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Supplemental Figure S1. Quantification of RYK-V5 immunoblots.

Supplemental Methods, Figures and Legends

ERK1 and p38 are not activated by alphavbeta6 integrin upon ligand binding.

Full-size scans of PSA immunoblots shown in Figure 5.

Full-size scans of PSA and androgen receptor immunoblots shown in Figure 4.

Increased alphavbeta6 integrin and JNK expression and enhanced JNK nuclear localization in castrated mice.

alphavbeta6 integrin, but not alphavbeta3 integrin, promotes tumor growth.

Androgen receptor signaling fuels prostate cancer and is a major therapeutic target. However, mechanisms of resistance to therapeutic androgen ablation are not well understood. Here, using a prostate cancer mouse model, Ptenpc−/−, carrying a prostate epithelial-specific Pten deletion, we show that the αvβ6 integrin is required for tumor growth in vivo of castrated as well as of noncastrated mice. We describe a novel signaling pathway that couples the αvβ6 integrin cell surface receptor to androgen receptor via activation of JNK1 and causes increased nuclear localization and activity of androgen receptor. This downstream kinase activation by αvβ6 is specific for JNK1, with no involvement of p38 or ERK kinase. In addition, differential phosphorylation of Akt is not observed under these conditions, nor is cell morphology affected by αvβ6 expression. This pathway, which is specific for αvβ6, because it is not regulated by a different αv-containing integrin, αvβ3, promotes upregulation of survivin, which in turn supports anchorage-independent growth of αvβ6-expressing cells. Consistently, both αvβ6 and survivin are significantly increased in prostatic adenocarcinoma, but are not detected in normal prostatic epithelium. Neither XIAP nor Bcl-2 is affected by αvβ6 expression. In conclusion, we show that αvβ6 expression is required for prostate cancer progression, including castrate-resistant prostate cancer; mechanistically, by promoting activation of JNK1, the αvβ6 integrin causes androgen receptor–increased activity in the absence of androgen and consequent upregulation of survivin. These preclinical results pave the way for further clinical development of αvβ6 antagonists for prostate cancer therapy. Cancer Res; 76(17); 5163–74. ©2016 AACR.

alphavbeta6 integrin promotes prostate cancer growth in castrated athymic nu/nu mice.

alphavbeta6 enhances AR transcriptional activity in LNCaP cells.

Progranulin is a pleiotropic growth factor with important physiological roles in embryogenesis and maintenance of adult tissue homeostasis. While-progranulin deficiency is associated with a broad range of pathological conditions affecting the brain, such as frontotemporal dementia and neuronal ceroid lipofuscinosis, progranulin upregulation characterizes many tumors, including brain tumors, multiple myeloma, leiomyosarcoma, mesothelioma and epithelial cancers such as ovarian, liver, breast, bladder, adrenal, prostate and kidney carcinomas. The increase of progranulin levels in tumors might have diagnostic and prognostic significance. In cancer, progranulin has a pro-tumorigenic role by promoting cancer cell proliferation, migration, invasiveness, anchorage-independent growth and resistance to chemotherapy. In addition, progranulin regulates the tumor microenvironment, affects the function of cancer-associated fibroblasts, and modulates tumor immune surveillance. However, the molecular mechanisms of progranulin oncogenic function are not fully elucidated. In bladder cancer, progranulin action relies on the activation of its functional signaling receptor EphA2. Notably, more recent data suggest that progranulin can also modulate a functional crosstalk between multiple receptor-tyrosine kinases, demonstrating a more complex and context-dependent role of progranulin in cancer. Here, we will review what is currently known about the function of progranulin in tumors, with a focus on its molecular mechanisms of action and regulation.

Hyaluronan plays a key role in regulating inflammation and tumor angiogenesis. Of the three transmembrane hyaluronan synthases, HAS2 is the main pro-angiogenic enzyme responsible for excessive hyaluronan production. We discovered that HAS2 was degraded in vascular endothelial cells via autophagy evoked by nutrient deprivation, mTOR inhibition, or pro-autophagic proteoglycan fragments endorepellin and endostatin. Using live-cell and super-resolution confocal microscopy, we found that protracted autophagy evoked a dynamic interaction between HAS2 and ATG9A, a key transmembrane autophagic protein. This regulatory axis of HAS2 degradation occurred in various cell types and species and in vivo upon nutrient deprivation. Inhibiting in vivo autophagic flux via chloroquine showed increased levels of HAS2 in the heart and aorta. Functionally, autophagic induction via endorepellin or mTOR inhibition markedly suppressed extracellular hyaluronan production in vascular endothelial cells and inhibited ex vivo angiogenic sprouting. Thus, we propose autophagy as a novel catabolic mechanism regulating hyaluronan production in endothelial cells and demonstrate a new link between autophagy and angiogenesis that could lead to potential therapeutic modalities for angiogenesis.

OBJECTIVE To elucidate the role of decorin, a small leucine-rich proteoglycan, in the degradation of cartilage matrix during the progression of post-traumatic osteoarthritis (OA). METHODS Three-month-old decorin-null (Dcn-/- ) and inducible decorin-knockout (DcniKO ) mice were subjected to surgical destabilization of the medial meniscus (DMM) to induce post-traumatic OA. The OA phenotype that resulted was evaluated by assessing joint morphology and sulfated glycosaminoglycan (sGAG) staining via histological analysis (n = 6 mice per group), surface collagen fibril nanostructure via scanning electron microscopy (n = 4 mice per group), tissue modulus via atomic force microscopy-nanoindentation (n = 5 or more mice per group) and subchondral bone structure via micro-computed tomography (n = 5 mice per group). Femoral head cartilage explants from wild-type and Dcn-/- mice were stimulated with the inflammatory cytokine interleukin-1β (IL-1β) in vitro (n = 6 mice per group). The resulting chondrocyte response to IL-1β and release of sGAGs were quantified. RESULTS In both Dcn-/- and DcniKO mice, the absence of decorin resulted in accelerated sGAG loss and formation of highly aligned collagen fibrils on the cartilage surface relative to the control (P < 0.05). Also, Dcn-/- mice developed more salient osteophytes, illustrating more severe OA. In cartilage explants treated with IL-1β, loss of decorin did not alter the expression of either anabolic or catabolic genes. However, a greater proportion of sGAGs was released to the media from Dcn-/- mouse explants, in both live and devitalized conditions (P < 0.05). CONCLUSION In post-traumatic OA, decorin delays the loss of fragmented aggrecan and fibrillation of cartilage surface, and thus, plays a protective role in ameliorating cartilage degeneration.

Decorin is a stromal-derived prototype member of the small leucine-rich proteoglycan gene family. In addition to its functions as a regulator of collagen fibrillogenesis and TGF-β activity soluble decorin acts as a pan-receptor tyrosine kinase (RTK) inhibitor. Decorin binds to various RTKs including EGFR HER2 HGFR/Met VEGFR2 TLR and IGFR. Although the molecular mechanism for the action of decorin on these receptors is not entirely elucidated overall decorin evokes transient activation of these receptors with suppression of downstream signaling cascades culminating in growth inhibition followed by their physical downregulation via caveosomal internalization and degradation. In the case of Met decorin leads to decreased β-catenin signaling pathway and growth suppression. As most of these RTKs are responsible for providing a growth advantage to cancer cells the result of decorin treatment is oncosuppression. Another decorin-driven mechanism to restrict cancer growth and dissemination is by impeding angiogenesis via vascular endothelial growth factor receptor 2 (VEGFR2) and the concurrent activation of protracted endothelial cell autophagy. In this review we will dissect the multiple roles of decorin in cancer biology and its potential use as a next-generation protein-based adjuvant therapy to combat cancer.

Proteoglycans are emerging as critical regulators of intracellular catabolism. This rise in prominence has transformed our basic understanding and alerted us to the existence of non-canonical pathways, independent of nutrient deprivation, that potently control the autophagy downstream of a cell surface receptor. As a member of the small leucine-rich proteoglycan gene family, decorin has single-handedly pioneered the connection between extracellular matrix signaling and autophagy regulation. Soluble decorin evokes protracted endothelial cell autophagy via Peg3 and breast carcinoma cell mitophagy via mitostatin by interacting with VEGFR2 or the MET receptor tyrosine kinase, respectively. In this paper, we give a mechanistic perspective of the vital factors underlying the nutrient-independent, SLRP-dependent programs utilized for autophagic and/or mitophagic progression in breast cancer. Future protein therapies based on decorin (or fellow proteoglycan members) will represent a quantum leap forward in transforming autophagic progression into a powerful tool to control intracellular cell catabolism from the outside.

Aims Although heart failure (HF) is one of the most common conditions affecting the heart, little attention has been placed on the role of arteries in contributing to the progression of this disease. We sought to determine the haemodynamic change of arteries in HF patients subdivided according to left ventricular ejection fraction. The major goal was to establish the active compensatory role of arteries in HF. Methods and results Using sphygmography, we systematically studied a cohort of 228 HF patients and 52 healthy controls. We focused on the common carotid as the main elastic artery and the posterior tibial as the main muscular artery (Figure 1). Moreover, we categorized the three HF groups, HFrEF, HFmrEF, HFpEF, into two subgroups (A and B) according to the presence or absence of HF signs at baseline. We discovered that all the parameters of measured arterial kinetics, i.e. work, power, acceleration, and speed, were significantly increased (P Conclusions We provide the first documentation of an active compensatory role of arteries during HF. Mechanistically, we explain these findings by a dual activity of large arteries accomplished via an active propulsive work and a concurrent haemodynamic suction. These underestimated arterial functions partially compensate for the heart dysfunction in HF, underlining a key interplay between the heart and the vessels. We propose a new paradigm that we define as ‘heart and vessels failure’ that explicitly focuses on both heart and vessels’ interaction during the progression of HF.

Cervical remodeling is critical for a healthy pregnancy. The proper regulation of extracellular matrix (ECM) turnover leads to remodeling throughout gestation, transforming the tissue from a stiff material to a compliant, extensible, viscoelastic tissue prepared for delivery. Small leucine-rich proteoglycans (SLRPs) regulate structural fiber assembly in the cervical ECM and overall tissue material properties. To quantify the SLRPs' mechanical role in the cervix, whole cervix specimens from nonpregnant and late pregnant knockout mice of SLRPs, decorin and biglycan, were subjected to cyclic load-unload, ramp-hold, and load-to-failure mechanical tests. Further, a fiber composite material model, accounting for collagen fiber bundle waviness, was developed to describe the cervix's three-dimensional large deformation equilibrium behavior. In nonpregnant tissue, SLRP knockout cervices have the same equilibrium material properties as wild-type tissue. In contrast, the load-to-failure and ramp-hold tests reveal SLRPs impact rupture and time-dependent relaxation behavior. Loss of decorin in nonpregnant (NP) cervices results in inferior rupture properties. After extensive remodeling, cervical strength is similar between all genotypes, but the SLRP-deficient tissue has a diminished ability to dissipate stress during a ramp-hold. In mice with a combined loss of decorin and biglycan, the pregnant cervix loses its extensibility, compliance, and viscoelasticity. These results suggest that decorin and biglycan are necessary for crucial extensibility and viscoelastic material properties of a healthy, remodeled pregnant cervix.

Background Although heart failure (HF) is one of the most common conditions affecting the heart, little attention has been placed on the role of arteries in contributing to the progression of this disease. Purpose We sought to determine the hemodynamic change of arteries in HF patients subdivided according to left ventricular ejection fraction. The major goal was to establish the active compensatory role of arteries in HF Methods Using sphygmography, we systematically studied a cohort of 228 HF patients and 52 healthy controls. We focused on the common carotid as the main elastic artery and the posterior tibial as the main muscular artery (Figure 1). Moreover, we categorized the three HF groups, HFrEF, HFmrEF, HFpEF, into two subgroups (A and B) according to the presence or absence of HF signs at baseline. Results We discovered that all the parameters of measured arterial kinetics, i.e., work, power, acceleration, and speed, were significantly increased (p Conclusions We provide the first documentation of an active compensatory role of arteries during HF. Mechanistically, we explain these findings by a dual activity of large arteries accomplished via an active propulsive work and a concurrent hemodynamic suction. These underestimated arterial functions partially compensate for the heart dysfunction in HF, underlining a key interplay between the heart and the vessels. We propose a new paradigm that we define as “heart and vessels failure” that explicitly focuses on both heart and vessels' interaction during the progression of HF. Funding Acknowledgement Type of funding sources: None. Figure 1. Sphygmograms of CCA and PTAFigure 2. Patients disposition & Results

The cervix undergoes rapid and dramatic shifts in collagen and elastic fiber structure to achieve its disparate physiological roles of competence during pregnancy and compliance during birth. An understanding of the structure-function relationships of collagen and elastic fibers to maintain extracellular matrix (ECM) homeostasis requires an understanding of the mechanisms executed by non-structural ECM molecules. Small-leucine rich proteoglycans (SLRPs) play key functions in biology by affecting collagen fibrillogenesis and regulating enzyme and growth factor bioactivities. In the current study, we evaluated collagen and elastic fiber structure-function relationships in mouse cervices using mice with genetic ablation of decorin and/or biglycan genes as representative of Class I SLRPs, and lumican gene representative of Class II SLRP. We identified structural defects in collagen fibril and elastic fiber organization in nonpregnant mice lacking decorin, or biglycan or lumican with variable resolution of defects noted during pregnancy. The severity of collagen and elastic fiber defects was greater in nonpregnant mice lacking both decorin and biglycan and defects were maintained throughout pregnancy. Loss of biglycan alone reduced tissue extensibility in nonpregnant mice while loss of both decorin and biglycan manifested in decreased rupture stretch in late pregnancy. Collagen cross-link density was similar in the Class I SLRP null mice as compared to wild-type nonpregnant and pregnant controls. A broader range in collagen fibril diameter along with an increase in mean fibril spacing was observed in the mutant mice compared to wild-type controls. Collectively, these findings uncover functional redundancy and hierarchical roles of Class I and Class II SLRPs as key regulators of cervical ECM remodeling in pregnancy. These results expand our understating of the critical role SLRPs play to maintain ECM homeostasis in the cervix.

During the pathogenesis of glaucoma, optic nerve (ON) axons become continuously damaged at the optic nerve head (ONH). This often is associated with reactive astrocytes and increased transforming growth factor (TGF-β) 2 levels. In this study we tested the hypothesis if the presence or absence of decorin (DCN), a small leucine-rich proteoglycan and a natural inhibitor of several members of the TGF family, would affect the expression of the TGF-βs and connective tissue growth factor (CTGF/CCN2) in human ONH astrocytes and murine ON astrocytes. We found that DCN is present in the mouse ON and is expressed by human ONH and murine ON astrocytes. DCN expression and synthesis was significantly reduced after 24 h treatment with 3 nM CTGF/CCN2, while treatment with 4 pM TGF-β2 only reduced expression of DCN significantly. Conversely, DCN treatment significantly reduced the expression of TGF-β1, TGF-β2 and CTGF/CCN2 vis-a-vis untreated controls. Furthermore, DCN treatment significantly reduced expression of fibronectin (FN) and collagen IV (COL IV). Notably, combined treatment with DCN and triciribine, a small molecule inhibitor of protein kinase B (AKT), attenuated effects of DCN on CTGF/CCN2, TGF-β1, and TGF-β2 mRNA expression. We conclude (1) that DCN is an important regulator of TGF-β and CTGF/CCN2 expression in astrocytes of the ON and ONH, (2) that DCN thereby regulates the expression of extracellular matrix (ECM) components and (3) that DCN executes its negative regulatory effects on TGF-β and CTGF/CCN2 via the pAKT/AKT signaling pathway in ON astrocytes.

The extracellular matrix is a network of secreted macromolecules that provides a harmonious meshwork for the growth and homeostatic development of organisms. It conveys multiple signaling cascades affecting specific surface receptors that impact cell behavior. During cancer growth, this bioactive meshwork is remodeled and enriched in newly formed blood vessels, which provide nutrients and oxygen to the growing tumor cells. Remodeling of the tumor microenvironment leads to the formation of bioactive fragments that may have a distinct function from their parent molecules, and the balance among these factors directly influence cell viability and metastatic progression. Indeed, the matrix acts as a gatekeeper by regulating the access of cancer cells to nutrients. Here, we will critically evaluate the role of selected matrix constituents in regulating tumor angiogenesis and provide up-to-date information concerning their primary mechanisms of action.

Joint biomechanical functions rely on the integrity of cartilage extracellular matrix. Understanding the molecular activities that govern cartilage matrix assembly is critical for developing effective cartilage regeneration strategies. This study elucidated the role of decorin, a small leucine-rich proteoglycan, in the structure and biomechanical functions of cartilage. In decorin-null cartilage, we discovered a substantial reduction of aggrecan content, the major proteoglycan of cartilage matrix, and mild changes in collagen fibril nanostructure. This loss of aggrecan resulted in significantly impaired biomechanical properties of cartilage, including decreased modulus, elevated hydraulic permeability, and reduced energy dissipation capabilities. At the cellular level, we found that decorin functions to increase the retention of aggrecan in the neo-matrix of chondrocytes, rather than to directly influence the biosynthesis of aggrecan. At the molecular level, we demonstrated that decorin significantly increases the adhesion between aggrecan and aggrecan molecules and between aggrecan molecules and collagen II fibrils. We hypothesize that decorin plays a crucial structural role in mediating the matrix integrity and biomechanical functions of cartilage by providing physical linkages to increase the adhesion and assembly of aggrecan molecules at the nanoscale.

The αvβ3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvβ3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvβ3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvβ3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvβ3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to β3-negative recipient cells. We also demonstrate the intracellular localization of β3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvβ3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvβ3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvβ3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvβ3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvβ3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvβ3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. In vitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. In vivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44+/+ and Cd44−/− mice but not Cd14−/− mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.

The overall structure and architecture of the extracellular matrix undergo dramatic alterations in composition, form, and functionality over time. The stochasticity begins during development, essential for maintaining organismal homeostasis and is heavily implicated in many pathobiological states including fibrosis and cancer. Modeling and remodeling of the matrix is driven by the local cellular milieu and secreted and cell-associated components in a framework of dynamic reciprocity. This collection of expertly-written reviews aims to relay state-of-the-art information concerning the mechanisms of matrix modeling and remodeling in physiological development and disease.

BACKGROUND: Although heart failure (HF) is one of the most common conditions affecting the heart, little attention has been placed on the role of arteries in contributing to the progression of this disease. We sought to determine the hemodynamic change of arteries in HF patients subdivided according to left ventricular ejection fraction. The major goal was to establish the active compensatory role of arteries in HF. METHODS: Using sphygmography, we systematically studied a cohort of 228 HF patients and 52 healthy controls. We focused on the common carotid as the main elastic artery and the posterior tibial as the main muscular artery. Moreover, we categorized the three HF groups, HFrEF, HFmrEF, HFpEF, into two subgroups (A and B) according to the presence or absence of HF signs at baseline. RESULTS: We discovered that all the parameters of measured arterial kinetics, i.e., work, power, acceleration, and speed, were significantly increased (P

Proteoglycans and selected extracellular matrix constituents are emerging as intrinsic and critical regulators of evolutionarily conversed, intracellular catabolic pathways. Often, these secreted molecules evoke sustained autophagy in a variety of cell types, tissues, and model systems. The unique properties of proteoglycans have ushered in a paradigmatic shift to broaden our understanding of matrix-mediated signaling cascades. The dynamic cellular pathway controlling autophagy is now linked to an equally dynamic and fluid signaling network embedded in a complex meshwork of matrix molecules. A rapidly emerging field of research encompasses multiple matrix-derived candidates, representing a menagerie of soluble matrix constituents including decorin, biglycan, endorepellin, endostatin, collagen VI and plasminogen kringle 5. These matrix constituents are pro-autophagic and simultaneously anti-angiogenic. In contrast, perlecan, laminin α2 chain, and lumican have anti-autophagic functions. Mechanistically, each matrix constituent linked to intracellular catabolic events engages a specific cell surface receptor that often converges on a common core of the autophagic machinery including AMPK, Peg3 and Beclin 1. We consider this matrix-evoked autophagy as non-canonical given that it occurs in an allosteric manner and is independent of nutrient availability or prevailing bioenergetics control. We propose that matrix-regulated autophagy is an important outside-in signaling mechanism for proper tissue homeostasis that could be therapeutically leveraged to combat a variety of diseases.

Mesothelioma is an aggressive tumor with very poor prognosis. The limited availability of therapeutic options for mesothelioma patients suggests the need for a better understanding of mesothelioma biology and the identification of novel therapeutic targets. Progranulin is a pluripotent growth factor whose dysregulation is implicated in several human pathologies including cancer. The pro-tumorigenic activity of progranulin depends on the activation of its functional receptor EphA2. Indeed, in bladder cancer, progranulin promotes EphA2 phosphorylation at Ser897 and triggers EphA2 oncogenic action resulting in increased tumor cell migration, invasion and in vivo tumor growth. Both progranulin and EphA2 are expressed in mesothelioma, with progranulin promoting tumor angiogenesis in a VEGF-independent manner and EphA2 being often mutated or overexpressed. However, in mesothelioma, the potential functional interaction between progranulin and EphA2 is unexplored. Using a panel of mesothelioma cell lines covering all mesothelioma subtypes, we observed increased levels of phosphorylated EphA2 at Ser897 when compared to immortalized normal mesothelial cells, and the levels of phosphorylation positively correlated with progranulin expression. Progranulin-dependent activation of EphA2 in mesothelioma cells promoted the activation of the MAPK and AKT pathways and ERK1/2-dependent EphA2 phosphorylation at Ser897. Transient depletion of progranulin by siRNA approaches as well as GRN gene knock-out by CRISP/Cas9 strategies in mesothelioma cells reduced the activation of the AKT signaling pathway and inhibited the migratory and invasive capabilities of progranulin-depleted cells when compared to parental cells, suggesting that the progranulin/EphA2 axis is oncogenic in mesothelioma. However, EphA2 genetic ablation resulted in an increased basal activation of AKT and MAPK and in progranulin-evoked EphA2-independet AKT/MAPK activation, indicating that alternative membrane receptors might compensate for EphA2 loss and sustain progranulin action in mesothelioma. Using several pharmacological and genetic approaches, we uncovered a role for EGFR and FAK in compensating for EphA2 loss by triggering progranulin-dependent oncogenic signaling in mesothelioma, highlighting a complex functional cross-talk between EphA2, EGFR and FAK in mediating progranulin-dependent tumorigenesis. Thus, these results suggest the need for combinatorial therapeutic approaches to blunt progranulin oncogenic action in mesothelioma. Citation Format: Elisa Ventura, Simone Buraschi, Stephen J. Williams, Antonino Belfiore, Renato V. Iozzo, Antonio Giordano, Andrea Morrione. Mechanisms of action and regulation of the progranulin/EphA2 axis in mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 97.

OBJECTIVE: To delineate the activities of decorin and biglycan in the progression of post-traumatic osteoarthritis (PTOA). DESIGN: Three-month-old inducible biglycan (Bgn(iKO)) and decorin/biglycan compound (Dcn/Bgn(iKO)) knockout mice were subjected to the destabilization of the medial meniscus (DMM) surgery to induce PTOA. The OA phenotype was evaluated by assessing joint structure and sulfated glycosaminoglycan (sGAG) staining via histology, surface collagen fibril nanostructure and calcium content via scanning electron microscopy, tissue modulus via atomic force microscopy-nanoindentation, as well as subchondral bone structure and meniscus ossification via micro-computed tomography. Outcomes were compared with previous findings in the inducible decorin (Dcn(iKO)) knockout mice. RESULTS: In the DMM model, Bgn(iKO) mice developed similar degree of OA as the control (0.44 [−0.18 1.05] difference in modified Mankin score), different from the more severe OA phenotype observed in Dcn(iKO) mice (1.38 [0.91 1.85] difference). Dcn/Bgn(iKO) mice exhibited similar histological OA phenotype as Dcn(iKO) mice (1.51 [0.97 2.04] difference versus control), including aggravated loss of sGAGs, salient surface fibrillation and formation of osteophyte. Meanwhile, Dcn/Bgn(iKO) mice showed further cartilage thinning than Dcn(iKO) mice, resulting in the exposure of underlying calcified tissues and aberrantly high surface modulus. Bgn(iKO) and Dcn/Bgn(iKO) mice developed altered subchondral trabecular bone structure in both Sham and DMM groups, while Dcn(iKO) and control mice did not. CONCLUSION: In PTOA, decorin plays a more crucial role than biglycan in regulating cartilage degeneration, while biglycan is more important in regulating subchondral bone structure. The two have distinct activities and modest synergy in the pathogenesis of PTOA.

In cartilage tissue engineering, one key challenge is for regenerative tissue to recapitulate the biomechanical functions of native cartilage while maintaining normal mechanosensitive activities of chondrocytes. Thus, it is imperative to discern the micromechanobiological functions of the pericellular matrix, the ~ 2–4 µm-thick domain that is in immediate contact with chondrocytes. In this study, we discovered that decorin, a small leucine-rich proteoglycan, is a key determinant of cartilage pericellular matrix micromechanics and chondrocyte mechanotransduction in vivo. The pericellular matrix of decorin-null murine cartilage developed reduced content of aggrecan, the major chondroitin sulfate proteoglycan of cartilage and a mild increase in collagen II fibril diameter vis-a-vis wild-type controls. As a result, decorin-null pericellular matrix showed a significant reduction in micromodulus, which became progressively more pronounced with maturation. In alignment with the defects of pericellular matrix, decorin-null chondrocytes exhibited decreased intracellular calcium activities, [Ca2+]i, in both physiologic and osmotically evoked fluidic environments in situ, illustrating impaired chondrocyte mechanotransduction. Next, we compared [Ca2+]i activities of wild-type and decorin-null chondrocytes following enzymatic removal of chondroitin sulfate glycosaminoglycans. The results showed that decorin mediates chondrocyte mechanotransduction primarily through regulating the integrity of aggrecan network, and thus, aggrecan-endowed negative charge microenvironment in the pericellular matrix. Collectively, our results provide robust genetic and biomechanical evidence that decorin is an essential constituent of the native cartilage matrix, and suggest that modulating decorin activities could improve cartilage regeneration.

Primary open-angle glaucoma a neurodegenerative disorder characterized by degeneration of optic nerve axons, is a frequent cause of vision loss and blindness worldwide. Several randomized multicenter studies have identified intraocular pressure as the major risk factor for its development, caused by an increased outflow resistance to the aqueous humor within the trabecular meshwork. However, the molecular mechanism for increased outflow resistance in POAG has not been fully established. One of the proposed players is the pro-fibrotic transforming growth factor (TGF)-β2, which is found in higher amounts in the aqueous humor of patients with POAG. In this study we elucidated the role of decorin, a small leucine-rich proteoglycan and known antagonist of TGF-β, in the region of aqueous humor outflow tissue. Utilizing decorin deficient mice, we discovered that decorin modulated TGF-β signaling in the canonical outflow pathways and the lack of decorin in vivo caused an increase in intraocular pressure. Additionally, the Dcn-/- mice showed significant loss of optic nerve axons and morphological changes in the glial lamina, typical features of glaucoma. Moreover, using human trabecular meshwork cells we discovered that soluble decorin attenuated TGF-β2 mediated synthesis and expression of typical downstream target genes including CCN2/CTGF, FN and COL IV. Finally, we found a negative reciprocal regulation of decorin and TGF-β, with a dramatic downregulation of decorin in the canonical outflow pathways of patients with primary open-angle glaucoma. Collectively, our results indicate that decorin plays an important role in the pathogenesis of primary open-angle glaucoma and offers novel perspectives in the treatment of this serious disease.

The tumor microenvironment encompasses a complex cellular network that includes cancer-associated fibroblasts, inflammatory cells, neo-vessels, and an extracellular matrix enriched in angiogenic growth factors. Decorin is one of the main components of the tumor stroma, but it is not expressed by cancer cells. Lack of this proteoglycan correlates with down-regulation of E-cadherin and induction of β-catenin signaling. In this study, we investigated the role of a decorin-deficient tumor microenvironment in colon carcinoma progression and metastasis. We utilized an established model of colitis-associated cancer by administering Azoxymethane/Dextran sodium sulfate to adult wild-type and Dcn(−/−) mice. We discovered that after 12 weeks, all the animals developed intestinal tumors independently of their genotype. However, the number of intestinal neoplasms was significantly higher in the Dcn(−/−) microenvironment vis-à-vis wild-type mice. Mechanistically, we found that under unchallenged basal conditions, the intestinal epithelium of the Dcn(−/−) mice showed a significant increase in the protein levels of epithelial-mesenchymal transition associated factors including Snail, Slug, Twist, and MMP2. In comparison, in the colitis-associated cancer evoked in the Dcn(−/−) mice, we found that intercellular adhesion molecule 1 (ICAM-1) was also significantly increased, in parallel with epithelial-mesenchymal transition signaling pathway-related factors. Furthermore, a combined Celecoxib/decorin treatment revealed a promising therapeutic efficacy in treating human colorectal cancer cells, in decorin-deficient animals. Collectively, our results shed light on colorectal cancer progression and provide a protein-based therapy, i.e., treatment using recombinant decorin, to target the tumor microenvironment.

Proteoglycans are rapidly emerging as versatile regulators of intracellular catabolic pathways. This is predominantly achieved via the non-canonical induction of autophagy, a fundamentally and evolutionarily conserved eukaryotic pathway necessary for maintaining organismal homeostasis. Autophagy facilitated by either decorin, a small leucine-rich proteoglycan, or perlecan, a basement membrane heparan sulfate proteoglycan, proceeds independently of ambient nutrient conditions. We found that soluble decorin evokes endothelial cell autophagy and breast carcinoma cell mitophagy by directly interacting with vascular endothelial growth factor receptor 2 (VEGFR2) or the Met receptor tyrosine kinase, respectively. Endorepellin, a soluble, proteolytic fragment of perlecan, induces autophagy and endoplasmic reticulum stress within the vasculature, downstream of VEGFR2. These potent matrix-derived cues transduce key biological information via receptor binding to converge upon a newly discovered nexus of core autophagic machinery comprised of Peg3 (paternally expressed gene 3) for autophagy or mitostatin for mitophagy. Here, we give a mechanistic overview of the nutrient-independent, proteoglycan-driven programs utilized for autophagic or mitophagic progression. We propose that catabolic control of cell behavior is an underlying basis for proteoglycan versatility and may provide novel therapeutic targets for the treatment of human disease

The extracellular matrix encompasses a reservoir of bioactive macromolecules that modulates a cornucopia of biological functions. A prominent body of work posits matrix constituents as master regulators of autophagy and angiogenesis and provides molecular insight into how these two processes are coordinated. Here, we review current understanding of the molecular mechanisms underlying hyaluronan and HAS2 regulation and the role of soluble proteoglycan in affecting autophagy and angiogenesis. Specifically, we assess the role of proteoglycan-evoked autophagy in regulating angiogenesis via the HAS2-hyaluronan axis and ATG9A, a novel HAS2 binding partner. We discuss extracellular hyaluronan biology and the post-transcriptional and post-translational modifications that regulate its main synthesizer, HAS2. We highlight the emerging group of proteoglycans that utilize outside-in signaling to modulate autophagy and angiogenesis in cancer microenvironments and thoroughly review the most up-to-date understanding of endorepellin signaling in vascular endothelia, providing insight into the temporal complexities involved.

Approximately, 25% of all preterm births are due to preterm premature rupture of membranes. Mice deficient in proteoglycans biglycan (Bgn) and decorin (Dcn) display abnormal fetal membranes and increased incidence of preterm birth. We conducted RNA-Seq to profile fetal membranes and identify molecular pathways that may lead to preterm birth in double knockout (DKO) mice (Bgn−/−; Dcn−/−) compared to wild-type (WT) at two different gestational stages, E12 and E18 (n = 3 in each group). 3264 transcripts were differentially regulated in E18 DKO vs. WT fetal membranes, and 96 transcripts differentially regulated in E12 DKO vs. WT fetal membranes (FDR 10) for transcription factor binding sites for Nr2f1 at E18. We propose that in DKO mice, cell cycle arrest results in lack of cell proliferation in fetal membranes, inability to contain the growing fetus, and preterm birth.

Extracellular matrix-evoked angiostasis and autophagy within the tumor microenvironment represent two critical, but unconnected, functions of the small leucine-rich proteoglycan, decorin. Acting as a partial agonist of vascular endothelial growth factor 2 (VEGFR2), soluble decorin signals via the energy sensing protein, AMP-activated protein kinase (AMPK), in the autophagic degradation of intracellular vascular endothelial growth factor A (VEGFA). Here, we discovered that soluble decorin evokes intracellular catabolism of endothelial VEGFA that is mechanistically independent of mTOR, but requires an autophagic regulator, paternally expressed gene 3 (PEG3). We found that administration of autophagic inhibitors such as chloroquine or bafilomycin A1, or depletion of autophagy-related 5 (ATG5), results in accumulation of intracellular VEGFA, indicating that VEGFA is a basal autophagic substrate. Mechanistically, decorin increased the VEGFA clearance rate by augmenting autophagic flux, a process that required RAB24 member RAS oncogene family (RAB24), a small GTPase that facilitates the disposal of autophagic compartments. We validated these findings by demonstrating the physiological relevance of this process in vivo. Mice starved for 48 h exhibited a sharp decrease in overall cardiac and aortic VEGFA that could be blocked by systemic chloroquine treatment. Thus, our findings reveal a unified mechanism for the metabolic control of endothelial VEGFA for autophagic clearance in response to decorin and canonical pro-autophagic stimuli. We posit that the VEGFR2/AMPK/PEG3 axis integrates the anti-angiogenic and pro-autophagic bioactivities of decorin as the molecular basis for tumorigenic suppression. These results support future therapeutic use of decorin as a next-generation protein therapy to combat cancer.

The growth factor progranulin plays a critical role in bladder cancer by modulating tumor cell motility and invasion. Progranulin regulates remodeling of the actin cytoskeleton by interacting with drebrin, an actin binding protein that regulates tumor growth. We previously discovered that progranulin depletion inhibits epithelial-to-mesenchymal transition and markedly reduces in vivo tumor growth. Moreover, progranulin depletion sensitizes urothelial cancer cells to cisplatin treatment, further substantiating a pro-survival function of progranulin. Until recently, the progranulin signaling receptor remained unidentified, precluding a full understanding of progranulin action in tumor cell biology. We recently identified EphA2, a member of a large family of receptor tyrosine-kinases, as the functional receptor for progranulin. However, it is not established whether EphA2 plays an oncogenic role in bladder cancer. Here we demonstrate that progranulin, and not ephrin-A1, the canonical ligand for EphA2, is the predominant EphA2 ligand in bladder cancer. Progranulin evoked Akt- and Erk1/2-mediated EphA2 phosphorylation at Ser897, which could drive bladder tumorigenesis. We discovered that EphA2 depletion severely blunted progranulin-dependent motility and anchorage-independent growth, and sensitized bladder cancer cells to cisplatin treatment. We further defined the mechanisms of progranulin/EphA2-dependent motility by identifying liprin-α1 as a novel progranulin-dependent EphA2 interacting protein and establishing its critical role in cell motility. The discovery of EphA2 as the functional signaling receptor for progranulin and the identification of novel downstream effectors offer a new avenue for understanding the underlying mechanism of progranulin action and may constitute novel clinical and therapeutic targets in bladder cancer.