Expression Of TGF-alpha Research Articles

Abstract 1838: TGFalpha (an EGFR ligand) promotes growth of EGFR-mutant lung tumors in airway regions but not in alveolar regions in a transgenic mouse model

Abstract Although an exogenous EGF is dispensable for EGFRL858R-mediated NIH3T3 cell transformation in vitro (Greulich et al., PLoS Med 2005), an exogenous EGF promotes growth of H1650 and H1975 lung adenocarcinoma cells that carry EGFR mutations in vitro (Sordella et al., Science 2004). However, it is unknown whether an exogenous EGFR ligand plays a role in growth of EGFR-mutant lung tumors in vivo. Here, using conditional transgenic mice expressing EGFRL858R or KRASG12D along with an exogenous TGFalpha (an EGFR ligand) in lung epithelium, we sought to determine the role of an EGFR ligand in EGFRL858R-lung tumors or wild type EGFR-lung tumors (KRASG12D-lung tumors) in vivo. As previously reported, conditional expression of TGFalpha (an EGFR ligand) in lung epithelium caused pulmonary fibrosis but not lung tumors (Korfhagen et al., J Clin Invest 1994; Hardie et al., AJP-LCMP 2004), indicating that the expression of an exogenous EGFR ligand is not sufficient to induce lung tumors. Conditional expression of EGFRL858R or KRASG12D in lung epithelium caused lung tumors as previously reported (Fisher et al., Genes Dev 2001; Politi et al., Gene Dev 2006). In our study using a new rtTA driver, Scgb1a1-rtTA line 2 (Perl et al., AJRCMB 2006), the majority of EGFRL858R-lung tumors occurred in alveolar regions while KRASG12D-lung tumors occurred in both alveolar and airway regions. Importantly, co-expression of EGFRL858R along with TGFalpha in lung epithelium induced the growth of lung tumors in airway regions but not in alveolar regions while the expression of EGFRL858R alone did not induce lung tumors in airway regions. Co-expression of EGFRL858R along with TGFalpha also shortened survival of the mice compared to the expression of either EGFRL858R or TGFalpha alone (median 32 days for EGFRL858R/TGFalpha vs 118 days for EGFRL858R alone or 248 days for TGFalpha alone). Co-expression of KRASG12D with TGFalpha did not influence tumor location or survival of the mice compared to the expression of either KRASG12D or TGFalpha alone. Consistent with the mouse study, TGFalpha expression in human EGFR-mutant lung cancer but not wild-type EGFR lung cancer was associated with worse overall survival (median 44 months for TGFalpha high vs 75 months for TGFalpha low; p<0.05) and recurrence free survival (median 21 months for TGFalpha high vs 61 months for TGFalpha low; p<0.05). Collectively, our results demonstrate that TGFalpha (an EGFR ligand) promotes not only the growth of EGFRL858R-lung tumors but also influences the regions where the EGFRL858R-lung tumors form. Citation Format: Koichi Tomoshige, Yutaka Maeda. TGFalpha (an EGFR ligand) promotes growth of EGFR-mutant lung tumors in airway regions but not in alveolar regions in a transgenic mouse model [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 1838. doi:10.1158/1538-7445.AM2017-1838

S&T-35 EXOSOMAL MIRNAS AND SERUM CYTOKINES AS PREDICTORS FOR TREATMENT OUTCOMES IN PATIENTS WITH METASTATIC RENAL CELL CARCINOMA TREATED WITH AXITINIB

You have accessJournal of UrologyScience & Technology Posters1 Apr 2016S&T-35 EXOSOMAL MIRNAS AND SERUM CYTOKINES AS PREDICTORS FOR TREATMENT OUTCOMES IN PATIENTS WITH METASTATIC RENAL CELL CARCINOMA TREATED WITH AXITINIB Naoko Suzuki-Honma, Norihiko Tsuchiya, Takamitsu Inoue, Shintaro Narita, Mitsuru Saito, Hiroshi Tsuruta, Atsushi Maeno, Shigeru Satoh, and Tomonori Habuchi Naoko Suzuki-HonmaNaoko Suzuki-Honma More articles by this author , Norihiko TsuchiyaNorihiko Tsuchiya More articles by this author , Takamitsu InoueTakamitsu Inoue More articles by this author , Shintaro NaritaShintaro Narita More articles by this author , Mitsuru SaitoMitsuru Saito More articles by this author , Hiroshi TsurutaHiroshi Tsuruta More articles by this author , Atsushi MaenoAtsushi Maeno More articles by this author , Shigeru SatohShigeru Satoh More articles by this author , and Tomonori HabuchiTomonori Habuchi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2864AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Axitinib (AXI), which selectively inhibits VEGF-Rs, is one of the promising molecular targeted drugs for metastatic renal cell carcinoma (mRCC). However, novel biomarkers to predict for patient outcomes are urgently needed because there are individual differences in treatment response. Here, we investigated the relationship between exosomal microRNAs, serum cytokines, and clinical outcomes in patients with mRCC treated with AXI. METHODS Serum samples at baseline and at 4 weeks after AXI treatment were extracted from total of 32 patients with mRCC treated with AXI between September 2012 and October 2014 at Akita University Hospital. The expression levels of 4 exosomal microRNAs (miR-21, miR-155, miR-210, miR-378) were evaluated by quantitative RT-PCR. The levels of 34 cytokines were measured by Bio-Plex Pro TM Human Cancer Biomarker Panels. The relationship between exosomal microRNAs, serum cytokines and clinical outcomes including response rate and progression free survival (PFS) after AXI treatment was statistically assessed. RESULTS 43.8% patients had a partial response to AXI. Median PFS was 7.4 months. The patients with a higher expression level of miR-378 at baseline had a significantly better tumor response rate than those with a lower expression level of miR-378 (p = 0.048), while the patients with a lower expression level of miR-21 at baseline had a significantly longer PFS than those with a higher expression level of miR-21 (p = 0.019). The patients with higher expression levels of serum HGF, IL-6R, and PECAM-1 at baseline had a significantly longer PFS than those with a lower expression of these cytokines (p = 0.043, p = 0.033, p = 0.002, respectively). Decreased expression of serum IL-8 and TGF-alpha at 4weeks after AXI treatment were significantly associated with longer PFS (p = 0.020, p = 0.025, respectively). The exosomal microRNAs at 4 weeks after AXI treatment were not significantly associated with clinical outcomes in patients with mRCC. CONCLUSIONS Pretreatment exosomal microRNAs and serum cytokines may be potential predictive biomarkers in patients with mRCC treated with AXI. Personalized medicine using these biomarkers is useful for selecting effective drugs in patient with mRCC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e322-e323 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Naoko Suzuki-Honma More articles by this author Norihiko Tsuchiya More articles by this author Takamitsu Inoue More articles by this author Shintaro Narita More articles by this author Mitsuru Saito More articles by this author Hiroshi Tsuruta More articles by this author Atsushi Maeno More articles by this author Shigeru Satoh More articles by this author Tomonori Habuchi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Study of the biologic behavior of odontogenic keratocyst and orthokeratinaized odontogenic cyst using TGF-alpha and P53 markers

Odontogenic keratocyst (OKC) is an aggressive cyst, and its recurrence rate is higher than that of other odontogenic cysts. Orthokeratinized odontogenic cyst (OOC) is less aggressive than OKC, but bears the probability of carcinomatous changes. In this study, we evaluated the expression and intensity of P53 and TGF-alpha in order to compare the biologic behavior or probable carcinomatous changes of these two cysts.In this cross-sectional study, 15 OKC and 15 OOC were stained immunohistochemically for P53 and TGF-alpha using the Novolink polymer method. Then, all slides were examined by an optical microscope with 400× magnification, and the stained cells in the basal and parabasal layers were counted. Finally, the results were analyzed by the Mann–Whitney and Wilcoxon tests (P-value<0.05).The difference between the expression of P53 and TGF alpha in the basal layer of OKC and OOC was not statistically significant (P-value>0.05), but the expression of P53 and TGF-alpha in the parabasal layer in OKC was statistically higher compared to OOC (P<0.05).Considering the known role of P53 and TGF-alpha in malignant changes and the higher expression of P53 and TGF-alpha in OKC compared to those in OOC, the probability of carcinomatous changes was higher in OKC than in OOC.

Quantitative analysis of the expression of TGF-alpha and EGFR in papillary thyroid carcinoma: clinicopathological relevance

There are no quantitative data on the mRNA expression of epidermal growth factor receptor (EGFR) and transformation growth factor alpha (TGF-α) in thyroid carcinoma. The aims of this study were to detect, quantify and analyse the clinicopathological correlations of the expression of these genes in a large cohort of patients with thyroid carcinoma. EGFR and TGF-α expression were investigated using real time quantitative polymerase chain reaction and immunohistochemistry on 71 papillary thyroid carcinomas (PTCs), 68 paired non-cancer thyroid tissues adjacent to the PTC and 20 benign thyroid lesions. TGF-α and EGFR mRNA increased in PTC when compared with benign thyroid lesions. In many PTCs with high level of expression of TGF-α and EGFR mRNA, the morphological non-cancer tissue adjacent to the cancer also showed high levels of expression of these mRNAs. The levels of expression of mRNA of TGF-α and EGFR correlated with each other and with the level of protein expression. The level of expression of TGF-α mRNA was significantly related to lymphovascular permeation while the expression of EGFR mRNA was related to the pathological subtype of PTC and cancer recurrence. TGF-α and EGFR were overexpressed, correlated with each other and associated with the pathological parameters in papillary thyroid carcinoma. The results provide information for management of thyroid cancer in the era of gene targeting therapy.

NADPH Oxidase NOX1 Controls Autocrine Growth of Liver Tumor Cells through Up-regulation of the Epidermal Growth Factor Receptor Pathway

FaO rat hepatoma cells proliferate in the absence of serum through a mechanism that requires activation of the epidermal growth factor receptor (EGFR) pathway. The aim of this work was to analyze the molecular mechanisms that control EGFR activation in these and other liver tumor cells. Reactive oxygen species production is observed a short time after serum withdrawal in FaO cells, coincident with up-regulation of the NADPH oxidase NOX1. NOX1-targeted knockdown, the use of antioxidants, or pharmacological inhibition of NADPH oxidase attenuates autocrine growth, coincident with lower mRNA levels of EGFR and its ligand transforming growth factor-alpha (TGF-alpha) and a decrease in phosphorylation of EGFR. EGFR-targeted knockdown induces similar effects on cell growth and downstream signals to those observed in NOX1-depleted cells. Early NOX1 activation induces both a feedback-positive loop via an Src-ERK pathway that up-regulates its own levels, and a parallel signaling pathway through p38 MAPK and AKT resulting in EGFR and TGF-alpha up-regulation. Human hepatocellular carcinoma cell lines, but not non-tumoral hepatocytes, show autocrine growth upon serum withdrawal, which is also coincident with NOX1 up-regulation that mediates EGFR and TGF-alpha expression. The use of antioxidants, or pharmacological inhibition of NADPH oxidase, effectively attenuates autocrine growth in hepatocellular carcinoma cell lines. In summary, results presented in this study indicate that NOX1 might control autocrine cell growth of liver tumor cells through regulation of the EGFR pathway.

Abstract LB-43: M402, a novel heparin sulphate mimetic, synergizes with gemcitabine to improve survival and reduce metastasis and epithelial-to-mesenchymal transition (EMT) in a genetically engineered mouse model for pancreatic cancer

Abstract Introduction: Heparan sulphate proteoglycans (HSPGs) play a central role in tumor progression and metastasis by presenting and modulating growth factors, cytokines, and other soluble factors. A novel heparin sulphate mimetic (M402), engineered from heparin to have low anti-coagulant activity, has shown promising anti-tumor efficacy in several pre-clinical tumor models. This study was designed to probe the efficacy and mechanism of action of M402 in a genetically engineered mouse (GEM) model for pancreatic cancer. Methods: Mice that spontaneously develop pancreatic cancer (LSL-KRASG12D/+; Trp53 LSL-R172H/flox; pdx-CRE) were treated with twice weekly i.p. doses of saline or gemcitabine (50 mg/kg) starting at Day 30, or with saline or M402 (40 mg/kg/day) administered by a subcutaneous osmotic minipump from Day 30-90, or with a combination of gemcitabine plus M402. Results: Treatment with M402 alone did not prolong survival and gemcitabine alone showed only a modest improvement in survival; however the combination of M402 and gemcitabine significantly improved survival. Moreover, mice treated with the combination of M402 and gemcitabine showed a substantially lower incidence of metastasis. RT-qPCR analysis revealed that M402 treated mice had significantly lower levels of TGF-alpha mRNA than the saline control group, which is corroborated by a corresponding decrease in tumor cell proliferation. Immunohistochemical analysis revealed that M402 treated mice developed reduced areas of epithelial-to-mesenchymal transition (EMT), as defined by negative staining for E-Cadherin, strongly positive staining for vimentin and positive nuclear staining for SNAIL. As there is a direct link between EGFR activation and the nuclear localisation of SNAIL we propose that M402 affects gemcitabine sensitivity and metastasis formation by reducing the expression of TGF-alpha. Discussion: M402 increased the anti-tumor efficacy of gemcitabine in a GEM model for pancreatic cancer resulting in increased survival and, interestingly, decreased incidence of metastasis. One potential mechanism is that the observed reduction in EMT may be due to the reduced expression of TGF-alpha, a cognate ligand for EGFR. These data suggest that the EGFR pathway is active in KRAS mutant tumors. Overall, these results provide a rationale for investigating the clinical use of M402 in combination with gemcitabine in the treatment of human pancreatic cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-43.

Abstract 2608: An intronic polymorphism determines TGF-alpha expression and is associated with cellular resistance to erlotinib

Abstract An intronic polymorphism determines TGF-alpha expression and is associated with cellular resistance to erlotinib Wanqing Liu, Mark J Ratain Section of Hematology/Oncology, Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. Background Responsiveness to EGFR-targeting agents has been demonstrated in lung cancer patients bearing EGFR mutations. However, the detailed mechanism underlying both sensitivity and resistance to these agents has not been completely revealed. Expression of transforming growth factor alpha (TGFA), one of the major EGFR ligands, appears to affect the inhibition potency of EGFR inhibitors. The aim of this study is to identify the genetic variants that may affect TGFA expression and confer susceptibility to response to EGFR-targeting agents. Methods Genome-wide genotyping of 100K single nucleotide polymorphisms (SNPs) had been previously performed in the NCI-60 cancer cell panel. SNPs in the TGFA gene region were selected to perform correlations with TGFA mRNA expression measured by real-time PCR. Confirmation of the SNP genotypes was performed by both sequencing and restriction enzyme digestion. The SNP genotype was correlated with cytotoxicity data of erlotinib and other EGFR inhibitors. Results A total of 10 SNPs in the TGFA gene were genotyped on the 100K SNP Chip. The genotypes were confirmed. A SNP (rs3821262 C/T) in the second intron of TGFA was significantly associated with decreased TGFA gene expression and cellular resistance to both erlotinib (p=0.01) and other 11 EGFR inhibitors (p=0.05) tested in the NCI-60. Conclusion The SNP rs3821262 C/T may contribute to cellular resistance to EGFR inhibitors by modulating the gene expression. However, since this is a hypothesis-generating study and the association was not adjusted for multiple testing, the finding must be validated further in both preclinical and clinical settings, in particular combined with mechanistic studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2608.

Nuclear factor‐kappa B as a promising target for selenium chemoprevention in rat hepatocarcinogenesis

Selenium's molecular mechanism for cancer chemoprevention remains unknown. We aimed to study the gene expression of nuclear factor-kappaB (NF-kappaB), tumor growth factor-alpha (TGF-alpha) and cyclin D1 and the effects of sodium selenite using preventive and therapeutic approaches in chemically-induced hepatocarcinogenesis in rats. Rats were divided randomly into six groups: negative control, positive control (diethyl nitrosamine [DEN] + 2-acetylaminofluorene [2-AAF]), preventive group, preventive control (respective control for preventive group), therapeutic group and therapeutic control (respective control for therapeutic group). The relative gene expression of NF-kappaB, TGF-alpha and cyclin D1 in liver tissues were measured using real-time polymerase chain reaction. The findings showed that the gene expression of NF-kappaB in the preventive group and its respective control was significantly lower (P < 0.05) when compared with both the negative and positive controls. However, the expression of NF-kappaB in the positive controls and therapeutic group was significantly higher (P < 0.05) when compared with the negative controls. The expression of TGF-alpha and cyclin D1 was insignificant in all groups. The inhibition of the NF-kappaB pathway in the initiation phase of hepatocarcinogenesis could be a promising target for selenium chemoprevention. However, further studies are required.

Inhibition of PI3K by PX-866 Prevents Transforming Growth Factor-α–Induced Pulmonary Fibrosis

Transforming growth factor-alpha (TGFalpha) is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. EGFR signaling activates several intracellular signaling pathways including phosphatidylinositol 3'-kinase (PI3K). We previously showed that induction of lung-specific TGFalpha expression in transgenic mice caused progressive pulmonary fibrosis over a 4-week period. The increase in levels of phosphorylated Akt, detected after 1 day of doxycycline-induced TGFalpha expression, was blocked by treatment with the PI3K inhibitor, PX-866. Daily administration of PX-866 during TGFalpha induction prevented increases in lung collagen and airway resistance as well as decreases in lung compliance. Treatment of mice with oral PX-866 4 weeks after the induction of TGFalpha prevented additional weight loss and further increases in total collagen, and attenuated changes in pulmonary mechanics. These data show that PI3K is activated in TGFalpha/EGFR-mediated pulmonary fibrosis and support further studies to determine the role of PI3K activation in human lung fibrotic disease, which could be amenable to targeted therapy.

Substance P and calcitonin gene related peptide induce TGF-alpha expression in epithelial cells via mast cells and fibroblasts

Objective/background Signals for the expression of EGF, TGFα and related proteins are largely unknown. Having shown earlier, that activation of the afferent sensory nervous system by capsaicin induced epithelial cell proliferation and TGFα expression in vivo the aim of this study was to demonstrate, that neurotransmitters of the afferent sensory nervous system induce TGFα expression via mast cells and fibroblasts. Results A significant increase in TGFα-mRNA and protein expression was detected in epithelial cells exposed to supernatants of CGRP-stimulated mast cells and SP-stimulated fibroblasts. Epithelial cell proliferation was only detected in epithelial cells exposed to supernatants of CGRP-stimulated mast cells but not in epithelial cells exposed to supernatants of SP-stimulated fibroblasts. TGFβ mRNA expression was increased in epithelial cells exposed to fibroblasts but not to mast cells. No increase in EGF-receptor expression was detected in epithelial cells exposed to either fibroblast or mast cell supernatants. Conclusion Neurotransmitters of the afferent sensory nervous system induce TGFα expression in epithelial cells mediated by mast cells and fibroblasts. Release of CGRP and SP from neuroeffector junctions is an important signal for the TGFα expression after mucosal injury. Mucosal expression of TGFα may contribute to the protective effects of the sensory nervous system after mucosal injury.

Carbachol induces TGF-alpha expression and colonic epithelial cell proliferation in sensory-desensitised rats

Signals for the expression of the peptide growth factors epidermal growth factor and transforming growth factor-alpha (TGFalpha) in the gastrointestinal mucosa are largely unknown. We have shown earlier that extrinsic afferents in the gastrointestinal tract induce TGFalpha expression in colonic mucosa via the deliberation of neurotransmitters substance P and calcitonin gene-related peptide. The aim of our present study was to determine the effects of carbachol on mucosal TGFalpha expression and epithelial cell proliferation in vivo. Rats were divided in three groups. Group 1 was treated with vehicle only, group 2 received one single subcutaneous injection of 250 microg/kg of carbachol and animals in group 3 were sensory-desensitised prior to the injection of 250 microg/kg carbachol. TGFalpha expression and epithelial cell proliferation was evaluated by polymerase chain reaction, Western blot analysis and bromodeoxyuridine staining. Carbachol induced a significant increase in mucosal epithelial cell proliferation and TGFalpha expression. Sensory desensitisation did neither abolish the increased TGFalpha expression nor the increase in epithelial cell proliferation. Parasympathetic pathways are involved in the control of TGFalpha expression in gastrointestinal mucosa as well as in epithelial cell proliferation.

The studies of hepatitis B virus X gene induces oval cell malignant transformation

To find out the mechanisms of HBx gene inducing oval cell malignant transformation into hepatoma carcinoma cell. The changes of morphology, cell cycle, differentiated markers, c-myc and TGF-alpha in pEGFP-HBx oval cells strain, which stably expressed HBx gene, were studied by inversion phase contrast microscope and transmission electron microscopy, flow cytometry, periodic acid-schiff (PAS) staining, soft agar growth assay, real-time PCR, immunocytochemistry. pEGFP-oval cells and LE/6 oval cells were used as control groups. (1) The pEGFP-HBx oval cells showed bigger in size with malformed nucleus as compared with control groups. (2) Flow cytometry showed that, in contrast with the control groups, the proportion of pEGFP-HBx oval cells arrested in G0/G1 phase decreased but in S or G2/M phase rose. Moreover, the population of aneuploid cells increased obviously. (3) PAS staining showed that there were many glycogen granules in the cytoplasm of pEGFP-HBx oval cell. (4) The pEGFP-HBx oval cell formed colonies in the soft agar. (5) Compared with the control groups, the expression of HNF-4 alpha, AFP, c-myc and TGF-alpha rose obviously, whereas the expression of CK-7 and CK-19 decreased. And the expression of cps1 mRNA was not in the extent of detection. The HBx gene can provoke abnormal differentiation of oval cell and induce oval cell malignant transformation.

The haematopoietic specific signal transducer Vav1 is aberrantly expressed in lung cancer and plays a role in tumourigenesis

Lung cancer is the leading cause of cancer death worldwide. The spectrum of aberrations affecting signalling pathways in lung cancer pathogenesis has not been fully elucidated. Physiological expression of Vav1 is restricted to the haematopoietic system, where its best-known function is as a GDP/GTP nucleotide exchange factor for Rho/RacGTPases, an activity strictly controlled by tyrosine phosphorylation downstream of cell surface receptors. Here we find Vav1 expression in 42% of 78 lung cancer cell lines analysed. Moreover, immunohistochemical analysis of primary human lung cancer tissue samples revealed Vav1 expression in 26/59 malignant samples, including adenocarcinoma, squamous cell carcinoma and bronchioloalveolar carcinoma. Stronger Vav1 staining was associated with larger tumour size. siRNA-mediated knockdown of Vav1 in lung cancer cells reduced proliferation in agar and tumour growth in nude mice, while control siRNA had no effect, suggesting that Vav1 plays a critical role in the tumorigenicity of lung cancer cells. Vav1 is tyrosine-phosphorylated in lung cancer cells following activation by the growth factors EGF and TGFalpha, suggesting its participation in signalling events in these cells. Depletion of Vav1 reduced Rac-GTP activation and decreased expression of TGFalpha, an autocrine growth factor. These data suggest that Vav1 plays a role in the neoplastic process in lung cancer, identifying it as a potential therapeutic target for lung cancer therapy.

Rapamycin Prevents Transforming Growth Factor-α–Induced Pulmonary Fibrosis

Transforming growth factor (TGF)-alpha is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. Overexpression of TGF-alpha in transgenic mice causes progressive and severe pulmonary fibrosis; however, the intracellular signaling pathways downstream of EGFR mediating this response are unknown. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we observed increased PCNA protein and phosphorylation of Akt and p70S6K in whole lung homogenates in association with induction of TGF-alpha. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over a 7-week period. Daily administration of rapamycin prevented accumulation of total lung collagen, weight loss, and changes in pulmonary mechanics. Treatment of mice with rapamycin 4 weeks after the induction of TGF-alpha prevented additional weight loss, increases in total collagen, and changes in pulmonary mechanics. Rapamycin prevented further increases in established pulmonary fibrosis induced by EGFR activation. This study demonstrates that mammalian target of rapamycin (mTOR) is a major effector of EGFR-induced pulmonary fibrosis, providing support for further studies to determine the role of mTOR in the pathogenesis and treatment of pulmonary fibrosis.

Early Growth Response-1 Suppresses Epidermal Growth Factor Receptor–Mediated Airway Hyperresponsiveness and Lung Remodeling in Mice

Transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor receptor, are induced after lung injury and are associated with remodeling in chronic pulmonary diseases, such as pulmonary fibrosis and asthma. Expression of TGF-alpha in the lungs of adult mice causes fibrosis, pleural thickening, and pulmonary hypertension, in addition to increased expression of a transcription factor, early growth response-1 (Egr-1). Egr-1 was increased in airway smooth muscle (ASM) and the vascular adventitia in the lungs of mice conditionally expressing TGF-alpha in airway epithelium (Clara cell secretory protein-rtTA(+/-)/[tetO](7)-TGF-alpha(+/-)). The goal of this study was to determine the role of Egr-1 in TGF-alpha-induced lung disease. To accomplish this, TGF-alpha-transgenic mice were crossed to Egr-1 knockout (Egr-1(ko/ko)) mice. The lack of Egr-1 markedly increased the severity of TGF-alpha-induced pulmonary disease, dramatically enhancing airway muscularization, increasing pulmonary fibrosis, and causing greater airway hyperresponsiveness to methacholine. Smooth muscle hyperplasia, not hypertrophy, caused the ASM thickening in the absence of Egr-1. No detectable increases in pulmonary inflammation were found. In addition to the airway remodeling disease, vascular remodeling and pulmonary hypertension were also more severe in Egr-1(ko/ko) mice. Thus, Egr-1 acts to suppress epidermal growth factor receptor-mediated airway and vascular muscularization, fibrosis, and airway hyperresponsiveness in the absence of inflammation. This provides a unique model to study the processes causing pulmonary fibrosis and ASM thickening without the complicating effects of inflammation.

Tumor necrosis factor‐α‐stimulated membrane type 1‐matrix metalloproteinase production is modulated by epidermal growth factor receptor signaling in human gingival fibroblasts

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a collagenolytic enzyme involved in connective tissue remodeling. In periodontal tissues, either cytokines or growth factors regulate the production of proteolytic enzymes. Mice deficient in epidermal growth factor receptor (EGFR) show a reduced expression of MT1-MMP, suggesting that this receptor may play an important role in MT1-MMP production. The present study evaluated the role of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and EGFR in the production of MT1-MMP in gingival fibroblasts. Primary cultures of human gingival fibroblasts were cultured over plastic or a type I collagen matrix and stimulated with TNF-alpha and EGF. A selective EGFR inhibitor (AG1478) was used to interfere with this signaling pathway. Production of MT1-MMP and activation of proMMP-2 were studied using Western blot and gelatin zymography, respectively. Activation of EGFR signaling was assessed through immunoprecipitation and Western blot. Expression of EGFR ligands was determined through reverse transcriptase-polymerase chain reaction. Treatment of gingival fibroblasts cultured over a collagen matrix with TNF-alpha stimulated proMMP-2 activation and MT1-MMP production. However, after using AG1478, both responses were inhibited. Tumor necrosis factor-alpha induced EGFR transactivation and stimulated the expression of the mRNA for the EGFR ligands heparin binding-epidermal growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha). The present study shows that TNF-alpha may stimulate MT1-MMP production through transactivation of EGFR. Tumor necrosis factor-alpha may also modulate the expression of the EGFR ligands TGF-alpha and HB-EGF. Production of MT1-MMP by TNF-alpha requires interaction with EGFR, suggesting that tissue remodeling is controlled by cross-communication between diverse signaling pathways in gingival fibroblasts.

TGF-alpha as a candidate tumor antigen for renal cell carcinomas.

Patients with renal cell carcinomas (RCC) have few treatment options, underscoring the importance of developing new approaches such as immunotherapy. However, few tumor associated antigens (TAA), which can be targeted by immunotherapy, have been identified for this type of cancer. von Hippel-Lindau clear cell RCC (VHL(-/-)RCC) are characterized by mutations in the VHL tumor suppressor gene. Loss of VHL function causes the overexpression of transforming growth factor (TGF)-alpha, leading us to hypothesize that TGF-alpha could be a potential TAA for immunotherapy of kidney cancer, which was evaluated in this study. We first confirmed the absent or weak expression of TGF-alpha in important normal tissues as well as its overexpression in 61% of renal tumors in comparison to autologous normal kidney tissues. In addition, we demonstrated the immunogenicity of TGF-alpha, by expanding many T cell lines specific for certain TGF-alpha peptides or the mature TGF-alpha protein, when presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. Interestingly, some of these TGF-alpha-specific T cells were polyfunctionals and secreted IFN-gamma, TNF-alpha and IL-2. We have shown that TGF-alpha is a valid candidate TAA, which should allow the development of a targeted immunotherapy.

Transgenic Induction of Vascular Endothelial Growth Factor-C Is Strongly Angiogenic in Mouse Embryos but Leads to Persistent Lymphatic Hyperplasia in Adult Tissues

Vascular endothelial growth factor-C (VEGF-C) is the quintessential lymphangiogenic growth factor that is required for the development of the lymphatic system and is capable of stimulating lymphangiogenesis in adults by activating its receptor, VEGFR-3. Although VEGF-C is a major candidate molecule for the development of prolymphangiogenic therapy for defective lymphatic vessels in lymphedema, the stability of lymph vessels generated by exogenous VEGF-C administration is not currently known. We studied VEGF-C-stimulated lymphangiogenesis in inducible transgenic mouse models in which growth factor expression can be spatially and temporally controlled without side effects, such as inflammation. VEGF-C induction in adult mouse skin for 1 to 2 weeks caused robust lymphatic hyperplasia that persisted for at least 6 months. VEGF-C induced lymphangiogenesis in numerous tissues and organs when expressed in the vascular endothelium in either neonates or adult mice. Very few or no effects were observed in either blood vessels or collecting lymph vessels. Additionally, VEGF-C stimulated lymphangiogenesis in embryos after the onset of lymphatic vessel development. Strikingly, a strong angiogenic effect was observed after VEGF-C induction in vascular endothelium at any point before embryonic day 16.5. Our results indicate that blood vessels can undergo VEGF-C-induced angiogenesis even after down-regulation of VEGFR-3 in embryos; however, transient VEGF-C expression in adults can induce long-lasting lymphatic hyperplasia with no obvious side effects on the blood vasculature.

Impaired liver regeneration in acute liver failure: the significance of cross‐communication of growth associated factors in liver regeneration

Liver regeneration is considered to be retarded or suppressed in patients with acute liver failure. However, the mechanisms for this suppression remain to be elucidated. In order to evaluate deficiencies in liver regeneration in patients with acute liver failure, we focused on the cross-communication of growth-associated factors in experimental models. In primary cultured rat hepatocytes, the levels of cellular p53 and transforming growth factor-alpha (TGF-alpha), as well as DNA synthesis increased by the addition of hepatocyte growth factor (HGF). When p53 activity was suppressed, TGF-alpha expression and DNA synthesis were reduced. DNA synthesis was also reduced when TGF-alpha activity was suppressed. In rats after partial hepatectomy, hepatic HGF and p53 protein levels increased, followed by an increase of hepatic TGF-alpha levels and hepatocyte proliferation. Circulating levels of HGF and TGF-alpha correlated with changes in the hepatic levels. The suppression of TGF-alpha activity reduced the proliferation of hepatocytes in these rats. In patients suffering from acute hepatitis, serum HGF levels increased followed by an increase of serum TGF-alpha levels. In contrast, in patients with acute liver failure, the increase of serum TGF-alpha levels was suppressed depending on the severity of hepatic failure, even though serum HGF levels markedly increased. The patients' hepatic levels were consistent with serum levels. Cross-communication of growth associated factors may be important in the progression of liver regeneration, and impaired regenerative capacity in patients with acute liver failure may be attributable, at least in part, to a disruption of communication of growth-associated factors.

Up-regulation of the hypoxia-inducible factor–1 transcriptional pathway in colorectal carcinomas

The aim of the study was to identify the impact on prognosis of hypoxia-inducible factor-1 genetic program in colorectal carcinomas and to develop an experimental procedure that would allow a reliable quantitative gene expression analysis in formalin-fixed and paraffin-embedded tissue. The expression of hypoxia-inducible factor-1alpha and 13 hypoxia-inducible factor-1 target genes (AMF, CAIX, VEGF, VEGFR1, VEGFR2, HGF, MET, TGFalpha, EGFR, IGF2, MMP2, PLAUR, NIX) was quantified by real-time polymerase chain reaction in 18 colorectal, poorly differentiated neuroendocrine carcinomas and in 60 invasive colorectal carcinomas. Moreover, hypoxia-inducible factor-1alpha protein expression was evaluated by immunohistochemistry. High levels of hypoxia-inducible factor-1alpha were positively associated with poorly differentiated neuroendocrine carcinoma histology (P < .005), poor differentiation (P < .025), presence of necrosis, and presence of microsatellite instability (P < .05). AMF, TGFalpha, IGF2, NIX, VEGF, and VEGFR2 transcripts were significantly higher in the very aggressive poorly differentiated neuroendocrine carcinomas than in exocrine colorectal carcinomas and TGFalpha expression was significantly associated with presence of lymph nodal metastases (P < .05). High levels of TGFalpha and NIX were significantly associated with decreased overall survival (P < .001; P < .01). The multivariate analysis showed that advanced stage, presence of lymph node metastases, and high TGFalpha expression had an independent effect on survival (P < .006; P < .01; P < .0006). Our study suggests an up-regulation of the hypoxia-inducible factor-1 transcriptional pathway in colorectal carcinomas. hypoxia-inducible factor-1alpha overexpression alone, has no impact on the prognosis of colorectal carcinomas likely because the consequences of hypoxia-inducible factor-1alpha expression/stabilization strongly depend on the genetic background of the transformed cells. Mechanisms leading to increased synthesis of hypoxia-inducible factor-1alpha mRNA via autocrine growth factor loops may play a crucial role in invasive growth in this site.