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Integrin αvβ8 binds with exquisite specificity to latenttransforming growth factor-β (L-TGF-β). This binding is essential for activating L-TGF-β presented by a variety of cell types. Inhibiting αvβ8-mediated TGF-β activation blocks immunosuppressive regulatory Tcell differentiation, which is a potential therapeuticstrategy in cancer. Using cryo-electron microscopy, structure-guided mutagenesis, and cell-based assays, we reveal the binding interactions between the entire αvβ8 ectodomain and its intact natural ligand, L-TGF-β, as well as two different inhibitory antibody fragments to understand the structural underpinnings of αvβ8 binding specificity and TGF-β activation. Our studies reveal a mechanism of TGF-β activation where mature TGF-β signals within the confines of L-TGF-β and the release and diffusion of TGF-β are not required. The structural details of this mechanism provide a rational basis for therapeutic strategies to inhibit αvβ8-mediated L-TGF-β activation.

The ability to disseminate, invade and successfully colonise other tissues is a critical hallmark of cancer that involves remodelling of the extracellular matrix (ECM) laid down by fibroblasts 1. Moreover, Cancer-Associated-Fibroblasts (CAFs) produce key growth factors and cytokines as components of the ECM that fuel tumour growth, metastasis and chemoresistance, and immune response 2-4. ECM changes also predict prognosis in pancreatic 5 and colorectal cancers 6,7. Here, we examine the landscape of ECM-gene dysregulation pan-cancer and find that a subset of ECM genes is (i) dysregulated specifically in cancer, (ii) adversely prognostic, (iii) linked to TGF-beta signalling and transcription in Cancer-Associated-Fibroblasts, (iv) enriched in immunologically active cancers, and (v) predicts responses to Immune checkpoint blockade better than mutation burden, cytolytic activity, or an interferon signature, thus identifying a novel mechanism of immune evasion for patient stratification in precision immunotherapy and pharmacological modulation.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with high mortality, uncertain cause, and few treatment options. Studies have identified a significant genetic risk associated with the development of IPF; however, mechanisms by which genetic risk factors promote IPF remain unclear. We aimed to identify genetic variants associated with IPF susceptibility and provide mechanistic insight using gene and protein expression analyses.Methods: We used a two-stage approach: a genome-wide association study in patients with IPF of European ancestry recruited from nine different centres in the UK and controls selected from UK Biobank (stage 1) matched for age, sex, and smoking status; and a follow-up of associated genetic variants in independent datasets of patients with IPF and controls from two independent US samples from the Chicago consortium and the Colorado consortium (stage 2). We investigated the effect of novel signals on gene expression in large transcriptomic and genomic data resources, and examined expression using lung tissue samples from patients with IPF and controls.Findings: 602 patients with IPF and 3366 controls were selected for stage 1. For stage 2, 2158 patients with IPF and 5195 controls were selected. We identified a novel genome-wide significant signal of association with IPF susceptibility near A-kinase anchoring protein 13 (AKAP13; rs62025270, odds ratio [OR] 1·27 [95% CI 1·18–1·37], p=1·32 × 10−9) and confirmed previously reported signals, including in mucin 5B (MUC5B; rs35705950, OR 2·89 [2·56–3·26], p=1·12 × 10−66) and desmoplakin (DSP; rs2076295, OR 1·44 [1·35–1·54], p=7·81 × 10−28). For rs62025270, the allele A associated with increased susceptibility to IPF was also associated with increased expression of AKAP13 mRNA in lung tissue from patients who had lung resection procedures (n=1111). We showed that AKAP13 is expressed in the alveolar epithelium and lymphoid follicles from patients with IPF, and AKAP13 mRNA expression was 1·42-times higher in lung tissue from patients with IPF (n=46) than that in lung tissue from controls (n=51).Interpretation: AKAP13 is a Rho guanine nucleotide exchange factor regulating activation of RhoA, which is known to be involved in profibrotic signalling pathways. The identification of AKAP13 as a susceptibility gene for IPF increases the prospect of successfully targeting RhoA pathway inhibitors in patients with IPF.Funding: UK Medical Research Council, National Heart, Lung, and Blood Institute of the US National Institutes of Health, Agencia Canaria de Investigación, Innovación y Sociedad de la Información, Spain, UK National Institute for Health Research, and the British Lung Foundation.

lntegrins are a group of transmembrane heterodimeric proteins that mediate cell-cell and cell-extracellular matrix (ECM) interactions. lntegrins have been under intense investigation for their role in inflammation in asthma. Clinical trials investigating integrin antagonists, however, have shown that these compounds are relatively ineffective. Airway remodelling is another pathological feature of asthma that is thought to make an important contribution to airway hyperresponsiveness (AHR) and lung function decline. Recent studies have identified integrins as important players in this process, with a particular role for beta(1) and alpha(v) integrins. Here we review the role of these integrins in airway remodelling and hyperresponsiveness in obstructive airway disease and their potential as pharmacological targets for future treatment.

Airway diseases such as chronic rhinosinusitis, asthma, and chronic obstructive pulmonary disorder are characterized by inflammation and remodeling. Among inflammatory and extracellular matrix regulatory cytokines, transforming growth factor-beta (TGF-β) stands central, as it possesses both important immunomodulatory and fibrogenic activities, and should be considered a key for understanding inflammation and remodeling processes. This review will briefly summarize the recent findings on the role of TGF-β1, from the view points of inflammation and remodeling, and discuss the role of TGF-β in the upper and lower airway diseases. This may reveal new perspectives in the understanding of airway inflammation and remodeling processes and may open innovative treatment strategies for the regulation of TGF-β1.

Integrins are heterodimeric cell-surface proteins that regulate cell growth, migration and survival. We have shown previously that the epithelial-restricted integrin αvβ6 has another critical function; that is, it binds and activates latent transforming growth factor-β (TGF-β)1,2. Through a global analysis of pulmonary gene expression in the lungs of mice lacking this integrin (Itgb6 null mice) we have identified a marked induction of macrophage metalloelastase (Mmp12)—a metalloproteinase that preferentially degrades elastin and has been implicated in the chronic lung disease emphysema3. Here we report that Itgb6-null mice develop age-related emphysema that is completely abrogated either by transgenic expression of versions of the β6 integrin subunit that support TGF-β activation, or by the loss of Mmp12. Furthermore, we show that the effects of Itgb6 deletion are overcome by simultaneous transgenic expression of active TGF-β1. We have uncovered a pathway in which the loss of integrin-mediated activation of latent TGF-β causes age-dependent pulmonary emphysema through alterations of macrophage Mmp12 expression. Furthermore, we show that a functional alteration in the TGF-β activation pathway affects susceptibility to this disease.

Transforming growth factor‐p (TGF‐P)s are multifunctional small polype‐ptides synthesized in most cell types. TGF‐P exerts pivotal effects on both bone formation and resorption. In addition, increasing lines of evidence implicate TGF‐p as a potential coupling factor between these two processes during bone remodeling. In the present study, the expression form and the activation mechanism of latent‐TGF‐β were investigated using specific antibodies for each isoform. TGF‐βs were observed to be synthesized and accumulated in a large amount in cultured osteoblastic cells. The estimated molecular weights of intracellular TGF‐β2 and ‐β3 were 49 and 55 kDa, respectively. Based on proteolytic digestion study and immunofluorescence observation, these precursor forms seemed to be accumulated in distinct intracellular compartments. To examine whether the internal pool of TGF‐β was possiblely regulated by external signals, their biological activites were examined in a conditioned media of this cell. Although the intact...

Cancer remains one of the leading causes of death in the developed countries and cancer mortality is expected to rise globally. Despite encouraging developments regarding targeted drugs, the most prevalent cancer mortality remains metastatic disease. Therefore, drugs that target cancer progression, invasion and metastasis are clearly needed. One of the most interesting targets in this setting is transforming growth factor beta (TGF-beta). TGF-beta can promote tumor growth, invasion and metastasis. However, TGF-beta also has a physiological, opposing role: maintaining tissue homeostasis and suppression of tumor progression. The window of effective TGF-beta targeting is therefore evidently small, which poses a clear challenge in selecting patients at the right time. Despite this complexity, several TGF-beta inhibitors are currently in clinical development, modulating TGF-beta production, activation or signaling. Still, specificity and long term toxicity remain unclear, emphasizing the importance of careful monitoring of clinical trials. Development and application of these drugs in the clinic require adequate insight and evaluation methods for the role of TGF-beta during tumor invasion and metastasis. In this review, presently available methods for clinical evaluation will be discussed, such as an ex vivo stimulation assay, TGF-beta response signature and molecular imaging techniques. Future clinical trials incorporating the validation of these evaluation methods will show which method will be most predictive and suitable for clinical application. (C) 2012 Elsevier Inc. All rights reserved.

Transforming growth factor-beta (TGF beta) is a multifunctional growth factor in bone that is secreted as a latent complex and must be activated in order to influence cellular activity. We have investigated the influence of dexamethasone (Dex; a potent glucocorticoid) on the secretion and activation of latent TGF beta by normal human osteoblast-like cells (hOBs). Dex had no significant effect on TGF beta mRNA or total protein production, but treatment with Dex resulted in a steroid dose-dependent activation of up to 90% of the TGF beta produced by the hOBs. Dex-treated hOBs activated multiple latent forms of TGF beta. Conditioned medium from Dex-treated hOBs retained the ability to activate latent TGF beta when incubated at 37 C in the absence of cells. Unlike other cell systems, Dex-induced hOB-mediated TGF beta activation did not involve binding to the mannose-6-phosphate receptor. However, the activation was prevented by treatment of hOB cells with microtubual disrupting agents, by the addition of protease inhibitors, or by weak base treatment of the medium. Dex treatment of hOBs was shown to induce a dose-dependent increase in the mRNA levels of cathepsin-B and -D and in the levels of cathepsin-B protein secreted by the cells. Taken together, these data suggest that Dex treatment of hOBs induces the production and secretion of lysosomal proteases that, when secreted, activate latent TGF beta which is secreted by the hOB cells. There is evidence for an involvement of more than one type of protease in this activation process. This activation of TGF beta may, therefore, play a role in glucocorticoid regulation of bone cell functions. Furthermore, TGF beta is most likely involved in autocrine and paracrine effects on bone cells.

Background/Aims: Despite advances in stent design, in-stent restenosis (ISR) remains a significant clinical problem. All implant metals exhibit corrosion, which results in release of metal ions. Stainless steel (SS), a metal alloy widely used in stents, releases ions to the vessel wall and induces reactive oxygen species, inflammation and fibroproliferative responses. The molecular mechanisms are unknown. TGF-β is known to be involved in the fibroproliferative responses of vascular smooth muscle cells (VSMCs) in restenosis, and TGF-β antagonists attenuate ISR. We hypothesized that SS ions induce the latent TGF-β activator, thrombospondin-1 (TSP1), through altered oxidative signaling to stimulate increased TGF-β activation and VSMC phenotype change. Methods: VSMCs were treated with SS metal ion cocktails, and morphology, TSP1, extracellular matrix production, desmin and TGF-β activity were assessed by immunoblotting. Results: SS ions stimulate the synthetic phenotype, increased TGF-β activity, TSP1, increased extracellular matrix and downregulation of desmin in VSMCs. Furthermore, SS ions increase hydrogen peroxide and decrease cGMP-dependent protein kinase (PKG) signaling, a known repressor of TSP1 transcription. Catalase blocks SS ion attenuation of PKG signaling and increased TSP1 expression. Conclusions: These data suggest that ions from stent alloy corrosion contribute to ISR through stimulation of TSP1-dependent TGF-β activation.

The ability of laser light to modulate specific biological processes has been well documented but the precise mechanism mediating these photobiological interactions remains an area of intense investigation. We recently published the results of our clinical trial with 30 patients in an oral tooth-extraction wound healing model using a 904nm GaAs laser (Oralaser 1010, Oralia, Konstnaz, Germany), assessing healing parameters using routine histopathology and immunostaining (Arany et al Wound Rep Regen 2007, 15, 866). We observed a better organized healing response in laser irradiated oral tissues that correlated with an increased expression of TGF-beta1 immediately post laser irradiation. Our data suggested the source of latent TGF-beta1 might be from the degranulating platelets in the serum, an abundant source of in vivo latent TGF-beta, in the freshly wounded tissues. Further, we also demonstrated the ability of the low power near-infrared laser irradiation to activate the latent TGF-beta complexes in vitro at varying fluences from 10sec (0.1 J/cm 2 ) to 600secs (6 J/cm 2 ). Using serum we observed two isoforms, namely TGF-beta1 and TGF-beta3, were capable of being activated by laser irradiation using an isoform-specific ELISA and a reporter based (p3TP) assay system. We are presently pursuing the precise photomolecular mechanisms focusing on potential chromophores, wavelength and fluence parameters affecting the Latent TGF-beta activation process in serum. As ROS mediated TGF-beta activation has been previously demonstrated and we are also exploring the role of Laser generated-ROS in this activation process. In summary, we present evidence of a potential molecular mechanism for laser photobiomodulation in its ability to activate latent TGF-beta complexes.

Secreted transforming growth factor-βs (TGF-βs) are rendered biologically inactive by binding proteins that also target and concentrate them to the extracellular matrix. Specific, but still poorly understood, activation is required for disassembly of the extracellular matrix–bound protein complex to liberate the mature growth factor and to obtain a correct biological effect. A new study shows that fibrillin-1, the protein defective in Marfan syndrome, has a biologically important role in controlling TGF-β activation in the lung.

eIF4E is frequently overexpressed in tumors of the breast, colon, prostate, and lung, and expression correlates with disease progression, increased tissue invasion and metastasis. It has been suggested that overexpression of eIF4E contributes to malignancy by selectively increasing translation of mRNAs containing significant secondary structure in the their 5' noncoding regions. A limited group of mRNAs encode key proteins involved in cellular growth, survival, angiogenesis and malignancy and contain long GC-rich highly structured 5' noncoding regions. The effect of increased levels of eIF4E in tumor cells, however, is still not well understood, nor is the role of its mTOR regulated repressor, 4E-BP1. Here we demonstrate the surprising finding that overexpression of eIF4E acts primarily to increase breast cancer cell invasion by significantly increasing translation of a single mRNA: β1 integrin. eIF4E overexpression at levels found in breast cancers (3-4 fold) results in a 10-fold increased level of tumor cell invasion. Analysis of polysomal mRNA found increased translation of β1 integrin mRNA with physiological levels of eIF4E overexpression observed in breast cancers. We found that increased β1 integrin expression was due to increased read-through of the complex GC-rich 5' noncoding region of β1 integrin mRNA following eIF4E overexpression, the first time this has been described physiologically, and that this effect is blocked by co-overexpression of 4E-BP1. The modest increase in β1 integrin protein levels is amplified by a disproportionately large increase in the formation of the cell surface heterodimer α3β1, an integrin frequently implicated in breast tumor invasion. These and other results can account for the large increase in invasive ability in eIF4E overexpressing breast cancer cells, suggesting that β1 integrin levels may be limiting in the cell. In addition, we found that increased expression of eIF4E results in increased transcription and consequent protein levels of many canonical TGFβ-responsive genes, including β1 integrin. TGFβ is partially responsible for the increased invasion seen with eIF4E overexpression, although cells are still 4.5 fold more invasive even in the absence of the cytokine, consistent with the TGFβ-independent increase in β1 integrin levels observed with eIF4E overexpresison. The TGFβ transcriptional effect is likely due to an increased rate of activation of latent TGFβ (typically present in serum or the stroma), mediated by the eIF4E-driven increased levels of β1 integrin and its subsequent heterodimerization with α integrins capable of activating TGFβ, including αvβ1. TGFβ transcriptional effects are due to TGFβ ligation with its receptor, and not eIF4E-mediated increased translation of a downstream TGFβ effector, as they are blocked with the use of a TGFβ receptor I kinase inhibitor. Additionally, eIF4E overexpression does not facilitate the translation of TGFβ mRNA, as protein levels of TGFβ are not increased with elevated levels of eIF4E. Co-overexpression of 4E-BP1 blocks all TGFβ effects. Our results suggest a new but prevalent model whereby eIF4E level is a master regulator that determines the direction of TGFβ activity. Increased expression of eIF4E at typical human breast cancer levels mediates increased tumor cell invasion through the selective increased translation of β1 integrin mRNA. Higher levels of β1 integrin promotes increased heterodimerization with α-integrins which activate latent TGFβ present in the breast stroma, thereby directing TGFβ to pro-invasive function. Citation Format: Celine Mestel, Mary-Helen Barcelos-Hoff, Jiri Zavadil, Silvia C. Formenti, Robert J. Schneider. eIF4E in breast cancer is a master regulator of tumor cell invasion through increased translation of beta-1 integrin mRNA and TGF-beta activation [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY35-03

Transforming growth factor-beta (TGF beta) is produced by most cultured cells in an inactive form. Potential activation mechanisms of latent TGF beta were studied using fibroblastic (NRK-49F and AKR-MCA) cell-conditioned medium as a model. Active TGF beta was monitored by radioreceptor and soft agar assays as well as by antibody inhibition and immunoprecipitation. Little or no TGF beta was detected in untreated conditioned medium. Treatment of the medium with extremes of pH (1.5 or 12) resulted in significant activation of TGF beta as shown by radioreceptor assays, while mild acid treatment (pH 4.5) yielded only 20-30% of the competition achieved by pH 1.5. In an effort to define more physiological means of TGF beta activation, the effects of some proteases were tested. Plasmin and cathepsin D were found to generate 25-kD bands corresponding to the active form of TGF beta as shown by immunoprecipitation analysis of radiolabeled cell-conditioned medium. Plasmin treatment of the medium resulted in activity that was quantitatively similar to that of mild acid treatment as measured by radioreceptor and soft agar assays. In addition, the plasmin-generated activity was inhibited by anti-TGF beta antibodies. Sequential treatments of AKR-MCA cell-conditioned medium with mild acid followed by plasmin or plasmin followed by mild acid gave activation comparable to either treatment alone. The data suggest that conditioned medium may contain at least two different pools of latent TGF beta. One pool is resistant to mild acid and/or plasmin and requires strong acid or alkali treatment for activation. A second pool is activated by mild pH change and/or plasmin. Activation of this form of latent TGF beta may take place by dissociation or proteolytic digestion from a precursor molecule or hypothetical TGF beta-binding protein complex.