Growth Factor-β Receptor Signaling Research Articles

Targeting hyperactive platelet-derived growth factor receptor-β signaling in T-cell acute lymphoblastic leukemia and lymphoma.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are rare aggressive hematologic malignancies. Current treatment consists of intensive chemotherapy leading to 80% overall survival but is associated with severe toxic side effects. Furthermore, 10-20% of patients still die from relapsed or refractory disease providing a strong rationale for more specific, targeted therapeutic strategies with less toxicities. Here, we report a novel MYH9::PDGFRB fusion in a T-LBL patient, and demonstrate that this fusion product is constitutively active and sufficient to drive oncogenic transformation in vitro and in vivo. Expanding our analysis more broadly across T-ALL, we found a T-ALL cell line and multiple patient-derived xenograft models with PDGFRB hyperactivation in the absence of a fusion, with high PDGFRB expression in TLX3 and HOXA T-ALL molecular subtypes. To target this PDGFRB hyperactivation, we evaluated the therapeutic effects of a selective PDGFRB inhibitor, CP-673451, both in vitro and in vivo and demonstrated sensitivity if the receptor is hyperactivated. Altogether, our work reveals that hyperactivation of PDGFRB is an oncogenic driver in T-ALL/T-LBL, and that screening T-ALL/T-LBL patients for phosphorylated PDGFRB levels can serve as a biomarker for PDGFRB inhibition as a novel targeted therapeutic strategy in their treatment regimen.

LAMC2 is a potential prognostic biomarker for cholangiocarcinoma.

Cholangiocarcinoma is a common malignancy with increasing incidence worldwide. Most patients are diagnosed at the advanced stage with poor survival rate. Laminin subunit γ2 (LAMC2) is a heparin binding-associated gene involved in tumorigenesis and has been implicated in the prognosis of various types of cancers. However, it is unclear whether expression of LAMC2 is associated with the clinical outcome of patients with cholangiocarcinoma. In the present study, the role and prognostic value of LAMC2 expression in patients with cholangiocarcinoma was investigated. Clinical information and pathological characteristics were analyzed and the association between LAMC2 expression and clinical characteristics, pathological findings and patient outcomes, including metastasis-free and disease-specific survival, were investigated. Data from 182 patients with cholangiocarcinoma were evaluated. High LAMC2 expression was associated with higher tumor stage (P<0.001), large duct type (P=0.024) and poor histological grade (P=0.002). Kaplan-Meier analysis showed high LAMC2 expression was associated with lower overall (P=0.003), disease-specific (P=0.0025), local recurrence-free (P<0.0001) and metastasis-free survival (P<0.0001). Moreover, multivariate analysis demonstrated that increased LAMC2 expression was a significant predictive risk factor for overall [hazard ratio (HR) 1.713; P=0.034], disease-specific (HR 2.011; P=0.039), local recurrence-free (HR 2.721; P<0.001) and metastasis-free survival (HR 3.117; P<0.001). Gene enrichment analysis using Gene Ontology showed that terms associated with LAMC2 upregulation were 'regulation of platelet-derived growth factor receptor-βsignaling pathway' and 'platelet-derived growth factor receptor-β signaling pathway'. The present study indicated that LAMC2 was upregulated in cholangiocarcinoma tumor tissue and had an inverse association with overall, disease-specific, local recurrence-free and metastasis-free survival in patients with cholangiocarcinoma. These results suggested that LAMC2 may serve as a potential biomarker for cholangiocarcinoma.

Stromal Platelet-Derived Growth Factor Receptor-β Signaling Promotes Breast Cancer Metastasis in the Brain.

Platelet-derived growth factor receptor-beta (PDGFRβ) is a receptor tyrosine kinase found in cells of mesenchymal origin such as fibroblasts and pericytes. Activation of this receptor is dependent on paracrine ligand induction, and its preferred ligand PDGFB is released by neighboring epithelial and endothelial cells. While expression of both PDGFRβ and PDGFB has been noted in patient breast tumors for decades, how PDGFB-to-PDGFRβ tumor-stroma signaling mediates breast cancer initiation, progression, and metastasis remains unclear. Here we demonstrate this paracrine signaling pathway that mediates both primary tumor growth and metastasis, specifically, metastasis to the brain. Elevated levels of PDGFB accelerated orthotopic tumor growth and intracranial growth of mammary tumor cells, while mesenchymal-specific expression of an activating mutant PDGFRβ (PDGFRβD849V) exerted proproliferative signals on adjacent mammary tumor cells. Stromal expression of PDGFRβD849V also promoted brain metastases of mammary tumor cells expressing high PDGFB when injected intravenously. In the brain, expression of PDGFRβD849V was observed within a subset of astrocytes, and aged mice expressing PDGFRβD849V exhibited reactive gliosis. Importantly, the PDGFR-specific inhibitor crenolanib significantly reduced intracranial growth of mammary tumor cells. In a tissue microarray comprised of 363 primary human breast tumors, high PDGFB protein expression was prognostic for brain metastases, but not metastases to other sites. Our results advocate the use of mice expressing PDGFRβD849V in their stromal cells as a preclinical model of breast cancer-associated brain metastases and support continued investigation into the clinical prognostic and therapeutic use of PDGFB-to-PDGFRβ signaling in women with breast cancer. SIGNIFICANCE: These studies reveal a previously unknown role for PDGFB-to-PDGFRβ paracrine signaling in the promotion of breast cancer brain metastases and support the prognostic and therapeutic clinical utility of this pathway for patients.See related article by Wyss and colleagues, p. 594.

Abstract PD9-11: Platelet derived growth factor receptor-β signaling: A novel therapeutic target for breast cancer associated brain metastasis

Abstract PDGFRβ is a receptor tyrosine kinase found in cells of mesenchymal origin such as fibroblasts and pericytes. Activation of this receptor is dependent on paracrine ligand induction, and its preferred ligand, PDGFB, is released by neighboring epithelial and endothelial cells. While expression of both PDGFRβ and PDGFB has been noted in patient breast tumors for decades, how PDGFB-to-PDGFRβ tumor-stromal signaling mediates breast cancer initiation, progression, and metastasis remains unclear. To test this important research question, we developed a mouse model of mesenchymal-specific PDGFRβ hyper-activation. PDGFRβ mutant mammary glands exhibit increased tertiary side-branching and epithelial proliferation confirming a stromal-specific PDGFRβ effect on neighboring epithelium during normal development. To test the effect of hyper-active mesenchymal PDGFRβ on disease progression, experimental tail vein metastasis assays were performed where we observed prominent brain metastases in 50% of the PDGFRβ mutantmice (n=5/10) with no brain lesions seen in controls (n=0/19). There was no difference in the incidence of lung or liver metastases in the mutant mice suggesting a pro-metastatic function for PDGFRβ in the brain metastatic niche. To rule out dysfunction of the blood brain barrier contributing to the observed metastatic spread, we then intracranially injected mammary tumor cells, and as expected based on our metastasis assay, found that larger tumors formed in the brains of PDGFRβ mutant mice versus controls. To our knowledge, these combined findings are the first example where genetic manipulation of the stroma increases breast cancer associated brain metastases (BCBM). Given that these pre-clinical data suggest that primary breast tumors expressing high PDGFB could preferentially metastasize to the brain, we analyzed PDGFB protein expression in a tissue microarray comprised of HER2-positive and triple negative breast cancer (TNBC) primary tumors (total n=425). While high PDGFB did not correlate with site-independent metastatic recurrence, it was prognostic of brain metastasis, mirroring our mouse data. Evaluation of PDGFB in a small cohort of matched primary breast tumors with associated brain (n=5) and lung metastases (n=2) revealed intense PDGFB staining in 100% of the brain metastases, but only 50% of the lung metastases. These findings further suggest that high primary tumor PDGFBexpression defines a subset of breast cancer patients predisposed to brain metastases and that these patients may benefit from therapeutic inhibition of PDGFRβ signaling. To test this pre-clinically, we treated mice harboring intracranial tumors with the PDGFR specific inhibitor crenolanib. Excitingly, crenolanib treatment significantly inhibited the brain tumor burden in these mice. Combined, our findings to date (1) advocate that primary tumor expression of PDGFB is a novel prognostic biomarker for the development of BCBM and (2) support clinical trial evaluation of PDGFR inhibitors for the prevention and treatment of BCBM. Ongoing studies are evaluating how the PDGFRβ-expressing mesenchymal cells within the brain promote a pro-metastatic niche. Citation Format: Sizemore GM, Hammer AM, Thies KA, Hildreth BE, Russell LO, Sizemore ST, Trimboli AJ, Kladney RD, Steck SA, Das M, Bolyard CM, Pilarski R, Schoenfield L, Otero J, Chakravarti A, Ringel M, Kaur B, Leone G, Ostrowski MC. Platelet derived growth factor receptor-β signaling: A novel therapeutic target for breast cancer associated brain metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-11.

Regional early and progressive loss of brain pericytes but not vascular smooth muscle cells in adult mice with disrupted platelet-derived growth factor receptor-β signaling.

Pericytes regulate key neurovascular functions of the brain. Studies in pericyte-deficient transgenic mice with aberrant signaling between endothelial-derived platelet-derived growth factor BB (PDGF-BB) and platelet-derived growth factor receptor β (PDGFRβ) in pericytes have contributed to better understanding of the role of pericytes in the brain. Here, we studied PdgfrβF7/F7 mice, which carry seven point mutations that disrupt PDGFRβ signaling causing loss of pericytes and vascular smooth muscle cells (VSMCs) in the developing brain. We asked whether these mice have a stable or progressive vascular phenotype after birth, and whether both pericyte and VSMCs populations are affected in the adult brain. We found an early and progressive region-dependent loss of brain pericytes, microvascular reductions and blood-brain barrier (BBB) breakdown, which were more pronounced in the cortex, hippocampus and striatum than in the thalamus, whereas VSMCs population remained unaffected at the time when pericyte loss was already established. For example, compared to age-matched controls, PdgfrβF7/F7 mice between 4–6 and 36–48 weeks of age developed a region-dependent loss in pericyte coverage (22–46, 24–44 and 4–31%) and cell numbers (36–49, 34–64 and 11–36%), reduction in capillary length (20–39, 13–46 and 1–30%), and an increase in extravascular fibrinogen-derived deposits (3.4–5.2, 2.8–4.1 and 0–3.6-fold) demonstrating BBB breakdown in the cortex, hippocampus and thalamus, respectively. Capillary reductions and BBB breakdown correlated with loss of pericyte coverage. Our data suggest that PdgfrβF7/F7 mice develop an aggressive and rapid vascular phenotype without appreciable early involvement of VSMCs, therefore providing a valuable model to study regional effects of pericyte loss on brain vascular and neuronal functions. This model could be a useful tool for future studies directed at understanding the role of pericytes in the pathogenesis of neurological disorders associated with pericyte loss such as vascular dementia, Alzheimer’s disease, amyotrophic lateral sclerosis, stroke and human immunodeficiency virus-associated neurocognitive disorder.

The Role of Platelet-Derived Growth Factor-B/Platelet-Derived Growth Factor Receptor-β Signaling in Chronic Atrial Fibrillation

Objective: To explore the role of platelet-derived growth factor-B (PDGF-B)/platelet-derived growth factor receptor-β (PDGFR-β) signaling in chronic atrial fibrillation (AF). Methods: Thirty-nine AF patients and 33 patients with sinus rhythm (SR) were enrolled. Twenty canines were randomized into 5 groups: control, sham and AF lasting 1, 2 or 4 weeks. The AF canine models were made by rapid atrial pacing. Rat atrial fibroblasts were treated with PDGF-BB or PDGF-BB + PDGFR inhibitor AG1295, respectively. Gene expression in the right atrial appendage of patients, the left atrium of canines and rat atrial fibroblasts was measured by quantitative real-time PCR and Western blot, respectively. The degree of atrial fibrosis was evaluated by Masson trichrome staining. Results: The degree of atrial fibrosis and the expression of PDGF-B, PDGFR-β and collagen type I (COL1) in AF patients significantly increased compared to patients with SR. The degree of atrial fibrosis and the expression of PDGF-B and COL1 in canines increased progressively with the increased duration of AF. The expression of PDGFR-β increased progressively 2 weeks after AF. PDGF-BB promoted the proliferation and COL1 secretion of rat atrial fibroblasts. AG1295 attenuated these effects. Conclusions: Our study suggests that PDGF-B/PDGFR-β signaling, which promotes the proliferation and COL1 secretion of atrial fibroblasts, is an important contributor to atrial fibrosis in AF and may represent a novel target for the intervention of AF.

Platelet‐derived growth factor‐BB increases expression of connexin 43 in an extracellular‐regulated protein kinase‐dependent manner in bladder smooth muscle cells

To investigate whether platelet-derived growth factor-BB induces the upregulation of connexin 43 in bladder smooth muscle cells and to examine the involved signaling pathway. Bladder smooth muscle cells were exposed to platelet-derived growth factor-BB in the presence or absence of p38 mitogen-activated protein kinase, c-jun amino-terminal kinase or extracellular-regulated protein kinase inhibitors. Transfection of bladder smooth muscle cells with specific small interference ribonucleic acid against platelet-derived growth factor receptor-β gene expression was also carried out to investigate whether platelet-derived growth factor receptor-β was involved in the signaling pathway. Expression of messenger ribonucleic acid and protein for connexin 43 was measured by real time polymerase chain reaction and western blot. The addition of platelet-derived growth factor-BB in cultured bladder smooth muscle cells caused the significant upregulation of connexin 43, and the activation of extracellular-regulated protein kinase, c-jun amino-terminal kinase and p38 mitogen-activated protein kinase compared with the control group. This action of platelet-derived growth factor-BB could be abolished by the pretreatment of bladder smooth muscle cells with the extracellular-regulated protein kinase inhibitor PD98059, whereas p38 mitogen-activated protein kinase and c-jun amino-terminal kinase inhibitors did not have any effect on this. Platelet-derived growth factor-BB could induce the activation of platelet-derived growth factor receptor-β. Transfection of bladder smooth muscle cells with small interference ribonucleic acid specific for platelet-derived growth factor receptor-β gene resulted in the potent suppression of gene expression and inhibition of extracellular-regulated protein kinase activation, as well as upregulation of connexin 43 induced by platelet-derived growth factor-BB. Platelet-derived growth factor-BB upregulates connexin 43 expression through the activation of extracellular-regulated protein kinase and platelet-derived growth factor receptor-β signaling pathways. This finding suggests that this signaling pathway might provide a potential target to manipulate detrusor overactivity.

Potentiation of Platelet-Derived Growth Factor Receptor-β Signaling Mediated by Integrin-Associated MFG-E8

Pericytes/pericyte precursors produce milk fat globule-associated protein with epidermal growth factor and factor VIII-like domains (MFG-E8) in vivo, and this α(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 β (PDGFRβ) signaling in 10T1/2 cells. Small interfering RNA depletion of MFG-E8 from 10T1/2 cells or antibody inhibition of MFG-E8 action enhanced PDGF-BB-dependent degradation of PDGFRβ and attenuated signaling. Coimmunoprecipitation revealed transient association of MFG-E8 with PDGFRβ in PDGF-BB-treated 10T1/2 cells and reduced PDGFRβ-focal adhesion kinase association in MFG-E8-depleted cells. Confocal microscopy demonstrated that MFG-E8 binding to 10T1/2 cells was RGD motif and α(v) dependent but PDGF-BB treatment independent, whereas colocalization of MFG-E8 with PDGFRβ was enhanced by PDGF-BB. Ubiquitination of PDGFRβ was also increased in MFG-E8 small interfering RNA-transfected cells. Integrin α(v)-bound MFG-E8 associates with PDGFRβ and focal adhesion kinase after PDGF-BB treatment, results in cell surface retention of PDGFRβ, 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:PDGFRβ signaling mediated via integrin-growth factor receptor cross-talk.

Platelet-derived growth factor receptor-α is a key determinant of smooth muscle α-actin filaments in bone marrow-derived mesenchymal stem cells

Smooth muscle α-actin filaments are a defining feature of mesenchymal stem cells, and of mesenchyme-derived contractile smooth muscle cells, pericytes and myofibroblasts. Here, we show that adult bone marrow-derived mesenchymal stem cells express abundant cell surface platelet-derived growth factor receptor-α, having a high ratio to platelet-derived growth factor receptor-β. Signaling through platelet-derived growth factor receptor-α increases smooth muscle α-actin filaments by activating RhoA, which results in Rho-associated kinase (ROCK)-dependent cofilin phosphorylation, enhancing smooth muscle α-actin filament polymerization, and also upregulates smooth muscle α-actin expression. In contrast, platelet-derived growth factor receptor-β signaling strongly upregulates RhoE, which inhibits ROCK activity, promoting smooth muscle α-actin filament depolymerization. This study thus provides new insights into the distinct roles of platelet-derived growth factor receptor-α and -β signaling in regulating the adult mesenchymal stem cell contractile cytoskeleton.

Shb promotes blood vessel formation in embryoid bodies by augmenting vascular endothelial growth factor receptor-2 and platelet-derived growth factor receptor-β signaling

The mechanisms controlling blood vessel formation during early embryonal development have only partly been elucidated. Shb is an adaptor protein previously implicated in the angiogenic response to vascular endothelial growth factor (VEGF). To elucidate a possible role of Shb in embryonic vascular development, wild-type and SH2 domain mutated (R522K) Shb were overexpressed in murine embryonic stem (ES) cells. Embryoid bodies (EBs) differentiating from Shb-overexpressing ES cells in vitro were stained for CD31 or VEGFR-2 to visualize the formation of vascular structures. We found that Shb promotes the outgrowth of blood vessels in EBs both in the absence and presence of growth factors. This response may be the consequence of an increased number of VEGFR-2 positive cells at an early stage of EB development, a finding corroborated by both immunostaining and real-time RT-PCR. In addition, Shb overexpression upregulated the expression of PDGFR-β, CD31, CD41 and Tal1. Cells co-expressing VEGFR-2 and PDGFR-β were commonly observed when Shb was overexpressed and inhibition of PDGF-BB signaling reduced the amount of VEGFR-2 mRNA under these conditions. EBs expressing the Shb R522K-mutant did not form vascular structures. Microarray analysis of VEGFR-2/CD31 positive cells after 6 days of differentiation revealed numerous changes of expression of genes relating to an endothelial/hematopoietic phenotype in response to Shb overexpression. The findings suggest that Shb may play a crucial role during early ES cell differentiation to vascular structures by transducing VEGFR-2 and PDGFR-β signals.