Imatinib blocks migration and invasion of medulloblastoma cells by concurrently inhibiting activation of platelet-derived growth factor receptor and transactivation of epidermal growth factor receptor.
ABSTRACT: Platelet-derived growth factor (PDGF) receptor (PDGFR) expression correlates with metastatic medulloblastoma. PDGF stimulation of medulloblastoma cells phosphorylates extracellular signal-regulated kinase (ERK) and promotes migration. We sought to determine whether blocking PDGFR activity effectively inhibits signaling required for medulloblastoma cell migration and invasion. DAOY and D556 human medulloblastoma cells were treated with imatinib mesylate (Gleevec), a PDGFR tyrosine kinase inhibitor, or transfected with small interfering RNA (siRNA) to PDGFRB to test the effects of blocking PDGFR phosphorylation and expression, respectively. PDGFR cell signaling, migration, invasion, survival, and proliferation following PDGF-BB stimulation, with and without PDGFR inhibition, were measured. PDGF-BB treatment of cells increased PDGFRB, Akt and ERK phosphorylation, and transactivated epidermal growth factor receptor (EGFR), which correlated with enhanced migration, survival, and proliferation. Imatinib (1 ?mol/L) treatment of DAOY and D556 cells inhibited PDGF-BB- and serum-mediated migration and invasion at 24 and 48 h, respectively, and concomitantly inhibited PDGF-BB activation of PDGFRB, Akt, and ERK but increased PTEN expression and activity. Imatinib treatment also induced DAOY cell apoptosis at 72 h and inhibited DAOY and D556 cell proliferation at 48 h. siRNA silencing of PDGFRB similarly inhibited signaling, migration, and survival and both siRNA and imatinib treatment inhibited PDGF-BB-mediated EGFR transactivation, indicating that the effects of imatinib treatment are specific to PDGFRB target inhibition. These results indicate that PDGFRB tyrosine kinase activity is critical for migration and invasion of medulloblastoma cells possibly by transactivating EGFR; thus, imatinib may represent an important novel therapeutic agent for the treatment of medulloblastoma.
Project description:Oral squamous cell carcinoma (OSCC) is a cancerous disease with poor prognosis. According to the statistics, the 5-year survival rate has not improved significantly over the past 20 years. The platelet-derived growth factor (PDGF) and its signaling pathway is a key regulator of angiogenesis and tumorigenesis. High level of PDGF and its receptor (PDGFR) have been reported in several types of malignancies. In this study, we investigated the relationship of the molecular expression levels of PDGF and PDGFR with clinicopathological parameters in OSCC. To this end, we measured the mRNA and protein levels of PDGF and PDGFR by real-time quantitative PCR (qRT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA), respectively. We found positive correlations of the mRNA levels of PDGFA, PDGFB, and PDGFRB with lymph node metastasis and poor overall survival (OS). High expression of PDGF, PDGFRA, and PDGFRB were remarkably associated with lymph node metastasis and poor OS, as determined by immunohistochemistry. Preoperative serum levels of PDGF-AA and PDGF-BB had a positive correlation with preoperative platelet count. Elevated serum levels of PDGF-AA. PDGF-BB, and platelet count correlated with lymph node metastasis and an unfavorable outcome. In multivariate Cox regression analysis, PDGFA mRNA, PDGFB mRNA, PDGFRB mRNA, PDGF immunoexpression, PDGFRB immunoexpression, serum PDGF-AA, serum PDGF-BB, and platelet count emerged as significant independent prognostic factors for OS. In vitro, we found that elevated PDGF promotes colony formation, migration, and invasiveness of SAS and OECM-1 cancer cell lines. Our results suggest that the expression level of serum PDGF has the potential to become a useful diagnostic marker for the prognosis of OSCC. In addition, PDGFR should be considered as a potential therapeutic target for OSCC. Furthermore, research should be undertaken to elucidate the role of PDGF and PDGFR regarding the behavior of tumor cells in OSCC.
Project description:PDGF-BB/PDGFR? signaling plays an important role during vascularization by mediating pericyte recruitment to the vasculature, promoting the integrity and function of vessels. Until now it has not been possible to assess the specific role of PDGFR? signaling in tumor progression and angiogenesis due to lack of appropriate animal models and molecular tools. Methods: In the present study, we used a transgenic knock-in mouse strain carrying a silent mutation in the PDGFR? ATP binding site that allows specific targeting of PDGFR? using the compound 1-NaPP1. To evaluate the impact of selective PDGFR? inhibition of stromal cells on tumor growth we investigated four tumor cell lines with no or low PDGFR? expression, i.e. Lewis lung carcinoma (LLC), EO771 breast carcinoma, B16 melanoma and a version of B16 that had been engineered to overexpress PDGF-BB (B16/PDGF-BB). Results: We found that specific impairment of PDGFR? kinase activity by 1-NaPP1 treatment efficiently suppressed growth in tumors with high expression of PDGF-BB, i.e. LLC and B16/PDGF-BB, while the clinically used PDGFR? kinase inhibitor imatinib did not suppress tumor growth. Notably, tumors with low levels of PDGF-BB, i.e. EO771 and B16, neither responded to 1-NaPP1 nor to imatinib treatment. Inhibition of PDGFR? by either drug impaired tumor vascularization and also affected pericyte coverage; however, specific targeting of PDGFR? by 1-NaPP1 resulted in a more pronounced decrease in vessel function with increased vessel apoptosis in high PDGF-BB expressing tumors, compared to treatment with imatinib. In vitro analysis of PDGFR? ASKA mouse embryo fibroblasts and the mesenchymal progenitor cell line 10T1/2 revealed that PDGF-BB induced NG2 expression, consistent with the in vivo data. Conclusion: Specific targeting of PDGFR? signaling significantly inhibits tumor progression and angiogenesis depending on PDGF-BB expression. Our data suggest that targeting PDGFR? in the tumor stroma could have therapeutic value in patients with high tumor PDGF-BB expression.
Project description:Pericytes and vascular smooth muscle cells (VSMCs), which are recruited to developing blood vessels by platelet-derived growth factor BB, support endothelial cell survival and vascular stability. Here, we report that imatinib, a tyrosine kinase inhibitor of platelet-derived growth factor receptor ? (PDGFR?), impaired growth of lymphoma in both human xenograft and murine allograft models. Lymphoma cells themselves neither expressed PDGFR? nor were growth inhibited by imatinib. Tumor growth inhibition was associated with decreased microvascular density and increased vascular leakage. In vivo, imatinib induced apoptosis of tumor-associated PDGFR?(+) pericytes and loss of perivascular integrity. In vitro, imatinib inhibited PDGFR?(+) VSMC proliferation and PDGF-BB signaling, whereas small interfering RNA knockdown of PDGFR? in pericytes protected them against imatinib-mediated growth inhibition. Fluorescence-activated cell sorter analysis of tumor tissue revealed depletion of pericytes, endothelial cells, and their progenitors following imatinib treatment. Compared with imatinib, treatment with an anti-PDGFR? monoclonal antibody partially inhibited lymphoma growth. Last, microarray analysis (Gene Expression Omnibus database accession number GSE30752) of PDGFR?(+) VSMCs following imatinib treatment showed down-regulation of genes implicated in vascular cell proliferation, survival, and assembly, including those representing multiple pathways downstream of PDGFR?. Taken together, these data indicate that PDGFR?(+) pericytes may represent a novel, nonendothelial, antiangiogenic target for lymphoma therapy.
Project description:Stromal cell-derived growth factor (SDF)-1? acts as a ligand to C-X-C chemokine receptors 4 (CXCR4) and 7 (CXCR7), which are involved in the formation of choroidal neovascularization. Previous studies have demonstrated crosstalk between the platelet-derived growth factor (PDGF)-BB/PDGF receptor (PDGFR)-? and SDF-1?/CXCR4 axes during tumor neovascularization by increasing the recruitment of pericytes. However, the effects of interactions between these two signaling pathways in retinal microvascular pericytes remain poorly understood. Western blotting and reverse transcription-quantitative PCR were used to measure CXCR4 and CXCR7 expression in PDGF-BB-treated pericytes, whilst Cell Counting Kit-8 and Transwell migration assays were used to investigate cell viability and migration following PDGF-BB pretreatment on SDF-1?-treated pericytes. Exogenous PDGF-BB enhanced CXCR4 and CXCR7 expression through PDGFR-? in a dose- and time-dependent manners. In addition, PDGF-BB increased cell viability and migration in SDF-1?-treated pericytes, which were inhibited by AMD3100 and niclosamide, inhibitors for CXCR4 and STAT3 respectively. Crosstalk between PDGF-BB/PDGFR-? and SDF-1?/CXCR4/CXCR7 were involved in the JAK2/STAT3 signaling pathway. PDGF-BB treatment enhanced CXCR4, CXCR7 and PDGFR-?expression, which may be associated with the phosphorylation of STAT3. siRNA-PDGFR-? transfection reduced CXCR4 and CXCR7 expression in pericytes. Therefore, PDGF-BB directly targets PDGFR-? and serves an important role in regulating CXCR4 and CXCR7 expression, ultimately affecting viability and migration in SDF-1?-treated pericytes. Therefore, targeting CXCR4/CXCR7 may serve as a potential therapeutic strategy for fundus diseases.
Project description:Three causal genes for idiopathic basal ganglia calcification (IBGC) have been identified. Most recently, mutations in PDGFRB, encoding a member of the platelet-derived growth factor receptor family type ?, and PDGFB, encoding PDGF-B, the specific ligand of PDGFR?, were found implicating the PDGF-B/PDGFR? pathway in abnormal brain calcification. In this study, we aimed to identify and study mutations in PDGFRB and PDGFB in a series of 26 patients from the Mayo Clinic Florida Brain Bank with moderate to severe basal ganglia calcification (BCG) of unknown etiology. No mutations in PDGFB were found. However, we identified one mutation in PDGFRB, p.R695C located in the tyrosine kinase domain, in one BGC patient. We further studied the function of p.R695C mutant PDGFR? and two previously reported mutants, p.L658P and p.R987W PDGFR? in cell culture. We show that, in response to PDGF-BB stimulation, the p.L658P mutation completely suppresses PDGFR? autophosphorylation, whereas the p.R695C mutation results in partial loss of autophosphorylation. For the p.R987W mutation, our data suggest a different mechanism involving reduced protein levels. These genetic and functional studies provide the first insight into the pathogenic mechanisms associated with PDGFRB mutations and provide further support for a pathogenic role of PDGFRB mutations in BGC.
Project description:We previously identified that overexpression of the platelet-derived growth factor receptor (PDGFR) is associated with metastatic medulloblastoma (MB) and showed that PDGF treatment increases ERK activity and promotes MB cell migration. In this study, we investigated whether ERK regulates Rac1/Pak1 signaling and is critically linked to MB cell migration. Herein we demonstrate that PDGF-BB treatment of MB cells induces concomitant activation of PDGFR?, MEK1/ERK, Rac1 and Pak1, but suppresses Rho activity, which together significantly promotes cell migration. Conversely, cells transfected with either PDGFR? or Pak1 siRNA or treated with an inhibitor of Rac1 (NSC23766) or N-myristoyltransferase-1 (Tris-dipalladium) are unable to activate Rac1 or Pak1 in response to PDGF, and consequently, are unable to undergo PDGF-mediated cell migration. Furthermore, we also demonstrate that either chemical inhibition of MEK/ERK (U0126) or stable downregulation of PDGFR? by shRNA similarly results in the loss of PDGF-induced ERK phosphorylation and abolishes Rac1/Pak1 activation and cell migration in response to PDGF. However, specific depletion of Pak1 by siRNA has no effect on PDGF-induced ERK phosphorylation, indicating that in MB cells ERK signaling is Pak1-independent, but PDGF-induced migration is dependent on ERK-mediated activation of Pak1. Finally, using tissue microarrays, we detect phosphorylated Pak1 in 53% of medulloblastomas and show that immunopositivity is associated with unfavorable outcome. We conclude that Rac1/Pak1 signaling is critical to MB cell migration and is functionally dependent on PDGFR?/ERK activity.
Project description:Endothelial barrier dysfunction is a critical pathophysiological process of sepsis. Impaired endothelial cell migration is one of the main reasons for endothelial dysfunction. Statins may have a protective effect on endothelial barrier function. However, the effect and mechanism of statins on lipopolysaccharide (LPS)-induced endothelial barrier dysfunction remain unclear. Simvastatin (SV) was loaded in nanostructured lipid carriers to produce SV nanoparticles (SV-NPs). Normal SV and SV-NPs were used to treat human umbilical vein vascular endothelial cells (HUVECs) injured by LPS. Barrier function was evaluated by monitoring cell monolayer permeability and transendothelial electrical resistance, and cell migration ability was measured by a wound healing assay. LY294002 and imatinib were used to inhibit the activity of PI3K/Akt and platelet-derived growth factor receptor (PDGFR) ?. IQ-GTPase-activating protein 1 (IQGAP1) siRNA was used to knockdown endogenous IQGAP1, which was used to verify the role of the PDGFR?/PI3K/Akt/IQGAP1 pathway in SV/SV-NPs-mediated barrier protection in HUVECs injured by LPS. The results show that SV/SV-NPs promoted the migration and decreased the permeability of HUVECs treated with LPS, and the efficacy of the SV-NPs exceeded that of SV significantly. LY294002, imatinib and IQGAP1 siRNA all suppressed the barrier protection of SV/SV-NPs. SV/SV-NPs promoted the secretion of platelet-derived growth factor-BB (PDGF-BB) and activated the PDGFR?/PI3K/Akt/IQGAP1 pathway. SV preparations restored endothelial barrier function by restoring endothelial cell migration, which is involved in the regulation of the PDGFR?/PI3K/Akt/IQGAP1 pathway and PDGF-BB secretion. As an appropriate formulation for restoring endothelial dysfunction, SV-NPs may be more effective than SV.
Project description:Cholangiocarcinomas (CCAs) are highly desmoplastic neoplasms with a tumour microenvironment plentiful in myofibroblasts (MFBs). MFB-derived PDGF-BB survival signalling is a mediator of CCA cell resistance to apoptotic stimuli. This raises the concept that targeting PDGFR-?, a cognate receptor of PDGF-BB, represents a potential strategy for the treatment of human CCA.Herein, we examine a role for inhibiting PDGFR-? in restoring CCA cell sensitivity to apoptotic stimuli in vitro and in vivo.We employed human CCA samples from 41 patients (19 intrahepatic and 22 extrahepatic CCA samples), the human CCA cell lines KMCH-1 and HUCCT-1 as well as shPDGFR-?-KMCH-1 and human myofibroblastic LX-2 cells for these studies. In vivo-experiments were conducted using a syngeneic rat orthotopic CCA model.Of several MFB-derived growth factors profiled, PDGF-BB and CTGF were most abundantly expressed; however, only PDGF-BB attenuated tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Co-culturing CCA cells with PDGF-BB-secreting MFBs significantly decreased TRAIL-induced CCA cell apoptosis when compared with monoculture conditions; this cytoprotective effect was abrogated in the presence of the tyrosine kinase inhibitors imatinib mesylate or linifanib, which inhibit PDGFR-?. Consistent with these findings, MFB-imparted cytoprotection also was abolished when PDGFR-? was knocked down as demonstrated in shPDGFR-?-KMCH-1 cells. Finally, administration of imatinib mesylate increased CCA cell apoptosis and reduced tumour growth in a rodent in vivo-CCA model that mimics the human disease.Targeting PDGFR-? sensitizes CCA cells to apoptotic stimuli and appears to be therapeutic in vivo.
Project description:OBJECTIVE:Pericytes/pericyte precursors produce milk fat globule-associated protein with epidermal growth factor and factor VIII-like domains (MFG-E8) in vivo, and this ?(v) integrin ligand enhances angiogenesis in tumors and in oxygen-induced retinopathy in mice. Inhibition of MFG-E8 production or function attenuates platelet-derived growth factor-BB (PDGF-BB)-induced migration of pericyte/pericyte precursor-like 10T1/2 cells in vitro. Herein, we describe mechanisms by which MFG-E8 modulates PDGF-BB:PDGF receptor ? (PDGFR?) signaling in 10T1/2 cells. METHODS AND RESULTS:Small interfering RNA depletion of MFG-E8 from 10T1/2 cells or antibody inhibition of MFG-E8 action enhanced PDGF-BB-dependent degradation of PDGFR? and attenuated signaling. Coimmunoprecipitation revealed transient association of MFG-E8 with PDGFR? in PDGF-BB-treated 10T1/2 cells and reduced PDGFR?-focal adhesion kinase association in MFG-E8-depleted cells. Confocal microscopy demonstrated that MFG-E8 binding to 10T1/2 cells was RGD motif and ?(v) dependent but PDGF-BB treatment independent, whereas colocalization of MFG-E8 with PDGFR? was enhanced by PDGF-BB. Ubiquitination of PDGFR? was also increased in MFG-E8 small interfering RNA-transfected cells. CONCLUSION:Integrin ?(v)-bound MFG-E8 associates with PDGFR? and focal adhesion kinase after PDGF-BB treatment, results in cell surface retention of PDGFR?, delays receptor degradation, potentiates downstream signaling, and enhances migration of 10T1/2 cells. MFG-E8 may promote angiogenesis, in part, via cell autonomous actions on pericytes or pericyte precursors that result in enhanced PDGF-BB:PDGFR? signaling mediated via integrin-growth factor receptor cross-talk.
Project description:NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1?C). NRP1 co-immunoprecipitated with PDGFR? (PDGF receptor ?), and immunofluorescent staining indicated that NRP1 and PDGFR? co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFR? activation. NRP1-specific siRNA, Ad.NRP1?C and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFR? and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.