EGF Signaling Research Articles

The Akt-SRPK-SR Axis Constitutes a Major Pathway in Transducing EGF Signaling to Regulate Alternative Splicing in the Nucleus

Pre-mRNA splicing is regulated by developmental and environmental cues, but little is known about how specific signals are transduced in mammalian cells to regulate this critical gene expression step. Here, we report massive reprogramming of alternative splicing in response to EGF signaling. By blocking individual branches in EGF signaling, we found that Akt activation plays a major role, while other branches, such as the JAK/STAT and ERK pathways, make minor contributions to EGF-induced splicing. Activated Akt next branches to SR protein-specific kinases, rather than mTOR, by inducing SRPK autophosphorylation that switches the splicing kinases from Hsp70- to Hsp90-containing complexes. This leads to enhanced SRPK nuclear translocation and SR protein phosphorylation. These findings reveal a major signal transduction pathway for regulated splicing and place SRPKs in a central position in the pathway, consistent with their reputed roles in a large number of human cancers.

Correlation between miRNA (miR) and gene expression profiles (GEP) and response to neoadjuvant chemotherapy (NT) in patients with locally advanced and inflammatory breast cancer (BC).

10545 Background: GEP may predict for pathologic complete response (pCR). Correlation between miRs, GEP, and pCR may reveal novel targets. Methods: Patients (pts) with HER2- BC were randomized to receive docetaxel, doxorubicin, cyclophosphamide (TAC, arm A) or A and C given every 2 weeks x 4, followed by carboplatin and nab-paclitaxel (arm B). Pts with HER2+ BC received NT per arm B, with trastuzumab added (arm C). Core biopsies were snap-frozen prior to NT, and RNA extracted for gene array analysis (Agilent 44K microarray) and deep sequence miRNA analysis (Solexa/Illumina platform). HER2 testing by IHC [3+] or FISH and/or gene array was carried out (Blueprint). MiRs were measured in 72 of 119 enrolled pts. Of 882 miRNAs reported by Solexa sequencing, 487 were assessed (observed at raw counts > 10 in at least 3 pts). TargetScan was used to obtain miRNA targets and negative correlation between miR and mRNA expression was applied. Results: In 44 HER2- cases, we found 17 miRs differentially expressed between pts who achieved pCR vs. pts who did not (< pCR), 13 with predicted targets by TargetScan (TS). We found 28 canonical pathways (including Embryonic Cell, EGF signaling, Estrogen-dependent signaling) affected by the identified miRs, and inclusive of 74 target mRNAs identified (Agilent) and correlating with pCR. The top overexpressed miRs are 31 [gene targets: FZD4]; 18a [IGF1, ALDH5A1]; 19a [PIK3R, IGF1]) and 3 lowest expressors are miR-190b [MMP1]; 29c [FOS, WNT5B]; 342-3p [BMP7]. Among the 28 HER2+ cases there were 14 miRNAs differentially expressed between BCs with pCR vs <pCR, and 8 had predicted targets from TS. We found 10 canonical pathways (including p53, HER-2, PTEN) affected by miRs, which included 44 target mRNAs (Agilent) correlating with pCR. The most overexpressed miRs are miR-708 [gene target: RRM2B; 193a-p5 [BMPR1B, RPRM] ; 92b [CCNE2, DKK2]. The lowest expressors are: miR-30a [FZD1, SMAD1, TP63]; miR-450-5p [FOXO1, WINT5A, ERBB2]; miR-497 [PIK3R1, FGFR1, FOXO1]. Conclusions: Combined assessment of miRNA and gene expression patterns is feasible, and may yield information leading to individualized and improved NT.

A phase II study of capecitabine-oxaliplatin and cetuximab in patients (pts) with advanced/metastatic gastric cancer (G) or gastroesophageal junction (GEJ) adenocarcinoma.

4096 Background: There have been limited advances in the treatment (tx) of pts with metastatic G or GEJ adenocarcinoma. The combination of fluroropyrimidines and platinums are standard regimens for this disease. The epidermal growth factor receptor pathway has been identified as a target and KRAS mutations are rare. Methods: The primary objective was to assess the proportion of pts with progression within 4 months. Secondary objectives include response (RR), progression free survival (PFS) overall survival (OS), toxicity and molecular correlates. A two-stage Simon phase II design was used to distinguish between a 30% vs. 50% 4 month PFS; with analysis planned at 27 and 53 pts. Eligible pts were chemonaive, metastatic/unresectable with measurable disease. Pts received capecitabine 850 mg/m2 BID D1-14, oxaliplatin 130 mg/m2 IV D1, cetuximab 400 mg/m2 (load) then 250 mg/m2 IV D1, 8, 15, q21 days. Blood and tissue were analyzed for genes in the DNA repair and EGFR pathways. Results: 61 pts were accrued, 2 GEJ and 59 G; 42 male, 19 female; median age 56 years (28-86); 54 pts were evaluable. The 4 month PFS rate 61% (95%CI 47-73%), PFS 5.4 months (95% CI 3.5-6.7); RR 50% (95%CI: 36-64%), 1 (1.9%) complete, 26 (48.1%) partial, 22 (40.7%) stable disease; 2 (3.7%) pts became resectable; median OS 14.9 months (95% CI 8.8-22.2) with median follow-up of 21.8 months. 65% (39/60) of pts had grade 3 (36 pts) or 4 (3 pts) toxicity; most common, anorexia (13.3%), diarrhea (13.3%), fatigue (13.3%), nausea (15%), vomiting (15%); grade 3 hand/foot (5.3%), acneiform rash (3.3%). SNP’s in LRIG4, (regulator of EGF signaling) and PAI1 (tumor angiogenesis, cell proliferation, migration and adhesion) were significantly associated with RR (67% G/G vs 17% A/A ,p=0.044) and PFS (5.8 months 5G/4G vs 3.4 months 5G/5G, p=0.017) respectively. Conclusions: XELOX and cetuximab has promising activity, comparable to existing regimens. Novel SNP’s were identified correlating with RR and PFS which may allow for optimization of pt selection.

Abstract 219: Glycosylation-dependent effect of CD109 on TGF-beta1 and EGF signaling in human glioblastoma cells

Abstract CD109, a glycosylphosphatidylinositol-anchored cell surface glycoprotein, is a negative regulator of TGF-beta1 signaling, whose expression is predominantly detected in squamous cell carcinomas of esophagus, lung, uterus and oral cavity. In the Northern blot analysis using various human tumor cell lines, we found CD109 expression in cell lines derived from human glioblastomas, however, the significance of CD109 in glioblastoma cells has not yet been elucidated. Here, we present the glycosylation-dependent effect of CD109 on TGF-beta1 and EGF signaling in glioblastoma cells. SK-MG-1 cells overexpressing CD109 exhibited downregulation of Smad2 phosphorylation after TGF-beta1 stimulation and upregulation of EGFR and ERK1/2 phosphorylation after EGF stimulation, indicating that CD109 is involved in not only negative regulation of TGF-beta1 signaling but also positive regulation of EGF signaling. However, in other glioblastoma cell lines, U251MG and MG178, CD109 overexpression showed no significant effect on TGF-beta1 and EGF signaling. CD109 overexpression also enhanced cell proliferation and migration in SK-MG-1 cells, but not in the other two cells. Western blot analysis showed that molecular mass of CD109 of SK-MG-1 cells was a bit high compared with those of the other two cell lines, and deglycosylation analysis revealed enhanced glycosylation of CD109 in SK-MG-1 cells. TGF-beta1 signal in U251MG and MG178 was suppressed when the medium with hyperglycosylated soluble form of CD109 was used for TGF-beta1 stimulation. Our results suggest the glycosylation-dependent effect of CD109 on cell biology of glioblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 219. doi:1538-7445.AM2012-219

Abstract 1262: Simple western and NanoPro analysis of key EGF signaling cascade proteins

Abstract Aberrant expression and signaling in the EGF signaling pathway is a common occurrence in a variety of cancers including breast cancer. Understanding how EGF signaling impacts disease progression is key to the development of novel therapeutics. Detection of important protein targets in cancer samples frequently employs Western blot analysis. In-depth phosphorylation analysis often requires 2D gels which are extremely variable and labor intensive, followed by MS analysis. Each of these methods have their advantages and challenges. This study employs the use of two novel nano-scale capillary-based electrophoresis and immunodetection technologies to characterize and validate subtle changes in key protein targets involved in the regulation of disease progression. The Simon system performs size-based separation of proteins, known as Simple Western assays. Alternatively, NanoPro systems utilize charge-based separation via isoelectric focusing (IEF). Both methods utilize capillary electrophoresis (CE) to perform the separation events. Both Simon and NanoPro instruments provide automated workflow eliminating the need for manual processing of multiple steps traditionally needed to carry out either of these separation and detection processes. Proteins from a single sample preparation were analyzed on two platforms: either in a native conformation using charge-based separation (NanoPro assays) or in a denatured state followed by size-based separation (Simple Western assays). Both methods incorporate immunodetection to monitor both subtle and specific changes to key signaling protein targets. Utilizing both platforms allowed for detailed analysis of post-translational modifications (charge-based) and protein abundance (size-based) within a prepared sample. As a validation of synergy between the platforms, we investigated key members of the EGF signaling cascade in different phosphorylation states. We showed that the same cellular lysate preparation can be used for charge- or size-based separation. Advantages of Simple Western and NanoPro assays over gels (1D and 2D) and traditional Westerns include ease of use, minimal user intervention, automatic analysis and excellent reproducibility which ultimate results in better data quality to characterize key regulatory events involved with cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1262. doi:1538-7445.AM2012-1262

Abstract 2308: MUC1 modulation of E-cadherin through miR-200c and ZEB1

Abstract MUC1, a cell-surface glycoprotein implicated in tumorigenesis and metastasis, has a cytoplasmic tail (MUC1.CT) that is phosphorylated by different receptor tyrosine kinases and conducts these signals by binding and altering the function or stability of transcription factors, thereby influencing gene transcription. MicroRNAs (miRNAs), short RNAs (19-22 nucleotides), regulate gene expression at the transcriptional level and are differentially regulated in cancer and metastasis. We discovered that MUC1.CT influences expression of miRNAs that contribute to metastasis, including miR-200c. MiR-200c is a known regulator of epithelial-to-mesenchymal transition (EMT) through regulation of ZEB1, a transcriptional repressor of E-cadherin. In addition, ZEB1 is a repressor of miR-200c in a feedback loop. Overexpression of MUC1 results in a dramatic decrease in miR-200c levels, resulting in increased levels of ZEB1. Chromatin immunoprecipitation (ChIP) was used to demonstrate that MUC1.CT occupies the ZEB1 binding motif upstream of the miR-200c start site. In addition, we found that ZEB1 occupancy of the miR-200c promoter region was enhanced 3.5-fold in MUC1 overexpressing cells, indicating that MUC1.CT may influence the expression and stability of ZEB1 at the miR-200c promoter. Co-immunoprecipitation experiments determined that MUC1.CT directly interacts with ZEB1. We evaluated levels of proteins affected by the miR-200c pathway in MUC1 overexpressing cells and observed an increase in ZEB1 levels and a decrease in E-cadherin levels, consistent with induction of EMT. We also confirmed an increase of ZEB1 occupancy at the E-cadherin promoter in MUC1 overexpressing cells, further confirming our hypothesis. In addition, MUC1.CT is differentially phosphorylated by several receptor tyrosine kinases. Antibodies against different phospho-isoforms were used with ChIP to investigate the signaling cascades that influence localization of the MUC1.CT to the miR-200c promoter. The phospho-YEKV form of MUC1.CT, which is representative of signaling through epidermal growth factor/receptor (EGF/EGFR), was the only phospho-MUC1.CT detected at the ZEB1 binding motif of the miR-200c promoter. EGF signaling is linked to a loss of E-cadherin, and our work is the first to link EGF signaling through the MUC1.CT as a direct regulator of miR-200c expression. These data portray a novel pathway of EMT modulation. EGF signaling via MUC1 regulates levels of miR-200c, altering regulation E-cadherin. Grant support was provided by grants from the NCI (Training Grant CA09476, R01CA57362, U01 CA111294) and student assistantships from UNMC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2308. doi:1538-7445.AM2012-2308

Abstract LB-298: EGF induces EMT through PI3K/Akt and MAPK singnaling pathways in breast cancer cell lines

Abstract EMT, epithelial-mesenchymal transition, is defined by the loss of epithelial characteristics and the acquisition of mesenchymal properties, such as reduced cell to cell contact, spindle morphology, expression mesenchymal markers and migratory function. In carcinogenesis, EMT is known to be associated with increased aggressiveness, and invasive and metastatic potential. Although various factor, such as EGF signaling pathways, cytokines, miRNAs, Wnt signaling and TGF-ß signaling have been suggested as inducers of EMT signaling pathway, detailed mechanism haven't been clarified except TGF-ß induced EMT. This study was designed to evaluate if and how EGF can induce EMT in breast cancer cell lines. We showed EGF induced EMT, such as elongation of cancer cells, down regulation of E-cadherin, and upregulation of vimentin and fibronectin in MCF-7 and MDA-MB-231 cell lines. In addition, we confirmed that EMT by EGF was induced via MAPK and PI3K/Akt signaling pathway using LY294002 (PI3K inhibitor) and PD98059 (MAPK inhibitor). PI3K inhibitor and MAPK inhibitor also blocked phosphorylation of Smad2 and inhibited nuclear localization of p-Smad2. With using SB203580 (p-Smad2 inhibitor) inhibited expression of Snail. In summary, EGF induces EMT through PI3K/Akt and MAPK signaling pathways, which activate downstream of Smad2 and Sanil signaling in MCF-7 and MDA-MB-231 cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-298. doi:1538-7445.AM2012-LB-298

Abstract 927: The ligand-gated Cl- channel GABA inhibits colon cancer invasion following activation by pharmacological treatments

Abstract Gamma-aminobutyric acid (GABA) receptors are ligand-gated ion channels that are essential for neurotransmitter function and neuronal cell homeostasis. Activation of these channels triggers the opening of a chloride-sensitive pore, which causes a hyperpolarization via an influx of chloride ions into the cell. The presence of these channels was recently implicated in several cancers, and the neurotransmitter GABA has been cited as a tumor suppressor. We propose that commercially available pharmacological treatments that manipulate GABA receptor function could be repurposed to target cancer. We have identified functional GABA receptors in a spectrum of colon cancer cell lines and examined the effects of the channels on invasion of the metastatic cell line SW620. Electrophysiological techniques were used to confirm that these channels have functional expression. Treatment with propofol, an agonist which slows the channel closing time, and GABA led to a decrease in invasive potential. Conversely, treatment with the antagonist gabazine, which allosterically blocks GABA from binding in the channel, facilitated invasion by preventing chloride influx. Curiously, treating the cells with bicuculline, which blocks GABA's inhibitory action, led to a decrease in invasion. In some situations bicuculline can also act as a GABA agonist by causing spontaneous gating of homomeric β3 channels. The expression of the β3 subunit was also confirmed through immunocytochemistry. In addition to colon cancer cell lines, β3 expression was detected at higher quantities in colon cancer patient samples compared to matched controls. To further understand how GABA signaling is implicated in cancer, we have looked at the association of GABA with the EGF signaling pathway, which is commonly dysregulated in cancer. The EGF pathway has also been linked with a loss of the glucagon receptor in liver cancer, suggesting that a common pathway may exist between GABA and glutamate that can be exploited for treatment. Altogether, our data suggest that ligand-gated GABA channels inhibit the metastatic potential of cancer cells and can be targeted to treat colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 927. doi:1538-7445.AM2012-927

1071 SINGLE CELL NETWORK PROFILING (SCNP) ASSAY APPLICATION TO BLADDER CANCER SAMPLES IDENTIFYING RARE EGF RESPONSIVE EPITHELIAL CANCER CELLS SENSITIVE TO PI3K INHIBITION FROM BLADDER WASHINGS

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20121071 SINGLE CELL NETWORK PROFILING (SCNP) ASSAY APPLICATION TO BLADDER CANCER SAMPLES IDENTIFYING RARE EGF RESPONSIVE EPITHELIAL CANCER CELLS SENSITIVE TO PI3K INHIBITION FROM BLADDER WASHINGS Zhamshid Okhunov, Todd M. Covey, Michael Gulrajani, Helen Levey, Michelle Cholankeril, Carol Marimpietri, Scott Fields, Alessandra Cesano, and Manish Vira Zhamshid OkhunovZhamshid Okhunov New Hyde Park, NY More articles by this author , Todd M. CoveyTodd M. Covey South San Francisco, CA More articles by this author , Michael GulrajaniMichael Gulrajani South San Francisco, CA More articles by this author , Helen LeveyHelen Levey Rochester, NY More articles by this author , Michelle CholankerilMichelle Cholankeril New Hyde Park, NY More articles by this author , Carol MarimpietriCarol Marimpietri South San Francisco, CA More articles by this author , Scott FieldsScott Fields New Hyde Park, NY More articles by this author , Alessandra CesanoAlessandra Cesano South San Francisco, CA More articles by this author , and Manish ViraManish Vira New Hyde Park, NY More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1177AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES SCNP is a multi-parametric flow cytometry-based assay that simultaneously provides measurements of extracellular surface markers and quantitative changes in intracellular signaling proteins in response to extracellular modulators at the single cell level. The aim of this study was to assess the feasibility of applying SCNP to the examination of signaling networks in bladder cancer (BC) using urine samples and EGF +/- the PI3K pathway inhibitor GDC-0941. METHODS Saline bladder washes were obtained from 28 patients undergoing surgical treatment for BC and 15 control patients without evidence of BC. Samples were mixed with fetal bovine serum, shipped overnight on ice, and processed within 24 hrs. Cells were pelleted, rested for 1 hr at 37C, and then stimulated with EGF +/- GDC0941. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of phospho-AKT and phospho-ERK both at baseline and in response to pathway modulation. The epithelial bladder carcinoma cell line HT-1376 was used as a control for the epithelial phenotype and EGF signaling. After cell fixation and permeabilization, samples were stained with fluorophore-conjugated antibodies/ DAPI cocktail, and the data was acquired using multi-parametric flow cytometry. RESULTS 64% (N=18) of the BC samples and 27% (N=4) of the NC samples met the “evaluable” criteria, i.e. at least 4 x 105 total cells upon sample receipt and >2% of cells with an epithelial phenotype (DAPI+, CD45 low, CK+, and EpCam+). The majority of transitional cells detected in the BC samples were non-apoptotic (i.e. >76% cleaved PARP negative). Signaling:In 3/18 BC evaluable samples a quantifiable increase in EGF-induced p-AKT and p-ERK signaling was identified (2-3 fold over baseline) and was inhibited by GDC-0941 incubation. 2 additional samples, while not responsive to EGF, had their basal pAKT state inhibited by GDC-0941. No EGF-induced signaling was observed in the control specimens. DNA Content In 8/18 BC evaluable samples a DNA index (MFIEpithelial / MFILymphoid) > 1.30 was observed by DAPI staining, suggesting aneuploidy. Of the 3 EGF-responsive samples, 2 were aneuploid. No aneuploidy was observed in the control specimens. CONCLUSIONS Our results demonstrate the feasibility of applying SCNP to the functional characterization of BC. Correlation of in vitro network signaling profiles with clinical outcomes has the potential not only as a prediction tool but also to move toward individualized treatment strategy. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e435 Peer Review Report Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Zhamshid Okhunov New Hyde Park, NY More articles by this author Todd M. Covey South San Francisco, CA More articles by this author Michael Gulrajani South San Francisco, CA More articles by this author Helen Levey Rochester, NY More articles by this author Michelle Cholankeril New Hyde Park, NY More articles by this author Carol Marimpietri South San Francisco, CA More articles by this author Scott Fields New Hyde Park, NY More articles by this author Alessandra Cesano South San Francisco, CA More articles by this author Manish Vira New Hyde Park, NY More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Integration of an abdominal Hox complex with Pax2 yields cell-specific EGF secretion from Drosophila sensory precursor cells

Cis-regulatory modules (CRMs) ensure specific developmental outcomes by mediating both proper spatiotemporal gene expression patterns and appropriate transcriptional levels. In Drosophila, the precise transcriptional control of the serine protease rhomboid regulates EGF signaling to specify distinct cell types. Recently, we identified a CRM that activates rhomboid expression and thereby EGF secretion from a subset of abdominal sensory organ precursor cells (SOPs) to induce an appropriate number of lipid-processing cells called oenocytes. Here, we use scanning mutagenesis coupled with reporter assays, biochemistry and genetics to dissect the transcriptional mechanisms regulating SOP-specific rhomboid activation. Our results show that proper spatial activity of the rhomboid CRM is dependent upon direct integration of the abdomen-specific Hox factor Abdominal-A and the SOP-restricted Pax2 factor. In addition, we show that the Extradenticle and Homothorax Hox co-factors are differentially integrated on the rhomboid CRM by abdominal versus thoracic Hox proteins in the presence of Pax2. Last, we show that Abdominal-A uses both Pax2-dependent and Pax2-independent mechanisms to stimulate rhomboid CRM activity to induce proper oenocyte numbers. Thus, these data demonstrate how a CRM integrates Hox and neural transcriptional inputs to regulate the appropriate spatial pattern and levels of EGF secretion to specify an essential cell fate.

Geometry, epistasis, and developmental patterning

Developmental signaling networks are composed of dozens of components whose interactions are very difficult to quantify in an embryo. Geometric reasoning enumerates a discrete hierarchy of phenotypic models with a few composite variables whose parameters may be defined by in vivo data. Vulval development in the nematode Caenorhabditis elegans is a classic model for the integration of two signaling pathways; induction by EGF and lateral signaling through Notch. Existing data for the relative probabilities of the three possible terminal cell types in diverse genetic backgrounds as well as timed ablation of the inductive signal favor one geometric model and suffice to fit most of its parameters. The model is fully dynamic and encompasses both signaling and commitment. It then predicts the correlated cell fate probabilities for a cross between any two backgrounds/conditions. The two signaling pathways are combined additively, without interactions, and epistasis only arises from the nonlinear dynamical flow in the landscape defined by the geometric model. In this way, the model quantitatively fits genetic experiments purporting to show mutual pathway repression. The model quantifies the contributions of extrinsic vs. intrinsic sources of noise in the penetrance of mutant phenotypes in signaling hypomorphs and explains available experiments with no additional parameters. Data for anchor cell ablation fix the parameters needed to define Notch autocrine signaling.

Salvianolic Acid A Inhibits PDGF-BB Induced Vascular Smooth Muscle Cell Migration and Proliferation While Does Not Constrain Endothelial Cell Proliferation and Nitric Oxide Biosynthesis

Proliferation and migration of vascular smooth muscle cells (VSMCs) are critical events in the initiation and development of restenosis upon percutaneous transluminal coronary angioplasty (PTCA). Polyphenols have been suggested to ameliorate post-angioplasty restenosis. Salvianolic A (SalA) is one of the most abundant polyphenols extracted from salvia. In this study, we investigated the effect of salvianolic A (SalA) on the migration and proliferation of VSMCs. We found a preferential interaction of SalA with cellular systems that rely on the PDGF signal, but not on the EGF and bFGF signal. SalA inhibits PDGF-BB induced VSMC proliferation and migration in the concentration range from 0.01 to 0.1 μM. The inhibition of SalA on VSMC proliferation is associated with cell cycle arrest. We also found that SalA inhibits the PDGFRβ-ERK1/2 signaling cascade activated by PDGF-BB in VSMCs. In addition, SalA does not influence the proliferation of endothelial cells, the synthesis of NO and eNOS protein expression. Our results suggest that SalA inhibits migration and proliferation of VSMCs induced by PDGF-BB via the inhibition of the PDGFRβ-ERK1/2 cascade, but that it does not constrain endothelial cell proliferation and nitric oxide biosynthesis. Thus, the present study suggests a novel adjunct pharmacological strategy to prevent angioplasty-related restenosis.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract A16: Aberrant signaling pathways in squamous cell lung carcinoma

Abstract Lung cancer is the second most commonly occurring non-cutaneous cancer in the United States with the highest mortality rate among both men and women. In this study, we utilized three lung cancer microarray datasets generated by previous researchers to identify differentially expressed genes, altered signaling pathways, and assess the involvement of Hedgehog (Hh) pathway. The three datasets contain the expression levels of tens of thousands genes in normal lung tissues and squamous cell lung carcinoma. The datasets were combined and analyzed. The dysregulated genes and altered signaling pathways were identified using statistical methods. We then performed Fisher's exact test on the significance of the association of Hh pathway downstream genes and squamous cell lung carcinoma. 395 genes were found commonly differentially expressed in squamous cell lung carcinoma. The genes encoding fibrous structural protein keratins and cell cycle dependent genes encoding cyclin-dependent kinases were significantly up-regulated while the ones encoding LIM domains were down. Over 100 signaling pathways were implicated in squamous cell lung carcinoma, including cell cycle regulation pathway, p53 tumor-suppressor pathway, IL-8 signaling, Wnt-β-catenin pathway, mTOR signaling and EGF signaling. In addition, 37 out of 223 downstream molecules of Hh pathway were altered. The pvalue from the Fisher's exact test indicates that Hh signaling is implicated in squamous cell lung carcinoma. Numerous genes were altered and multiple pathways were dysfunctional in squamous cell lung carcinoma. Many of the altered genes have been implicated in different types of carcinoma while some are organ-specific. Hh signaling is implicated in squamous cell lung cancer, opening the door for exploring new cancer therapeutic treatment using GLI antagonist GANT 61.

SIRT1 induces EMT by cooperating with EMT transcription factors and enhances prostate cancer cell migration and metastasis

The epithelial-to-mesenchymal transition (EMT) is a crucial program for the invasion and metastasis of epithelial tumors that involves loss of cell-cell adhesion and increased cell mobility; however, mechanisms underlying this transition are not fully elucidated. Here, we propose a novel mechanism through which the nicotinamide adenine dinucleotide-dependent histone deacetylase SIRT1 regulates EMT in prostate cancer cells through cooperation with the EMT inducing transcription factor ZEB1. We found that forced expression of SIRT1 in non-transformed PZ-HPV-7 prostate epithelial cells disrupts the epithelial morphology concomitant with decreased expression of the epithelial marker, E-cadherin, and increased expression of mesenchymal markers. In contrast, silencing SIRT1 in metastatic prostate tumor cells restores cell-cell adhesion and induces a shift toward an epithelial morphology concomitant with increased expression of E-cadherin and decreased expression of mesenchymal markers. We also found that SIRT1 has a physiologically relevant role in endogenous EMT induced by EGF signaling in prostate cancer cells. We propose that the regulation of EMT by SIRT1 involves modulation of, and cooperation with, the EMT inducing transcription factor ZEB1. Specifically, we show that SIRT1 silencing reduces expression of ZEB1 and that SIRT1 is recruited to the E-cadherin proximal promoter by ZEB1 to deacetylate histone H3 and to reduce binding of RNA polymerase II, ultimately suppressing E-cadherin transcription. We thus identify a necessary role for ZEB1 in SIRT1-mediated EMT. Finally, we show that reduction of SIRT1 decreases prostate cancer cell migration in vitro and metastasis in vivo in immunodeficient mice, which is largely independent of any general effects of SIRT1 on prostate cancer growth and survival. We therefore identify SIRT1 as a positive regulator of EMT and metastatic growth of prostate cancer cells and our findings implicate overexpressed SIRT1 as a potential therapeutic target to reverse EMT and to prevent prostate cancer progression.

Egf Signaling Regulates Hematopoietic Regeneration Following Total Body Irradiation

Egf Signaling Regulates Hematopoietic Regeneration Following Total Body Irradiation

EGFR signaling is required for regenerative proliferation in the cochlea: Conservation in birds and mammals

Proliferation and transdifferentiaton of supporting cells in the damaged auditory organ of birds lead to robust regeneration of sensory hair cells. In contrast, regeneration of lost auditory hair cells does not occur in deafened mammals, resulting in permanent hearing loss. In spite of this failure of regeneration in mammals, we have previously shown that the perinatal mouse supporting cells harbor a latent potential for cell division. Here we show that in a subset of supporting cells marked by p75, EGFR signaling is required for proliferation, and this requirement is conserved between birds and mammals. Purified p75+ mouse supporting cells express receptors and ligands for the EGF signaling pathway, and their proliferation in culture can be blocked with the EGFR inhibitor AG1478. Similarly, in cultured chicken basilar papillae, supporting cell proliferation in response to hair cell ablation requires EGFR signaling. In addition, we show that EGFR signaling in p75+ mouse supporting cells is required for the down-regulation of the cell cycle inhibitor p27Kip1 (CDKN1b) to enable cell cycle re-entry. Taken together, our data suggest that a conserved mechanism involving EGFR signaling governs proliferation of auditory supporting cells in birds and mammals and may represent a target for future hair cell regeneration strategies.

Rhomboid Proteolysis: Why inside the Membrane?

Rhomboid proteins are membrane-immersed enzymes with the remarkable ability to hydrolyze protein segments within the membrane. This form of intramembrane proteolysis plays key roles in cell communication including EGF signalling during animal development, and quorum sensing during bacterial growth. We have been investigating rhomboid protease mechanism using a pure enzyme reconstitution system that we developed, coupled with high-resolution crystal structures of a model bacterial rhomboid protease (1, 2). Intramembrane proteolysis is catalyzed within a hydrated cavity that opens to the outside of the cell and allows water to enter, but is protected laterally from membrane lipids by a ring of protein segments. Using a structure-function approach, we subsequently identified one transmembrane helix and overlying loop that provide a rate-limiting movement to gate substrate entry laterally from the membrane (3). By integrating reconstitution into defined bilayer environments with spectroscopic interrogation, we recently discovered that rhomboid proteases rely on the natural biophysical constraints of the membrane to achieve site-specific proteolysis. Since rhomboid proteins play critical roles in the infective cycles of several protozoan parasites (4,5,6), understanding the protease mechanism may provide therapeutic opportunities (7).1. Urban, S. and M.S. Wolfe. (2005). Proc. Natl. Acad. Sci. USA, 102(6):1883-1888.2. Wu Z., Yan N., Feng L., Oberstein A., Yan H., Baker R. P., Gu L., Jeffrey P.D., Urban S., and Y. Shi. (2006). Nature Structural and Molecular Biology 13: 1084-1091.3. Baker, R.P., Young K., Feng L., Shi Y. and S. Urban. (2007). Proc. Natl. Acad. Sci. USA, 104: 8257-62.4. Baker, R.P., Wijetilaka R, and S. Urban. (2006). PLoS Pathogens 10(2): e113; 922-32.5. Baxt, L., Baker, R.P., Singh, U. and S. Urban. (2008). Genes & Development 22(12): 1636-46.6. Urban S. (2009). Nature Reviews Microbiology 7: 411-23.7. Urban S. (2010). The Biochemical Journal 425(3): 501-512.

Molecular mechanisms of EGF signaling-dependent regulation of pipe, a gene crucial for dorsoventral axis formation in Drosophila

During Drosophila oogenesis the expression of the sulfotransferase Pipe in ventral follicle cells is crucial for dorsoventral axis formation. Pipe modifies proteins that are incorporated in the ventral eggshell and activate Toll signaling which in turn initiates embryonic dorsoventral patterning. Ventral pipe expression is the result of an oocyte-derived EGF signal which down-regulates pipe in dorsal follicle cells. The analysis of mutant follicle cell clones reveals that none of the transcription factors known to act downstream of EGF signaling in Drosophila is required or sufficient for pipe regulation. However, the pipe cis-regulatory region harbors a 31-bp element which is essential for pipe repression, and ovarian extracts contain a protein that binds this element. Thus, EGF signaling does not act by down-regulating an activator of pipe as previously suggested but rather by activating a repressor. Surprisingly, this repressor acts independent of the common co-repressors Groucho or CtBP.

Functions of Rhomboid Family Protease RHBDL2 and Thrombomodulin in Wound Healing

The expression of thrombomodulin (TM), a membrane glycoprotein, is upregulated in neoepidermis during cutaneous wound healing. Rhomboid-like-2 (RHBDL2), an intramembrane serine protease, specifically cleaves TM at the transmembrane domain and causes the release of soluble TM (sTM). However, the physiological functions of TM and RHBDL2 in wound healing remain unclear. We demonstrated that both TM and RHBDL2 are upregulated in HaCaT cells stimulated by scratch wounds; furthermore, increased sTM was found in culture medium. Conversely, inhibition of RHBDL2 by 3,4-dichloroisocoumarin (DCI) or short hairpin RNA significantly inhibited wound-induced TM ectodomain shedding and wound healing. Both conditioned media from multiple-scratch-wounded HaCaT and recombinant sTM accelerated wound healing in HaCaT cells; such effects were abrogated by anti-TM antibodies. The RNA released from injured cells is involved in the induction of TM and RHBDL2. RHBDL2 and sTM were upregulated in ex vivo tissue culture of the injured skin. Furthermore, DCI inhibited sTM production and wound healing; this was reversed by recombinant sTM in mice. Thus, RHBDL2 and TM have important roles in wound healing via the release of sTM from keratinocytes; this may function as an autocrine/paracrine signal promoting wound healing.