Platelet-derived Growth Factor Ligands Research Articles

Abstract B066: PDGFR upregulation functions as a bypass mechanism contributing to FGFR inhibitor resistance in metastatic breast cancer

Abstract Breast cancer (BC) is the most diagnosed cancer in females in the United States and the second leading cause of cancer related deaths among women. The fibroblast growth factor receptor (FGFR) signaling pathway is frequently activated in BC, making it an attractive therapeutic target, especially in metastatic disease. However, clinical trials using FGFR inhibitors in BC have been disappointing in comparison to other cancer types treated with these therapies. As a result, BC patients with activated FGFR do not have a targeted therapeutic option. Failure of FGFR inhibitors is indicative of intrinsic or acquired resistance mechanisms in metastatic BC. Herein we demonstrate significant efficacy of the FGFR inhibitor, pemigatinib, in the 4T07 murine model of BC utilizing a tail vein model to induce pulmonary tumors. Despite this initial response, pemigatinib fails to eliminate minimal residual disease (MRD) and allows for accelerated tumor return after pemigatinib cessation. Characterizing the MRD by IHC following pemigatinib treatment shows that expression of platelet-derived growth factor receptor (PDGFR) is significantly increased on tumor cells. This rise in PDGFR expression following pemigatinib treatment increases the responsiveness of 4T07 cells to platelet-derived growth factor ligand indicating PDGFR may act as an alternate signaling pathway bypassing FGFR signaling. Mechanistically, previous studies indicate that PDGFR protein expression is transcriptionally repressed by restriction of enhancer positioning by the CTCF insulator. DNA methylation surrounding CTCF causes its dissociation enhancing protein expression of PDGFR. Along these lines cotreatment with a DNA methyl transferase inhibitor prevents the increase of PDGFR protein expression induced by pemigatinib. Overall, our study determined that a highly potent FGFR inhibitor can inhibit tumor growth in a metastatic site, but it fails to eliminate MRD giving rise to tumor recurrence. Upregulation of PDGFR may act as a bypass mechanism of resistance allowing for tumor cell survival during pemigatinib treatment. Finally, our findings suggest that dual inhibition of DNA methylation and FGFR could improve patient response in metastatic BC. Citation Format: Mitchell Ayers, Michael Wendt. PDGFR upregulation functions as a bypass mechanism contributing to FGFR inhibitor resistance in metastatic breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B066.

Stiffness-induced cancer-associated fibroblasts are responsible for immunosuppression in a platelet-derived growth factor ligand-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a vast stromal reaction that arises mainly from cancer-associated fibroblasts (CAFs) and promotes both immune escape and tumor growth. Here, we used a mouse model with deletion of the activin A receptor ALK4 in the context of the KrasG12D mutation, which strongly drives collagen deposition that leads to tissue stiffness. By ligand-receptor analysis of single-cell RNA-sequencing data, we identified that, in stiff conditions, neoplastic ductal cells instructed CAFs through sustained platelet-derived growth factor (PDGF) signaling. Tumor-associated tissue rigidity resulted in the emergence of stiffness-induced CAFs (siCAFs) in vitro and in vivo. Similar results were confirmed in human data. siCAFs were able to strongly inhibit CD8+ T-cell responses in vitro and in vivo, promoting local immunosuppression. More importantly, targeting PDGF signaling led to diminished siCAF and reduced tumor growth. Our data show for the first time that early paracrine signaling leads to profound changes in tissue mechanics, impacting immune responses and tumor progression. Our study highlights that PDGF ligand neutralization can normalize the tissue architecture independent of the genetic background, indicating that finely tuned stromal therapy may open new therapeutic avenues in pancreatic cancer.

PDGF signaling inhibits mitophagy in glioblastoma stem cells through N6-methyladenosine.

Dysregulated growth factor receptor pathways, RNA modifications, and metabolism each promote tumor heterogeneity. Here, we demonstrate that platelet-derived growth factor (PDGF) signaling induces N6-methyladenosine (m6A) accumulation in glioblastoma (GBM) stem cells (GSCs) to regulate mitophagy. PDGF ligands stimulate early growth response 1 (EGR1) transcription to induce methyltransferase-like 3 (METTL3) to promote GSC proliferation and self-renewal. Targeting the PDGF-METTL3 axis inhibits mitophagy by regulating m6A modification of optineurin (OPTN). Forced OPTN expression phenocopies PDGF inhibition, and OPTN levels portend longer survival of GBM patients; these results suggest a tumor-suppressive role for OPTN. Pharmacologic targeting of METTL3 augments anti-tumor efficacy of PDGF receptor (PDGFR) and mitophagy inhibitors invitro and invivo. Collectively, we define PDGF signaling as an upstream regulator of oncogenic m6A regulation, driving tumor metabolism to promote cancer stem cell maintenance, highlighting PDGF-METTL3-OPTN signaling as a GBM therapeutic target.

Role of PDGF-A/B Ligands in Cardiac Repair After Myocardial Infarction.

Platelet-derived growth factors (PDGFs) are powerful inducers of cellular mitosis, migration, angiogenesis, and matrix modulation that play pivotal roles in the development, homeostasis, and healing of cardiac tissues. PDGFs are key signaling molecules and important drug targets in the treatment of cardiovascular disease as multiple researchers have shown that delivery of recombinant PDGF ligands during or after myocardial infarction can reduce mortality and improve cardiac function in both rodents and porcine models. The mechanism involved cannot be easily elucidated due to the complexity of PDGF regulatory activities, crosstalk with other protein tyrosine kinase activators, and diversity of the pathological milieu. This review outlines the possible roles of PDGF ligands A and B in the healing of cardiac tissues including reduced cell death, improved vascularization, and improved extracellular matrix remodeling to improve cardiac architecture and function after acute myocardial injury. This review may highlight the use of recombinant PDGF-A and PDGF-B as a potential therapeutic modality in the treatment of cardiac injury.

PDGF-PDGFR network differentially regulates the fate, migration, proliferation, and cell cycle progression of myogenic cells

Platelet-derived growth factors (PDGFs) regulate embryonic development, tissue regeneration, and wound healing through their binding to PDGF receptors, PDGFRα and PDGFRβ. However, the role of PDGF signaling in regulating muscle development and regeneration remains elusive, and the cellular and molecular responses of myogenic cells are understudied. Here, we explore the PDGF-PDGFR gene expression changes and their involvement in skeletal muscle myogenesis and myogenic fate. By surveying bulk RNA sequencing and single-cell profiling data of skeletal muscle stem cells, we show that myogenic progenitors and muscle stem cells differentially express PDGF ligands and PDGF receptors during myogenesis. Quiescent adult muscle stem cells and myoblasts preferentially express PDGFRβ over PDGFRα. Remarkably, cell culture- and injury-induced muscle stem cell activation altered PDGF family gene expression. In myoblasts, PDGF-AB and PDGF-BB treatments activate two pro-chemotactic and pro-mitogenic downstream transducers, RAS-ERK1/2 and PI3K-AKT. PDGFRs inhibitor AG1296 inhibited ERK1/2 and AKT activation, myoblast migration, proliferation, and cell cycle progression induced by PDGF-AB and PDGF-BB. We also found that AG1296 causes myoblast G0/G1 cell cycle arrest. Remarkably, PDGF-AA did not promote a noticeable ERK1/2 or AKT activation, myoblast migration, or expansion. Also, myogenic differentiation reduced the expression of both PDGFRα and PDGFRβ, whereas forced PDGFRα expression impaired myogenesis. Thus, our data highlight PDGF signaling pathway to stimulate satellite cell proliferation aiming to enhance skeletal muscle regeneration and provide a deeper understanding of the role of PDGF signaling in non-fibroblastic cells.

Expression and prognostic significance of PDGF ligands and receptors across soft tissue sarcomas

BackgroundWhile the anti-PDGFRA antibody olaratumab failed to confirm an impact on survival in unselected advanced soft tissue sarcoma (STS) patients, the level of expression and the prognosis of platelet-derived growth factor (PDGF) receptors and ligands in STS remain unclear.Patients and methodsWe analyzed PDGF ligands and receptors' expression levels in a series of 255 patients with different histologies of STS [gastrointestinal stromal tumor (GIST), myxoid liposarcoma (MLPS), sarcoma with complex genomics, synovial sarcoma (SyS)] with Agilent single-color micro-arrays. We explored expression levels as prognostic values in univariate and multivariate analysis using R software (version 3.4.2).ResultsComplex patterns of correlation of expression between ligands and receptors were observed for each histotype. PDGFA levels were highest in SyS and lowest in MLPS (P < 4 × 10−9), PDGFB and C levels were lower in GIST (P < 2 × 10−15 and P < 3 × 10−9) while PDGFD expression was similar across histological subtypes. PDGF receptor (PDGFR) A expression was lowest in MLPS (P < 0.002), whereas PDGFRB and L expressions were lowest in GIST and SyS (P < 0.0004). Interestingly, high PDGFA expression levels were associated with higher risk of metastasis (P = 0.006), whereas PDGFD levels above average were associated with a reduced risk of metastasis (P = 0.01) in univariate and multivariate analysis.ConclusionsThe expression of PDGF ligands and receptors varies across sarcoma histological subtypes. PDGFA and D expression levels independently and inversely correlate with the risk of metastatic relapse.

Platelet‑derived growth factor‑BB mediates pancreatic cancer malignancy via regulation of the Hippo/Yes‑associated protein signaling pathway.

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant that serves a role in the development of several types of solid cancer, and abnormal PDGF activity has been reported in numerous human tumors. Tumor-derived PDGF ligands are considered to act in either a paracrine or autocrine manner, serving roles in the phosphorylation of receptors on tumor and stromal cells in the tumor microenvironment. Despite the well-established association between PDGF and tumor progression, the precise mechanisms of autocrine PDGF signaling in pancreatic tumor cells remain elusive. Therefore, the present study aimed to analyze the influence of PDGF-BB in pancreatic cancer. Pancreatic adenocarcinoma BxPC-3 cells were cultured and treated with recombinant human PDGF-BB in vitro. Cell proliferation was tested using an MTT assay. Cell apoptosis was measured using flow cytometry. Tumor cell migration and invasion were examined via wound-healing and Transwell assays, respectively. The expression and subcellular localization of Yes-associated protein (YAP) was determined using western blotting and immunofluorescence. The transcriptional activity of target genes was tested using a luciferase assay and reverse transcription-quantitative PCR. The present study revealed that PDGF-BB significantly promoted cell proliferation in pancreatic adenocarcinoma BxPC-3 cells and enhanced the aggressiveness of this cell line, as demonstrated by Transwell and wound-healing assays. Anoikis resistance is an important mechanism by which metastatic cells avoid apoptosis when detaching from adjacent cells or the extracellular matrix. PDGF-BB treatment inhibited anoikis under anchorage-independent conditions. Mechanistic experiments revealed that PDGF-BB promoted the upregulation and activation of the transcriptional coactivator YAP, an effector of the Hippo signaling pathway. RhoA or protein phosphatase-1 (PP-1) inhibition partially abolished the accumulation and activation of YAP, suggesting PDGF-BB-mediated YAP dephosphorylation and transactivation via the RhoA/PP-1 cascade. Pharmacologic inhibition of the PDGF receptor directly downregulated YAP activity and the expression levels of downstream genes. Furthermore, verteporfin, a small molecular inhibitor of the Hippo/YAP signaling pathway, partially reversed the effects of PDGF-BB on cell proliferation, anoikis resistance and cell migration. In conclusion, the present study revealed that the Hippo/YAP signaling pathway may be involved in the tumor-promoting activity of PDGF-BB in pancreatic cancer.

Engineered receptors for human cytomegalovirus that are orthogonal to normal human biology.

A trimeric glycoprotein complex on the surface of human cytomegalovirus (HCMV) binds to platelet-derived growth factor (PDGF) receptor α (PDGFRα) to mediate host cell recognition and fusion of the viral and cellular membranes. Soluble PDGFRα potently neutralizes HCMV in tissue culture, and its potential use as an antiviral therapeutic has the benefit that any escape mutants will likely be attenuated. However, PDGFRα binds multiple PDGF ligands in the human body as part of developmental programs in embryogenesis and continuing through adulthood. Any therapies with soluble receptor therefore come with serious efficacy and safety concerns, especially for the treatment of congenital HCMV. Soluble virus receptors that are orthogonal to human biology might resolve these concerns. This engineering problem is solved by deep mutational scanning on the D2-D3 domains of PDGFRα to identify variants that maintain interactions with the HCMV glycoprotein trimer in the presence of competing PDGF ligands. Competition by PDGFs is conformation-dependent, whereas HCMV trimer binding is independent of proper D2-D3 conformation, and many mutations at the receptor-PDGF interface are suitable for functionally separating trimer from PDGF interactions. Purified soluble PDGFRα carrying a targeted mutation succeeded in displaying wild type affinity for HCMV trimer with a simultaneous loss of PDGF binding, and neutralizes trimer-only and trimer/pentamer-expressing HCMV strains infecting fibroblasts or epithelial cells. Overall, this work makes important progress in the realization of soluble HCMV receptors for clinical application.

Abstract P6-06-06: Platelet derived growth factor-b (PDGFB) promotes breast cancer progression

Abstract Objectives of the Study: Platelet-derived growth factor (PDGF) mediated signaling is pro-tumorigenic in many types of cancer, including breast cancer. However, the requirement of individual PDGF ligands in mediating breast cancer progression remains unclear. Our evaluation of publicly available breast cancer patient datasets shows that high primary tumor expression of PDGFB correlates with reduced overall survival (OS) and reduced metastasis free survival (MFS). This is in contrast to the lack of prognostic power of PDGFA and PDGFC and the association with improved OS and MFS for PDGFD. Based on our analysis of patient datasets, we set out to test the requirement of PDGFB in mammary tumor growth. Methods Used: Gain-of-function and loss-of-function experiments were performed wherein PDGFB was either overexpressed or knocked-down in mammary tumor cells. Manipulated tumor cells were injected directly into the mammary fat pads of adult female mice, and tumor growth was monitored over time. PDGFB is produced by tumor cells whereas the corresponding receptor, PDGFRβ, is expressed by mesenchymal cells in the stroma. As another way to mimic oncogenic PDGFB-to-PDGFRβ signaling in the breast, we developed a mouse model of stroma-specific PDGFRβ activation using the Fsp-cre transgene. We evaluated changes mammary gland development upon mesenchymal specific PDGFRβ activation, and performed orthotopic injections with mammary tumor cells in these animals to test the functional role of receptor activation in mammary tumor growth. Results and Conclusions: We evaluated expression of PDGF ligands in FVB/N murine mammary tumor cell lines and found that the PDGFB is dramatically higher in DB7 tumor cells compared to other syngeneic cell lines. In the high-PDGFB expressing DB7 cells, we used shRNA technology to knockdown the ligand. At the same time, we overexpressed the ligand in an isogenic cell line that expresses low levels of PDGFB. These cells were injected orthotopically into the mammary fat pads of adult female mice, and in both cases, expression of PDGFB dictated tumor growth. There was a significant reduction in tumor growth with shRNA-mediated knockdown of PDGFB whereas overexpression of the ligand accelerated tumor growth. In our mouse model of mesenchymal-specific PDGFRβ activation, we reveal that activation of the receptor exerts pro-proliferative signals on adjacent mammary epithelial cells and accelerates orthotopic tumor growth of PDGFB-expressing cells. These findings indicate that PDGFB-to-PDGFRβ signaling is a viable therapeutic target for breast cancer. In fact, treatment with the PDGFR inhibitor, imatinib, impedes tumor cell proliferation when mouse mammary fibroblasts are co-injected orthotopically with DB7 cells. Significance: The requirement of other PDGF ligands in breast cancer remains to be evaluated, but our data support a pro-tumorigenic role for PDGFB in the breast. Importantly, PDGFR inhibitors are being used in clinical trials for several cancer types. Our data advocate for (1) the potential utility of PDGFB as a prognostic biomarker and (2) the pre-clinical evaluation of PDGFR inhibitors in breast cancer models. Citation Format: Katie A Thies, Anisha M Hammer, Sarah A Steck, Manjusri Das, Raleigh D Kladney, Steven T Sizemore, Michael C Ostrowski, Gina M Sizemore. Platelet derived growth factor-b (PDGFB) promotes breast cancer progression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-06.

PDGF signaling from pharyngeal pouches promotes arch artery morphogenesis

The great vessels of the heart originate from the pharyngeal arch arteries (PAAs). Anomalies of the PAAs often occur together with pharyngeal pouch malformations, but the reasons for this phenomenon are not fully understood. In the current study, we show that platelet-derived growth factor (PDGF) signaling derived from the pharyngeal pouches plays an important function in PAA vasculogenesis. During PAA development in zebrafish embryos, pdgfαa and pdgfαb are expressed in the developing pharyngeal pouches. Results from loss-of-function experiments revealed a critical role of these genes in PAA formation. We found that nitroreductase (NTR)-mediated pouch ablation distinctly decreased PDGF receptor tyrosine phosphorylation, yielding a severe loss of PAAs. Importantly, pouch-specific overexpression of pdgfαa in pdgfαa−/−; pdgfαb−/− mutants significantly relieved the PAA defects, which indicated a primary role of pharyngeal pouch-expressed PDGF ligands in signal activation and PAA morphogenesis. Our findings further showed that PDGF signaling was indispensable for the proliferation of PAA angioblasts. Together, these results established a role for PDGFαa- and PDGFαb-mediated tissue-tissue interaction during PAA development.

Lysosomal Pathways and Autophagy Distinctively Control Endothelial Cell Behavior to Affect Tumor Vasculature

Cancer cell-stromal cell crosstalk is orchestrated by a plethora of ligand-receptor interactions generating a tumor microenvironment (TME) which favors tumor growth. The high pro-angiogenic nature of the TME perpetuates the chaotic network of structurally immature, low pericyte-covered vessels characteristic of the tumor vasculature. We previously demonstrated that chloroquine (CQ) -a lysosomotropic agent used as first-generation autophagy blocker in clinical trials- induced tumor vessel normalization and reduced tumor hypoxia. CQ improved both vessel structure and maturation, whereas the conditional knockout of the crucial autophagy gene Atg5 in endothelial cells (ECs) did not, thus highlighting a potential differential role for EC-associated autophagy and the lysosomes in pathological tumor angiogenesis. However, how CQ or ATG5-deficiency in ECs affect angiogenic signals regulating EC-pericyte interface and therefore vessel maturation, remains unknown. Here, we show that in ECs CQ constrained VEGF-A-mediated VEGF receptor (VEGFR)2 phosphorylation, a driver of angiogenic signaling. In the presence of CQ we observed increased expression of the decoy receptor VEGFR1 and of a lower molecular weight form of VEGFR2, suggesting receptor cleavage. Consequently, VEGF-A-driven EC spheroid sprouting was reduced by CQ treatment. Furthermore, CQ significantly affected the transcription and secretion of platelet-derived growth factor (PDGF)-AB/BB (upregulated) and Endothelin-1 (EDN1, downregulated), both modulators of perivascular cell (PC) behavior. In contrast, silencing of ATG5 in ECs had no effect on VEGFR2 to VEGFR1 ratio nor on PDGFB and EDN1 expression. Accordingly, mice harboring B16F10 melanoma tumors treated with CQ, displayed both an increased number of αSMA+ PCs covering tumor vessels and co-expressed PDGF receptor-β, enabling PDGF ligand dependent recruitment. Moreover, upon CQ treatment the tumoral expression of angiopoietin-1 (Angpt1), which retains mural cells, and induces vessel stabilization by binding to the EC-localized cognate receptor (TIE2), was increased thus supporting the vessel normalization function of CQ. These features associated with improved tumor vasculature were not phenocopied by the specific deletion of Atg5 in ECs. In conclusion, this study further unravels endothelial cell autonomous and non-autonomous mechanisms by which CQ “normalizes” the intercellular communication in the tumor vasculature independent of autophagy.

Involvement of platelet-derived growth factor ligands and receptors in tumorigenesis.

Platelet-derived growth factor (PDGF) isoforms and their receptors have important roles during embryogenesis, particularly in the development of various mesenchymal cell types in different organs. In the adult, PDGF stimulates wound healing and regulates tissue homeostasis. However, overactivity of PDGF signalling is associated with malignancies and other diseases characterized by excessive cell proliferation, such as fibrotic conditions and atherosclerosis. In certain tumours, genetic or epigenetic alterations of the genes for PDGF ligands and receptors drive tumour cell proliferation and survival. Examples include the rare skin tumour dermatofibrosarcoma protuberance, which is driven by autocrine PDGF stimulation due to translocation of a PDGF gene, and certain gastrointestinal stromal tumours and leukaemias, which are driven by constitute activation of PDGF receptors due to point mutations and formation of fusion proteins ofthe receptors, respectively. Moreover, PDGF stimulates cells in tumour stroma and promotes angiogenesis as well as the development of cancer-associated fibroblasts, both of which promote tumour progression. Inhibitors of PDGF signalling may thus be of clinical usefulness in the treatment of certain tumours.

Abstract 3911: Stromal platelet derived growth factor receptor-beta (PDGFRbeta) promotes breast brain metastasis

Abstract The platelet derived growth factor (PDGF) pathway is a prime example of tumor-stroma signaling in a number of cancer types. Others have shown that PDGF receptors are expressed in breast fibroblasts and pericytes while PDGF ligands are often expressed in breast cancer cells and tumor-associated endothelium; however, how PDGF signaling mediates breast cancer initiation, progression and metastasis remains unclear. Importantly, our evaluation of publicly available datasets revealed that PDGFB expression correlates with breast cancer patient metastatic recurrence leading to the hypothesis that PDGF-B to PDGFR signaling promotes metastatic progression of breast cancer. Given that PDGF-B preferentially activates PDGFRβ, we established an in vivo system to investigate this pathway during breast cancer progression. We utilized a mesenchymal-specific promoter to drive Cre recombinase and conditionally activate PDGFRβ by way of the endogenous Pdgfrb promoter (hereafter “PDGFRβ mutant”). A murine mammary tumor cell line which expresses high levels of PDGF-B was injected either by tail vein or intracranially to evaluate metastatic seeding and distant tumor growth. Following tail vein injection of tumor cells, we observed 50% incidence of brain metastases in the PDGFRβ mutant mice while no brain lesions were seen in the controls. There was no difference in incidence of lung, liver or bone metastases (other common sites of breast cancer metastasis). Not surprisingly, larger tumors formed in the brains of PDGFRβ mutant mice when cells were injected intracranially. Brains were stained for phospho-PLCγ as a way to confirm activation of PDGFRβ. To our knowledge, this is the first example where genetic manipulation of the stroma leads to an increased incidence of breast brain metastases. Furthermore, this study highlights a role for stromal activation of PDGFRβ in the brain microenvironment and during metastatic progression. For the 20-30% of patients that develop breast cancer brain metastases, the one-year survival rate is sadly less than 20%, and how the brain microenvironment contributes to metastatic seeding and subsequent growth of tumor cells remains poorly understood. To confirm translational relevance, we analyzed a small cohort of matched primary breast tumors and brain metastases for PDGFRβ expression observing strong stromal staining in fibroblasts and pericytes within and around all of the primary tumors similar to previous studies. Importantly, high PDGFRβ expression was found in the perivasculature of all associated brain metastases suggesting a functional role in the establishment or growth at this site. Combined, our findings strongly suggest that high primary tumor expression of PDGF-B/PDGFRβ might define a subset of breast cancer patients predisposed to brain metastases. These patients may benefit from therapeutic targeting of PDGFR signaling as a means to thwart metastatic seeding in the brain. Citation Format: Katie A. Thies, Anisha M. Hammer, Anthony J. Trimboli, B. Eason Hildreth, Luke O. Russell, Chelsea M. Bolyard, Raleigh D. Kladney, Steven T. Sizemore, Robert Pilarski, Lynn Schoenfield, Jose Otero, Arnab Chakravarti, Balveen Kaur, Gustavo Leone, Michael C. Ostrowski, Gina M. Sizemore. Stromal platelet derived growth factor receptor-beta (PDGFRbeta) promotes breast brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3911. doi:10.1158/1538-7445.AM2017-3911

Abstract 2128: PDGFR beta signaling is required for hypoxia induced mitophagy in glioblastoma survival

Abstract BACKGROUND: Platelet-derived growth factor (PDGF) signaling plays a key role in gliomagenesis. Glioblastomas express all PDGF ligands and both cell membrane receptors, PDGFRα and PDGFRβ, that are involved in pro-survival autocrine and paracrine loops. Our previous work demonstrated that expression of PDGFRβ is enriched in hypoxic glioblastoma tumor tissues. Also, induction of mitophagy is a well-known critical coping response to tissue hypoxia. We therefore investigated the role of PDGFRβ signaling cascade in regulating hypoxia-induced mitophagy of glioblastoma. METHODS: Protein detection was performed by immunoblotting and immunofluorescence to determine changes in levels of target proteins upon RNA interference, treatment with PDGFRβ inhibitor, and ectopic gene transfection. MitoSox assay was used to measure mitochondrial reactive oxygen species (ROS). Tumor growth assays were performed in vitro and in vivo using subcutaneous tumor implantation model. RESULTS: PDGFRβ signaling was essential for induction of hypoxia-mediated mitophagy in glioblastoma cells. Nix, a hypoxic responsive protein and regulator of mitophagy, was necessary for PDGFRβ-mediated glioblastoma survival. Inhibition of PDGFRβ signaling pathway killed glioblastoma cells by suppressing mitophagy and leading to subsequent elevation of mitochondrial ROS. Furthermore, interference of mitophagic response by blocking PDGFRβ signaling enhanced cell death even in chemotherapy-resistant tumor cells. CONCLUSION: This work suggests a potential mechanism by which PDGFRβ inhibition could kill glioblastoma cells through suppression of NIX-mediated mitophagy. Manipulation of NIX and PDGFRβ pathway may provide novel therapeutic opportunities in chemoresistant tumor models. Citation Format: Jinkyu Jung, Mark R. Gilbert, Deric M. Park. PDGFR beta signaling is required for hypoxia induced mitophagy in glioblastoma survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2128. doi:10.1158/1538-7445.AM2017-2128

Platelet-derived growth factor signaling modulates adult hair follicle dermal stem cell maintenance and self-renewal

Hair follicle regeneration is dependent on reciprocal signaling between epithelial cells and underlying mesenchymal cells within the dermal papilla. Hair follicle dermal stem cells reside within the hair follicle mesenchyme, self-renew in vivo, and function to repopulate the dermal papilla and regenerate the connective tissue sheath with each hair cycle. The identity and temporal pattern of signals that regulate hair follicle dermal stem cell function are not known. Here, we show that platelet-derived growth factor signaling is crucial for hair follicle dermal stem cell function and platelet-derived growth factor deficiency results in a progressive depletion of the hair follicle dermal stem cell pool and their progeny. Using αSMACreERT2:RosaYFP:Pdgfrαflox mice, we ablated Pdgfrα specifically within the adult hair follicle dermal stem cell lineage. This led to significant loss of hair follicle dermal stem cell progeny in connective tissue sheath and dermal papilla of individual follicles, and a progressive reduction in total number of anagen hair follicles containing YFP+ve cells. As well, over successive hair cycles, fewer hair follicle dermal stem cells were retained within each telogen hair follicle suggesting an impact on hair follicle dermal stem cell self-renewal. To further assess this, we grew prospectively isolated hair follicle dermal stem cells (Sox2GFP+ve αSMAdsRed+ve) in the presence or absence of platelet-derived growth factor ligands. Platelet-derived growth factor-BB enhanced proliferation, increased the frequency of Sox2+ve hair follicle dermal stem cell progeny and improved inductive capacity of hair follicle dermal stem cells in an ex vivo hair follicle formation assay. Similar effects on proliferation were observed in adult human SKPs. Our findings impart novel insights into the signals that comprise the adult hair follicle dermal stem cell niche and suggest that platelet-derived growth factor signaling promotes self renewal, is essential to maintain the hair follicle dermal stem cell pool and ultimately their regenerative capacity within the hair follicle.

Olaratumab: First Global Approval.

Olaratumab (Lartruvo™) is a fully human IgG1 monoclonal antibody targeted against the human platelet-derived growth factor (PDGF) receptor α (PDGFRα). It was developed by Eli Lilly and Co. (previously ImClone Systems) after PDGFRα was identified as a potential therapeutic target in a variety of cancers. Olaratumab acts by selectively binding PDGFRα, thereby blocking PDGF ligand binding and inhibiting PDGFRα activation and downstream signalling. In October 2016, olaratumab received its first global approval, in the USA, for use in combination with doxorubicin for the treatment of adult patients with soft tissue sarcoma. The approval was granted by the US FDA under its Accelerated Approval Program based on the results of the JGDG phase II trial (NCT01185964). In addition, the EMA granted conditional approval for olaratumab in this indication in November 2016 following a review under the EMA's Accelerated Assessment Program. An international, confirmatory phase III trial in patients with soft tissue sarcoma is ongoing (ANNOUNCE; NCT02451943). Olaratumab has also been investigated in phase II trials in several other cancers. This article summarizes the milestones in the development of olaratumab leading to this first approval, for use in combination with doxorubicin for the treatment of soft tissue sarcoma in adults.

PDGF-A and PDGF-B induces cardiac fibrosis in transgenic mice

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) contribute to normal heart development. Deficient or abnormal expression of Pdgf and Pdgfr genes have a negative impact on cardiac development and function. The cellular effects of PDGFs in the hearts of Pdgf/Pdgfr mutants and the pathogenesis of the resulting abnormalities are poorly understood, but different PDGF isoforms induce varying effects. Here, we generated three new transgenic mouse types which complete a set of studies, where all different PDGF ligands have been expressed under the same heart specific alpha-myosin heavy chain promoter.Transgenic expression of the natural isoforms of Pdgfa and Pdgfb resulted in isoform specific fibrotic reactions and cardiac hypertrophy. Pdgfa overexpression resulted in a severe fibrotic reaction with up to 8-fold increase in cardiac size, leading to lethal cardiac failure within a few weeks after birth. In contrast, Pdgfb overexpression led to focal fibrosis and moderate cardiac hypertrophy. As PDGF-A and PDGF-B have different affinity for the two PDGF receptors, we analyzed the expression of the receptors and the histology of the fibrotic hearts. Our data suggest that the stronger fibrotic effect generated by Pdgfa overexpression was mediated by Pdgfrα in cardiac interstitial mesenchymal cells, i.e. the likely source of extracellular matrix depostion and fibrotic reaction. The apparent sensitivity of the heart to ectopic PDGFRα agonists supports a role for endogenous PDGFRα agonists in the pathogenesis of cardiac fibrosis.

Increased expression of platelet-derived growth factor associated protein-1 is associated with PDGF-B mediated glioma progression

The current treatment therapies available for malignant gliomas are inadequate. There is an urgent need to develop more effective therapies by characterizing the molecular pathogenesis of the disease. Over expression of platelet-derived growth factor (PDGF) ligands and receptors have been reported in malignant gliomas. Platelet-derived growth factor associated protein-1 (PDAP-1) is reported to modulate the mitogenic activity of PDGF ligands, but to date, there is no information concerning its role in PDGF-mediated glioma cell proliferation. This study aimed to characterize the role of PDAP-1 in PDGF-mediated glioma proliferation. The expression of PDAP-1 was observed to be significantly increased (p<0.05) in grade IV glioma tissue and cell lines compared to grade III. siRNA-mediated knockdown of PDAP-1 reduced the expression of PDGF-B and its downstream genes (Akt1/Protein kinase B (PKB) and phosphoinositide-dependent kinase-1 (PDK1) by up to 50%. In PDAP-1 knockdown glioma cells, more than a twofold reduction was also observed in the level of phosphorylated Akt. Interestingly, knockdown of PDAP-1 in combination with PDGF-B antibody inhibited glioma cell proliferation through activation of Caspase 3/7 and 9. We also demonstrate that PDAP-1 co-localizes with PDGF-B in the cytoplasm of glioma cells, and an interaction between both of the proteins was established. Collectively, these findings suggest that the expression of PDAP-1 is associated with disease malignancy, and its inhibition reduced the proliferation of malignant glioma cells through down-regulation of PDGF-B/Akt/PDK1 signaling. Thus, this study establishes PDAP-1 as an effecter of PDGF signaling in glioma cells and suggests that it could also be a promising therapeutic target.

152. Tumor Inhibition by Using Chitosan:siRNA PDGFR-β in Breast Cancer Model of Rat

Platelet-derived-growth factors (PDGFs) may represent a novel target in human cancer because they regulate many cellular processes, including cell proliferation, transformation, apoptosis and angiogenesis. PDGF-D's exert their cellular effects by two receptors PDGFR-α and PDGFR-β. Moreover PDGF ligands and receptors are proto-oncogenes that can be activated by various types of alternatives in cancer cells. Breast cancer is the most common and fatal type of cancer. The PDGF pathway is essential in tumor angiogenesis. It is known that the expression of PDGF receptors is altered and upregulated in breast cancer. Delivery is very important for success in gene therapy. Among the different non-viral gene delivery system, chitosan has very useful properties as a gene carrier. In this study; we investigated the effect of chitosan:siRNA complexes on tumor angiogenesis in breast tumor models of rats. Firstly chitosan:siRNA complexes (1/5-1/20 rate) targeting to PDGFR-β were prepared and characterized in vitro. Transfection efficiency of these nanoplexes in breast cancer cells such MCF-7, MDA-MB-231 and MDA-MB-435 was assayed. In in vitro transfection studies, 54%-65% of PDGFR-β inhibition was measured. Then these chitosan:siRNA complexes were injected intratumorally to tumor bearing Sprague Dawley rats. After tumor reached to constant size, complexes (20/1) were injected (40µg siRNA) and tumor volumes were measured periodically. After sacrification of animals, tissue and blood samples were taken and investigated histologically and immuno-histochemically. Expression levels of PDGFR-β determined by ELISA, mRNA levels were measured by RT-PCR also. Nanoplexes having 207 ± 3.5 nm size and 14.2 mV surface charge were used in this study. Tumor volume of nanoplex treated rats decreased at 92.49% at the end of the experiment (28th day). After free siRNA treatment, however, tumor volume increased very slowly until 21 days (68.1 % of decreasing value) then went up fastly. RT-PCR, Western-blot studies and immuno-histochemical data showed similarity with tumor volume measurements. Similar IFN response with tumor (untreated) was obtained after nanoplex administration. In conclusion, chitosan can be complexed with siRNA targeting PDGFR-β and chitosan:siRNA PDGFR-β nanocomplexes may be useful alternative in the treatment of breast cancer. The receptors of PDGF may be also suitable target in breast cancer.

Collective migration in tissues.

The Minisymposium on “Cell Migration in Tissues” focused on analysis of dynamic collective cell migration in several model systems and was cochaired by Alissa M. Weaver (Vanderbilt University Medical Center) and Celeste M. Nelson (Princeton University). The program highlighted both the physical forces exerted during migration and the signaling pathways involved in the process. Celeste Nelson (Princeton University) presented results suggesting that cells migrate collectively through fibrous extracellular matrix (ECM) by exerting tensile forces at the leading edge of the cohort. Three-dimensional traction force microscopy was used to quantify the physical forces exerted by normal and malignant mammary epithelial cells undergoing collective migration through collagen gels (Gjorevski et al., 2015 ). The forces and displacements were surprisingly dynamic, with the cohort periodically relaxing as it migrated forward. Jessica Konen (Marcus laboratory, Emory University) described a new technique called spatiotemporal genomic analysis, which was used to select rare populations of leader cells from three-dimensional (3D) lung cancer spheroids and compare their gene expression patterns to that of follower cells. Leader cells were found to express higher levels of genes related to cell adhesion and vascular sprouting. These gene expression changes persisted in purified cultures. Eliah Shamir (Ewald laboratory, Johns Hopkins University) showed that in 3D culture of mammary epithelial organoids, the myoepithelial layer was required to prevent invasion and dissemination of Twist1-expressing luminal epithelial cells. Surprisingly, Twist1-induced invasion required the expression of E-cadherin, and knocking down expression of this cell–cell adhesion protein prevented dissemination downstream of Twist1 (Shamir et al., 2014 ). Joshua Bloomekatz (Yelon laboratory, University of California, San Diego) showed a critical role for platelet-derived growth factor (PDGF) in the movement of cardiomyocytes during cardiac fusion. A forward genetic screen was used to identify a mutation called refuse-to-fuse, which causes inhibition of cardiac fusion and the subsequent formation of a bifurcated ventricle. This mutation was found to disrupt splicing of pdgfra, leading to expression of a truncated form of the PDGF receptor. Expression of PDGF ligands in relatively medial locations suggested a model in which PDGF signaling coordinates the movement of cardiomyocytes toward the midline during cardiac fusion. Bong Hwan Sung (Weaver laboratory, Vanderbilt University) showed that directionally persistent migration of cancer cells is promoted by autocrine secretion of exosomes, late endosome–derived extracellular vesicles (Sung et al., 2015 ). Exosomes were shown to contain ECM proteins as cargo and to be secreted at sites of nascent adhesions. Accordingly, exosome secretion promoted adhesion assembly, migrational polarity, and the stability of lamellipodia. David Matus (Stony Brook University) used an RNA interference screen to identify a transcriptional program that maintains invasive anchor cells in a cell cycle–arrested state during uterine-vulval attachment in Caenorhabditis elegans. Nuclear hormone receptor nhr-67 (TLX) expression leads to up-regulation of cyclin-dependent kinase inhibitor cki-1, causing cell cycle arrest in G1 (Matus et al., 2015 ). Cells that lack nhr-67 are proliferative and noninvasive, failing to express matrix or form invadopodia. The Minisymposium concluded with three lightning talks. Micah Webster (Fan laboratory, Carnegie Institution for Science) showed a role for “ghost fibers,” ECM remnants of damaged muscle fibers, in organizing skeletal muscle progenitor migration, division, and fusion during regeneration in mice (Webster et al., 2016 ). Rebecca Green (Oegema laboratory, University of California, San Diego) developed a four-dimensional high-content imaging approach to screen for developmentally important genes within C. elegans embryos. Yutaka Matsubayashi (Stramer laboratory, King’s College London) showed that formation of the basement membrane in the Drosophila embryo requires rapid anterior–posterior dispersal of macrophages (hemocytes), which first appear in the head of the embryo.