Expression of Collagen XIII in Tissues of the Thyroid and Orbit With Relevance to Thyroid-Associated Ophthalmopathy.

Purpose: Antibodies against collagen XIII have previously been identified in patients with active thyroid-associated ophthalmopathy (TAO). Although collagen XIII expression has been described in extraocular muscles and orbital fat, its detailed localization in extraocular and thyroid tissues and the connection to autoimmunity for collagen XIII remain unclear. Our objective was to map the potential targets for these antibodies in the tissues of the orbit and thyroid. Methods: We evaluated the expression of collagen XIII in human patient and mouse orbital and thyroid tissues with immunostainings and RT-qPCR using Col13a1-/- mice as negative controls. COL13A1 expression in Graves' disease and goiter thyroid samples was compared with TGF-ß1 and TNF, and these were also studied in human thyroid epithelial cells and fibroblasts. Results: Collagen XIII expression was found in the neuromuscular and myotendinous junctions of extraocular muscles, blood vessels of orbital connective tissue and fat and the thyroid, and in the thyroid epithelium. Thyroid expression was also seen in germinal centers in Graves' disease and in neoplastic epithelium. The expression of COL13A1 in goiter samples correlated with levels of TGF-B1. Upregulation of COL13A1 was reproduced in thyroid epithelial cells treated with TGF-ß1. Conclusions: We mapped the expression of collagen XIII to various locations in the orbit, demonstrated its expression in the pathologies of the Graves' disease thyroid and confirmed the relationship between collagen XIII and TGF-ß1. Altogether, these data add to our understanding of the targets of anti-collagen XIII autoantibodies in TAO.

Targeting collagen XVIII improves the efficiency of ErbB inhibitors in breast cancer models.

The tumor extracellular matrix (ECM) critically regulates cancer progression and treatment response. Expression of the basement membrane component collagen XVIII (ColXVIII) is induced in solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII was markedly upregulated in human breast cancer (BC) and was closely associated with a poor prognosis in high-grade BCs. We discovered a role for ColXVIII as a modulator of epidermal growth factor receptor tyrosine kinase (ErbB) signaling and show that it forms a complex with ErbB1 and -2 (also known as EGFR and human epidermal growth factor receptor 2 [HER2]) and α6-integrin to promote cancer cell proliferation in a pathway involving its N-terminal portion and the MAPK/ERK1/2 and PI3K/AKT cascades. Studies using Col18a1 mouse models crossed with the mouse mammary tumor virus-polyoma virus middle T antigen (MMTV-PyMT) mammary carcinogenesis model showed that ColXVIII promoted BC growth and metastasis in a tumor cell-autonomous manner. Moreover, the number of mammary cancer stem cells was significantly reduced in the MMTV-PyMT and human cell models upon ColXVIII inhibition. Finally, ablation of ColXVIII substantially improved the efficacy of ErbB-targeting therapies in both preclinical models. In summary, ColXVIII was found to sustain the stemness properties of BC cells and tumor progression and metastasis through ErbB signaling, suggesting that targeting ColXVIII in the tumor milieu may have important therapeutic potential.

Contribution of collagen XIII to lung function and development of pulmonary fibrosis.

BACKGROUND: Collagen XIII is a transmembrane collagen associated with neuromuscular junction development, and in humans its deficiency results in congenital myasthenic syndrome type 19 (CMS19), which leads to breathing difficulties. CMS19 patients usually have restricted lung capacity and one patient developed chronic lung disease. In single-cell RNA sequencing studies, collagen XIII has been identified as a marker for pulmonary lipofibroblasts, which have been implicated in the resolution of pulmonary fibrosis. METHODS: We investigated the location and function of collagen XIII in the lung to understand the origin of pulmonary symptoms in human CMS19 patients. Additionally, we performed immunostainings on idiopathic pulmonary fibrosis (IPF) samples (N=5) and both normal and fibrotic mouse lung. To study whether the lack of collagen XIII predisposes to restrictive lung disease, we exposed Col13a1-modified mice to bleomycin-induced pulmonary fibrosis. RESULTS: Apparently normal alveolar septum sections of IPF patients' lungs stained faintly for collagen XIII, and its expression was pinpointed to the septal fibroblasts in the mouse lung. Lung capacity was increased in mice lacking collagen XIII by over 10%. In IPF samples, collagen XIII was expressed by basal epithelial cells, hyperplastic alveolar epithelial cells and stromal cells in fibrotic areas, but the development of pulmonary fibrosis was unaffected in collagen XIII-deficient mice. CONCLUSIONS: Changes in mouse lung function appear to represent a myasthenic manifestation of collagen XIII deficiency. We suggest that respiratory muscle myasthenia is the primary cause of the breathing problems suffered by CMS19 patients in addition to skeletal deformities. Induction of collagen XIII expression in the IPF patients' lungs warrants further studies to reveal collagen XIII-dependent disease mechanisms.

Upregulated integrin α11 in the stroma of cutaneous squamous cell carcinoma promotes skin carcinogenesis.

Integrin α11ß1 is a collagen-binding integrin that is needed to induce and maintain the myofibroblast phenotype in fibrotic tissues and during wound healing. The expression of the α11 is upregulated in cancer-associated fibroblasts (CAFs) in various human neoplasms. We investigated α11 expression in human cutaneous squamous cell carcinoma (cSCC) and in benign and premalignant human skin lesions and monitored its effects on cSCC development by subjecting α11-knockout (Itga11-/- ) mice to the DMBA/TPA skin carcinogenesis protocol. α11-deficient mice showed significantly decreased tumor cell proliferation, leading to delayed tumor development and reduced tumor burden. Integrin α11 expression was significantly upregulated in the desmoplastic tumor stroma of human and mouse cSCCs, and the highest α11 expression was detected in high-grade tumors. Our results point to a reduced ability of α11-deficient stromal cells to differentiate into matrix-producing and tumor-promoting CAFs and suggest that this is one causative mechanism underlying the observed decreased tumor growth. An unexpected finding in our study was that, despite reduced CAF activation, the α11-deficient skin tumors were characterized by the presence of thick and regularly aligned collagen bundles. This finding was attributed to a higher expression of TGFß1 and collagen crosslinking lysyl oxidases in the Itga11-/- tumor stroma. In summary, our data suggest that α11ß1 operates in a complex interactive tumor environment to regulate ECM synthesis and collagen organization and thus foster cSCC growth. Further studies with advanced experimental models are still needed to define the exact roles and molecular mechanisms of stromal α11ß1 in skin tumorigenesis.

Analysis of extracellular matrix network dynamics in cancer using the MatriNet database.

The extracellular matrix (ECM) is a three-dimensional network of proteins of diverse nature, whose interactions are essential to provide tissues with the correct mechanical and biochemical cues they need for proper development and homeostasis. Changes in the quantity of extracellular matrix (ECM) components and their balance within the tumor microenvironment (TME) accompany and fuel all steps of tumor development, growth and metastasis, and a deeper and more systematic understanding of these processes is fundamental for the development of future therapeutic approaches. The wealth of "big data" from numerous sources has enabled gigantic steps forward in the comprehension of the oncogenic process, also impacting on our understanding of ECM changes in the TME. Most of the available studies, however, have not considered the network nature of ECM and the possibility that changes in the quantity of components might be regulated (co-occur) in cancer and significantly "rebound" on the whole network through its connections, fundamentally altering the matrix interactome. To facilitate the exploration of these network-scale effects we have implemented MatriNet (www.matrinet.org), a database enabling the study of structural changes in ECM network architectures as a function of their protein-protein interaction strengths across 20 different tumor types. The use of MatriNet is intuitive and offers new insights into tumor-specific as well as pan-cancer features of ECM networks, facilitating the identification of similarities and differences between cancers as well as the visualization of single-tumor events and the prioritization of ECM targets for further experimental investigations.

Deletion of Col15a1 Modulates the Tumour Extracellular Matrix and Leads to Increased Tumour Growth in the MMTV-PyMT Mouse Mammary Carcinoma Model.

Basement membrane (BM) zone-associated collagen XV (ColXV) has been shown to suppress the malignancy of tumour cells, and its restin domain can inhibit angiogenesis. In human breast cancer, as well as in many other human carcinomas, ColXV is lost from the epithelial BM zone prior to tumour invasion. Here, we addressed the roles of ColXV in breast carcinogenesis using the transgenic MMTV-PyMT mouse mammary carcinoma model. We show here for the first time that the inactivation of Col15a1 in mice leads to changes in the fibrillar tumour matrix and to increased mammary tumour growth. ColXV is expressed by myoepithelial and endothelial cells in mammary tumours and is lost from the ductal BM along with the loss of the myoepithelial layer during cancer progression while persisting in blood vessels and capillaries, even in invasive tumours. However, despite the absence of anti-angiogenic restin domain, neovascularisation was reduced rather than increased in the ColXV-deficient mammary tumours compared to controls. We also show that, in robust tumour cell transplantation models or in a chemical-induced fibrosarcoma model, the inactivation of Col15a1 does not affect tumour growth or angiogenesis. In conclusion, our results support the proposed tumour suppressor function of ColXV in mammary carcinogenesis and reveal diverse roles of this collagen in different cancer types.

The Burden of Post-Translational Modification (PTM)-Disrupting Mutations in the Tumor Matrisome.

BACKGROUND: To evaluate the occurrence of mutations affecting post-translational modification (PTM) sites in matrisome genes across different tumor types, in light of their genomic and functional contexts and in comparison with the rest of the genome. METHODS: This study spans 9075 tumor samples and 32 tumor types from The Cancer Genome Atlas (TCGA) Pan-Cancer cohort and identifies 151,088 non-silent mutations in the coding regions of the matrisome, of which 1811 affecting known sites of hydroxylation, phosphorylation, N- and O-glycosylation, acetylation, ubiquitylation, sumoylation and methylation PTM. RESULTS: PTM-disruptive mutations (PTMmut) in the matrisome are less frequent than in the rest of the genome, seem independent of cell-of-origin patterns but show dependence on the nature of the matrisome protein affected and the background PTM types it generally harbors. Also, matrisome PTMmut are often found among structural and functional protein regions and in proteins involved in homo- and heterotypic interactions, suggesting potential disruption of matrisome functions. CONCLUSIONS: Though quantitatively minoritarian in the spectrum of matrisome mutations, PTMmut show distinctive features and damaging potential which might concur to deregulated structural, functional, and signaling networks in the tumor microenvironment.

The Amino-Terminal Oligomerization Domain of Angiopoietin-2 Affects Vascular Remodeling, Mammary Gland Tumor Growth, and Lung Metastasis in Mice.

Angiopoietin-2 (ANGPT2) is a context-dependent TIE2 agonistic or antagonistic ligand that induces diverse responses in cancer. Blocking ANGPT2 provides a promising strategy for inhibiting tumor growth and metastasis, yet variable effects of targeting ANGPT2 have complicated drug development. ANGPT2443 is a naturally occurring, lower oligomeric protein isoform whose expression is increased in cancer. Here, we use a knock-in mouse line (mice expressing Angpt2443), a genetic model for breast cancer and metastasis (MMTV-PyMT), a syngeneic melanoma lung colonization model (B16F10), and orthotopic injection of E0771 breast cancer cells to show that alternative forms increase the diversity of Angpt2 function. In a mouse retina model of angiogenesis, expression of Angpt2443 caused impaired venous development, suggesting enhanced function as a competitive antagonist for Tie2. In mammary gland tumor models, Angpt2443 differentially affected primary tumor growth and vascularization; these varying effects were associated with Angpt2 protein localization in the endothelium or in the stromal extracellular matrix as well as the frequency of Tie2-positive tumor blood vessels. In the presence of metastatic cells, Angpt2443 promoted destabilization of pulmonary vasculature and lung metastasis. In vitro, ANGPT2443 was susceptible to proteolytical cleavage, resulting in a monomeric ligand (ANGPT2DAP) that inhibited ANGPT1- or ANGPT4-induced TIE2 activation but did not bind to alternative ANGPT2 receptor α5ß1 integrin. Collectively, these data reveal novel roles for the ANGPT2 N-terminal domain in blood vessel remodeling, tumor growth, metastasis, integrin binding, and proteolytic regulation. SIGNIFICANCE: This study identifies the role of the N-terminal oligomerization domain of angiopoietin-2 in vascular remodeling and lung metastasis and provides new insights into mechanisms underlying the versatile functions of angiopoietin-2 in cancer.See related commentary by Kamiyama and Augustin, p. 35.

Machine Learning Identifies Robust Matrisome Markers and Regulatory Mechanisms in Cancer.

The expression and regulation of matrisome genes-the ensemble of extracellular matrix, ECM, ECM-associated proteins and regulators as well as cytokines, chemokines and growth factors-is of paramount importance for many biological processes and signals within the tumor microenvironment. The availability of large and diverse multi-omics data enables mapping and understanding of the regulatory circuitry governing the tumor matrisome to an unprecedented level, though such a volume of information requires robust approaches to data analysis and integration. In this study, we show that combining Pan-Cancer expression data from The Cancer Genome Atlas (TCGA) with genomics, epigenomics and microenvironmental features from TCGA and other sources enables the identification of "landmark" matrisome genes and machine learning-based reconstruction of their regulatory networks in 74 clinical and molecular subtypes of human cancers and approx. 6700 patients. These results, enriched for prognostic genes and cross-validated markers at the protein level, unravel the role of genetic and epigenetic programs in governing the tumor matrisome and allow the prioritization of tumor-specific matrisome genes (and their regulators) for the development of novel therapeutic approaches.

Collagen XV, a multifaceted multiplexin present across tissues and species.

Type XV collagen is a non-fibrillar collagen that is associated with basement membranes and belongs to the multiplexin subset of the collagen superfamily. Collagen XV was initially studied because of its sequence homology with collagen XVIII/endostatin whose anti-angiogenic and anti-tumorigenic properties were subjects of wide interest in the past years. But during the last fifteen years, collagen XV has gained growing attention with increasing number of studies that have attributed new functions to this widely distributed collagen/proteoglycan hybrid molecule. Despite the cumulative evidence of its functional pleiotropy and its evolutionary conserved function, no review compiling the current state of the art about collagen XV is currently available. Here, we thus provide the first comprehensive view of the knowledge gathered so far on the molecular structure, tissue distribution and functions of collagen XV in development, tissue homeostasis and disease with an evolutionary perspective. We hope that our review will open new roads for promising research on collagen XV in the coming years.

Exploring the roles of MACIT and multiplexin collagens in stem cells and cancer.

The extracellular matrix (ECM) is ubiquitously involved in neoplastic transformation, tumour growth and metastatic dissemination, and the interplay between tumour and stromal cells and the ECM is now considered crucial for the formation of a tumour-supporting microenvironment. The 28 different collagens (Col) form a major ECM protein family and display extraordinary functional diversity in tissue homeostasis as well as in pathological conditions, with functions ranging from structural support for tissues to regulatory binding activities and storage of biologically active cryptic domains releasable through ECM proteolysis. Two subfamilies of collagens, namely the plasma membrane-associated collagens with interrupted triple-helices (MACITs, including ColXIII, ColXXIII and ColXXV) and the basement membrane-associated collagens with multiple triple-helix domains with interruptions (multiplexins, including ColXV and ColXVIII), have highly interesting regulatory functions in tissue and organ development, as well as in various diseases, including cancer. An increasing, albeit yet sparse, data suggest that these collagens play crucial roles in conveying regulatory signals from the extracellular space to cells. We summarize here the current knowledge about MACITs and multiplexins as regulators of stemness and oncogenic processes, as well as their roles in influencing cell fate decisions in healthy and cancerous tissues. In addition, we present a bioinformatic analysis of the impacts of MACITs and multiplexins transcript levels on the prognosis of patients representing a wide array of malignant diseases, to aid future diagnostic and therapeutic efforts.

Stromal integrin α11 regulates PDGFR-ß signaling and promotes breast cancer progression.

Cancer-associated fibroblasts (CAFs) are key actors in modulating the progression of many solid tumors such as breast cancer (BC). Herein, we identify an integrin α11/PDGFRß+ CAF subset displaying tumor-promoting features in BC. In the preclinical MMTV-PyMT mouse model, integrin α11-deficiency led to a drastic reduction of tumor progression and metastasis. A clear association between integrin α11 and PDGFRß was found at both transcriptional and histological levels in BC specimens. High stromal integrin α11/PDGFRß expression was associated with high grades and poorer clinical outcome in human BC patients. Functional assays using five CAF subpopulations (one murine, four human) revealed that integrin α11 promotes CAF invasion and CAF-induced tumor cell invasion upon PDGF-BB stimulation. Mechanistically, integrin α11 pro-invasive activity relies on its ability to interact with PDGFRß in a ligand-dependent manner and to promote its downstream JNK activation, leading to the production of tenascin C, a pro-invasive matricellular protein. Pharmacological inhibition of PDGFRß and JNK impaired tumor cell invasion induced by integrin α11-positive CAFs. Collectively, our study uncovers an integrin α11-positive subset of pro-tumoral CAFs that exploits PDGFRß/JNK signalling axis to promote tumor invasiveness in BC.

Pan-Cancer analysis of the expression and regulation of matrisome genes across 32 tumor types.

The microenvironment plays a central role in cancer, and neoplastic cells actively shape it to their needs by complex arrays of extracellular matrix (ECM) proteins, enzymes, cytokines and growth factors collectively referred to as the matrisome. Studies on the cancer matrisome have been performed for single or few neoplasms, but a more systematic analysis is still missing. Here we present a Pan-Cancer study of matrisome gene expression in 10,487 patients across 32 tumor types, supplemented with transcription factors (TFs) and driver genes/pathways regulating each tumor's matrisome. We report on 919 TF-target pairs, either used specifically or shared across tumor types, and their prognostic significance, 40 master regulators, 31 overarching regulatory pathways and the potential for druggability with FDA-approved cancer drugs. These results provide a comprehensive transcriptional architecture of the cancer matrisome and suggest the need for development of specific matrisome-targeting approaches for future therapies.

Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance.

BACKGROUND: Increased collagen expression and deposition are associated with cancer progression and poor prognosis in breast cancer patients. However, function and regulation of membrane-associated collagen in breast cancer have not been determined. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse models show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. In the present study, we determined roles of collagen XIII in breast cancer progression and metastasis. METHODS: We analyzed the association of collagen XIII expression with breast cancer development and metastasis using published gene expression profiles generated from human breast cancer tissues. Utilizing gain- and loss- of function approaches and 3D culture assays, we investigated roles of collagen XIII in regulating invasive tumor growth. Using the tumorsphere/mammosphere formation assay and the detachment cell culture assay, we determined whether collagen XIII enhances cancer cell stemness and induces anoikis resistance. We also inhibited collagen XIII signaling with ß1 integrin function-blocking antibody. Finally, using the lung colonization assay and the orthotopic mammary tumor model, we investigated roles of collagen XIII in regulating breast cancer colonization and metastasis. Cox proportional hazard (log-rank) test, two-sided Student's t-test (two groups) and one-way ANOVA (three or more groups) analyses were used in this study. RESULTS: Collagen XIII expression is significantly higher in human breast cancer tissue compared with normal mammary gland. Increased collagen XIII mRNA levels in breast cancer tissue correlated with short distant recurrence free survival. We showed that collagen XIII expression promoted invasive tumor growth in 3D culture, enhanced cancer cell stemness, and induced anoikis resistance. Collagen XIII expression induced ß1 integrin activation. Blocking ß1 integrin activation significantly reduced collagen XIII-induced invasion and mammosphere formation. Importantly, silencing collagen XIII in MDA-MB-231 cells reduced lung colonization and metastasis. CONCLUSIONS: Our results demonstrate a novel function of collagen XIII in promoting cancer metastasis, cell invasion, and anoikis resistance.

2-Phenylquinazolinones as dual-activity tankyrase-kinase inhibitors.

Tankyrases (TNKSs) are enzymes specialized in catalyzing poly-ADP-ribosylation of target proteins. Several studies have validated TNKSs as anti-cancer drug targets due to their regulatory role in Wnt/ß-catenin pathway. Recently a lot of effort has been put into developing more potent and selective TNKS inhibitors and optimizing them towards anti-cancer agents. We noticed that some 2-phenylquinazolinones (2-PQs) reported as CDK9 inhibitors were similar to previously published TNKS inhibitors. In this study, we profiled this series of 2-PQs against TNKS and selected kinases that are involved in the Wnt/ß-catenin pathway. We found that they were much more potent TNKS inhibitors than they were CDK9/kinase inhibitors. We evaluated the compound selectivity to tankyrases over the ARTD enzyme family and solved co-crystal structures of the compounds with TNKS2. Comparative structure-based studies of the catalytic domain of TNKS2 with selected CDK9 inhibitors and docking studies of the inhibitors with two kinases (CDK9 and Akt) revealed important structural features, which could explain the selectivity of the compounds towards either tankyrases or kinases. We also discovered a compound, which was able to inhibit tankyrases, CDK9 and Akt kinases with equal µM potency.

Autoimmune antibodies to collagen XIII in myasthenia gravis patients.

INTRODUCTION: Evaluation of the nerve fascicular structure can be useful in diagnosing nerve damage, but it is a very challenging task with 3T MRI because of limited resolution. In this pilot study, we present the feasibility of high-resolution 7T MRI for examining the nerve fascicular structure. METHODS: A 3-dimensional (3D) gradient-spoiled sequence was used for imaging peripheral nerves in extremities. Images acquired with different in-plane resolutions (0.42 × 0.42 mm vs. 0.12 × 0.12 mm), and different main field strengths (7T vs. 3T) were compared. RESULTS: The individual nerve fascicles were identified at 0.12 × 0.12 mm resolution in both field strengths but not at 0.42 × 0.42 mm resolution. The fascicular structure was more sharply depicted in 7T images than in 3T images. DISCUSSION: High-resolution 3D imaging with 7T MRI demonstrated feasibility for imaging nerve fascicular structures. Muscle Nerve 57: 506-510, 2018.

Expression of a specific extracellular matrix signature is a favorable prognostic factor in acute myeloid leukemia.

Relapse of acute myeloid leukemia (AML) is still dramatically frequent, imposing the need for early markers to quantify such risk. Recent evidence point to a prominent role for extracellular matrix (ECM) in AML, but its prognostic value has not yet been investigated. Here we have investigated whether the expression of a 15-ECM gene signature could be applied to clinical AML research evaluating a retrospective cohort of 61 AML patients and 12 healthy donors. Results show that patients whose ECM signature expression is at least twice as that of healthy donors have considerably longer relapse-free survival, with further stage-specific therapy outcomes.

Hypoxia-inducible factor prolyl-4-hydroxylase-1 is a convergent point in the reciprocal negative regulation of NF-κB and p53 signaling pathways.

Hypoxia-inducible factor 1α (HIF1α) induces the expression of several hundred genes in hypoxia aiming at restoration of oxygen homeostasis. HIF prolyl-4-hydroxylases (HIF-P4Hs) regulate the stability of HIF1α in an oxygen-dependent manner. Hypoxia is a common feature in inflammation and cancer and the HIF pathway is closely linked with the inflammatory NF-κB and tumor suppressor p53 pathways. Here we show that genetic inactivation or chemical inhibition of HIF-P4H-1 leads to downregulation of proinflammatory genes, while proapoptotic genes are upregulated. HIF-P4H-1 inactivation reduces the inflammatory response under LPS stimulus in vitro and in an acute skin inflammation model in vivo. Furthermore, HIF-P4H-1 inactivation increases p53 activity and stability and hydroxylation of proline 142 in p53 has an important role in this regulation. Altogether, our data suggest that HIF-P4H-1 inhibition may be a promising therapeutic candidate for inflammatory diseases and cancer, enhancing the reciprocal negative regulation of the NF-κB and p53 pathways.