DeannaLee M. Beauvais

The syndecan-1 ectodomain regulates αvβ3 integrin activity in human mammary carcinoma cells

The αvβ3 integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the αvβ3 integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to αvβ3 requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances αvβ3 recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt αvβ3-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated αvβ3 integrins.

Syndecan-1 regulates αvβ3 and αvβ5 integrin activation during angiogenesis and is blocked by synstatin, a novel peptide inhibitor

Syndecan-1 (Sdc1) is a matrix receptor shown to associate via its extracellular domain with the αvβ3 and αvβ5 integrins, potentially regulating cell adhesion, spreading, and invasion of cells expressing these integrins. Using Sdc1 deletion mutants expressed in human mammary carcinoma cells, we identified the active site within the Sdc1 core protein and derived a peptide inhibitor called synstatin (SSTN) that disrupts Sdc1s interaction with these integrins. Because the αvβ3 and αvβ5 integrins are critical in angiogenesis, a process in which a role for Sdc1 has been uncertain, we used human vascular endothelial cells in vitro to show that the Sdc1 regulatory mechanism is also required for integrin activation on these cells. We found Sdc1 expressed in the vascular endothelium during microvessel outgrowth from aortic explants in vitro and in mouse mammary tumors in vivo. Moreover, we show that SSTN blocks angiogenesis in vitro or when delivered systemically in a mouse model of angiogenesis in vivo, and impairs mammary tumor growth in an orthotopic mouse tumor model. Thus, Sdc1 is a critical regulator of these two important integrins during angiogenesis and tumorigenesis, and is inhibited by the novel SSTN peptide.

Syndecan-1-mediated cell spreading requires signaling by αvβ3 integrins in human breast carcinoma cells

Abstract Syndecans are cell surface heparan sulfate proteoglycans with regulatory roles in cell adhesion, proliferation, and differentiation [Annu. Rev. Biochem. 68 (1999) 729]. While the syndecan heparan sulfate chains are essential for matrix binding, less is known about the signaling role of their core proteins. To mimic syndecan-specific adhesion, MDA-MB-231 mammary carcinoma cells were plated on antibodies against syndecan-4 or syndecan-1. While cells adherent via syndecan-4 spread, cells adherent via syndecan-1 do not. However, cells adherent via syndecan-1 can be induced to spread by Mn2+, suggesting that activation of a β1 or β3 integrin partner is required. Surprisingly, pretreatment of cells with a function-activating β1 antibody does not induce spreading, whereas function-blocking β1 integrin antibodies do, suggesting involvement of a β1-to-β3 integrin cross-talk. Indeed, blockade of β1 integrin activation induces αvβ3 integrin activation detectable by soluble fibrinogen binding. Spreading in response to syndecan-1 is independent of integrin-ligand binding. Furthermore, competition with soluble murine syndecan-1 ectodomain, which does not disrupt cell adhesion, nonetheless blocks the spreading mechanism. These data suggest that the ectodomain of the syndecan-1 core protein directly participates in the formation of a signaling complex that signals in cooperation with αvβ3 integrins; signaling via this complex is negatively regulated by β1 integrins.

Syndecan-1 couples the insulin-like growth factor-1 receptor to inside-out integrin activation

Syndecan-1 (Sdc1) engages and activates the αvβ3 (and/or αvβ5) integrin when clustered in human carcinoma and endothelial cells. Although the engagement is extracellular, the activation mechanism is cytoplasmic. This talin-dependent, inside-out signaling pathway is activated downstream of the insulin-like growth factor-1 receptor (IGF1R), whose kinase activity is triggered by Sdc1 clustering. In vitro binding assays using purified receptors suggest that association of the Sdc1 ectodomain with the integrin provides a ‘docking face’ for IGF1R. IGF1R docking and activation of the associated integrin is blocked by synstatin (SSTN92–119), a peptide derived from the integrin engagement site in Sdc1. IGF1R colocalizes with αvβ3 integrin and Sdc1 in focal contacts, but fails to associate with or activate the integrin in cells either lacking Sdc1 or expressing Sdc1Δ67–121, a mutant that is unable to form the Sdc1–integrin–IGF1R ternary complex. Integrin activation is also blocked by IGF1R inhibitors or by silencing IGF1R or talin expression with small-interfering RNAs (siRNAs). In both cases, expression of the constitutively active talin F23 head domain rescues integrin activation. We recently reported that SSTN92–119 blocks angiogenesis and impairs tumor growth in mice, therefore this Sdc1-mediated integrin regulatory mechanism might be a crucial regulator of disease processes known to rely on these integrins, including tumor cell metastasis and tumor-induced angiogenesis.

Vascular endothelial‐cadherin stimulates syndecan‐1‐coupled insulin‐like growth factor‐1 receptor and cross‐talk between αVβ3 integrin and vascular endothelial growth factor receptor 2 at the onset of endothelial cell dissemination during angiogenesis

Vascular endothelial growth factor (VEGF)‐stimulated angiogenesis depends on a cross‐talk mechanism involving VEGF receptor 2 (VEGFR2), vascular endothelial (VE)‐cadherin and the αVβ3 integrin. Because we have shown that αVβ3 integrin activation is dependent on its incorporation, along with the insulin‐like growth factor‐1 receptor (IGF1R) kinase, into a ternary receptor complex organized by the matrix receptor syndecan‐1 (Sdc1), we questioned the role of this core complex in VEGF‐stimulated angiogenesis. We find that the Sdc1‐coupled ternary receptor complex is required for VEGF signalling and for stimulation of vascular endothelial cell migration by vascular endothelial cadherin (VE‐cadherin) engagement. VE‐cadherin binding to Fc/VE‐cadherin extracellular domain chimera activates Sdc1‐coupled IGF1R and αvβ3 integrin; this depends on VEGFR2 and c‐Src activated by the cadherin. Blocking homotypic VE‐cadherin engagement disrupts VEGF‐stimulated cell migration, which is restored by clustering the cadherin in the absence of cell–cell adhesion. This cadherin‐dependent stimulation requires VEGFR2 and IGF1R and is blocked by synstatin (SSTN)92–119, a peptide that competitively disrupts the Sdc1‐coupled ternary complex and prevents the αVβ3 integrin activation required for VEGFR2 activation. VEGFR2‐stimulated angiogenesis in the mouse aortic ring explant assay is disrupted by SSTN, although only early in the process, suggesting that IGF1R coupling to Sdc1 and αVβ3 integrin comprises a core activation mechanism activated by VE‐cadherin that is necessary for VEGFR2 and integrin activation in the initial stages of endothelial cell dissemination during angiogenesis.

Cytoplasmic Domain Interactions of Syndecan-1 and Syndecan-4 with α6β4 Integrin Mediate Human Epidermal Growth Factor Receptor (HER1 and HER2)-dependent Motility and Survival

Background: The α6β4 integrin assembles via an unknown mechanism with receptor tyrosine kinases. Results: HER2-dependent activation of α6β4 depends on capture of the β4 cytoplasmic domain by syndecan-1, whereas HER1 (EGFR) relies on syndecan-4. Conclusion: Cell invasion and survival mediated by the α6β4 integrin depend on its assembly with kinase-specific syndecans. Significance: These novel interactions may provide targets for new therapeutics to combat carcinogenesis. Epithelial cells are highly dependent during wound healing and tumorigenesis on the α6β4 integrin and its association with receptor tyrosine kinases. Previous work showed that phosphorylation of the β4 subunit upon matrix engagement depends on the matrix receptor syndecan (Sdc)-1 engaging the cytoplasmic domain of the β4 integrin and coupling of the integrin to human epidermal growth factor receptor-2 (HER2). In this study, HER2-dependent migration activated by matrix engagement is compared with migration stimulated by EGF. We find that whereas HER2-dependent migration depends on Sdc1, EGF-dependent migration depends on a complex consisting of human epidermal growth factor receptor-1 (HER1, commonly known as EGFR), α6β4, and Sdc4. The two syndecans recognize distinct sites at the extreme C terminus of the β4 integrin cytoplasmic domain. The binding motif in Sdc1 is QEEXYX, composed in part by its syndecan-specific variable (V) region and in part by the second conserved (C2) region that it shares with other syndecans. A cell-penetrating peptide containing this sequence competes for HER2-dependent epithelial migration and carcinoma survival, although it is without effect on the EGFR-stimulated mechanism. β4 mutants bearing mutations specific for Sdc1 and Sdc4 recognition act as dominant negative mutants to block cell spreading or cell migration that depends on HER2 or EGFR, respectively. The interaction of the α6β4 integrin with the syndecans appears critical for it to be utilized as a signaling platform; migration depends on α3β1 integrin binding to laminin 332 (LN332; also known as laminin 5), whereas antibodies that block α6β4 binding are without effect. These findings indicate that specific syndecan family members are likely to have key roles in α6β4 integrin activation by receptor tyrosine kinases.

Syndecan-1 (CD138) Suppresses Apoptosis in Multiple Myeloma by Activating IGF1 Receptor: Prevention by SynstatinIGF1R Inhibits Tumor Growth

Syndecan-1 (Sdc1/CD138) expression is linked to disease severity in multiple myeloma, although the causal basis for this link remains unclear. Here we report that capture of the IGF1 receptor (IGF1R) by Sdc1 suppresses ASK1-dependent apoptosis in multiple myeloma cells. Sdc1 binds two different fractions of IGF1R, one that is constitutively active and a second that is activated by IGF1 ligand. Notably, IGF1R kinase activity in both fractions is blocked by synstatinIGF1R (SSTNIGF1R), a peptide that inhibits IGF1R capture by Sdc1, as well as by a truncated peptide (SSTNIGF1R-T) that appears to be specific for multiple myeloma cells. Mechanistically, we show that ASK1 is bound to active IGF1R and inhibited by Tyr and Ser83/Ser966 phosphorylation. When IGF1R engagement with Sdc1 is blocked by SSTNIGF1R, ASK1 becomes activated, and initiates JNK- and caspase-3-mediated apoptosis. In pharmacologic tests, we find SSTNIGF1R is highly stable in human plasma and displays a half-life of 27 hours in mice, wherein it significantly reduces both the size and neovascularization of CAG myeloma tumor xenografts. Taken together, our results offer a preclinical proof of concept and mechanistic rationale for the exploration of SSTNIGF1R as an experimental therapeutic to dually attack multiple myeloma tumor cell survival and tumor angiogenesis. Cancer Res; 76(17); 4981-93. ©2016 AACR.

MST1R/RON and EGFR in a complex with syndecans sustain carcinoma S-phase progression by preventing p38MAPK activation

Syndecan-4 (Sdc4) organizes a complex of receptors consisting of its homologue, Sdc2, the receptor tyrosine kinases EGFR and MST1R/RON, and the laminin-binding α3β1 and α6β4 integrins that depends on a docking site within its extracellular domain. A peptide mimetic of the extracellular docking site, synstatin-EGFR (SSTNEGFR), disrupts the receptor complex and prevents the invasion of non-transformed or carcinoma cells that relies on active EGFR. However, the peptide also prevents DNA replication that relies on active MST1R/RON and c-Abl kinase within the complex, resulting in rapid S-phase arrest of head & neck (HN) and breast carcinoma cells. SSTNEGFR does not affect DNA replication in non-transformed oral or breast epithelial cells, but it does block their EGF-dependent invasion. Although EGFR is required as a component of the complex, its kinase activity is not required to sustain S-phase progression in the carcinoma cells, perhaps explaining why many HN and breast carcinomas that overexpress EGFR are nonetheless refractory to EGFR inhibitors. The syndecan-organized receptor complex (Sdc:RTK:ITG complex) appears to suppress stress signals that would otherwise disrupt the replisome engaged in DNA synthesis. SSTNEGFR-treatment of carcinoma cells, or normal oral epithelial cells expressing stress-inducing HPV oncogenes, causes rapid activation of the p38 stress MAPK leading to loss of PCNA from the chromatin and cessation of DNA synthesis. This arrest is independent of the common DNA damage response (DDR) known to activate an S-phase checkpoint, revealing a novel arrest mechanism and a novel receptor complex that is activated on tumor cells to suppress stress-induced proliferation arrest.