Platelet-derived Growth Factor Receptor Signaling Research Articles

Phase II pilot results of imatinib mesylate with weekly docetaxel in metastatic breast cancer

1090 Background: Imatinib mesylate (G) is a potent tyrosine kinase inhibitor of platelet derived growth factor receptor (PDGFR), c-KIT, and bcr/abl. PDGFR signaling regulates interstitial tumor pressure enhancing tumor drug delivery and effects. This phase II pilot evaluates the feasibility, toxicity, and efficacy of imatinib with weekly docetaxel as a strategy to potentiate docetaxel in metastatic breast cancer (MBC). Methods: Eligibility: 0–1 regimens for MBC, > 6 months from prior adjuvant taxanes, measurable disease, ECOG PS 0–2, adequate organ function, < G2 neuropathy. Treatment: Docetaxel 30 mg/m2 IV days 1, 8 and 15 q28 with G 600 mg QD. After the first 14 pts, G was decreased to 400 mg in the remaining 19 pts. Response evaluations were every q 8 wks. Following 6 cycles of therapy, pts with SD or better continued maintenance G until PD. Results: 33 pts are evaluable for toxicity. Median age 58 (38–82), ECOG PS 0–18, PS 1–14 pts. 49% ER-/PR- and 79% HER2 neg. 61% had metastases in > 2 visceral sites with lung predominating 42%. 55% received therapy 1st line and 42% as 2nd-line. 13 pts had prior taxanes of which 70% were treated 2nd-line. Hematologic toxicity was mild with G3/4 neutropenia 12%, anemia 9%; 1 pt with febrile neutropenia. G3/4 nonhematologic toxicity consisted of fatigue 27% and GI toxicity prompting G dose modification to 400 mg after the first 14 pts. Despite the lower G dose, the GI events remained distributed equally between the 600/400 mg G doses: G3/4 diarrhea 21% (4/3), nausea 18% (4/2), vomiting 12% (2/3), anorexia 6% (1/1) 22 pts required hospitalizations, 9 treatment related (GI toxicity 4, febrile neutropenia 1, pleural effusion 2, pneumonia 2), 2 other, and 11 due to progression. Median # of cycles was 3 (1.5 - 12.5). 5 pts went on to maintenance G with a median of 6 cycles and 2 remain on maintenance G. 4 of 18 evaluable pts (12%) had PRs, 8 pts had SD, and 6 pts progressed. 11 pts were not evaluable and responses unknown in 4 pts. Median time to progression and overall survival were 3 and 10 months respectively. Conclusions: Imatinib with docetaxel is associated with significant GI toxicity, despite G dose modification to 400 mg daily. No therapeutic advantage for the addition of imatinib to weekly docetaxel in MBC was evident. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Bayer, Genentech Bristol-Myers Squibb, Eli Lilly and Company, Genentech, GlaxoSmithKline, GPC Biotech, Novartis, sanofi-aventis

A Novel Mechanism of Vascular Smooth Muscle Cell Regulation by Notch

See related article, pages 1483–1491 The Notch family of receptors, Notch1 to -4, are heterodimer transmembrane proteins, consisting of an extracellular domain and a noncovalently linked intracellular domain (ICD). Upon interaction with the DSL family of proteins (Jagged, Delta-like) on neighboring cells, Notch undergoes proteolytic cleavage, which frees the ICD from the plasma membrane. This results in translocation of the ICD into the nucleus, where it forms a complex with the CSL family of transcriptional repressors (CBF1/RBP-Jk), removing the repression and allowing for target gene (Hes, Hey) transcription.1,2 Tissue distribution of the Notch proteins varies widely. Notch1 and -4 are predominantly endothelial, prominent in both arteries and veins, and present in all stages of development (embryonic to adult); the expression of Notch2 is typically confined to pulmonary endothelium, but Notch3 is primarily expressed in adult arterial vascular smooth muscle cells (VSMCs) in large conduit, pulmonary, and systemic resistance arteries.3 This specific pattern of temporal and spatial distribution correlate to diverse functions of the Notch family in vascular development and physiology in vertebrates reported to date.4 In the cardiovascular system, Notch signaling plays a role in several aspects of vascular development, including vasculogenesis, angiogenesis, differentiation, vascular remodeling, and VSMC maturation. Notch1 and -4 signaling appears critical in vasculogenesis and angiogenesis during early development, when it interacts with vascular endothelial growth factor signaling to specify artery–vein differentiation of endothelial cells (ECs). Transgenic mice deficient for Notch1 fail to undergo embryonic angiogenic remodeling, and vascular development is arrested at the primitive undifferentiated plexus, resulting in an embryonic lethal phenotype. Notch2-null mutations likewise result in embryonic lethality characterized by multiple large and small vessel aneurysms. Notch4 deletion results in no obvious phenotypic alteration. Notch3−/− mice are viable; however, they fail to develop proper arterial VSMC phenotype, resulting in vein-like …

RNA Interference Screen Identifies Abl Kinase and PDGFR Signaling in Chlamydia trachomatis Entry

To elucidate the mechanisms involved in early events in Chlamydia trachomatis infection, we conducted a large scale unbiased RNA interference screen in Drosophila melanogaster S2 cells. This allowed identification of candidate host factors in a simple non-redundant, genetically tractable system. From a library of 7,216 double stranded RNAs (dsRNA), we identified ∼226 host genes, including two tyrosine kinases, Abelson (Abl) kinase and PDGF- and VEGF-receptor related (Pvr), a homolog of the Platelet-derived growth factor receptor (PDGFR). We further examined the role of these two kinases in C. trachomatis binding and internalization into mammalian cells. Both kinases are phosphorylated upon infection and recruited to the site of bacterial attachment, but their roles in the infectious process are distinct. We provide evidence that PDGFRβ may function as a receptor, as inhibition of PDGFRβ by RNA interference or by PDGFRβ neutralizing antibodies significantly reduces bacterial binding, whereas depletion of Abl kinase has no effect on binding. Bacterial internalization can occur through activation of PDGFRβ or through independent activation of Abl kinase, culminating in phosphorylation of the Rac guanine nucleotide exchange factor (GEF), Vav2, and two actin nucleators, WAVE2 and Cortactin. Finally, we show that TARP, a bacterial type III secreted actin nucleator implicated in entry, is a target of Abl kinase. Together, our results demonstrate that PDGFRβ and Abl kinases function redundantly to promote efficient uptake of this obligate intracellular parasite.

Dual inhibition of c‐abl and PDGF receptor signaling by dasatinib and nilotinib for the treatment of dermal fibrosis

Abelson kinase (c-abl) and platelet-derived growth factor (PDGF) are key players in the pathogenesis of systemic sclerosis (SSc). The aim of the present study was to evaluate the antifibrotic potential of dasatinib and nilotinib, 2 novel inhibitors of c-abl and PDGF, which are well tolerated and have recently been approved. Dasatinib and nilotinib dose-dependently reduced the mRNA and protein levels of extracellular matrix proteins in human stimulated dermal fibroblasts from SSc patients (IC(50) of 0.5-2.0 nM for dasatinib and 0.8-2.5 nM for nilotinib). In a mouse model of bleomycin-induced dermal fibrosis, dasatinib and nilotinib potently reduced the dermal thickness, the number of myofibroblasts, and the collagen content of the skin in a dose-dependent manner at well-tolerated doses. These data indicate that dasatinib and nilotinib potently inhibit the synthesis of extracellular matrix in vitro and in vivo at biologically relevant concentrations. Thus, we provide the first evidence that dasatinib and nilotinib might be promising drugs for the treatment of patients with SSc.

Imatinib mesylate attenuates fibrosis in coxsackievirus b3-induced chronic myocarditis

Coxsackievirus B3 (CVB3)-induced chronic myocarditis in mice is accompanied by severe fibrosis and by sustained elevation of platelet-derived growth factor (PDGF)-A, -B, and -C levels in the cardiac tissue. To test if PDGF stimulation of resident fibroblasts causally contributes to fibrosis, we employed inhibition of PDGF receptor signalling with the orally available kinase inhibitor Imatinib. Chronic myocarditis was induced by CVB3 infection of major histocompatibility complex (MHC) class II knockout (B6Aa(0)/Aa(0)) mice. The mice were treated with 100 mg/kg Imatinib or vehicle, respectively, twice daily for 34 days. Expression of PDGF-C and of inflammatory cytokines were analysed by semi-quantitative RT-PCR. PDGFalpha receptor phosphorylation was detected by immunoblotting of cardiac tissue extracts and in situ by immunohistochemistry. Fibrosis formation was analysed by Sirius-Red staining and hydroxyproline (HP) determination. Fibronectin, and tenascin expression was analysed by RT-PCR and immunohistochemistry. Matrix metalloproteinase (MMP) activity was assessed with collagen, synthetic peptides, and gelatine as substrates. Imatinib significantly inhibited the myocarditis-related PDGFalpha receptor activation in the heart tissue. The virus titres in the hearts, inflammatory infiltrations, and elevated PDGF levels were unaffected by the Imatinib treatment. A significant attenuation of fibrosis occurred in Imatinib-treated animals. The Sirius Red-stained fibrotic area was reduced from 5.30 +/- 0.50 to 3.21 +/- 0.35%, and the HP content was reduced from 362 +/- 43 to 238 +/- 32 microMol/10 mg dry weight vs. 190 +/- 27 in uninfected controls. The expression of fibronectin, EIIIA+ fibronectin, and tenascin C were likewise reduced. The diminished matrix protein deposition was not caused by elevated MMP activity, since MMP activity was not changed or even reduced under Imatinib. The data suggest a causal role for elevated PDGF expression and PDGF receptor activity in the pathogenesis of cardiac fibrosis.

Platelet-derived growth factor receptor expression and amplification in choroid plexus carcinomas

Platelet-derived growth factor (PDGF) receptor signaling has been implicated in the development of glial tumors, but not yet been examined in choroid plexus carcinomas, pediatric tumors with dismal prognosis for which novel treatment options would be desirable. Therefore, protein expression of PDGF receptors α and β as well as amplification status of the respective genes, PDGFRA and PDGFRB, were examined in a series of 22 patients harboring choroid plexus carcinoma using immunohistochemistry and chromogenic in situ hybridization (CISH). The majority of choroid plexus carcinomas expressed PDGF receptors with 6 cases (27%) displaying high staining scores for PDGF receptor α and 13 cases (59%) showing high staining scores for PDGF receptor β. Correspondingly, copy-number gains of PDGFRA were observed in 8 cases out of 12 cases available for CISH and 1 case displayed amplification (six or more signals per nucleus). The proportion of choroid plexus carcinomas with amplification of PDGFRB was even higher (5/12 cases). PDGFRB amplification status and PDGF receptor β protein expression scores were significantly correlated (P=0.01, Spearman). Expression status of PDGF receptor α or PDGF receptor β was not significantly associated with progression-free survival. To conclude, expression and amplification of PDGF receptors, particularly PDGF receptor β, are frequent in choroid plexus carcinomas, providing a first rationale for the development of treatments targeting PDGF receptor signaling in these rare malignant pediatric tumors.

Effects of osmotic stress on the activity of MAPKs and PDGFR-β-mediated signal transduction in NIH-3T3 fibroblasts

Signaling in cell proliferation, cell migration, and apoptosis is highly affected by osmotic stress and changes in cell volume, although the mechanisms underlying the significance of cell volume as a signal in cell growth and death are poorly understood. In this study, we used NIH-3T3 fibroblasts in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-beta-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most likely via a pathway independent of PDGFR-beta and MEK1/2. Conversely, hyperosmolarity (cell shrinkage at 582 mosM) moved nuclear and phosphorylated ERK1/2 to the cytoplasm and induced the phosphorylation and nuclear translocation of p38 and phosphorylation of JNK1/2. In a series of parallel experiments, hypoosmolarity did not affect PDGF-BB-induced activation of PDGFR-beta, whereas hyperosmolarity strongly inhibited ligand-dependent PDGFR-beta activation as well as downstream mitogenic signal components of the receptor, including Akt and the MEK1/2-ERK1/2 pathway. Based on these results, we conclude that ligand-dependent activation of PDGFR-beta and its downstream effectors Akt, MEK1/2, and ERK1/2 is strongly modulated (inhibited) by hyperosmotic cell shrinkage, whereas cell swelling does not seem to affect the activation of the receptor but rather to activate ERK1/2 via a different mechanism. It is thus likely that cell swelling via activation of ERK1/2 and cell shrinkage via activation of the p38 and JNK pathway and inhibition of the PDGFR signaling pathway may act as key players in the regulation of tissue homeostasis.

The Src-like adaptor protein regulates PDGF-induced actin dorsal ruffles in a c-Cbl-dependent manner

The Src-like adaptor protein (SLAP) belongs to the subfamily of adapter proteins that negatively regulate cellular signalling initiated by tyrosine kinases. SLAP has a unique, myristylated N-terminus, followed by SH3 and SH2 domains with high homology to Src family tyrosine kinases (SFK) and a unique C-terminal tail, which is important for c-Cbl binding. We have previously shown that SLAP negatively regulates platelet-derived growth factor (PDGF)-induced mitogenesis in fibroblasts and we now report that it regulates F-actin assembly for dorsal ruffles formation. c-Cbl mediated SLAP inhibition towards actin remodelling. Moreover, SLAP enhanced PDGF-induced c-Cbl phosphorylation by SFK. In contrast, SLAP mitogenic inhibition was not mediated by c-Cbl, but it rather involved a competitive mechanism with SFK for PDGF-receptor (PDGFR) association and mitogenic signalling. Accordingly, phosphorylation of the Src mitogenic substrates Stat3 and Shc were reduced by SLAP. Thus, we concluded that SLAP regulates PDGFR signalling by two independent mechanisms: a competitive mechanism for PDGF-induced Src mitogenic signalling and a non-competitive mechanism for dorsal ruffles formation mediated by c-Cbl.

Molecular mechanisms linking wound inflammation and fibrosis: knockdown of osteopontin leads to rapid repair and reduced scarring

Previous studies of tissue repair have revealed osteopontin (OPN) to be up-regulated in association with the wound inflammatory response. We hypothesize that OPN may contribute to inflammation-associated fibrosis. In a series of in vitro and in vivo studies, we analyze the effects of blocking OPN expression at the wound, and determine which inflammatory cells, and which paracrine factors from these cells, may be responsible for triggering OPN expression in wound fibroblasts. Delivery of OPN antisense oligodeoxynucleotides into mouse skin wounds by release from Pluronic gel decreases OPN protein levels at the wound and results in accelerated healing and reduced granulation tissue formation and scarring. To identify which leukocytic lineages may be responsible for OPN expression, we cultured fibroblasts in macrophage-, neutrophil-, or mast cell–conditioned media (CM), and found that macrophage- and mast cell–secreted factors, specifically platelet-derived growth factor (PDGF), induced fibroblast OPN expression. Correspondingly, Gleevec, which blocks PDGF receptor signaling, and PDGF-Rβ–neutralizing antibodies, inhibited OPN induction by macrophage-CM. These studies indicate that inflammation-triggered expression of OPN both hinders the rate of repair and contributes to wound fibrosis. Thus, OPN and PDGF are potential targets for therapeutic modulation of skin repair to improve healing rate and quality.

Gene expression-based screening for inhibitors of PDGFR signaling.

Inhibitors of the platelet derived growth factor receptor (PDGFR) signaling pathway are isolated using gene expression-based high-throughput screening (GE-HTS), a method that is applicable to other pathways.

Modulation of Circadian Rhythm of DNA Synthesis in Tumor Cells by Inhibiting Platelet-Derived Growth Factor Signaling

Circadian synchronization of cell proliferation is observed not only in normal healthy tissues but also in malignant solid tumors. However, the proliferation rhythm of tumor cells is often different from that of normal cells. We reported here that the peculiar rhythm of tumor cell proliferation was modulated by inhibition of platelet-derived growth factor (PDGF) signaling. DNA synthesis in tumor cells implanted in mice showed a 24-h oscillation apparently differing from that of normal bone marrow cells. Continuous administration of AG1295 (10 μg/h, s.c.), a PDGF receptor tyrosine kinase inhibitor, substantially suppressed DNA synthesis in the implanted tumor cells but not in the healthy bone marrow cells. During the administration of this drug, the rhythm of DNA synthesis in the tumor cells was synchronized with that in bone marrow cells. The present results suggest that the circadian rhythm of DNA synthesis in tumor cells is modulated by PDGF receptor signaling, which is activated following tumor progression. Because the rhythmic patterns of clock gene expression in tumor cells did not differ significantly from those in other healthy tissues, the enhanced signal transduction of PDGF receptor may cause an alteration in the rhythmicity of tumor cell proliferation without changing in the intracellular molecular clockwork.

Functions of Paracrine PDGF Signaling in the Proangiogenic Tumor Stroma Revealed by Pharmacological Targeting

BackgroundImportant support functions, including promotion of tumor growth, angiogenesis, and invasion, have been attributed to the different cell types populating the tumor stroma, i.e., endothelial cells, cancer-associated fibroblasts, pericytes, and infiltrating inflammatory cells. Fibroblasts have long been recognized inside carcinomas and are increasingly implicated as functional participants. The stroma is prominent in cervical carcinoma, and distinguishable from nonmalignant tissue, suggestive of altered (tumor-promoting) functions. We postulated that pharmacological targeting of putative stromal support functions, in particular those of cancer-associated fibroblasts, could have therapeutic utility, and sought to assess the possibility in a pre-clinical setting.Methods and FindingsWe used a genetically engineered mouse model of cervical carcinogenesis to investigate platelet-derived growth factor (PDGF) receptor signaling in cancer-associated fibroblasts and pericytes. Pharmacological blockade of PDGF receptor signaling with the clinically approved kinase inhibitor imatinib slowed progression of premalignant cervical lesions in this model, and impaired the growth of preexisting invasive carcinomas. Inhibition of stromal PDGF receptors reduced proliferation and angiogenesis in cervical lesions through a mechanism involving suppression of expression of the angiogenic factor fibroblast growth factor 2 (FGF-2) and the epithelial cell growth factor FGF-7 by cancer-associated fibroblasts. Treatment with neutralizing antibodies to the PDGF receptors recapitulated these effects. A ligand trap for the FGFs impaired the angiogenic phenotype similarly to imatinib. Thus PDGF ligands expressed by cancerous epithelia evidently stimulate PDGFR-expressing stroma to up-regulate FGFs, promoting angiogenesis and epithelial proliferation, elements of a multicellular signaling network that elicits functional capabilities in the tumor microenvironment.ConclusionsThis study illustrates the therapeutic benefits in a mouse model of human cervical cancer of mechanism-based targeting of the stroma, in particular cancer-associated fibroblasts. Drugs aimed at stromal fibroblast signals and effector functions may prove complementary to conventional treatments targeting the overt cancer cells for a range of solid tumors, possibly including cervical carcinoma, the second most common lethal malignancy in women worldwide, for which management remains poor.

Regulation of Platelet-derived Growth Factor Receptor Activation by Afadin through SHP-2: IMPLICATIONS FOR CELLULAR MORPHOLOGY

Upon binding of platelet-derived growth factor (PDGF), PDGF receptor is autophosphorylated at tyrosine residues in its cytoplasmic region, which induces the activation of diverse intracellular signaling pathways such those involving Ras-ERK, c-Src, and Rap1-Rac. Signaling through activated Ras-ERK promotes cell cycle and cell proliferation. The sequential activation of Rap1 and Rac affects cellular morphology and induces the formation of leading-edge structures, including lamellipodia, peripheral ruffles, and focal complexes, resulting in the enhancement of cell movement. In addition to the promotion of cell proliferation, the Ras-ERK signaling is involved in the regulation of cellular morphology. Here, we showed a novel role of afadin in the regulation of PDGF-induced intracellular signaling and cellular morphology in NIH3T3 cells. Afadin was originally identified as an actin filament-binding protein, which binds to a cell-cell adhesion molecule nectin and is involved in the formation of cell-cell junctions. When afadin was tyrosine-phosphorylated by c-Src activated in response to PDGF, afadin physically interacted with and increased the phosphatase activity of Src homology 2 domain-containing phosphatase-2 (SHP-2), a protein-tyrosine phosphatase that dephosphorylates PDGF receptor, leading to the prevention of hyperactivation of PDGF receptor and the Ras-ERK signaling. In contrast, knockdown of afadin or SHP-2 induced the hyperactivation of PDGF receptor and Ras-ERK signaling and consequently suppressed the formation of leading-edge structures. Thus, afadin plays a critical role in the proper regulation of the PDGF-induced activation of PDGF receptor and signaling by Ras-ERK. This effect, which is mediated by SHP-2, impacts cellular morphology.

Platelet-derived growth factor receptor-β, carrying the activating mutation D849N, accelerates the establishment of B16 melanoma

BackgroundPlatelet-derived growth factor (PDGF)-BB and PDGF receptor (PDGFR)-β are mainly expressed in the developing vasculature, where PDGF-BB is produced by endothelial cells and PDGFR-β is expressed by mural cells, including pericytes. PDGF-BB is produced by most types of solid tumors, and PDGF receptor signaling participates in various processes, including autocrine stimulation of tumor cell growth, recruitment of tumor stroma fibroblasts, and stimulation of tumor angiogenesis. Furthermore, PDGF-BB-producing tumors are characterized by increased pericyte abundance and accelerated tumor growth. Thus, there is a growing interest in the development of tumor treatment strategies by blocking PDGF/PDGFR function. We have recently generated a mouse model carrying an activated PDGFR-β by replacing the highly conserved aspartic acid residue (D) 849 in the activating loop with asparagine (N). This allowed us to investigate, in an orthotopic tumor model, the role of increased stromal PDGFR-β signaling in tumor-stroma interactions.MethodsB16 melanoma cells lacking PDGFR-β expression and either mock-transfected or engineered to express PDGF-BB, were injected alone or in combination with matrigel into mice carrying the activated PDGFR-β (D849N) and into wild type mice. The tumor growth rate was followed and the vessel status of tumors, i.e. total vessel area/tumor, average vessel surface and pericyte density of vessels, was analyzed after resection.ResultsTumors grown in mice carrying an activated PDGFR-β were established earlier than those in wild-type mice. In this early phase, the total vessel area and the average vessel surface were higher in tumors grown in mice carrying the activated PDGFR-β (D849N) compared to wild-type mice, whereas we did not find a significant difference in the number of tumor vessels and the pericyte abundance around tumor vessels between wild type and mutant mice. At later phases of tumor progression, no significant difference in tumor growth rate was observed between wild type mice and mutant mice, although the pericyte coverage was higher around tumor vessels from mutant mice.ConclusionOur findings suggest that the activated PDGFR-β (D849N) in the host animal increased the total vessel area and the average vessel surface even in PDGF-negative tumors, resulting in a shorter lag phase during tumor establishment.

Identification of a subset of pericytes that respond to combination therapy targeting PDGF and VEGF signaling

The aim of our study was to further explore the use of anti-angiogenic therapy targeting the vascular endothelial growth factor receptor (VEGFR) on endothelial cells while simultaneously targeting platelet-derived growth factor receptors (PDGFRs) on adjacent pericytes. B16 mouse melanoma tumors exogenously expressing PDGF-BB (B16/PDGF-BB) display higher pericyte coverage on the vasculature compared to the parental B16 tumors (B16/mock). These models were used to investigate the effects of combination therapy targeting VEGFR and PDGFR signaling on size-matched tumors. Combination therapy using 25 mg/kg/day of the VEGFR inhibitor PTK787 and 100 mg/kg/day of the PDGFR inhibitor STI571 decreased the tumor growth rate of both tumor types, but the inhibition was only significant in the B16/PDGF-BB tumors. Combination therapy induced vessel remodeling, primarily by reducing the vessel density in B16/mock tumors, and by reducing the vessel size in B16/PDGF-BB tumors. When analyzing the effects of combination therapy on tumor vessel pericytes, it was found to primarily reduce the subpopulation of alpha-smooth muscle actin and PDGFRbeta-positive pericytes partly detached from the tumor vessels, without affecting the number of pericytes closely attached to the endothelium, which also express desmin. Taken together, these data demonstrate an increased benefit of targeting both VEGFR and PDGFR pathways in B16/PDGF-BB tumors, and demonstrates that the increased tumor growth inhibition in this model is accompanied by a reduction in a specific subset of pericytes, characterized by being loosely attached to endothelial cells and negative for the pericyte marker desmin.

A phase I/II study of imatinib and docetaxel as neoadjuvant therapy in locally advanced breast cancer

11039 Background: Imatinib mestylate(Gleevec,G) is a tyrosine kinase inhibitor of platelet derived growth factor receptor(PDGFR) and KIT(CD117). In breast cancer(BC),PDGFR signaling regulates tumor interstitial fluid pressure(IFP). PDGFR inhibition lowers IFP enhancing tumor drug delivery and chemo effect. KIT’s role in BC growth is unclear.PDGFR and KIT signal pathways regulate proliferation and cell cycle progression.Receptor blockade may enhance anti-tumor activity combined with chemo.This study evaluated tolerability,toxicity and efficacy of imatinib and docetaxel and treatment changes in biomarker expression and downstream mediators. Methods: Eligibility:Total 12 women, invasive LABC(T2–4,No-2,Mo)IHC + for KIT and/or PDGF, ECOG 0–2,bidimensional disease.Treatment:Assigned to 1of 3 G dose levels(400,600,800mg) taken daily 2 weeks then combined with D30mg/m2 IV weekly x3 Q28 days. Three completed G(400mg D) and protocol modified to G600 and 800mg dose for 1 week then combined with D30mg/m2 IV weekly x12. Tumor(T)measured pretreatment and at definitive surgery at conclusion of G+D.T biopsy done preG, postG and at end of G+D for tissue markers. Three treated G400, 5G600 and 4G800mg dose. Results: 12 patients median age 51 (range 39–62), ECOG PS O, 4 pre/8 post menopausal treated. Clinical pretreatment (C-PRx) tumor size (TS)median 6cm (range 3- 12cm).C-PRx node status 7cN0 and 5cN1.PRx x-ray TS median 4.5 cm (range 2.5–8.5cm). Response:OR 25% 1 near CR+2 PR(3/12) at 400 and 600mg dose levels.Two SD(G800mg),3 progressed/off study,2 withdrew/toxicity(G3 rash/edema and G4 N/V), 2 NR.Surgery:5PM and 7MRM. Toxicity:Non- hematologic:nausea(1G4/G400mg),vomiting(1G4/G600mg),diarrhea(1G3/G800mg),fatigue(2G3/G800mg),myalgia(1G3/G800mg),rash(1G3/G600m g) and hypophosphatemia (2G3/G600mg). Hematologic toxicity mild neutropenia/anemia G1/2.One hospitalized for G3 odonophagia/stomatitis,G800mg.Dose Delay:2 missed 1 week of D+G (G600mg),2 missed 2 weeks of D+G (1G600mg and 1G800mg). Conclusion: Imatinib and docetaxel in neoadjuvant LABC is feasible and tolerable with low G3/4 toxicity and acceptable activity.G400mg dose best tolerated. Tissue IHC and molecular profiles to be reported separately.

Cooperation between the PDGF receptors in cardiac neural crest cell migration

Neural crest cells (NCCs) are essential components of the sympathetic nervous system, skin, craniofacial skeleton, and aortic arch. It has been known for many years that perturbation of migration, proliferation, and/or differentiation of these cells leads to birth defects such as cleft palate and persistent truncus arteriosus (PTA). Previously, we had shown that disruption of the platelet-derived growth factor receptor (PDGFR) α in NCCs resulted in defects in craniofacial and aortic arch development, the latter with variable penetrance. Because we observed ventricular septal defects in embryos that are null for the PDGFRβ, we hypothesized that both PDGF receptors are involved in NCC formation. Here, we show that both receptors are expressed in cardiac NCCs and that the combined loss of the PDGFRα and PDGFRβ in NCCs resulted in NCC-related heart abnormalities, including PTA and a ventricular septal defect (VSD). Using NCC lineage tracing, we observed that loss of PDGF receptor signaling resulted in reduced NCCs in the conotruncus region, leading to defects in aortic arch septation. These results indicate that while PDGFRα plays a predominant role in NCC development, the PDGFRβ is expressed by and functions in cardiac NCCs. Combined PDGF receptor signaling is required for sufficient recruitment of cardiac NCCs into the conotruncal region and for formation of the aortico-pulmonary and ventricular septum.

Osteoblast-conditioned medium promotes proliferation and sensitizes breast cancer cells to imatinib treatment

Inhibition of platelet-derived growth factor receptor (PDGFR) signaling restricts the growth of human breast cancer in the bone of nude mice. We hypothesized that osteoblast-secreted substances may alter the response capacity of breast cancer cells to the PDGFRs tyrosine kinase inhibitor imatinib mesylate. We found that osteoblast-conditioned medium (OCM) increases the proliferation rate of the estrogen receptor negative (ER-) MDA-MB-231 and of the ER+ MCF-7 human breast cancer cell lines and the growth-promoting effect on ER+ cells is independent from estrogen. OCM significantly improved the dose- and the time-dependent sensitivity of the tumor cells to the anti-proliferative effect of imatinib. We also found that MDA-MB-231 and MCF-7 cells express the two PDGFRs subtypes, PDGFR-alpha and PDGFR-beta, and OCM treatment increases the expression of the PDGFRs. Furthermore, imatinib inhibited the phosphorylation rate of its target tyrosine kinase receptors. We conclude that bone microenvironment, through osteoblast-secreted substances may cause estrogen-independent proliferation of breast cancer cells by a mechanism mediated by the induction of PDGFRs expression. The enhanced sensitivity of OCM-treated breast cancer cells to imatinib would justify investigation on the efficacy of imatinib in bone breast cancer metastasis.

Constitutive diffuse activation of phosphoinositide 3-kinase at the plasma membrane by v-Src suppresses the chemotactic response to PDGF by abrogating the polarity of PDGF receptor signalling

Cancer cells depend on chemotaxis for invasion and frequently overexpress and/or activate Src. We previously reported that v-Src accelerates motility by promoting phosphoinositide 3-kinase (PI3-K) signalling but abrogates chemotaxis. We here addressed the mechanism of the loss of chemotactic response to platelet-derived growth factor (PDGF) gradients in fibroblasts harbouring a thermosensitive v-Src kinase. At non-permissive temperature, PDGF receptor (PDGFR) signalling, assessed by phosphoY 751-specific antibodies (a docking site for PI3-K), was not detected without PDGF and showed a concentration-dependent PDGF response. Both immunolabeling of PI3-K (p110) and live cell imaging of its product (phosphatidylinositol 3,4,5 tris-phosphate) showed PI3-K recruitment and activation at lamellipodia polarized towards a PDGF gradient. Centrosomes and PDGFR- and Src-bearing endosomes were also oriented towards this gradient. Upon v-Src thermoactivation, (i) Y 751 phosphorylation was moderately induced without PDGF and synergistically increased with PDGF; (ii) PI3-K was recruited and activated all along the plasma membrane without PDGF and did not polarize in response to a PDGF gradient; and (iii) polarization of centrosomes and of PDGFR-bearing endosomes were also abrogated. Thus, PDGF can further increase PDGFR auto-phosphorylation despite strong Src kinase activity, but diffuse downstream activation of PI3-K by Src abrogates cell polarization and chemotaxis: “signalling requires silence”.

Functional Expression of IQGAP1 in Vasculature: Role in Reactive Oxygen Species‐dependent PDGF signaling and Vascular Migration

Vascular smooth muscle cell (VSMC) migration in response to platelet-derived growth factor (PDGF) is a key event in atherosclerosis and restenosis. Reactive oxygen species (ROS) derived from Rac1-dependent NADPH oxidase play an important role in PDGF receptor (PDGFR) signaling linked to VSMC migration. We previously identified IQGAP1 as a novel VEGF receptor binding protein expressed in endothelial cells. IQGAP1 controls cellular motility by interacting with actin and active Rac1; however, its role in VSMC remains unknown. Here we show that IQGAP1 expression is significantly increased after PDGF stimulation. Overexpression of IQGAP1 using adenovirus (Ad.IQGAP1) enhances PDGF-stimulated autophosphorylation of PDGFR, Rac1 binding to IQGAP1, ROS production and VSMC migration. Moreover, IQGAP1 promotes tyrosine phosphorylation of PLCgamma without affecting ERK1/2 phosphorylation. IQGAP1 siRNA inhibits PDGF-stimulated ROS production and VSMC migration. Wound assays reveal that IQGAP1 accumulates at the leading edge where it colocalizes with F-actin in actively migrating VSMCs. IQGAP1 expression is dramatically increased in the neointima in ApoE knockout mice with high fat-diet and mice with wire injury. In summary, IQGAP1 functions as a PDGFR-binding scaffold protein to organize ROS-dependent PDGF signaling, thereby promoting VSMC migration, which may contributes to formation of neointima in response to vascular injury.