Thomas Neill

Decorin Antagonizes the Angiogenic Network: CONCURRENT INHIBITION OF MET, HYPOXIA INDUCIBLE FACTOR 1α, VASCULAR ENDOTHELIAL GROWTH FACTOR A, AND INDUCTION OF THROMBOSPONDIN-1 AND TIMP3*

Background: Decorin antagonizes multiple receptor tyrosine kinases, such as Met, to suppress tumorigenesis. Results: Decorin promotes angiostasis by blocking hypoxia inducible factor-1α and β-catenin to inhibit vascular endothelial growth factor A and matrix metalloprotease-2/9 activity concurrent with thrombospondin-1 and TIMP3 induction. Conclusion: Decorin abrogates the pro-angiogenic HGF/Met signaling axis, thereby repressing vascular endothelial growth factor A-mediated angiogenesis under normoxia. Significance: Soluble decorin attenuates early tumor growth by preventing normoxic angiogenic signaling through the Met receptor. Decorin, a small leucine-rich proteoglycan, inhibits tumor growth by antagonizing multiple receptor tyrosine kinases including EGFR and Met. Here, we investigated decorin during normoxic angiogenic signaling. An angiogenic PCR array revealed a profound decorin-evoked transcriptional inhibition of pro-angiogenic genes, such as HIF1A. Decorin evoked a reduction of hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor A (VEGFA) in MDA-231 breast carcinoma cells expressing constitutively-active HIF-1α. Suppression of Met with decorin or siRNA evoked a similar reduction of VEGFA by attenuating downstream β-catenin signaling. These data establish a noncanonical role for β-catenin in regulating VEGFA expression. We found that exogenous decorin induced expression of thrombospondin-1 and TIMP3, two powerful angiostatic agents. In contrast, decorin suppressed both the expression and enzymatic activity of matrix metalloprotease (MMP)-9 and MMP-2, two pro-angiogenic proteases. Our data establish a novel duality for decorin as a suppressor of tumor angiogenesis under normoxia by simultaneously down-regulating potent pro-angiogenic factors and inducing endogenous anti-angiogenic agents.

Decorin causes autophagy in endothelial cells via Peg3.

Significance We identified a function for a member of the extracellular matrix in the regulation of autophagy. Decorin, a member of the small leucine-rich proteoglycan family and an established pan-receptor tyrosine kinase inhibitor, evokes endothelial cell autophagy and inhibits angiogenesis. This process is mediated by a high-affinity interaction with VEGFR2 which leads to increased levels of Peg3, a tumor-suppressor gene. We provide mechanistic evidence that Peg3 is required to maintain basal levels of Beclin 1, a major autophagic marker. These data provide a paradigmatic shift for other soluble matrix constituents to regulate autophagy. Soluble decorin affects the biology of several receptor tyrosine kinases by triggering receptor internalization and degradation. We found that decorin induced paternally expressed gene 3 (Peg3), an imprinted tumor suppressor gene, and that Peg3 relocated into autophagosomes labeled by Beclin 1 and microtubule-associated light chain 3. Decorin evoked Peg3-dependent autophagy in both microvascular and macrovascular endothelial cells leading to suppression of angiogenesis. Peg3 coimmunoprecipitated with Beclin 1 and LC3 and was required for maintaining basal levels of Beclin 1. Decorin, via Peg3, induced transcription of Beclin 1 and microtubule-associated protein 1 light chain 3 alpha genes, thereby leading to a protracted autophagic program. Mechanistically, decorin interacted with VEGF receptor 2 (VEGFR2) in a region overlapping with its natural ligand VEGFA, and VEGFR2 was required for decorin-evoked Beclin 1 and microtubule-associated protein 1 light chain 3 alpha expression as well as for Peg3 induction in endothelial cells. Moreover, decorin induced VEGFR2-dependent mitochondrial fragmentation and loss of mitochondrial membrane potential. Thus, we have unveiled a mechanism for a secreted proteoglycan in inducing Peg3, a master regulator of macroautophagy in endothelial cells.

Decorin Antagonizes Met Receptor Activity and Down-regulates β-Catenin and Myc Levels

A theme emerging during the past few years is that members of the small leucine-rich proteoglycan gene family affect cell growth by interacting with multiple receptor tyrosine kinases (RTKs), mostly by a physical down-regulation of the receptors, thereby depriving tumor cells of pro-survival signals. Decorin binds and down-regulates several RTKs, including Met, the receptor for hepatocyte growth factor. Here we demonstrate that decorin blocks several biological activities mediated by the Met signaling axis, including cell scatter, evasion, and migration. These effects were mediated by a profound down-regulation of noncanonical β-catenin levels. In addition, Myc, a downstream target of β-catenin, was markedly down-regulated by decorin, whereas phosphorylation of Myc at threonine 58 was markedly induced. The latter is known to destabilize Myc and target it for proteasomal degradation. We also discovered that systemic delivery of decorin using three distinct tumor xenograft models caused down-regulation of Met and a concurrent suppression of β-catenin and Myc levels. We found that decorin protein core labeled with the near infrared dye IR800 specifically targeted the tumor cells expressing Met. Even 68-h post-injection, decorin was found to reside within the tumor xenografts with little or no binding to other tissues. Collectively, our results indicate a role for a secreted proteoglycan in suppressing the expression of key oncogenic factors required for tumor progression.

Endorepellin, the angiostatic module of perlecan, interacts with both the α2β1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2): a dual receptor antagonism.

Endorepellin, the C-terminal module of perlecan, negatively regulates angiogenesis counter to its proangiogenic parental molecule. Endorepellin (the C-terminal domain V of perlecan) binds the α2β1 integrin on endothelial cells and triggers a signaling cascade that leads to disruption of the actin cytoskeleton. Here, we show that both perlecan and endorepellin bind directly and with high affinity to both VEGF receptors 1 and 2, in a region that differs from VEGFA-binding site. In both human and porcine endothelial cells, this interaction evokes a physical down-regulation of both the α2β1 integrin and VEGFR2, with concurrent activation of the tyrosine phosphatase SHP-1 and downstream attenuation of VEGFA transcription. We demonstrate that endorepellin requires both the α2β1 integrin and VEGFR2 for its angiostatic activity. Endothelial cells that express α2β1 integrin but lack VEGFR2, do not respond to endorepellin treatment. Thus, we provide a new paradigm for the activity of an antiangiogenic protein and mechanistically explain the specificity of endorepellin for endothelial cells, the only cells that simultaneously express both receptors. We hypothesize that a mechanism such as dual receptor antagonism could operate for other angiostatic fragments.

Instructive Roles of Extracellular Matrix on Autophagy

Autophagy plays an essential role in maintaining an intricate balance between nutrient demands and energetic requirements during normal homeostasis. Autophagy recycles metabolic substrates from nonspecific bulk degradation of proteins and excess or damaged organelles. Recent work posits an active and dynamic signaling role for extracellular matrix-evoked autophagic regulation, that is, allosteric and independent of prevailing nutrient conditions. Several candidates, representing a diverse repertoire of matrix constituents (decorin, collagen VI, laminin α2, endostatin, endorepellin, and kringle V), can modulate autophagic signaling pathways. Importantly, a novel principle indicates that matrix constituents can differentially modulate autophagic induction and repression via interaction with specific receptors. Most of the matrix-derived factors described here appear to control autophagy in a canonical manner but independent of nutrient deprivation. Because the molecular composition and structure of the extracellular matrix are dynamically remodeled during various physiological and pathological conditions, we propose that matrix-regulated autophagy is key for maintaining proper tissue homeostasis and disease prevention, such as cancer progression and muscular dystrophies.

Insights into the key roles of proteoglycans in breast cancer biology and translational medicine.

Proteoglycans control numerous normal and pathological processes, among which are morphogenesis, tissue repair, inflammation, vascularization and cancer metastasis. During tumor development and growth, proteoglycan expression is markedly modified in the tumor microenvironment. Altered expression of proteoglycans on tumor and stromal cell membranes affects cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Despite the high complexity and heterogeneity of breast cancer, the rapid evolution in our knowledge that proteoglycans are among the key players in the breast tumor microenvironment suggests their potential as pharmacological targets in this type of cancer. It has been recently suggested that pharmacological treatment may target proteoglycan metabolism, their utilization as targets for immunotherapy or their direct use as therapeutic agents. The diversity inherent in the proteoglycans that will be presented herein provides the potential for multiple layers of regulation of breast tumor behavior. This review summarizes recent developments concerning the biology of selected proteoglycans in breast cancer, and presents potential targeted therapeutic approaches based on their novel key roles in breast cancer.

Decorin protein core affects the global gene expression profile of the tumor microenvironment in a triple-negative orthotopic breast carcinoma xenograft model.

Decorin, a member of the small leucine-rich proteoglycan gene family, exists and functions wholly within the tumor microenvironment to suppress tumorigenesis by directly targeting and antagonizing multiple receptor tyrosine kinases, such as the EGFR and Met. This leads to potent and sustained signal attenuation, growth arrest, and angiostasis. We thus sought to evaluate the tumoricidal benefits of systemic decorin on a triple-negative orthotopic breast carcinoma xenograft model. To this end, we employed a novel high-density mixed expression array capable of differentiating and simultaneously measuring gene signatures of both Mus musculus (stromal) and Homo sapiens (epithelial) tissue origins. We found that decorin protein core modulated the differential expression of 374 genes within the stromal compartment of the tumor xenograft. Further, our top gene ontology classes strongly suggests an unexpected and preferential role for decorin protein core to inhibit genes necessary for immunomodulatory responses while simultaneously inducing expression of those possessing cellular adhesion and tumor suppressive gene properties. Rigorous verification of the top scoring candidates led to the discovery of three genes heretofore unlinked to malignant breast cancer that were reproducibly found to be induced in several models of tumor stroma. Collectively, our data provide highly novel and unexpected stromal gene signatures as a direct function of systemic administration of decorin protein core and reveals a fundamental basis of action for decorin to modulate the tumor stroma as a biological mechanism for the ascribed anti-tumorigenic properties.

Decoding the matrix: Instructive roles of proteoglycan receptors

The extracellular matrix is a dynamic repository harboring instructive cues that embody substantial regulatory dominance over many evolutionarily conserved intracellular activities, including proliferation, apoptosis, migration, motility, and autophagy. The matrix also coordinates and parses hierarchical information, such as angiogenesis, tumorigenesis, and immunological responses, typically providing the critical determinants driving each outcome. We provide the first comprehensive review focused on proteoglycan receptors, that is, signaling transmembrane proteins that use secreted proteoglycans as ligands, in addition to their natural ligands. The majority of these receptors belong to an exclusive subset of receptor tyrosine kinases and assorted cell surface receptors that specifically bind, transduce, and modulate fundamental cellular processes following interactions with proteoglycans. The class of small leucine-rich proteoglycans is the most studied so far and constitutes the best understood example of proteoglycan-receptor interactions. Decorin and biglycan evoke autophagy and immunological responses that deter, suppress, or exacerbate pathological conditions such as tumorigenesis, angiogenesis, and chronic inflammatory disease. Basement membrane-associated heparan sulfate proteoglycans (perlecan, agrin, and collagen XVIII) represent a unique cohort and provide proteolytically cleaved bioactive fragments for modulating cellular behavior. The receptors that bind the genuinely multifactorial and multivalent proteoglycans represent a nexus in understanding basic biological pathways and open new avenues for therapeutic and pharmacological intervention.

Endorepellin evokes autophagy in endothelial cells.

Background: Endorepellin inhibits angiogenesis by simultaneously binding the α2β1 integrin and VEGFR2, attenuating the PI3K/Akt/mTOR and PKC/JNK/AP1 signaling pathways. Results: Endorepellin evokes autophagy by inducing Beclin 1 and LC3 downstream of VEGFR2 in a Peg3-dependent manner. Conclusion: Endorepellin causes endothelial cell autophagy through VEGFR2 independent of the α2β1 integrin. Significance: Endorepellin-evoked endothelial cell autophagy represents a promising strategy for angiostatic-based therapeutics. Endorepellin, the C-terminal fragment of the heparan sulfate proteoglycan perlecan, possesses angiostatic activity via dual receptor antagonism, through concurrent binding to the α2β1 integrin and vascular endothelial growth factor receptor 2 (VEGFR2). Here, we discovered that soluble endorepellin induced autophagy in endothelial cells by modulating the expression of Beclin 1, LC3, and p62, three established autophagic markers. Moreover, endorepellin evoked expression of the imprinted tumor suppressor gene Peg3 and its co-localization with Beclin 1 and LC3 in autophagosomes, suggesting a major role for this gene in endothelial cell autophagy. Mechanistically, endorepellin induced autophagy by down-regulating VEGFR2 via the two LG1/2 domains, whereas the C-terminal LG3 domain, the portion responsible for binding the α2β1 integrin, was ineffective. Endorepellin also induced transcriptional activity of the BECN1 promoter in endothelial cells, and the VEGFR2-specific tyrosine kinase inhibitor, SU5416, blocked this effect. Finally, we found a correlation between endorepellin-evoked inhibition of capillary morphogenesis and enhanced autophagy. Thus, we have identified a new role for this endogenous angiostatic fragment in inducing autophagy through a VEGFR2-dependent but α2β1 integrin-independent pathway. This novel mechanism specifically targets endothelial cells and could represent a promising new strategy to potentiate the angiostatic effect of endorepellin and perhaps other angiostatic matrix proteins.

A current view of perlecan in physiology and pathology: A mosaic of functions.

Perlecan, a large basement membrane heparan sulfate proteoglycan, is expressed in a wide array of tissues where it regulates diverse cellular processes including bone formation, inflammation, cardiac development, and angiogenesis. Here we provide a contemporary review germane to the biology of perlecan encompassing its genetic regulation as well as an analysis of its modular protein structure as it pertains to function. As perlecan signaling from the extracellular matrix converges on master regulators of autophagy, including AMPK and mTOR, via a specific interaction with vascular endothelial growth factor receptor 2, we specifically focus on the mechanism of action of perlecan in autophagy and angiogenesis and contrast the role of endorepellin, the C-terminal fragment of perlecan, in these cellular and morphogenic events.