The Glypican-1/HGF/C-Met and Glypican-1/VEGF/VEGFR2 Ternary Complexes Regulate Hair Follicle Angiogenesis.
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ABSTRACT:
SUBMITTER: Colin-Pierre C
PROVIDER: S-EPMC8692797 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) drive cancer through their respective receptors, MET and VEGF receptor 2 (VEGFR2). VEGFR2 inhibits MET by promoting MET dephosphorylation. However, whether MET conversely regulates VEGFR2 remains unknown. Here we show that MET suppresses VEGFR2 protein by inducing its endoplasmic-reticulum-associated degradation (ERAD), via intracrine VEGF action. HGF-MET signaling in epithelial cancer cells promoted VEGF biosynthesis through PI3-kinase. In turn, VEGF and VEGFR2 associated within the ER, activating inositol-requiring enzyme 1?, and thereby facilitating ERAD-mediated depletion of VEGFR2. MET disruption upregulated VEGFR2, inducing compensatory tumor growth via VEGFR2 and MEK. However, concurrent disruption of MET and either VEGF or MEK circumvented this, enabling more profound tumor inhibition. Our findings uncover unique cross-regulation between MET and VEGFR2-two RTKs that play significant roles in tumor malignancy. Furthermore, these results suggest rational combinatorial strategies for targeting RTK signaling pathways more effectively, which has potentially important implications for cancer therapy.
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Project description:The etiology and progression of a variety of human malignancies are linked to the deregulation of receptor tyrosine kinases (RTKs). To define the role of RTK-dependent signals in various oncogenic processes, we have previously engineered RTK oncoproteins that recruit either the Shc or Grb2 adaptor proteins. Although these RTK oncoproteins transform cells with similar efficiencies, fibroblasts expressing the Shc-binding RTK oncoproteins induced tumors with short latency (approximately 7 days), whereas cells expressing the Grb2-binding RTK oncoproteins induced tumors with delayed latency (approximately 24 days). The early onset of tumor formation correlated with the ability of cells expressing the Shc-binding RTK oncoproteins to produce vascular endothelial growth factor (VEGF) in culture and an angiogenic response in vivo. Consistent with this, treatment with a VEGF inhibitor, VEGF-Trap, blocked the in vivo angiogenic and tumorigenic properties of these cells. The importance of Shc recruitment to RTKs for the induction of VEGF was further demonstrated by using mutants of the Neu/ErbB2 RTK, where the Shc, but not Grb2, binding mutant induced VEGF. Moreover, the use of fibroblasts derived from ShcA-deficient mouse embryos, demonstrated that Shc was essential for the induction of VEGF by the Met/hepatocyte growth factor RTK oncoprotein and by serum-derived growth factors. Together, our findings identify Shc as a critical angiogenic switch for VEGF production downstream from the Met and ErbB2 RTKs.
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Project description:Inhibition of VEGF signaling leads to a proinvasive phenotype in mouse models of glioblastoma multiforme (GBM) and in a subset of GBM patients treated with bevacizumab. Here, we demonstrate that vascular endothelial growth factor (VEGF) directly and negatively regulates tumor cell invasion through enhanced recruitment of the protein tyrosine phosphatase 1B (PTP1B) to a MET/VEGFR2 heterocomplex, thereby suppressing HGF-dependent MET phosphorylation and tumor cell migration. Consequently, VEGF blockade restores and increases MET activity in GBM cells in a hypoxia-independent manner, while inducing a program reminiscent of epithelial-to-mesenchymal transition highlighted by a T-cadherin to N-cadherin switch and enhanced mesenchymal features. Inhibition of MET in GBM mouse models blocks mesenchymal transition and invasion provoked by VEGF ablation, resulting in substantial survival benefit.
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Project description:RATIONALE:We previously reported that vascular endothelial growth factor (VEGF)-induced binding of VEGF receptor 2 (VEGFR2) to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2. However, the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. OBJECTIVE:The goals for the present study were as follows: (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain whether such determinants were critical for physiological angiogenesis in vivo. METHODS AND RESULTS:Structural modeling uncovered 2 novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Furthermore, we found that the VEGF-induced VEGFR2-epsin interaction promoted casitas B-lineage lymphoma-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. CONCLUSIONS:Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.
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Project description:VEGF binding to VEGFR2 leads to VEGFR2 endocytosis and downstream signaling activation to promote angiogenesis. Methods: Using genetic strategies, we tested the requirement of ? subunits of heterotrimeric G proteins (G?i1/3) in the process. Results: G?i1/3 are located in the VEGFR2 endocytosis complex (VEGFR2-Ephrin-B2-Dab2-PAR-3), where they are required for VEGFR2 endocytosis and downstream signaling transduction. G?i1/3 knockdown, knockout or dominant negative mutation inhibited VEGF-induced VEGFR2 endocytosis, and downstream Akt-mTOR and Erk-MAPK activation. Functional studies show that G?i1/3 shRNA inhibited VEGF-induced proliferation, invasion, migration and vessel-like tube formation of HUVECs. In vivo, G?i1/3 shRNA lentivirus inhibited alkali burn-induced neovascularization in mouse cornea. Further, oxygen-induced retinopathy (OIR)-induced retinal neovascularization was inhibited by intravitreal injection of G?i1/3 shRNA lentivirus. Moreover, in vivo angiogenesis by alkali burn and OIR was significantly attenuated in G?i1/3 double knockout mice. Significantly, G?i1/3 proteins are upregulated in proliferative retinal tissues of proliferative diabetic retinopathy (PDR) patients. Conclusion: These results provide mechanistic insights into the critical role played by G?i1/3 proteins in VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis.
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Project description:BackgroundActivation of autocrine VEGF-VEGFR2 signalling in tumour cells activates cell proliferation, survival, and angiogenesis, all of which are crucial for tumour progression. Ovarian cancer-associated antigen 66 (OVA66) is now known to be overexpressed in multiple tumours and plays a role in tumour development, but the underlying mechanisms has not been fully investigated.MethodsWe employed ovarian and cervical cancer cells and mouse models to detect the role of OVA66 in angiogenesis, growth, and metastasis of cancer cells. Immunofluorescence and western blot were used to determine the function of OVA66 in regulating autocrine VEGF-VEGFR2 signalling. Immunohistochemistry and bioinformatics analysis were used to detect the correlation of OVA66 and VEGF expression.FindingsOVA66 overexpression in the cancer cell lines promoted VEGF secretion, tumour growth and angiogenesis in vitro and in vivo. Conversely, shRNA-mediated OVA66 knockdown had the opposite effects. Mechanistically, OVA66 overexpression was found to boost an autocrine VEGF-VEGFR2 positive-feedback signalling loop in the tumour cells, leading to amplified effect of VEGF on tumour angiogenesis and proliferation and increased migration in vitro and in vivo, respectively. Finally, we identified a significant positive correlation between the expression levels of OVA66 and VEGF in ovarian and cervical cancer specimens, and found that OVA66 was significantly associated with advanced ovarian cancer.InterpretationWe identify a novel function for OVA66 in regulating an autocrine VEGF-VEGFR2 feed-forward signalling loop that promotes tumour progression and angiogenesis. FUND: This work was supported by the National Natural Science Foundation of China (81602262); and Excellent Youth Scholar Program of Tongji University (2015KJ062).
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Project description:The molecular mechanisms that control the balance between antiangiogenic and proangiogenic factors and initiate the angiogenic switch in tumors remain poorly defined. By combining chemical genetics with multimodal imaging, we have identified an autocrine feed-forward loop in tumor cells in which tumor-derived VEGF stimulates VEGF production via VEGFR2-dependent activation of mTOR, substantially amplifying the initial proangiogenic signal. Disruption of this feed-forward loop by chemical perturbation or knockdown of VEGFR2 in tumor cells dramatically inhibited production of VEGF in vitro and in vivo. This disruption was sufficient to prevent tumor growth in vivo. In patients with lung cancer, we found that this VEGF:VEGFR2 feed-forward loop was active, as the level of VEGF/VEGFR2 binding in tumor cells was highly correlated to tumor angiogenesis. We further demonstrated that inhibition of tumor cell VEGFR2 induces feedback activation of the IRS/MAPK signaling cascade. Most strikingly, combined pharmacological inhibition of VEGFR2 (ZD6474) and MEK (PD0325901) in tumor cells resulted in dramatic tumor shrinkage, whereas monotherapy only modestly slowed tumor growth. Thus, a tumor cell-autonomous VEGF:VEGFR2 feed-forward loop provides signal amplification required for the establishment of fully angiogenic tumors in lung cancer. Interrupting this feed-forward loop switches tumor cells from an angiogenic to a proliferative phenotype that sensitizes tumor cells to MAPK inhibition.
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Project description:Alternative pathways to the VEGF, such as hepatocyte growth factor or HGF/c-met, are emerging as key players in tumor angiogenesis and resistance to anti-VEGF therapies. The aim of this study was to assess the effects of a combination strategy targeting the VEGF and c-met pathways in clear cell renal cell carcinoma (ccRCC) models. Male SCID mice (8/group) were implanted with 786-O tumor pieces and treated with either a selective VEGF receptor tyrosine kinase inhibitor, axitinib (36 mg/kg, 2×/day); a c-met inhibitor, crizotinib (25 mg/kg, 1×/day); or combination. We further tested this drug combination in a human ccRCC patient-derived xenograft, RP-R-01, in both VEGF-targeted therapy-sensitive and -resistant models. To evaluate the resistant phenotype, we established an RP-R-01 sunitinib-resistant model by continuous sunitinib treatment (60 mg/kg, 1×/day) of RP-R-01-bearing mice. Treatment with single-agent crizotinib reduced tumor vascularization but failed to inhibit tumor growth in either model, despite also a significant increase of c-met expression and phosphorylation in the sunitinib-resistant tumors. In contrast, axitinib treatment was effective in inhibiting angiogenesis and tumor growth in both models, with its antitumor effect significantly increased by the combined treatment with crizotinib, independently from c-met expression. Combination treatment also induced prolonged survival and significant tumor growth inhibition in the 786-O human RCC model. Overall, our results support the rationale for the clinical testing of combined VEGF and HGF/c-met pathway blockade in the treatment of ccRCC, both in first- and second-line setting.
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