Epidermal Growth Factor Signaling Research Articles

SPSB1-mediated HnRNP A1 ubiquitylation regulates alternative splicing and cell migration in EGF signaling.

Extracellular signals have been shown to impact on alternative pre-mRNA splicing; however, the molecular mechanisms and biological significance of signal-induced splicing regulation remain largely unknown. Here, we report that epidermal growth factor (EGF) induces splicing changes through ubiquitylation of a well-known splicing regulator, hnRNP A1. EGF signaling upregulates an E3 ubiquitin (Ub) ligase adaptor, SPRY domain-containing SOCS box protein 1 (SPSB1), which recruits Elongin B/C-Cullin complexes to conjugate lysine 29-linked polyUb chains onto hnRNP A1. Importantly, SPSB1 and ubiquitylation of hnRNP A1 have a critical role in EGF-driven cell migration. Mechanistically, EGF-induced ubiquitylation of hnRNP A1 together with the activation of SR protein kinases (SRPKs) results in the upregulation of a Rac1 splicing isoform, Rac1b, to promote cell motility. These findings unravel a novel crosstalk between protein ubiquitylation and alternative splicing in EGF/EGF receptor signaling, and identify a new EGF/SPSB1/hnRNP A1/Rac1 axis in modulating cell migration, which may have important implications for cancer treatment.

Porphyromonas gingivalis and its lipopolysaccharide differently modulate epidermal growth factor–dependent signaling in human gingival epithelial cells

ABSTRACTPeriodontitis is an inflammatory disease induced by pathogenic bacteria such as Porphyromonas gingivalis. Little is known about epidermal growth factor (EGF) signals in human gingival epithelial cells (HGEC), which are major targets of P. gingivalis, and how the expression of proteins participating in EGF signaling—that is, EGF-receptor (EGFR), suppressor of cytokine signaling-3 (SOCS-3), interferon regulatory factor-1 (IRF-1), and signal transducers and activators of transcription (STAT-3)—are modified. This study aimed to assess the effects of P. gingivalis and its purified lipopolysaccharide (LPS-Pg) on EGF signaling. HGEC were infected for 2 h in a dose-dependent manner with P. gingivalis and with heat-killed P. gingivalis, and activated for 2 and 24 h by 1 µg/mL of purified LPS-Pg. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed to measure mRNA and protein levels for SOCS-3, IRF-1 EGF, EGFR, and STAT-3. The tyrosine-phosphorylation status of STAT-3 was also examined. The results showed that infection of HGEC cells with P. gingivalis, but not with heat-killed P. gingivalis, led to significant reductions in expression levels of mRNAs and proteins for SOCS-3, IRF-1, and EGFR, while LPS-Pg over time significantly increased the expression of these mRNAs and proteins. Tyrosine-phosphorylation of STAT-3 was significantly increased during infection with P. gingivalis and activation by LPS-Pg but not modified during infection with heat-killed P. gingivalis. This study highlights that P. gingivalis and its purified LPS differentially modulated the expression of proteins (SOCS-3, IRF-1, EGFR, and STAT-3) interfering with EGF signaling.

Epidermal Growth Factor Signaling towards Proliferation: Modeling and Logic Inference Using Forward and Backward Search.

In biological systems, pathways define complex interaction networks where multiple molecular elements are involved in a series of controlled reactions producing responses to specific biomolecular signals. These biosystems are dynamic and there is a need for mathematical and computational methods able to analyze the symbolic elements and the interactions between them and produce adequate readouts of such systems. In this work, we use rewriting logic to analyze the cellular signaling of epidermal growth factor (EGF) and its cell surface receptor (EGFR) in order to induce cellular proliferation. Signaling is initiated by binding the ligand protein EGF to the membrane-bound receptor EGFR so as to trigger a reactions path which have several linked elements through the cell from the membrane till the nucleus. We present two different types of search for analyzing the EGF/proliferation system with the help of Pathway Logic tool, which provides a knowledge-based development environment to carry out the modeling of the signaling. The first one is a standard (forward) search. The second one is a novel approach based on narrowing, which allows us to trace backwards the causes of a given final state. The analysis allows the identification of critical elements that have to be activated to provoke proliferation.

MiR-200c Accelerates Hepatic Stellate Cell-Induced Liver Fibrosis via Targeting the FOG2/PI3K Pathway.

Background Although expression of miR-200s is aberrant in liver fibrosis, its role in liver fibrogenesis still remains unknown. Here, we investigated the role of miR-200c in the activation of human hepatic stellate cells (HSCs) and induction of liver fibrosis. Methods We engineered human HSCs (LX2 cell line) to stably express miR-200c (LX2-200c) or empty vector control (LX2-nc). Results miR-200c expression upregulated α-smooth muscle actin (SMA) and vimentin, enhanced HSCs growth and migration, increased expression of collagen type I (a main component of ECM) gene and secretion of epidermal growth factor (EGF), and upregulated the phosphorylation of Akt, a downstream effector of the PI3K pathway. As a target of miR-200s and inhibitor of PI3K pathway, FOG2 protein expression was significantly suppressed in LX2-200c cells. Moreover, LY294002, a highly selective inhibitor of PI3K, blocked phosphorylation of Akt and the effects of miR-200c. Conclusions These data suggest that miR-200c activates HSCs in liver fibrosis possibly by downregulating FOG2 protein expression and upregulating PI3K/Akt signaling. Autocrine activation of EGF signaling may also be a mechanism of miR-200c-mediated HSCs activation. So miR-200c can be a potential marker for HSCs activation and liver fibrosis progression, as well as a potential target to attenuate liver fibrosis.

Epidermal growth factor receptor (EGFR) involvement in successful growth hormone (GH) signaling in GH transduction defect

Growth hormone (GH) transduction defect (GHTD) is a growth disorder with impaired signal transducer and activator of transcription 3 (STAT3) phosphorylation mediated by overexpression of cytokine-inducible SH2-containing protein (CIS), which causes increased growth hormone receptor (GHR) degradation. This study investigated the role of epidermal growth factor (EGF) in the restoration of normal GH signaling in GHTD. Protein expression, cellular localization and physical contact of proteins of the GH and EGF signaling pathways were studied by Western immunoblotting, immunofluorescence and co-immunoprecipitation, respectively. These were performed in fibroblasts of one GHTD patient (P) and one control child (C) at the basal state and after induction with human GH (hGH) 200 μg/L (GH200), either with or without silencing of CIS mRNA, and after induction with hGH 1000 μg/L (GH1000) or 50 ng/mL EGF. The membrane availability of the EGF receptor (EGFR) and the activated EGFR (pEGFR) was increased in P only after simultaneous GH200 and silencing of CIS mRNA or with GH1000, whereas this occurred in C after GH200 alone. After EGF induction, the membrane localization of GHR, STAT3 and that of EGFR were increased in P more than in C. In conclusion, in GHTD, the EGFR seems to participate in successful GH signaling, but induction of GHTD fibroblasts with a higher dose of hGH is needed. The EGF/EGFR pathway, in contrast to the GH/GHR pathway, seems to function normally in P and is more primed compared to C. The involvement of the EGFR in successful GH signaling may explain the catch-up growth seen in the Ps when exogenous hGH is administered.

Abstract PR04: Small molecule-mediated activation of Ras elicits inhibition of MAPK and PI3K signaling though pathway feedback

Abstract Oncogenic mutation or hyper-activation of Ras results in aberrant cellular signaling and is responsible for approximately 30% of all human tumors. Therefore, Ras is an outstanding candidate for therapeutic inhibition. However, the discovery of potent inhibitors has been challenging. Our laboratory has recently discovered small molecules that perturb Son of Sevenless (SOS) -catalyzed nucleotide exchange on Ras (Burns et al. PNAS 2014). Here we describe experiments conducted to determine the mechanism of compound action. SOS activator compound 4 (C4) induces signaling flux through the MAPK pathway in response to elevated Ras-GTP levels, reminiscent of epidermal growth factor signaling. We used mass spectrometry to show that in response to C4 stimulation, phospho-modifications on SOS1 increased at ERK phosphorylation consensus sequences. We hypothesize that these phospho-modifications can cause delocalization of SOS and membrane bound Ras, and we are currently using proximity-ligation to assess whether the interaction between SOS1 and Ras is affected by C4 treatment. Cross-talk between the MAPK and PI3K signaling pathways may be responsible for the inhibition of phospho-AKT Ser473 levels in response to MAPK signaling flux, or vice versa. Here we show, using MEK and PI3K inhibitor pre-treatments, that events in both pathways are independent after C4 treatment. As both SOS1 and PI3K bind to Ras at a similar site, we are currently testing the hypothesis that C4 disrupts the interaction between Ras and PI3K, whilst allowing signaling flux through the MAPK pathway. Overall, we have discovered small-molecule activators of SOS that modulate Ras-GTP levels, resulting in signaling flux through the MAPK pathway and eventual downstream inhibition of both the MAPK and PI3K pathways in cancer cells. This abstract is also being presented as Poster A15. Citation Format: Jennifer E. Howes, Denis T. Akan, Bethany M. Alicie, Alex G. Waterson, Olivia W. Rossanese, Stephen W. Fesik. Small molecule-mediated activation of Ras elicits inhibition of MAPK and PI3K signaling though pathway feedback. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr PR04.

Pancreatic neuroendocrine tumor cancer stem cells: potential novel therapeutic targets?

New therapeutic treatments are urgently needed for patients with pancreatic neuroendocrine tumors (PanNETs) that are insensitive or resistant to current therapeutic methods. The cancer stem cells (CSCs) in PanNETs may imply a promising and novel therapeutic approach to treat patients with advanced stage tumor and improve patient survival through targeting various molecular pathways. In this review, we define the process of identifying CSCs in PanNETs and address the potential therapeutic implications of targeting CSCs. The CSCs in PanNETs are marked by aldehyde dehydrogenases (ALDH) or CD90. We discuss the relevant molecular pathways in ALDH+ and CD90+ CSCs, including the Src, Notch, Hedgehog, Wnt/β-catenin, mammalian target of rapamycin (mTOR), epidermal growth factor receptor (EGFR) and signal transducers and activators of transcription 3 (STAT3) signaling pathways. The function of Src signaling has been identified in a NET cell line, which suggests it could become a new therapeutic target in PanNETs. With the exception of the STAT3 signaling pathway, signaling pathways involved in the tumorigenicity of CSCs in many other cancers play a role in the pathophysiology of PanNETs. These molecular pathways may be potential targets for the treatment of PanNETs despite a lack of studies relating them to PanNET CSCs and more studies are needed to clarify the role of these signaling pathways in PanNETs. Additionally, targeting of the cellular surface markers CD47 and CD73 decreased the tumor growth of ALDH+ CSCs in PanNETs cell lines. In conclusion, the study of PanNET CSCs could aid in the development of novel therapeutic targets for PanNETs, and this review details the molecular pathways that might become new targets.

Paradoxical induction of growth arrest and apoptosis by EGF via the up-regulation of PTEN by activating Redox factor-1/Egr-1 in human lung cancer cells.

Epidermal growth factor (EGF) signaling promotes cell proliferation and survival in several types of cancer. Here, however, we showed that EGF inhibits proliferation and promotes programmed cell death in non-small cell lung cancer (NSCLC) cells. In A549 cells, EGF increased redox factor-1 (Ref-1) expression and the association of Ref-1 with zinc finger-containing transcriptional regulator (EGR1) via activation of p22phox, RAC1, and an NADPH oxidase subunit. EGF increased p22phox and RAC1 expression through activation of purinergic receptors (P2Y). Elevated Ref-1/EGR1 levels increased phosphatase and tensin homolog (PTEN) levels, leading to inhibition of the Akt pathway. EGF-induced PTEN upregulation increased apoptosis and autophagy-induced damage in A549 cells, whereas Ref-1 knockdown blocked EGF-induced PTEN upregulation in an NADPH oxidase p22phox subunit-independent manner. In addition, p22phox knockdown restored EGF-induced effects, implying that changes in P2Y activity caused by EGF, which activates NADPH oxidase via RAC1, influenced Ref-1-mediated redox regulation. Finally, EGF similarly attenuated cell proliferation and promoted autophagy and apoptosis in vivo in a xenograft model using A549 cells. These findings reveal that EGF-induced redox signaling is linked to Ref-1-induced death in NSCLC cells.

EGF Accelerates Hematopoietic Stem Cell Regeneration Following 5-FU Chemotherapy Via G-CSF Receptor Signaling

Hematopoietic stem cells (HSCs) reside in specialized bone marrow microenvironments adjacent to endothelial cells (BM ECs). BM ECs regulate both HSC self-renewal and regeneration by expressing soluble growth factors such as epidermal growth factor (EGF). EGF accelerates HSC regeneration following ionizing radiation injury (Doan et al., Nat Med, 2013), though its role in HSC reconstitution after chemotherapy is not yet defined. At 24 hours following administration of 5-fluorouracil (5-FU), the expression of EGF receptor is increased 10-fold (p<0.0001) compared to untreated control mice within ckit+Sca-1+Lineage- (KSL) cells. EGFR expression is preferentially induced 6.8-fold in KSL cells compared to whole bone marrow cells after 5-FU (p=0.01). When C57Bl6 mice are treated with a single injection of 150 mg/kg 5-FU followed by daily injections of EGF for 4 days, they demonstrated preserved BM cellularity (p=0.0003), increased BM EC density (p=0.002), and contained higher levels of donor engraftment at 16-weeks post-secondary transplantation in competitive repopulating assays (mean 21.9% vs 52.2%, p=0.0005) compared to saline-treated control mice. Following a lethal dose of 5-FU, EGF-treated mice displayed 70% survival compared to 20% survival in saline-treated control mice (p=0.04). These results suggest that EGF signaling may accelerate both hematopoietic progenitor and stem cell reconstitution following myelosuppressive and lethal-dose chemotherapy.Since the pro-apoptotic gene Bax is increased 2.8-fold in BM lin- cells following 5-FU compared to cells from untreated mice (p<0.0001), we sought to determine whether chemo-protection of BM ECs via deletion of Baxwould accelerate HSC reconstitution. We employed a genetic mouse model with Cre recombinase to delete Bax specifically in VECadherin-expressing ECs (VECadherinCre;BaxFL/+ and VECadherinCre;BaxFL/FL mice). These mice displayed no differences in hematopoiesis at baseline. When C57Bl6 KSL cells are in non-contact co-cultures with BaxFL/+ BM ECs and treated with 5-fluorodeoxyuridine monophosphate (FdUMP) + EGF for 48 hours, they displayed both increased colony-forming cells (CFCs) and decreased annexin+ cells compared to control cultures (p=0.001 and p=0.03, respectively). Conversely, when C57Bl6 KSL cells are cultured with BaxFL/FL ECs and FdUMP + erlotinib, a tyrosine kinase inhibitor that neutralizes EGFR signaling, they displayed decreased CFCs and increased annexin+ cells compared to control cultures (p=0.001 and p=0.0003, respectively). These data suggest that chemo-protection of BM ECs by deletion of Bax in vivo results in accelerated hematopoietic recovery.Following 5-FU, EGF promoted cell cycling with increased Ki67+ cells and cells in interphase (mean 4.8% vs 16.1%, p=0.03). Moreover, EGF decreased annexin+ cells within the KSL population both in vitro following FdUMP (mean 26.3 % vs 17%, p=0.002) and in vivo following 5-FU (mean 21.5% vs 11.5%, p<0.0001). Concordant with these results, KSL cells treated with FdUMP + EGF displayed lower levels of double-strand DNA (dsDNA) breaks, measured by γH2AX, compared to control cultures (mean 39% vs 16%, p<0.0001). Since EGF can repair dsDNA breaks via non-homologous end joining recombination, we measured the levels of phosphorylation of DNA pk cs. Following 24 hours with FdUMP and 15 minutes with EGF, KSL cells displayed a 4.8-fold increase in phospho-DNA pk cs (T2647) compared to control cultures (p=0.04). Finally, we sought to determine whether EGF could accelerate HSC reconstitution via the granulocyte colony-stimulating factor (G-CSF) receptor. Following 5-FU + EGF, G-CSF-R expression is increased 3.6-fold in mRNA levels (p<0.0001) and by flow cytometric analysis (mean 23.9% vs 42.4%, p=0.003) compared to 5-FU-treated mice. These results suggest that EGF may be a potent HSC growth factor and may exert its effect at least in part via G-CSF receptor signaling. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Novel 3-D cell culture system for in vitro evaluation of anticancer drugs under anchorage-independent conditions.

Anticancer drug discovery efforts have used 2‐D cell‐based assay models, which fail to forecast in vivo efficacy and result in a lower success rate of clinical approval. Recent 3‐D cell culture models are expected to bridge the gap between 2‐D and in vivo models. However, 3‐D cell culture methods that are available for practical anticancer drug screening have not yet been fully attained. In this study, we screened several polymers for their ability to suspend cells or cell spheroids homogeneously in a liquid medium without changing the viscosity behavior, and identified gellan gum (FP001), as the most potent polymer. FP001 promoted cell dispersion in the medium and improved the proliferation of a wide range of cancer cell lines under low attachment conditions by inhibiting the formation of large‐sized spheroids. In addition, cancer cells cultured with FP001‐containing medium were more susceptible to inhibitors of epidermal growth factor (EGF) signaling than those cultured under attachment conditions. We also showed that ligands of the EGF receptor family clearly enhance proliferation of SKOV3 ovarian carcinoma cells under anchorage‐independent conditions with FP001. Consistent with this result, the cells grown with FP001 showed higher EGF receptor content compared with cells cultured under attachment conditions. In conclusion, we developed a novel 3‐D cell culture system that is available for high throughput screening of anticancer agents, and is suitable for evaluation of molecular‐targeted anticancer drugs. Three‐dimensional cell culture using FP001 will be of value in the development of useful technologies for anticancer drug discovery.

Loss of EGFR-ASAP1 signaling in metastatic and unresectable hepatoblastoma.

Hepatoblastoma (HBL), the most common childhood liver cancer is cured with surgical resection after chemotherapy or with liver transplantation if local invasion and multifocality preclude resection. However, variable survival rates of 60–80% and debilitating chemotherapy sequelae argue for more informed treatment selection, which is not possible by grading the Wnt-β-catenin over activity present in most HBL tumors. A hypothesis-generating whole transcriptome analysis shows that HBL tumors removed at transplantation are enriched most for cancer signaling pathways which depend predominantly on epidermal growth factor (EGF) signaling, and to a lesser extent, on aberrant Wnt-β-catenin signaling. We therefore evaluated whether EGFR, ASAP1, ERBB2 and ERBB4, which signal downstream after ligation of EGF, and which show aberrant expression in several other invasive cancers, would also predict HBL tumor invasiveness. Immunohistochemistry of HBL tumors (n = 60), which are histologically heterogeneous, shows that compared with well-differentiated fetal cells, less differentiated embryonal and undifferentiated small cells (SCU) progressively lose EGFR and ASAP1 expression. This trend is exaggerated in unresectable, locally invasive or metastatic tumors, in which embryonal tumor cells are EGFR-negative, while SCU cells are EGFR-negative and ASAP1-negative. Loss of EGFR-ASAP1 signaling characterizes undifferentiated and invasive HBL. EGFR-expressing HBL tumors present novel therapeutic targeting opportunities.

Pancreatic neuroendocrine tumor cancer stem cells: potential novel therapeutic targets?

: New therapeutic treatments are urgently needed for patients with pancreatic neuroendocrine tumors (PanNETs) that are insensitive or resistant to current therapeutic methods. The cancer stem cells (CSCs) in PanNETs may imply a promising and novel therapeutic approach to treat patients with advanced stage tumor and improve patient survival through targeting various molecular pathways. In this review, we define the process of identifying CSCs in PanNETs and address the potential therapeutic implications of targeting CSCs. The CSCs in PanNETs are marked by aldehyde dehydrogenases (ALDH) or CD90. We discuss the relevant molecular pathways in ALDH+ and CD90+ CSCs, including the Src, Notch, Hedgehog, Wnt/β-catenin, mammalian target of rapamycin (mTOR), epidermal growth factor receptor (EGFR) and signal transducers and activators of transcription 3 (STAT3) signaling pathways. The function of Src signaling has been identified in a NET cell line, which suggests it could become a new therapeutic target in PanNETs. With the exception of the STAT3 signaling pathway, signaling pathways involved in the tumorigenicity of CSCs in many other cancers play a role in the pathophysiology of PanNETs. These molecular pathways may be potential targets for the treatment of PanNETs despite a lack of studies relating them to PanNET CSCs and more studies are needed to clarify the role of these signaling pathways in PanNETs. Additionally, targeting of the cellular surface markers CD47 and CD73 decreased the tumor growth of ALDH+ CSCs in PanNETs cell lines. In conclusion, the study of PanNET CSCs could aid in the development of novel therapeutic targets for PanNETs, and this review details the molecular pathways that might become new targets.

Modeling Cellular Noise Underlying Heterogeneous Cell Responses in the Epidermal Growth Factor Signaling Pathway.

Cellular heterogeneity, which plays an essential role in biological phenomena, such as drug resistance and migration, is considered to arise from intrinsic (i.e., reaction kinetics) and extrinsic (i.e., protein variability) noise in the cell. However, the mechanistic effects of these types of noise to determine the heterogeneity of signal responses have not been elucidated. Here, we report that the output of epidermal growth factor (EGF) signaling activity is modulated by cellular noise, particularly by extrinsic noise of particular signaling components in the pathway. We developed a mathematical model of the EGF signaling pathway incorporating regulation between extracellular signal-regulated kinase (ERK) and nuclear pore complex (NPC), which is necessary for switch-like activation of the nuclear ERK response. As the threshold of switch-like behavior is more sensitive to perturbations than the graded response, the effect of biological noise is potentially critical for cell fate decision. Our simulation analysis indicated that extrinsic noise, but not intrinsic noise, contributes to cell-to-cell heterogeneity of nuclear ERK. In addition, we accurately estimated variations in abundance of the signal proteins between individual cells by direct comparison of experimental data with simulation results using Apparent Measurement Error (AME). AME was constant regardless of whether the protein levels varied in a correlated manner, while covariation among proteins influenced cell-to-cell heterogeneity of nuclear ERK, suppressing the variation. Simulations using the estimated protein abundances showed that each protein species has different effects on cell-to-cell variation in the nuclear ERK response. In particular, variability of EGF receptor, Ras, Raf, and MEK strongly influenced cellular heterogeneity, while others did not. Overall, our results indicated that cellular heterogeneity in response to EGF is strongly driven by extrinsic noise, and that such heterogeneity results from variability of particular protein species that function as sensitive nodes, which may contribute to the pathogenesis of human diseases.

Transcription Factor Response Elements on Tip: A Sensitive Approach for Large-Scale Endogenous Transcription Factor Quantitative Identification.

The ability to map endogenous transcription factors (TFs) DNA binding activity at the proteome scale will greatly enhance our understanding of various biological processes. Here we report a highly sensitive, rapid, and high-throughput approach, transcription factor response elements on tip-mass spectrometry (TOT-MS), that allows for quantitative measurement of endogenous TFs. A total of 150 TFs from 1 μg of nuclear extracts can be quantified with single shot mass spectrometry detection in 1 h of machine time. Up to 755 TFs, which is comparable to the depth of RNA-seq, were identified by TOT coupled with on-tip small size reverse-phase liquid chromatography. We further demonstrated the capability of TOT-MS by interrogating the dynamic change of TFs in the epidermal growth factor (EGF) signaling pathway. This approach should find broad applications in elucidating the TF landscape from limited amounts of biological materials.

Failure to launch: aberrant cumulus gene expression during oocyte in vitro maturation.

In vitro maturation (IVM) offers significant benefits for human infertility treatment and animal breeding, but this potential is yet to be fully realised due to reduced oocyte developmental competence in comparison with in vivo matured oocytes. Cumulus cells occupy an essential position in determining oocyte developmental competence. Here we have examined the areas of deficient gene expression, as determined within microarrays primarily from cumulus cells of mouse COCs, but also other species, between in vivo matured and in vitro matured oocytes. By retrospectively analysing the literature, directed by focussing on downregulated genes, we provide an insight as to why the in vitro cumulus cells fail to support full oocyte potential and dissect molecular pathways that have important roles in oocyte competence. We conclude that the roles of epidermal growth factor signalling, the expanded extracellular matrix, cumulus cell metabolism and the immune system are critical deficiencies in cumulus cells of IVM COCs.

KITENIN functions as a fine regulator of ErbB4 expression level in colorectal cancer via protection of ErbB4 from E3-ligase Nrdp1-mediated degradation.

Understanding the complex biological functions of E3-ubiquitin ligases may facilitate the development of mechanism-based anti-cancer drugs. We recently identified that the KITENIN/ErbB4-Dvl2-c-Jun axis works as a novel unconventional downstream signal of epidermal growth factor (EGF) in colorectal cancer (CRC) tissues. Here we addressed whether E3-ubiquitin ligases are required for operation of this axis. We found that Nrdp1, an E3-ligase for ErbB3/ErbB4, interacted with KITENIN (KAI1 C-terminal interacting tetraspanin) to form a functional KITENIN/ErbB4/Nrdp1 complex and is responsible for down-regulating Dvl2 within this complex. Interestingly, ErbB4 was resistant to degradation by Nrdp1 in KITENIN/Nrdp1-co-transfected CRC cells, and KITENIN bound to the C-terminal coiled-coil domain of Nrdp1. Chemical blockade of ErbB kinase did not block the action of EGF to increase in total/phospho-ErbB4 and phospho-ERK in KITENIN/ErbB4-cotransfected cells, whereas it blocked the action of EGF in ErbB4 alone-transfected CRC cells. In human CRC tissues, higher expressions of ErbB4 and KITENIN and lower expression of Dvl2 was observed in stage IV samples than in stage I, but a low level of Nrdp1 was expressed in both stages and it did not differ significantly by stage. These results indicated that Nrdp1 is necessary for the reduction in Dvl2 to generate c-Jun in the EGF-KITENIN/ErbB4-c-Jun axis, but more importantly, elevated KITENIN protects KITENIN-bound ErbB4 from Nrdp1-mediated degradation via physical collaboration between the KITENIN/ErbB4 complex and Nrdp1, but not via modulation of ErbB kinase activity. Thus, KITENIN functions in the maintenance of a higher expression level of ErbB4 in advanced CRC tissues, independent of ubiquitin-mediated degradation via Nrdp1. © 2016 Wiley Periodicals, Inc.

Simultaneous Enrichment of Cysteine-containing Peptides and Phosphopeptides Using a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) in Combination with titanium dioxide (TiO2) Chromatography

Cysteine is a rare and conserved amino acid involved in most cellular functions. The thiol group of cysteine can be subjected to diverse oxidative modifications that regulate many physio-pathological states. In the present work, a Cysteine-specific Phosphonate Adaptable Tag (CysPAT) was synthesized to selectively label cysteine-containing peptides (Cys peptides) followed by their enrichment with titanium dioxide (TiO2) and subsequent mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to protein lysates from Hela cells, achieving high specificity and enrichment efficiency. In particular, for Cys proteome analysis, the method led to the identification of 7509 unique Cys peptides from 500 μg of HeLa cell lysate starting material. Furthermore, the method was developed to simultaneously enrich Cys peptides and phosphorylated peptides. This strategy was applied to SILAC labeled Hela cells subjected to 5 min epidermal growth factor (EGF) stimulation. In total, 10440 unique reversibly modified Cys peptides (3855 proteins) and 7339 unique phosphopeptides (2234 proteins) were simultaneously identified from 250 μg starting material. Significant regulation was observed in both phosphorylation and reversible Cys modification of proteins involved in EGFR signaling. Our data indicates that EGF stimulation can activate the well-known phosphorylation of EGFR and downstream signaling molecules, such as mitogen-activated protein kinases (MAPK1 and MAPK3), however, it also leads to substantial modulation of reversible cysteine modifications in numerous proteins. Several protein tyrosine phosphatases (PTPs) showed a reduction of the catalytic Cys site in the conserved putative phosphatase HC(X)5R motif indicating an activation and subsequent de-phosphorylation of proteins involved in the EGF signaling pathway. Overall, the CysPAT strategy is a straight forward, easy and promising method for studying redox proteomics and the simultaneous enrichment strategy offers an excellent solution for characterization of cross-talk between phosphorylation and redox induced reversible cysteine modifications.

Population Pharmacokinetics of Necitumumab in Cancer Patients.

Necitumumab is a second-generation, recombinant, human immunoglobulin G1, epidermal growth factor (EGFR) receptor antibody that specifically blocks the ligand binding site of EGFR. Necitumumab potentially acts by blocking ligand epidermal growth factor (EGF) binding-mediated activation of the EGFR signaling pathway, inhibiting tumor growth, angiogenesis, and anti-apoptotic mechanisms. Necitumumab inhibited the interaction of EGF and EGFR with a concentration that inhibits binding by 50 % of approximately 0.9 nM (0.13 mg/L) and demonstrated antitumor activity during in vivo experiments associated with trough plasma concentrations of approximately 40 mg/L. This work describes the population pharmacokinetics of necitumumab in cancer patients when administered with or without concomitant chemotherapy and evaluates patient characteristics that may guide dosing. Nonlinear mixed-effects modeling of serum concentration data across five clinical studies (phases I–III) indicated that necitumumab exhibited target-mediated drug disposition, commonly observed with monoclonal antibodies, and that pharmacokinetics were expected to be linear in the studied dose ranges when administered as repeated infusions. No age, sex, race, or concomitant medication factors were found influential, while weight was a statistically significant factor for both distribution and elimination. Simulations from the final model indicated that only a limited reduction in patient drug exposure variability would be achieved by weight- or body surface area-based dosing. Necitumumab effective half-life was estimated to approximately 2 weeks, and steady state was achieved within three to four cycles of treatment. The phase III dosing schedule of 800 mg dosed on days 1 and 8 of a 21-day schedule resulted in serum concentrations that exceeded the 40-mg/L threshold indicated by preclinical experiments.

Dietary selenium disrupts hepatic triglyceride stores and transcriptional networks associated with growth and Notch signaling in juvenile rainbow trout

Dietary Se has been shown to adversely affect adult fish by altering growth rates and metabolism. To determine the underlying mechanisms associated with these observations, we measured biochemical and transcriptomic endpoints in rainbow trout following dietary Se exposures. Treatment groups of juvenile rainbow trout were fed either control Lumbriculus variegatus worms or worms cultured on selenized yeast. Selenized yeast was cultured at four nominal doses of 5, 10, 20 or 40mg/kg Se dry weight (measured dose in the worms of 7.1, 10.7, 19.5, and 31.8mg/kgSedw respectively) and fish were fed for 60days. At 60 d, hepatic triglycerides, glycogen, total glutathione, 8-isoprostane and the transcriptome response in the liver (n=8/group) were measured. Fish fed the nominal dose of 20 and 40mg/kg Se dry weight had lower body weight and a shorter length, as well as lower triglyceride in the liver compared to controls. Evidence was lacking for an oxidative stress response and there was no change in total glutathione, 8-isoprostane levels, nor relative mRNA levels for glutathione peroxidase isoforms among groups. Microarray analysis revealed that molecular networks for long-chain fatty acid transport, lipid transport, and low density lipid oxidation were increased in the liver of fish fed 40mg/kg, and this is hypothesized to be associated with the lower triglyceride levels in these fish. In addition, up-regulated gene networks in the liver of 40mg/kg Se treated fish included epidermal growth factor receptor signaling, growth hormone receptor, and insulin growth factor receptor 1 signaling pathways. These molecular changes are hypothesized to be compensatory and related to impaired growth. A gene network related to Notch signaling, which is involved in cell–cell communication and gene transcription regulation, was also increased in the liver following dietary treatments with both 20 and 40mg/kg Se. Transcriptomic data support the hypothesis that dietary Se increases the expression of networks for growth-related signaling cascades in addition to those related to fatty acid synthesis and metabolism. We propose that the disruption of metabolites related to triglyceride processing and storage, as well as gene networks for epidermal growth factor and Notch signaling in the liver, represent key molecular initiating events for adverse outcomes related to growth and Se toxicity in fish.

SKN-1-independent transcriptional activation of glutathione S-transferase 4 (GST-4) by EGF signaling.

In C. elegans research, transcriptional activation of glutathione S-transferase 4 (gst-4) is often used as a read-out for SKN-1 activity. While many heed an assumed non-exclusivity of the GFP reporter signal driven by the gst-4 promoter to SKN-1, this is also often ignored. We here show that gst-4 can also be transcriptionally activated by EOR-1, a transcription factor mediating effects of the epidermal growth factor (EGF) pathway. Along with enhancing exogenous oxidative stress tolerance, EOR-1 inde-pendently of SKN-1 increases gst-4 transcription in response to augmented EGF signaling. Our findings caution researchers within the C. elegans community to always rely on sufficient experimental controls when assaying SKN-1 transcriptional activity with a gst-4p::gfp reporter, such as SKN-1 loss-of-function mutants and/or additional target genes next to gst-4.