains regions of variable length where no sulphation occurs, regions which are monosulphated mainly on GlcNAc and further regions of high sulphation where both of the repeat disaccharides are sulphated. Each of these respective regions in the KS chain can be of variable length leading to KS complexity in terms of chain length and charge localization along the KS chain. Like other GAGs, it is these variably sulphated regions in KS which define its interactive properties with ligands such as growth factors, morphogens and cytokines and which determine the functional properties of tissues containing KS. Further adding to KS complexity is the identification of three different linkage structures in KS to asparagine (N-linked) or to threonine or serine residues (O-linked) in proteoglycan core proteins which has allowed the categorization of KS into three types, namely KS-I (corneal KS, N-linked), KS-II (skeletal KS, O-linked) or KS-III (brain KS, O-linked). KS-I to -III are also subject to variable addition of L-fucose and sialic acid groups. Furthermore, the GlcNAc residues of some members of the mucin-like glycoprotein family can also act as acceptor molecules for the addition of D-Gal and GlcNAc residues which can also be sulphated leading to small low sulphation glycoforms of KS. These differ from the more heavily sulphated KS chains found on proteoglycans. Like other GAGs, KS has evolved molecular recognition and information transfer properties over hundreds of millions of years of vertebrate and invertebrate evolution which equips them with cell mediatory properties in normal cellular processes and in aberrant pathological situations such as in tumourogenesis. Two KS-proteoglycans in particular, podocalyxin and lumican, are cell membrane, intracellular or stromal tissue–associated components with roles in the promotion or regulation of tumour development, mucin-like KS glycoproteins may also contribute to tumourogenesis. A greater understanding of the biology of KS may allow better methodology to be developed to more effectively combat tumourogenic processes

Glycated collagen induces a11 integrin expression through TGF-ß2 and Smad3

The adhesion of cardiac fibroblasts to the glycated collagen interstitium in diabetics is associated with de novo expression of the a11 integrin, myofibroblast formation and cardiac fibrosis. We examined how methylglyoxal-glycated collagen regulates a11 integrin expression. In cardiac fibroblasts plated on glycated collagen but not glycated fibronectin, there was markedly increased a11 integrin and a-smooth muscle actin expression. Compared with native collagen, binding of purified a11b1 integrin to glycated collagen was reduced by fourfold, which was consistent with reduced fibroblast attachment to glycated collagen. Glycated collagen strongly enhanced the expression of TGF-b2 but not TGF-b1 or TGF-b3. The increased expression of TGF-b2 was inhibited by triple helical collagen peptides that mimic the a11b1 integrin binding site on type I collagen. In cardiac fibroblasts transfected with a11 integrin luciferase promoter constructs, glycated collagen activated the a11 integrin promoter. Analysis of a11 integrin promoter truncation mutants showed a novel Smad2/3 binding site located between 809 and 1300 nt that was required for promoter activation. We conclude that glycated collagen in the cardiac interstitium triggers an autocrine TGF-b2 signaling pathway that stimulates a11 integrin expression through Smad2/3 binding elements in the a11 integrin promoter, which is important for myofibroblast formation and fibrosis.This work was sponsored by the Canadian Institutes of Health Research

Integrin α11β1 in tumor fibrosis:more than just another cancer‑associated fibroblast biomarker?

Abstract There is currently an increased interest in understanding the role of the tumor microenvironment (TME) in tumor growth and progression. In this context the role of integrins in cancer-associated fibroblasts (CAFs) will need to be carefully re-evaluated. Fibroblast-derived cells are not only in the focus in tumors, but also in tissue fibrosis as well as in inflammatory conditions. The recent transcriptional profiling of what has been called “the pan-fibroblast cell lineage” in mouse and human tissues has identified novel transcriptional biomarker mRNAs encoding the secreted ECM proteins dermatopontin and collagen XV as well as the phosphatidylinositol-anchored membrane protein Pi16. Some of the genes identified in these fibroblasts scRNA-seq datasets will be useful for rigorous comparative characterizations of fibroblast-derived cell subpopulations. At the same time, it will be a challenge in the coming years to validate these transcriptional mRNA datasets at the protein-(expression) and at tissue-(distribution) levels and to find useful protein biomarker reagents that will facilitate fibroblast profiling at the cell level. In the current review we will focus on the role of the collagen-binding integrin α11β1 in CAFs, summarizing our own work as well as published datasets with information on α11 mRNA expression in selected tumors. Our experimental data suggest that α11β1 is more than just another biomarker and that it as a functional collagen receptor in the TME is playing a central role in regulating collagen assembly and matrix remodeling, which in turn impact tumor growth and metastasis

Overexpression of integrin α11 induces cardiac fibrosis in mice

AIM: To understand the role of the collagen-binding integrin α11 in vivo, we have used a classical approach of creating a mouse strain overexpressing integrin α11. A transgenic mouse strain overexpressing α11 in muscle tissues was analysed in the current study with special reference to the heart tissue. METHODS: We generated and phenotyped integrin α11 transgenic (TG) mice by echocardiography, magnetic resonance imaging and histology. Wild-type (WT) mice were subjected to aortic banding (AB) and the expression of integrin α11 was measured in flow cytometry-sorted cardiomyocytes and non-myocytes. RESULTS: TG mice developed left ventricular concentric hypertrophy by 6 months, with increased collagen deposition and reactivation of mRNA encoding foetal genes associated with cardiovascular pathological remodelling compared to WT mice. Masson's trichrome staining revealed interstitial fibrosis, confirmed additionally by magnetic resonance imaging and was found to be most prominent in the cardiac septum of TG but not WT mice. TG hearts expressed increased levels of transforming growth factor-β2 and transforming growth factor-β3 and upregulated smooth muscle actin. Macrophage infiltration coincided with increased NF-κB signalling in TG but not WT hearts. Integrin α11 expression was increased in both cardiomyocytes and non-myocyte cells from WT AB hearts compared to sham-operated animals. CONCLUSION: We report for the first time that overexpression of integrin α11 induces cardiac fibrosis and left ventricular hypertrophy. This is a result of changes in intracellular hypertrophic signalling and secretion of soluble factors that increase collagen production in the heart

Differential proteomic analysis of a human breast tumor and its matched bone metastasis identifies cell membrane and extracellular proteins associated with bone metastasis

The classical fate of metastasizing breast cancer cells is to seed and form secondary colonies in bones. The molecules closely associated with these processes are predominantly present at the cell surface and in the extracellular space, establishing the first contacts with the target tissue. In this study, we had the rare opportunity to analyze a bone metastatic lesion and its corresponding breast primary tumor obtained simultaneously from the same patient. Using mass spectrometry, we undertook a proteomic study on cell surface and extracellular protein-enriched material. We provide a repertoire of significantly modulated proteins, some with yet unknown roles in the bone metastatic process as well as proteins notably involved in cancer cell invasiveness and in bone metabolism. The comparison of these clinical data with those previously obtained using a human osteotropic breast cancer cell line highlighted an overlapping group of proteins. Certain differentially expressed proteins are validated in the present study using immunohistochemistry on a retrospective collection of breast tumors and matched bone metastases. Our exclusive set of selected proteins supports the set-up of further investigations on both clinical samples and experimental bone metastasis models that will help to reveal the finely coordinated expression of proteins that favor the development of metastases in the bone microenvironment