Platelet-derived Growth factor-A Research Articles

Prostate field cancerization: deregulated expression of macrophage inhibitory cytokine 1 (MIC-1) and platelet derived growth factor A (PDGF-A) in tumor adjacent tissue.

Prostate field cancerization denotes molecular alterations in histologically normal tissues adjacent to tumors. Such alterations include deregulated protein expression, as we have previously shown for the key transcription factor early growth response 1 (EGR-1) and the lipogenic enzyme fatty acid synthase (FAS). Here we add the two secreted factors macrophage inhibitory cytokine 1 (MIC-1) and platelet derived growth factor A (PDGF-A) to the growing list of protein markers of prostate field cancerization. Expression of MIC-1 and PDGF-A was measured quantitatively by immunofluorescence and comprehensively analyzed using two methods of signal capture and several groupings of data generated in human cancerous (n = 25), histologically normal adjacent (n = 22), and disease-free (n = 6) prostate tissues. A total of 208 digitized images were analyzed. MIC-1 and PDGF-A expression in tumor tissues were elevated 7.1x to 23.4x and 1.7x to 3.7x compared to disease-free tissues, respectively (p<0.0001 to p = 0.08 and p<0.01 to p = 0.23, respectively). In support of field cancerization, MIC-1 and PDGF-A expression in adjacent tissues were elevated 7.4x to 38.4x and 1.4x to 2.7x, respectively (p<0.0001 to p<0.05 and p<0.05 to p = 0.51, respectively). Also, MIC-1 and PDGF-A expression were similar in tumor and adjacent tissues (0.3x to 1.0x; p<0.001 to p = 0.98 for MIC-1; 0.9x to 2.6x; p<0.01 to p = 1.00 for PDGF-A). All analyses indicated a high level of inter- and intra-tissue heterogeneity across all types of tissues (mean coefficient of variation of 86.0%). Our data shows that MIC-1 and PDGF-A expression is elevated in both prostate tumors and structurally intact adjacent tissues when compared to disease-free specimens, defining field cancerization. These secreted factors could promote tumorigenesis in histologically normal tissues and lead to tumor multifocality. Among several clinical applications, they could also be exploited as indicators of disease in false negative biopsies, identify areas of repeat biopsy, and add molecular information to surgical margins.

The mRNA expressions and immunohistochemistry of factors involved in angiogenesis and lymphangiogenesis in the early stage of rat skin incision wounds.

Wound healing evaluation is important in forensic pathology, in which angiogenesis plays an important role. We have already shown that vascular endothelial growth factor A (VEGF) is produced in the rat skin incision wounds by neutrophils, endothelial cells, and fibroblasts. In this study, we assessed the changes in the mRNA expressions of various factors possibly involved in angiogenesis including angiopoietin (ANGPT) 1 and 2, cadherin 5 (CDH5), granulocyte-macrophage colony stimulating factor (CSF2/GM-CSF), granulocyte colony stimulating factor (CSF3/G-CSF), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand12 (CXCL12/SDF1), endothelin 1 (ET1), fibroblast growth factor 1 (FGF 1), hepatocyte growth factor (HGF), hypoxia inducible factor 1 alpha (HIF1a), leptin, matrix metallopepitidase 9 (MMP9), serpine/plasminogen activator inhibitor1 (PAI1), platelet-derived growth factor-A (PDGF-A), transforming growth factor alpha and beta 1 (TGFa and b1), tenomodulin (TNMD), and troponin I type 2 (TNNI2) in the early stage of the rat skin incision wounds by real time RT-PCR. Factors reported to be involved in lymphangiogenesis such as fibroblast growth factor 2 (FGF 2), c-fos induced growth factor (FIGF/VEGF-D), forkhead box C2 (FOXC2), and prospero homeobox 1 (PROX1) were also studied. One and 3 days after the dorsal skin incisions, wounds on male Sprague-Dawley rats showed the statistically significant increases in the mRNA expressions for CXCL2, CSF3, MMP9, PAI1, and CSF2, whereas TGFa, TNNI2, FGF1, TNMD, leptin, and CXCL12 showed the statistically significant decreases. Interestingly, lymphgangiogenic factors FOXC2, PROX1, and FGF2 also showed the statistically significant decreases. In situ hybridization and immunohistochemistry showed the mRNA and protein positivity in endothelial cells, fibroblasts, and some leukocytes at the bottom of the wound tissue for PAI1, CSF3, and MMP9, 1 day after the skin incisions. Our novel findings show the possible involvement of several factors involved in angiogenesis and lymphangiogenesis in the early stage of wound healing process, which may be useful for forensic wound evaluations.

Dependence of fibroblast infiltration in tumor stroma on type IV collagen-initiated integrin signal through induction of platelet-derived growth factor

Cancer-associated fibroblasts play a crucial role in accelerating tumor progression, but there is a knowledge gap regarding the chemotactic signal activated in a tumor microenvironment. In this study, the expression of type IV collagen was knocked down using a lentiviral-mediated short hairpin RNA strategy. Although there was no obvious effect on cell growth in vitro, silencing the Col4-α1 gene decreased the tumorigenicity of B16F10 in C57BL/6 mice, which was accompanied by a reduction in the infiltration of alpha-smooth muscle actin-positive (α-SMA+) fibroblasts. Silencing the Col4-α1 gene or disrupting integrin engagement by blocking the antibody reduced the expression of platelet-derived growth factor A (PDGF-A), a potent chemotactic factor for fibroblasts. Furthermore, ectopic expression of the autoclustering integrin mutant significantly stimulated PDGF-A expression in murine B16F10 and human U118MG and Huh7 cells. PDGF-A-specific sh-RNA and neutralizing anti-PDGF-A antibody effectively inhibited the transwell migration of fibroblasts. Adding recombinant PDGF-A back to shCol cell-conditioned media restored the fibroblast-attraction ability indicating that PDGF-A is a major chemotactic factor for fibroblasts in the current study model. The integrin-associated PDGF-A production correlated with the activation of Src and ERK. High type IV collagen staining intensity colocalized with elevated PDGF-A expression was observed in tumor tissues obtained from hepatoma and glioma patients. The integrin signal pathway was activated by collagen engagement through Src and ERK, leading to enhanced PDGF-A production, which serves as a key regulator of fibroblast recruitment.

Clear cell adenocarcinoma of ureter: A case report with immunohistochemical study

The author reports herein the fourth case of rare clear cell adenocarcinoma (CCA) of ureter. The present case is the first of ureter CCA with extensive immunohistochemical examination. The patient is a 61-year-old man. The CCA was composed of malignant cells with very clear ample cytoplasm with occasional glandular formations. No apparent hobnail cells were seen. The tumor cells were seen to invade the adventitia. No carcinoma in situ was seen in the surrounding urothelium. A lymph showed metastasis. Histochemical stains revealed that the clear cytoplasm was due to glycogen not mucins or lipids. Immunohistochemically, tumor cells were positive for cytokeratin (CK) AE1/3, CK CAM5.2, CK34BE12, CK8, CK18, EMA, CEA, CA19-9, p53, platelet-derived growth factor-α (PDGFRA), MUC-1, and Ki-67 (labeling index=26%). The tumor cells were negative for CK5/6, CK7, CK14, CK19, CK20, NSE, p63, vimentin, S100 protein, α-smooth muscle actin, c-erbB2, chromogranin, synaptophysin, KIT (CD117), CD56, CDX-2, MUC2, MUC5AC, MUC6, CD10 and RCC markers.

Phosphodiesterase type 5 inhibition attenuates cyclosporine A induced nephrotoxicity in mice

We investigated the renal protective effects of phophodiesterase type 5 (PDE5) inhibitors in mice with cyclosporine A (CyA; a calcineurin phosphatase inhibitor) induced nephrotoxicity. Fifty male mice were divided into five groups of 10. Group 1 received no treatment, group 2 received only saline orally, group 3 received 30 mg/kg/day CyA by subcutaneous injection, group 4 received only 30 mg/kg/day vardenafil orally, and group 5 received 30 mg/kg/day CyA by subcutaneous injection and 30 mg/kg/day vardenafil orally. At 28 days, platelet-derived growth factor A (PDGF-A) and C (PDGF-C), transforming growth factor-beta 1 (TGF-β1), cyclo-oxygenase 1 and 2 (COX-1 and COX-2), and P glycoprotein (Pgp) expression levels were measured in the renal tissues. In addition, expressions of COX-1 and COX-2 genes were determined using real-time PCR. PDE5 inhibitor administration ameliorated decreased PDGF-A and C, TGF-β1, COX-1 and -2, and Pgp expression levels by modulation of cyclic guanosine monophosphate (cGMP) activity in kidneys. The relative expressions of COX-1 and COX-2 genes to GAPDH revealed that the maximum increase was obtained in the group treated with CyA and vardenafil for both COX-1 and COX-2 genes. Our study revealed that long term oral treatment with vardenafil protects kidneys from CyA induced nephrotoxicity. We showed that long term oral treatment with PDE5 prevents pathological kidney changes caused by CyA induced nephrotoxicity.

Correction: Characterization of Platelet-Derived Growth Factor-A Expression in Mouse Tissues Using a lacZ Knock-In Approach

Correction: Characterization of Platelet-Derived Growth Factor-A Expression in Mouse Tissues Using a lacZ Knock-In Approach

SOX11 promotes tumor angiogenesis through transcriptional regulation of PDGFA in mantle cell lymphoma

AbstractSOX11 is overexpressed in several solid tumors and in the vast majority of aggressive mantle cell lymphomas (MCLs). We have recently proven that SOX11 silencing reduces tumor growth in a MCL xenograft model, consistent with the indolent clinical course of the human SOX11-negative mantle cell lymphoma (MCL). However, the direct oncogenic mechanisms and downstream effector pathways implicated in SOX11-driven transformation remain poorly understood. Here, we observed that SOX11-positive xenograft and human primary MCL tumors overexpressed angiogenic gene signatures and had a higher microvascular density compared with their SOX11-negative counterparts. Conditioned media of SOX11-positive MCL cell lines induced in vitro endothelial cell proliferation, migration, tube formation, and activation of downstream angiogenic pathways. We identified PDGFA as a SOX11 direct target gene upregulated in MCL cells whose inhibition impaired SOX11-enhanced in vitro angiogenic effects on endothelial cells. In addition, platelet-derived growth factor A (PDGFA) was overexpressed in SOX11-positive but not in SOX11-negative MCL. In vivo, imatinib impaired tumor angiogenesis and lymphoma growth in SOX11-positive MCL xenograft tumors. Overall, our results demonstrate a prominent role for SOX11 as a driver of proangiogenic signals in MCL, and highlight the SOX11-PDGFA axis as a potential therapeutic target for the treatment of this aggressive disease.

Abstract 1523: Molecular typing of Chinese gastrointestinal stromal tumors using a multigene next generation sequencing panel

Abstract Identifying mutations in individual tumor is critical to improve the efficacy of cancer therapy by matching targeted drugs to specific mutations. Gastrointestinal stromal tumor (GIST) is a stromal or mesenchymal subepithelial neoplasm affecting the gastrointestinal tract and it frequently contains an activating mutation in either the KIT or platelet-derived growth factor A (PDGFRA) gene. Although GIST is highly responsive to several selective tyrosine kinase inhibitors, combined use of inhibitors targeting other mutations is needed to further prolong survival. In this study, we aim to screen and identify genetic mutations in GIST for targeted therapy using the new Ion Torrent next-generation sequencing platform. By utilizing the Ion Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes from DNA extracted from formalin-fixed and paraffin-embedded (FFPE) samples of 125 human gastrointestinal stromal tumors, set up stringent parameters for reliable variant calling by filtering out potential raw base calling errors, and identified frequent mutations in PDGFRA, KIT, APC, MLH1, MET, RET, and PIK3CA. Moreover, frequent missense mutations were detected in MLH1 (12%), MET (16.80%), KIT (33.60%), and PIK3CA (41.60%) genes. The mutations in MLH1 gene were frequently detected in the exons 2, while MET gene was frequently mutated in the exon 12. In addition, we identified missense mutations in other genes, including FLT3, KRAS, PDGFRA, and STK11, at lower frequencies. This study demonstrates the utility of using Ion Torrent sequencing to efficiently identify human cancer mutations. With the finding of the frequent mutations in MLH1 and MET genes, in addition to KIT, PDGFRA and PIK3CA, in GISTs, this study provides a molecular basis for clinically developing new drugs targeting these gene mutations for GIST therapy. Citation Format: Zhi Xu, Zhibin Hu, Xinying Huo, Chuanning Tang, Si-Yi Chen, Jinfei Chen. Molecular typing of Chinese gastrointestinal stromal tumors using a multigene next generation sequencing panel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1523. doi:10.1158/1538-7445.AM2014-1523

Abstract 2220: Impact of single-nucleotide polymorphisms (SNPs) on thymic hyperplasia and tumors outcome

Abstract Background: Thymic Hyperplasia refers to an enlargement of the thymus, while thymomas display significant heterogeneity from morphologic point of view, clinical behaviour, expression of immunohistochemical markers and molecular profiling. Therefore improving our understanding of the molecular biology of thymic disorders represents a key challenge in the treatment of these rare diseases. Methods: The genomic DNA of 93 consecutive patients undergoing total thymectomy at our Institution was extracted from paraffin-embedded tissue. We selected the following SNPs: Hypoxia Inducible Factor-1 alpha (HIF1a: rs2057482T&amp;gt;C, rs1951795A&amp;gt;C, rs2301113C&amp;gt;A, rs10873142C&amp;gt;T, rs11158358G&amp;gt;C, rs12434438G&amp;gt;A, rs11549465C&amp;gt;T, rs11549467G&amp;gt;A), Vascular Endothelial Growth Factor-A (VEGF-A: rs2010963G&amp;gt;C, rs699947A&amp;gt;C), VEGF Receptor 2 (VEGFR-2: rs2305948C&amp;gt;T, rs1870377T&amp;gt;A), VEGFR-3 (rs307826T&amp;gt;C, rs307821C&amp;gt;A), Platelet-Derived Growth Factor-A (PDGFR-A: rs35597368C&amp;gt;T). Gene polymorphisms were determined by Real-Time PCR using TaqMan assays. Results: 93 patients were included into the study, 58 females and 35 males. The patients underwent surgery: 43 for thymomas, 14 for thymic carcinomas, 36 for thymic hyperplasia. The frequency of PDGFRa rs35597368T (95.24%) was significantly higher in thymic malignancies if compared with data available for population (86%, p=0.012), thus suggesting that it may represent a risk factor for this type of disease. On the other hand, the frequency of several HIF-1a polymorphisms (rs2057482C, rs11549465C, rs1951795C, rs2301113A, rs10873142T, rs11158358C, rs12434438A) resulted lower in our study than in the general population (p&amp;lt;0.05). In particular, frequencies of HIF1a rs1951795C, rs2301113A, rs12434438A were significantly lower in thymic malignancy than in thymic hyperplasia and moreover these were lower than population, according to available literature data on other types of tumors, suggesting a protective role. Furthermore, VEGFR3 rs307821C polymorphism was higher in thymoma than in thymic carcinoma (79.5% vs 72%, p=0.0371), and therefore it seems to be related to a lower grade of malignancy. Finally, the following factors were significantly correlated with a better overall survival: VEGFR-3 rs307826C, VEGFR-2 rs1870377A, PDGFR-A rs35597368T/C, HIF1a rs2301113C, rs2057482C/T, rs1951795C, rs11158358G/C and rs10873142T/C (p&amp;lt;0.05). Conclusion: To the best of our knowledge this is the largest monocentric study analyzing the angiogenetic variants in thymic hyperplasia and tumors representing a further asset in the definition of high-risk patients after curative resection, also driving the selection of more aggressive adjuvant treatment in this subgroup of patients. The selection tool deriving from this analysis may allow an optimal use of innovative treatment strategies in thymic malignancies including targeted agents such as sunitinib, sorafenib or pazopanib. Citation Format: Rossana Berardi, Silvia Pagliaretta, Alessandro Brunelli, Vittorio Paolucci, Gaia Goteri, Majed Refai, Cecilia Pompili, Agnese Savini, Giulia Marcantognini, Mariangela Torniai, Michela Tiberi, Consuelo Ferrini, Francesca Morgese, Miriam Caramanti, Silvia Rinaldi, Azzurra Onofri, Antonio Zizzi, Paola Mazzanti, Stefano Cascinu. Impact of single-nucleotide polymorphisms (SNPs) on thymic hyperplasia and tumors outcome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2220. doi:10.1158/1538-7445.AM2014-2220

Role of platelet-derived growth factor-α in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps.

The pathogenesis of human chronic rhinosinusitis with nasal polyps (CRSwNP) comprising eosinophilic CRSwNP (ECRSwNP) and non-eosinophilic (nECRSwNP) is not completely understood. Recent evidence has suggested that platelet-derived growth factor receptor alpha (PDGFRα) is implicated in cell growth, transformation, proliferation, migration, and vascular permeability and platelet-derived growth factor-A (PDGF-A) is a specific ligand for PDGFRα. However, little is known about their roles in CRSwNP. Therefore, we aimed to investigate the expression and role of PDGFRα and PDGF-A in CRSwNP. PDGFRα protein expression was investigated by immunohistochemistry method and messenger RNA (mRNA) expression of PDGFRα and PDGF-A were assessed by real-time polymerase chain reaction (PCR) in CRSwNP patients and control subjects. Moreover, the effects of various stimulators with different concentrations and time on PDGFRα were evaluated on nasal explant culture. Quantitative analysis of immunostaining for PDGFRα showed an obvious elevation in immunolabeling of PDGFRα in CRSwNP groups compared with controls. Furthermore, PDGFRα protein was significantly stronger expressed in ECRSwNP group than nECRSwNP group and atopic patients showed stronger expression of PDGFRα protein than nonatopic patients. The mRNA of PDGFRα and PDGF-A were overexpressed in CRSwNP, especially in ECRSwNP. PDGFRα mRNA expression was closely related to PDGF-A mRNA. In nasal explant culture and stimulation, PDGFRα mRNA was augmented by interleukin 4 (IL-4), IL-5, or IL-1β respectively, but suppressed by IL-27. PDGFRα may play a pivotal role in the pathophysiology of ECRSwNP and nECRSwNP by combining with PDGF-A. IL-4, IL-5, or IL-1β may be critical for PDGFRα gene expression.

Vasopressin regulation of epithelial colonic proliferation and permeability is mediated by pericryptal platelet-derived growth factor A.

Arginine vasopressin (AVP) has trophic effects on the rat distal colon, increasing the growth of pericryptal myofibroblasts and reducing the colonic crypt wall permeability. This study aimed to reproduce in vitro the effects of AVP observed in vivo using cultures of human CCD-18Co myofibroblasts and T84 colonic epithelial cells. Proliferation of myofibroblasts was quantified by bromodeoxyuridine incorporation; the expression of platelet-derived growth factorA (PDGFA), platelet-derived growth factorB, epidermal growth factor, transforming growth factor-β and vascular endothelial growth factor was measured by PCR and the expression of epithelial junction proteins by Western blot. Arginine vasopressin stimulated myofibroblast proliferation and the expression of PDGFA without affecting the expression of platelet-derived growth factorB, epidermal growth factor, transforming growth factor-β or vascular endothelial growth factor. These effects were prevented when AVP receptor inhibitors were present in the medium. Pre-incubation of CCD-18Co cells with anti-PDGF antibody or with an inhibitor of the PDGF receptor abolished the effects of AVP. When colonocytes were incubated with medium obtained from myofibroblasts incubated with AVP, both cell proliferation and the expression of epithelial junction proteins increased; however, direct incubation of colonocytes with AVP did not modify these variables. These results demonstrate that AVP stimulates myofibroblast proliferation and induces PDGFA secretion, implying that PDGFA mediates local myofibroblast proliferation by an autocrine feedback loop and regulates epithelial proliferation and permeability by a paracrine mechanism.

Characterization of platelet-derived growth factor-A expression in mouse tissues using a lacZ knock-in approach.

Expression of the platelet-derived growth factor A-chain gene (Pdgfa) occurs widely in the developing mouse, where it is mainly localized to various epithelial and neuronal structures. Until now, in situ mRNA hybridization (ISH) has been the only reliable method to identify Pdgfa expression in tissue sections or whole mount preparations. Validated protocols for in situ detection of PDGF-A protein by immunohistochemistry is lacking. In particular, this has hampered understanding of Pdgfa expression pattern in adult tissues, where ISH is technically challenging. Here, we report a gene targeted mouse Pdgfa allele, Pdgfaex4 COIN, which is a combined conditional knockout and reporter allele. Cre-mediated inversion of the COIN cassette inactivates Pdgfa coding while simultaneously activating a beta-galactosidase (lacZ) reporter under endogenous Pdgfa transcription control. The generated Pdgfaex4 COIN-INV-lacZ allele can next be used to identify cells carrying a Pdgfa null allele, as well as to map endogenous Pdgfa expression. We evaluated the Pdgfaex4 COIN-INV-lacZ allele as a reporter for endogenous Pdgfa expression patterns in mouse embryos and adults. We conclude that the expression pattern of Pdgfaex4 COIN-INV-lacZ recapitulates known expression patterns of Pdgfa. We also report on novel embryonic and adult Pdgfa expression patterns in the mouse and discuss their implications for Pdgfa physiology.

Imatinib mesylate lacks efficacy in relapsed/refractory peripheral T cell lymphoma

Platelet derived growth factor-α (PDGFR-α) is expressed in peripheral T cell lymphoma, not otherwise specified (PTCL, NOS). Imatinib mesylate demonstrated in vitro cytotoxicity against primary PTCL, NOS cells. We initiated a trial of imatinib in 12 patients with relapsed or refractory T-cell non-Hodgkin lymphoma (T-NHL). PDGFR-α expression by immunohistochemistry and fluorescence in situ hybridization (FISH) to assess for FIP1L1–PDGFR-α fusion and/or PDGFR-α amplification were not required for study entry. We documented no objective responses. The median progression-free survival was 21.0 days (90% confidence interval [CI] 15.0, 28.0) and median overall survival was 154 days (90% CI 35, 242). Four patients had tissue available for analysis of PDGFR-α by immunohistochemistry and three of these patients’ tumors expressed PDGFR-α. Imatinib was not effective for the treatment of peripheral T cell lymphoma in an unselected group of patients in which PDGFR-α expression was not required for study entry.

Frequent KIT mutations in human gastrointestinal stromal tumors.

Identifying gene mutations in individual tumors is critical to improve the efficacy of cancer therapy by matching targeted drugs to specific mutations. Gastrointestinal stromal tumors (GIST) are stromal or mesenchymal subepithelial neoplasms affecting the gastrointestinal tract and frequently contain activating gene mutations in either KIT or platelet-derived growth factor A (PDGFRA). Although GIST is highly responsive to several selective tyrosine kinase inhibitors, combined use of inhibitors targeting other mutations is needed to further prolong survival in patients with GIST. In this study, we aim to screen and identify genetic mutations in GIST for targeted therapy using the new Ion Torrent next-generation sequencing platform. Utilizing the Ion Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes using DNA extracted from formalin-fixed and paraffin-embedded (FFPE) samples of 121 human gastrointestinal stromal tumors, set up stringent parameters for reliable variant calling by filtering out potential raw base calling errors, and identified frequent mutations in the KIT gene. This study demonstrates the utility of using Ion Torrent sequencing to efficiently identify human cancer mutations. This may provide a molecular basis for clinically developing new drugs targeting these gene mutations for GIST therapy.

Primary Atypical Carcinoid of the Parietal Pleura Expressing KIT

The author reports an extremely rare case of atypical carcinoid of the pleura expressing KIT. An 81-year-old Japanese man with emphysema was found to have a mass (3×3×2 cm) in the left parietal pleura by various imaging modalities. Video-assisted tumorectomy was performed, but could not excise it completely. Histologically, the tumor was composed of atypical cells arranged in trabecular, ribbon-like, and rosette patterns. Mitotic figures and necrosis were present. The tumor cells were argyrophil. Immunohistochemically, the tumor cells were positive for pancytokeratins, cytokeratin (CK) 18, chromogranin (focal), p53, KIT, and Ki67 (labeling=17%). In contrast, the tumor cells were negative for CK5/6, CK7, CK19, CK20, CK34βE12, epithelial membrane antigen, vimentin, S100-protein, α-smooth muscle actin, desmin, CEA, TTF-1, CDX-2, AFP, HepPar-1, synaptophysin, CD56, CA19-9, CD15, neuron specific enolase, serotonin, CD34, platelet-derived growth factor-α, calretinin, thrombomodulin, WT-1, B72.3, and D2-40. The pathologic diagnosis was atypical carcinoid of the pleura. The patient was examined by whole body computed tomography, magnetic resonance imaging, and positron emission tomography, but there were no tumors other than the pleural tumor. Endoscopic examinations including upper and lower gastrointestinal tract revealed no tumors. Therefore, the pleural tumor was thought to be primary. Ten months later, the patient developed metastatic lesions in the vertebular and femoral bones, and died of respiratory failure. Autopsy was not performed. To the best of the author's knowledge, there are no cases of primary carcinoid in the pleura in the English literature. This case is also interesting in that the atypical carcinoid expressed KIT.

Prodomain of the proprotein convertase subtilisin/kexin Furin (ppFurin) protects from tumor progression and metastasis

Proteolytic maturation of various precursor proteins by the proprotein convertase Furin is now considered as a crucial step in tumor progression and metastasis. Here, we report the repression of the malignant and metastatic potential of carcinoma cells by the prodomain region of Furin (ppFurin), a naturally occurring inhibitor of this convertase. Overexpression of ppFurin in carcinoma cells in a stable manner significantly reduced their convertase activity and ability to mediate processing of the Furin cancer-related substrates platelet-derived growth factor (PDGF)-A and insulin-like growth factor-I receptor precursors. Unprocessed platelet-derived growth factor-A produced by ppFurin expressing cells failed to induce the activation of Akt in the platelet-derived growth factor receptor-expressing cells NIH BALB/c-3T3 and treatment of ppFurin expressing cells with insulin-like growth factor-I failed to induce Akt phosphorylation, compared with controls. The malignant potential of ppFurin expressing cells was significantly reduced as revealed by the loss of anchorage-independent growth and survival that associated their increased chemosensitivity. In vivo, comparative studies revealed that expression of ppFurin in the carcinoma cells MDA-MB-231 and CT-26 cells inhibited tumor growth when subcutaneously inoculated in nude mice. The use of an experimental liver colorectal metastasis model revealed the reduced ability of metastatic carcinoma CT-26 cells to colonize the liver in response to intrasplenic/portal inoculation. Further analyses revealed reduced Furin activity in tumors derived from intrasplenic inoculated mice with ppFurin expressing CT-26 cells. This finding highlights the role of Furin in the malignant and metastatic potential of tumor cells and suggests the possible consideration of using its naturally occurring inhibitor ppFurin in anticancer therapy.

Platelet-derived growth factor-A and sonic hedgehog signaling direct lung fibroblast precursors during alveolar septal formation

Alveolar septal formation is required to support the respiration of growing mammals; in humans effacement of the alveolar surface and impaired gas exchange are critical features of emphysema and pulmonary fibrosis. Platelet-derived growth factor-A (PDGF-A) and its receptor PDGF-receptor-α (PDGFRα) are required for secondary septal elongation in mice during postnatal days 4 through 12 and they regulate the proliferation and septal location of interstitial fibroblasts. We examined lung fibroblasts (LF) to learn whether PDGFRα expression distinguished a population of precursor cells, with enhanced proliferative and migratory capabilities. We identified a subpopulation of LF that expresses sonic hedgehog (Shh) and stem cell antigen-1 (Sca1). PDGF-A and Shh both increased cytokinesis and chemotaxis in vitro, but through different mechanisms. In primary LF cultures, Shh signaled exclusively through a noncanonical pathway involving generation of Rac1-GTP, whereas both the canonical and noncanonical pathways were used by the Mlg neonatal mouse LF cell line. LF preferentially oriented their primary cilia toward their anterior pole during migration. Furthermore, a larger proportion of PDGFRα-expressing LF, which are more abundant at the septal tips, bore primary cilia compared with other alveolar cells. In pulmonary emphysema, destroyed alveolar septa do not regenerate, in part because cells fail to assume a configuration that allows efficient gas exchange. Better understanding how LF are positioned during alveolar development could identify signaling pathways, which promote alveolar septal regeneration.

Frequent mutations in MLH1, MET, KIT, PDGFRA, and PIK3CA genes in human gastrointestinal stromal tumors.

10544 Background: Identifying mutations in individual tumor is critical to improve the efficacy of cancer therapy by matching targeted drugs to specific mutations. Gastrointestinal stromal tumor (GIST) is a stromal or mesenchymal subepithelial neoplasm affecting the gastrointestinal tract and it frequently contains an activating mutation in either the KIT or platelet-derived growth factor A (PDGFRA) gene. Although GIST is highly responsive to several selective tyrosine kinase inhibitors, combined use of inhibitors targeting other mutations is needed to further prolong survival. Methods: In this study, we aim to screen and identify genetic mutations in GIST for targeted therapy using the new Ion Torrent next-generation sequencing platform. Results: By utilizing the Ion Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes from DNA extracted from formalin-fixed and paraffin-embedded (FFPE) samples of 125 human gastrointestinal stromal tumors, set up stringent parameters for reliable variant calling by filtering out potential raw base calling errors, and identified frequent mutations in PDGFRA, KIT, APC, MLH1, MET, RET, and PIK3CA. Moreover, frequent missense mutations were detected in MLH1 (12%), MET (16.8%), KIT (51.2%), and PIK3CA (41.6%) genes. The mutations in MLH1 gene were frequently detected in the exon 14, while MET gene was frequently mutated in the exon 2. In addition, we identified missense mutations in other genes, including FLT3, KRAS, PDGFRA, and STK11, at lower frequencies. Conclusions: This study demonstrates the utility of using Ion Torrent sequencing to efficiently identify human cancer mutations. With the finding of the frequent mutations in MLH1 and MET genes, in addition to KIT, PDGFRA and PIK3CA, in GISTs, this study provides a molecular basis for clinically developing new drugs targeting these gene mutations for GIST therapy.

The Ah receptor regulates growth factor expression in head and neck squamous cell carcinoma cell lines

Previous studies in head and neck squamous cell carcinoma (HNSCC) cell lines have revealed that the Ah receptor (AHR) plays a significant role in mediating the "aggressive" phenotype of these cells, which includes enhanced inflammatory signaling (e.g., IL6) and migratory potential. Here we sought to identify putative novel targets of the AHR associated with enhanced tumor invasiveness. Global gene expression analysis identified a number of genes that are repressed upon treatment of OSC-19 or HN30 cells with an AHR antagonist. Three growth factors were targets of AHR activity; amphiregulin (AREG), epiregulin (EREG), and platelet-derived growth factor A (PDGFA) were repressed by an AHR antagonist and further examined. Quantitative PCR analysis, ELISA, and siRNA-mediated knock down of AHR revealed an attenuation of basal and/or induced levels of expression of these growth factors in two HNSCC lines, following AHR antagonism. In silico analysis revealed that these growth factors possess dioxin-like response elements. Two other AHR ligands, 6-formylindolo[3,2-b]carbazole and benzo(a)pyrene (BP) also elicited similar responses. In conclusion, this study identified AREG, EREG, and PDGFA as growth factor targets of AHR activity associated with metastatic phenotype of HNSCC cells, suggesting that attenuation of AHR activity may be a therapeutic strategy.

Abstract 4093: Growth factors regulated by the aryl hydrocarbon receptor in highly metastatic head and neck squamous cell carcinoma cell lines.

Abstract The aryl hydrocarbon receptor (AHR), a ligand activated transcription factor, is implicated in mediating pleotropic cellular effects in response to a variety of AHR ligands. Recent evidence has implicated the AHR in tumor initiation, promotion and progression as well. Enhanced AHR expression has been associated with dysregulated cellular proliferation, enhanced cell motility and loss of cell-cell contact. Previous studies from this laboratory have revealed considerable constitutive and ligand-inducible AHR transcriptional activity in head and neck squamous cell carcinoma cell lines (HNSCC). Subsequently, antagonism of AHR activity was found to greatly attenuate the highly metastatic and proliferative phenotype of these cells, thereby suggesting that the AHR plays a pivotal role in dictating the ‘aggressive’ phenotype of these cells. Hence, this study sought to use a whole genome expression profiling based approach to identify putative novel targets of the AHR associated with cancer cell survival and tumor invasiveness. Given previous documented evidence, from other studies in the literature, of the involvement of growth factors in dictating the aggressive phenotype of various cancers, this study sought to focus specifically on the growth factor targets regulated by AHR. Additionally, enhanced growth factor expression, along with high inflammatory cytokine expression, has been found to play a prominent role in dictating tumor cell malignancy. Given previous evidence from our laboratory of high inflammatory cytokine expression (IL1B, IL6) in HNSCC cell lines, the expression of growth factors was also monitored in the presence of an inflammatory signal (IL1B) and an AHR agonist (TCDD). Three growth factor targets were considered; amphiregulin (AREG), epiregulin (EREG) and platelet-derived growth factor A (PDGFA) based on expression profiling and from previous studies. Quantitative PCR analysis revealed an attenuation of basal and/or induced gene expression levels of these growth factors following AHR antagonism, either by AHR antagonist treatment or by siRNA mediated AHR knock down. ELISA analysis revealed an attenuation of both basal and induced protein levels of the growth factors examined in these cell lines. In silico analysis revealed these growth factors to possess dioxin-like response elements in regions upstream of their promoter. Two other AHR ligands, 6-formylindolo[3,2-b]carbazole and benzo(a)pyrene also exhibited induction of gene expression of the AHR growth factors examined. TCDD+IL1B in combination caused a significant increase in the expression of all growth factors examined with CH223191 causing an attenuation of their expression. In conclusion, AREG, EREG and PDGFA were identified as three key growth factor targets of the AHR associated with metastatic phenotype of head and neck cancers. Citation Format: Kaarthik John, Jarod Hughes, Tejas Lahoti, Wagner Kelly, Perdew Gary. Growth factors regulated by the aryl hydrocarbon receptor in highly metastatic head and neck squamous cell carcinoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4093. doi:10.1158/1538-7445.AM2013-4093