HB-EGF Research Articles

Cetuximab Inhibits T790M-Mediated Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor in a Lung Adenocarcinoma Patient-Derived Xenograft Mouse Model

BackgroundThe epidermal growth factor receptor (EGFR) kinase domain T790M (amino acid substitution at position 790 in EGFR from threonine [T] to methionine [M]) mutation in non–small-cell lung cancer (NSCLC) results in resistance to EGFR tyrosine kinase inhibitors (TKIs). We used a patient-derived tumor xenograft (PDX) model containing an EGFR exon 19 deletion/T790M mutation to assess response to the EGFR-directed antibody cetuximab. Changes in the EGFR signaling pathway and ligand expression after treatment were investigated. MethodsPDX were randomized into control and treatment arms. Pharmacodynamic studies were performed at 2 and 24 hours and at 4 days after a single administration of cetuximab, erlotinib, or dacomitinib. Changes in the EGFR signaling pathway were assessed using Western blot analysis, and baseline mRNA expression of EGFR ligands using microarray analysis. Relative changes after treatment were assessed using quantitative polymerase chain reaction. ResultsThe xenograft showed a dramatic response to cetuximab. A complete reduction of total EGFR and phosphorylated EGFR occurred after cetuximab treatment. The PDX had increased baseline levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF) compared with other PDX models with or without EGFR mutations. Amphiregulin was significantly reduced 2 hours after treatment with cetuximab. Compared with control mice, cetuximab- and EGFR–TKI-treated mice had significantly reduced HB-EGF gene expression at 2 hours, however, by day 4 the level of HB-EGF expression was higher. The effect of cetuximab compared with EGFR TKI on HB-EGF gene expression levels differed significantly at 2 and 24 hours but not at 4 days. ConclusionWe showed a dramatic tumor response with cetuximab in an exon 19 deletion/T790M EGFR mutant lung adenocarcinoma PDX model, which suggests a role for the autocrine feedback loop in the mutant EGFR signaling pathway. Further investigation using cetuximab in NSCLC with T790M mutation is warranted.

Switching of growth factor binding on heparin-functionalized thermoresponsive surface for hepatocyte sheet manipulation with maintenance of hepatic functions

Event Abstract Back to Event Switching of growth factor binding on heparin-functionalized thermoresponsive surface for hepatocyte sheet manipulation with maintenance of hepatic functions Jun Kobayashi1, Yoshikatsu Akiyama1*, Masayuki Yamato1* and Teruo Okano1* 1 Tokyo Women's Medical University, Institute of Advanced Biomedical Engineering and Science, Japan Introduction: Liver tissue engineering using primary hepatocytes is an attractive method for the treatment of liver diseases[1]. Engineered Hepatocyte sheets were effectively engrafted in pre-vascularized subcutaneous site and exhibited liver-specific functionalities. By contrast, cultured hepatocytes rapidly lose their viability and phenotypic functions on isolation from the native in vivo microenvironment of the liver. To overcome this problem, heparin-functionalized poly(N-isopropylacrylamide) (PIPAAm)-grafted cell culture surface[2],[3], which interacts with heparin-binding proteins such as heparin-binding EGF-like growth factor (HB-EGF), was been designed for maintaining hepatic functions during the cultivation (Figure 1, left). In addition, the detachment of the cultured hepatocytes as a sheet was examined when lowering temperature to 20°C (Figure 1, right). Figure 1. Schematic of affinity regulation between heparin-binding protein and immobilized heparin by conformational change of PIPAAm. Materials and Methods: Heparin-functionalized thermoresponsive surfaces were prepared as described previously[2]. Briefly, poly(IPAAm-co-2-carboxyisopropylacrylamide) (poly(IPAAm-co-CIPAAm))-grafted surfaces on tissue culture polystyrene (TCPS) dishes were prepared by electron beam irradiation. Heparin was covalently immobilized on the poly(IPAAm-co-CIPAAm)-grafted dishes by condensing reaction. Then, affinity binding of HB-EGF was performed on the heparinized thermoresponsive surfaces by incubation of HB-EGF solution at 37°C for 24 h. Amounts of bound HB-EGF on the heparinized surface were quantified using [125I]-labeled HB-EGF. Primary rat hepatocytes were seeded on the dishes in DMEM with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2. Results: The addition of soluble HB-EGF in the cell culture media was essential for the survival of hepatocytes. When the medium contained less than 10 ng/cm2 of soluble HB-EGF, the hepatocytes were not able to adhere and form their cell sheets. By contrast, hepatocytes adhered and formed their sheets on heparin-functionalized thermoresponsive surface with 10 ng/cm2 of bound HB-EGF. In addition, the secretion of albumin on bound HB-EGF was maintained and higher than that on PIPAAm-grafted surfaces with soluble HB-EGF. Therefore, bound HB-EGF gave a high activity of maintenance of hepatocyte adhesion and function compared with soluble HB-EGF. Finally, when lowering temperature to 20°C, the cultured hepatocyte sheets were detached from the surface through the reduction of affinity binding between HE-EGF and immobilized heparin with increasing the mobility of heparin and steric hindrance of the swollen PIPAAm chains (Figure 1, right). Conclusions: Heparin-functionalized thermoresponsive cell culture surfaces facilitated the manipulation of hepatic cell sheets with maintaining hepatic functions by changing temperature. Creation of transferable and functional liver tissues is considered to have a potential to treat liver disease. Part of this work was financially supported by JSPS KAKENHI Grant Numbers 23106009 and 15K01317

Abstract 43: HBEGF cooperates with Cdkn2a and Pten loss to form malignant gliomas

Abstract Malignant gliomas are aggressive brain tumors that carry a grim prognosis to over 10,000 patients a year in the United States. The highest grade tumor, glioblastoma, has an average survival of one year with the current standard of care, which includes surgical resection, radiotherapy, and temozolomide chemotherapy. Heparin-binding EGF-like growth factor (HBEGF), first discovered in macrophages as a mitogen for vascular smooth muscle cells, is a ligand for epidermal growth factor receptor, the most commonly amplified receptor-tyrosine kinase in glioblastoma. About 30% of glioblastomas express HBEGF, and it is also commonly upregulated in tumors that express the common EGFR mutant, EGFRvIII. To date, HBEGF has not been evaluated for its ability to promote the development of malignant gliomas in vivo. Using a method for somatic cell gene transfer, we show that HBEGF cooperates with loss of tumor suppressors Cdkn2a and Pten to form malignant gliomas in vivo. These tumors share many characteristics found in human glioblastoma such as necrosis and nuclear atypia. We also demonstrate using high-throughput sequencing that these tumors share a common gene signature to the human glioblastoma samples from The Cancer Genome Atlas study. No tumors were seen with loss of Cdkn2a and Pten alone, which are the two most common tumor suppressors altered in glioblastoma. Our findings demonstrate that HBEGF can drive the development of high grade gliomas in the context of Cdkn2a and Pten loss and that these tumors closely resemble the human disease. Citation Format: Clifford Shin, Sheri Holmen. HBEGF cooperates with Cdkn2a and Pten loss to form malignant gliomas. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 43.

Expression of Heparin-binding Epidermal Growth Factor-like Growth Factor (HB-EGF) in Human Renal Cell Carcinoma

Background: The levels of expression of Heparin binding-epidermal growth factor like growth factor (HB-EGF) mRNA in tumor tissues and normal tissues of the excised kidney were compared in order to clarify the role of HBEGF inrenal cell carcinoma (RCC) derived from the proximal tubule. Method: Normal and tumor tissues were collected from surgical specimens of 16 cases pathologically diagnosed with RCC. Total RNA was extracted from these samples, and the level of expression of HB-EGF mRNA was measured by real-time quantitative PCR using a TaqMan probe after reverse transcription. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal standard. The expression levels of HB-EGF mRNA in normal and tumor tissues of the same case were compared, and statistical analysis was performed to evaluate the association between the expression level and various clinical-pathological factors in RCC. Results: Expression of HB-EGF mRNA was detected in 82% (13/16) of the normal tissues and 63% (10/16) of the tumor tissues. The expression level in the normal tissues was significantly 7-fold higher than that in the tumor tissues. No significant association was detected between the expression of HB-EGF mRNA and the clinical stage or prognosis of RCC. However, the pathological findings indicated that negative expression of ratio of HB-EGF was higher in RCC with more-advanced malignant progression. Conclusion: Our results indicated that it was unlikely that HB-EGF might play a role in determining the aggressiveness or clinical features in RCC. However, the decreased expression of HB-EGF mRNA in RCC tissues indicates that tumorigenesis of RCC may disrupt the normal regulatory system of HB-EGF.

Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix.

Defective eyelid leading edge cell migration in C57BL/6-corneal opacity mice with an "eye open at birth" phenotype.

Development of the eyelid requires coordination of the cellular processes involved in proliferation, cell size alteration, migration, and cell death. C57BL/6J-corneal opacity (B6-Co) mice are mutant mice generated by the administration of N-ethyl-N-nitrosourea (100 mg/kg). They exhibit the eyelids open at birth phenotype, abnormal round cell shape from tightened F-actin bundles in leading edge keratinocytes at E16.5, and gradual corneal opacity with neovessels. The tip of the leading edge in B6-Co mice did not move forward, and demonstrated a sharp peak shape without obvious directionality. Analysis of the biological characteristics of B6-Co mice demonstrated that abnormal migration of keratinocytes could affect eyelid development, but proliferation and apoptosis in B6-Co mice had no effect. Mutant gene mapping and sequence analysis demonstrated that in B6-Co mice, adenosine was inserted into the untranslated regions, between 3030 and 3031, in the mRNA 3'-terminal of Fgf10. In addition, guanine 7112 was substituted by adenine in the Mtap1B mRNA, and an A2333T mutation was identified in Mtap1B. Quantitative real-time polymerase chain reaction analysis showed that expression of the Hbegf gene was significantly down-regulated in the eyelids of B6- Co mice at E16.5, compared to B6 mice. However, the expression of Rock1, Map3k1, and Jnk1 genes did not show any significant changes. Abnormal keratinocyte migration and down-regulated expression of the Hbegf gene might be associated with impaired eyelid development in B6-Co mice.

Genome-wide association study reveals regions associated with gestation length in two pig populations.

Reproduction traits, such as gestation length (GLE), play an important role in dam line breeding in pigs. The objective of our study was to identify single nucleotide polymorphisms (SNPs) that are associated with GLE in two pig populations. Genotypes and deregressed breeding values were available for 2081 Dutch Landrace-based (DL) and 2301 Large White-based (LW) pigs. We identified two QTL regions for GLE, one in each population. For DL, three associated SNPs were detected in one QTL region spanning 0.52Mbp on Sus scrofa chromosome (SSC) 2. For LW, four associated SNPs were detected in one region of 0.14Mbp on SSC5. The region on SSC2 contains the heparin-binding EGF-like growth factor (HBEGF) gene, which promotes embryo implantation and has been described to be involved in embryo survival throughout gestation. The associated SNP can be used for marker-assisted selection in the studied populations, and further studies of the HBEGF gene are warranted to investigate its role in GLE.

75 EXPRESSION OF GROWTH FACTOR GENES IN IN VITRO-PRODUCED BLASTOCYST CHANGES AFTER UTERINE PASSAGE, BUT ENDOMETRIAL EXPRESSION IS UNAFFECTED BY THE PRESENCE OF EMBRYOS

Growth factors (GFs) exert recognised roles in mammalian reproduction. However, little is known about the role of GFs in very early development. We examined here endometrial and embryonic transcription of genes coding for GF proteins identified in bovine uterine fluid (UF): artemin, stanniocalcin-1 (STC-1); heparin-binding EGF-like growth factor (HBEGF); connective tissue growth factor (CTGF); and stem cell factor (SCF). Embryos were in vitro produced (IVP) including culture in SOF+BSA. On Day 6, embryos (n = 50) were transferred into the uteri of oestrus-synchronized heifers (n = 10). On Day 8, embryos were flushed, and groups (n = 6) of expanded blastocysts (n = 10) were snap frozen in LN2 and stored at –145°C. Day 8 embryos cultured in vitro from Day 6 were used as controls (n = 6 × 10 embryos). Transcript levels were also analysed in endometrial tissue collected from Day 8 slaughtered females that were embryo (n = 4) or sham transferred (n = 4). Samples were incubated overnight at 4°C in RNAlater and stored at –145°C in an ultrafreezer. Samples analysed (n = 3 caruncle; n = 3 intercaruncle) were taken from ipsilateral middle horn region. Embryonic and endometrial samples were analysed in duplicate for RT-qPCR analysis. Data were analysed by Wilcoxon Mann-Whitney test. In embryos, uterine passage compared with IVP down-regulated artemin (P < 0.01) and STC1 (P < 0.05) expression (0.61 ± 0.04 and 0.81 ± 0.06 v. 1.54 ± 0.10 and 1.25 ± 0.17, respectively). Conversely, abundance of CTGF (P < 0.05) and HBEGF (P < 0.01) decreased in IVP v. uterus-exposed embryos (0.79 ± 0.04 and 0.81 ± 0.03 v. 1.38 ± 0.18 and 1.37 ± 0.23, respectively). No changes were observed for SCF (0.96 ± 0.16 v. 1.09 ± 0.05 in IVP embryos). Endometrial gene expression for each GF did not change in response to embryos v. sham transfer (P > 0.10). Caruncular expression was higher (P < 0.05) for SCF compared with intercaruncles (1.32 ± 0.13 v. 0.88 ± 0.13). However, strong positive correlations (P < 0.006 to P < 0.0001) were seen for HBEGF with STC-1 (r = 0.67) and SCF (r = 0.72); for SCF with STC-1 (r = 0.61); and for CTGF with STC-1 (r = 0.59) and SCF (r = 0.50), suggesting a common transcription regulation among some GFs. Changes in embryonic gene expression reflect a regulatory response to the cognate GFs in the UF, which suggests a relevant role for GFs in early embryo-maternal interactions. Within the endometrium, our observations are consistent with studies postulating faster, non-transcriptional responses to early embryos (Gómez and Muñoz, Reproduction 2015 150, R35–R43). Alternatively, it is possible that very local endometrial responses for the analysed GFs were not detected despite the presence of multiple embryos. Research was supported by MICINN, project AGL2012–37772, and FEDER. The authors are members of the COST Action FA1201 (Epigenetics and Periconception environment).

Biomarkers of Cardiotoxicity Among Multiple Myeloma Patients Subsequently Treated with Proteasome Inhibitor Therapy

BACKGROUNDCardiovascular (CV) events are a known complication to proteasome inhibitor therapy in myeloma. Underlying mechanisms are unknown. We recently completed an investigator initiated, single institution Phase II study of high dose carfilzomib (56mg/m2) in patients with relapsed/refractory MM (NCT01351623). Among 42 response evaluable patients, 11 patients (25%) developed treatment-emergent heart failure of any grade, and 5 patients (11%) developed severe heart failure requiring mechanical ventilation. We undertook a study to identify potential biomarkers that may point to underlying mechanisms of CV events among multiple myeloma patients treated with carfilzomib therapy. METHODSWe performed a nested case-control study with 7 patients who experienced a CV event on our high dose carfilzomib study and had pre-treatment (baseline) plasma stored and 19 case matched controls treated on the same study who did not have a CV event. We screened for 90 proteins known to be associated with CV disease using O-linked glycosylation. We used the Proseek Multiplex CVD I 96x96 platform which is based on the Proximity Extension Assay (PEA) technique. PEA is a 96-plex immunoassay that allows high throughput detection of protein biomarkers in liquid samples. For each biomarker, a matched pair of antibodies linked to unique oligonucleotides (proximity probes) binds to the respective protein target. Upon binding, the unique proximity probes can hybridize to each other and subsequently be detected and quantified by real-time PCR. Mean biomarker levels were compared using a t-test. False discovery rate (FDR) was used for multiple comparisons adjustment. RESULTSUsing samples collected prior to initiation of carfilzomib therapy, in an agnostic statistical model we identified the following four proteins to have altered levels in myeloma patients who developed CV events (p=0.002-0.004, unadjusted; p=0.089, after FDR correction): matrix metalloproteinase-1 (MMP-1, heparin-binding EGF-like growth factor (HB-EGF), TNF-related apoptosis-inducing ligand (TRAIL), and myoglobin (MB). Myeloma patients who developed CV events had 37% lower MMP-1, 15% lower MB, and 4% lower HB-EGF, while TRAIL was 7% higher in patients who developed CV events. Matrix metalloproteinases are a family of proteolytic enzymes responsible, among other functions, for myocardial extracellular protein degradation. Interestingly, several MMP species, including MMP-1, have been identified within the human myocardium and are thought to be dysregulated in congestive heart failure. HB-EGF is a mitogenic and chemotactic glycoprotein that is essential for maintaining normal cardiac function and is known to play an important role in myocardial remodeling. CONCLUSIONSWe found that there was a trend towards lower MMP-1, HB-EGF, and MB levels and higher TRAIL levels in patients with CV events while receiving proteasome therapy. MMP-1 appears to be the most promising potential biomarker based on our data. Our study supports further investigation of these proteins as potential biomarkers for patients at risk of CV events when treated with carfilzomib.Table 1CV eventNo CV eventN=7N=19CKD Proteins1Mean (SD)Mean(SD)Unadjusted P-valueAdjusted P-valueMMP_11.7 (0.5)2.7 (0.9)0.0020.089HB_EGF6.9 (0.2)7.2 (0.3)0.0040.089TRAIL8 (0.3)7.5 (0.5)0.0040.089MB5 (0.5)5.9 (0.8)0.0040.089HSP_272.2 (0.3)2.7 (0.8)0.0320.528PDGF_subunit_B4 (0.7)5 (1.5)0.0360.528CD40_L3.4 (0.6)4.2 (1.2)0.0420.533EGF3.7 (0.9)4.7 (1.4)0.0530.592CX3CL15.9 (0.2)5.6 (0.6)0.0920.895TRAIL_R24.2 (0.4)4.6 (0.6)0.1010.8951. Proteins are listed based on the p-value associated with the difference between patients who did and did not have CV events, with lowest p-value on the top. The top 10 biomarkers are shown. DisclosuresEkman:Olink Bioscience: Employment. Grundberg:Olink Bioscience: Employment. Hassoun:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Takeda: Research Funding; Novartis: Consultancy. Lesokhin:Aduro: Consultancy; Efranat: Consultancy; Genentech: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Landau:Janssen: Consultancy; Prothena: Consultancy, Honoraria; Janssen: Consultancy; Spectrum Pharmaceuticals: Honoraria; Onyx: Honoraria, Research Funding; Takeda: Research Funding. Giralt:TAKEDA: Consultancy, Honoraria, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; AMGEN: Consultancy, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding. Landgren:Onyx: Honoraria; Celgene: Honoraria; BMJ Publishing: Consultancy; International Myeloma Foundation: Research Funding; Bristol-Myers Squibb: Honoraria; Onyx: Research Funding; Medscape: Consultancy; Medscape: Honoraria; BMJ Publishing: Honoraria; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Onyx: Consultancy.

NMDA receptor agonists reverse impaired psychomotor and cognitive functions associated with hippocampal Hbegf-deficiency in mice.

BackgroundStructural and functional changes of the hippocampus are correlated with psychiatric disorders and cognitive dysfunctions. Genetic deletion of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is predominantly expressed in cortex and hippocampus, also causes similar psychiatric and cognitive dysfunctions, accompanying down-regulated NMDA receptor signaling. However, little is known of such dysfunctions in hippocampus-specific Hbegf cKO mice.ResultsWe successfully developed hippocampus-specific cKO mice by crossbreeding floxed Hbegf and Gng7-Cre knock-in mice, as Gng7 promoter-driven Cre is highly expressed in hippocampal neurons as well as striatal medium spiny neurons. In mice lacking hippocampus Hbegf gene, there was a decreased neurogenesis in the subgranular zone (SGZ) of the dentate gyrus as well as down-regulation of PSD-95/NMDA-receptor-NR1/NR2B subunits and related NMDA receptor signaling. Psychiatric, social-behavioral and cognitive abnormalities were also observed in hippocampal cKO mice. Interestingly, D-cycloserine and nefiracetam, positive allosteric modulators (PAMs) of NMDA receptor reversed the apparent reduction in NMDA receptor signaling and most behavioral abnormalities. Furthermore, decreased SGZ neurogenesis in hippocampal cKO mice was reversed by nefiracetam.ConclusionsThe present study demonstrates that PAMs of NMDA receptor have pharmacotherapeutic potentials to reverse down-regulated NMDA receptor signaling, neuro-socio-cognitive abnormalities and decreased neurogenesis in hippocampal cKO mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0176-0) contains supplementary material, which is available to authorized users.

Cross-reacting material 197 reverses the resistance to paclitaxel in paclitaxel-resistant human ovarian cancer.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been proven to be a promising chemotherapeutic target for ovarian cancer. Our previous studies have demonstrated that inhibition of HB-EGF by the special inhibitor, cross-reacting material 197 (CRM197), potently inhibits the anti-tumor activity in paclitaxel-resistant ovarian cancer. Here, we found that inhibition of HB-EGF by CRM197 significantly reverses the resistance to paclitaxel in paclitaxel-resistant ovarian carcinoma cell line (A2780/Taxol). A2780/Taxol cells over-expressed HB-EGF and epidermal growth factor receptor (EGFR) and CRM197 notably suppressed the expression of HB-EGF and EGFR. Experiments performed in vitro and in vivo further suggested that CRM197 markedly down-regulated the ATP-binding cassette sub-family B member 1 (ABCB1/MDR1) messenger RNA (mRNA) expression (P = 0.01), plasma membrane glycoprotein (P-gp) protein (P = 0.009), and P-gp-mediated efflux (P = 0.007) through inhibition of nuclear factor-κB (NF-κB) expression, which were classical chemoresistance-related targets with respect to paclitaxel therapy. Meanwhile, inhibition of HB-EGF enhanced caspase-3 activity to induce apoptosis via MDR1 inhibition in A2780/Taxol cells (P = 0.038). Collectively, HB-EGF is a molecular target for the resistance of ovarian cancer to paclitaxel and CRM197 as a HB-EGF-targeted agent might be a chemosensitizing agent for paclitaxel-resistant ovarian carcinoma. Our findings provide novel possible mechanisms for HB-EGF to be a target to restore the chemosensitivity to paclitaxel.

TMOD-11HBEGF ACTIVATES EGFR AND AXL TO FORM MALIGNANT GLIOMAS IN THE CONTEXT OF Ink4a/Arf AND Pten LOSS

Heparin-binding EGF-like growth factor (HBEGF) is a ligand for epidermal growth factor receptor (EGFR), the most frequently amplified receptor-tyrosine kinase in malignant gliomas. About 30% of high-grade gliomas express HBEGF, and it is also commonly upregulated in tumors that express the EGFR mutant, EGFRvIII. HBEGF has been associated with the progression of many solid tumors including breast, ovarian, bladder, and pancreatic. To date, HBEGF has not been evaluated for its ability to promote the development of high-grade gliomas in vivo. Using a retroviral method for somatic cell gene transfer, we show that HBEGF cooperates with loss of tumor suppressors Ink4a/Arf and Pten to form malignant gliomas in vivo. These mouse tumors share many characteristics found in human high-grade gliomas including necrosis and nuclear atypia. No tumors were seen with loss of two of the most common tumor suppressors altered in malignant gliomas, Ink4a/Arf and Pten. Our preliminary studies in astrocytes in vitro suggest that HBEGF activates not only EGFR but also AXL, a member of the TAM (Tyro3, AXL, Mer) family of RTKs. This cross activation may explain some of the inefficacy of targeted EGFR therapies that have been tested in malignant gliomas – even in those that have amplification of EGFR which would otherwise predict increased sensitivity to EGFR inhibition. Although unreported in the context of HBEGF or gliomas, a role for AXL in mediating EGFR signaling has been cited as a mechanism of resistance against targeted EGFR therapies in other solid tumors. Our ongoing research seeks to define the mechanism by which HBEGF activates AXL and EGFR to initiate high-grade gliomas.

Heparin-binding EGF-like growth factor (HB-EGF) stimulates the proliferation of Müller glia-derived progenitor cells in avian and murine retinas.

Müller glia can be stimulated to de-differentiate, proliferate and form Müller glia-derived progenitor cells (MGPCs) that regenerate retinal neurons. In the zebrafish retina, heparin-binding EGF-like growth factor (HB-EGF) may be one of the key factors that stimulate the formation of proliferating MGPCs. Currently nothing is known about the influence of HB-EGF on the proliferative potential of Müller glia in retinas of birds and rodents. In the chick retina, we found that levels of both hb-egf and egf-receptor are rapidly and transiently up-regulated following NMDA-induced damage. Although intraocular injections of HB-EGF failed to stimulate cell-signaling or proliferation of Müller glia in normal retinas, HB-EGF stimulated proliferation of MGPCs in damaged retinas. By comparison, inhibition of the EGF-receptor (EGFR) decreased the proliferation of MGPCs in damaged retinas. HB-EGF failed to act synergistically with FGF2 to stimulate the formation of MGPCs in the undamaged retina and inhibition of EGF-receptor did not suppress FGF2-mediated formation of MGPCs. In the mouse retina, HB-EGF stimulated the proliferation of Müller glia following NMDA-induced damage. Furthermore, HB-EGF not only stimulated MAPK-signaling in Müller glia/MGPCs, but also activated mTor- and Jak/Stat-signaling. We propose that levels of expression of EGFR are rate-limiting to the responses of Müller glia to HB-EGF and the expression of EGFR can be induced by retinal damage, but not by FGF2-treatment. We conclude that HB-EGF is mitogenic to Müller glia in both chick and mouse retinas, and HB-EGF is an important player in the formation of MGPCs in damaged retinas.

Preclinical Pharmacokinetics Evaluation of Anti-heparin-binding EGF-like Growth Factor (HB-EGF) Monoclonal Antibody Using Cynomolgus Monkeys via (89)Zr-immuno-PET Study and the Determination of Drug Concentrations in Serum and Cerebrospinal Fluid.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family and is an important therapeutic target in some types of human cancers. KHK2866 is a humanized anti-HB-EGF monoclonal antibody IgG that neutralizes HB-EGF activity by inhibiting the binding of HB-EGF to its receptors. The phase I study of KHK2866 was discontinued because of neuropsychiatric toxicity. In this study, the pharmacokinetics of KHK2866 was evaluated by (89)Zr-immuno-PET study and the determination of drug concentrations in serum and cerebrospinal fluid using cynomolgus monkeys was performed in order to predict neurotoxicity in a reverse-translational manner. KHK2866 was radiolabeled with (89)Zr for preclinical evaluations in normal cynomolgus monkeys and its distribution was analyzed. Furthermore, as a separate study, KHK2866 concentrations in serum and cerebrospinal fluid were determined after administration of a single dose. PET studies with monkeys revealed (89)Zr-KHK2866 accumulation in the liver, spleen and joints of multiple parts, but not in brain. In addition, the pharmacokinetic analyses in serum and CSF demonstrated a low penetration of KHK2866 into the brain. These studies indicate the difficulty of prediction for neuropsychiatric toxicity of monoclonal antibodies in human by means of pharmacokinetic evaluations using cynomolgus monkeys.

Fingolimod induces neuroprotective factors in human astrocytes

BackgroundFingolimod (FTY720) is the first sphingosine-1-phosphate (S1P) receptor modulator approved for the treatment of multiple sclerosis. The phosphorylated active metabolite FTY720-phosphate (FTY-P) interferes with lymphocyte trafficking. In addition, it accumulates in the CNS and reduces brain atrophy in multiple sclerosis (MS), and neuroprotective effects are hypothesized.MethodsHuman primary astrocytes as well as human astrocytoma cells were stimulated with FTY-P or S1P. We analyzed gene expression by a genome-wide microarray and validated induced candidate genes by quantitative PCR (qPCR) and ELISA. To identify the S1P-receptor subtypes involved, we applied a membrane-impermeable S1P analog (dihydro-S1P), receptor subtype specific agonists and antagonists, as well as RNAi silencing.ResultsFTY-P induced leukemia inhibitory factor (LIF), interleukin 11 (IL11), and heparin-binding EGF-like growth factor (HBEGF) mRNA, as well as secretion of LIF and IL11 protein. In order to mimic an inflammatory milieu as observed in active MS lesions, we combined FTY-P application with tumor necrosis factor (TNF). In the presence of this key inflammatory cytokine, FTY-P synergistically induced LIF, HBEGF, and IL11 mRNA, as well as secretion of LIF and IL11 protein. TNF itself induced inflammatory, B-cell promoting, and antiviral factors (CXCL10, BAFF, MX1, and OAS2). Their induction was blocked by FTY-P. After continuous exposure of cells to FTY-P or S1P for up to 7 days, the extent of induction of neurotrophic factors and the suppression of TNF-induced inflammatory genes declined but was still detectable. The induction of neurotrophic factors was mediated via surface S1P receptors 1 (S1PR1) and 3 (S1PR3).ConclusionsWe identified effects of FTY-P on astrocytes, namely induction of neurotrophic mediators (LIF, HBEGF, and IL11) and inhibition of TNF-induced inflammatory genes (CXCL10, BAFF, MX1, and OAS2). This supports the view that a part of the effects of fingolimod may be mediated via astrocytes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0393-6) contains supplementary material, which is available to authorized users.

Abstract 40: HB-EGF‑mediated CITED4 signaling in lung cancer

Abstract Abnormal activation of the EGF receptor (EGFR) signaling is a hallmark of lung cancer. However, the role of heparin-binding EGF-like growth factor (HB-EGF), one of EGFR ligands, in lung tumorigenesis has been unappreciated. In this study, we showed that HB-EGF was highly expressed in a subgroup of lung cancer cells, the proliferation of which was dependent on HB-EGF signaling. Members of CITED family proteins respond to various cytokine signalings, regulating cell proliferation; among them, we identified CITED4 as a downstream effecter of HB-EGF. We revealed that CITED4 was upregulated by HB-EGF through a STAT3 dependent pathway. Moreover, CITED4-silencing attenuated HB-EGF-mediated lung cancer cell proliferation. ChIP analysis and promoter reporter assay showed that STAT3 bound CITED4 promoter, enhancing CITED4 transcription. Co-immunoprecipitation analysis identified MYC as a CITED4-interacting protein. Furthermore, we found that CITED4 expression promoted MYC-mediated transcription of cyclin D1 promoter, leading to increased G1/S cell cycle transition. Thus, our findings support that HB-EGF-CITED4 signaling cascade plays a critical role in aberrant proliferation in lung cancer with potential as a biomarker for lung cancer progression. Citation Format: Cheng-Han Hsieh, Yu-Ting Chou, Cheng-Wen Wu. HB-EGF‑mediated CITED4 signaling in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 40. doi:10.1158/1538-7445.AM2015-40

Mitogen-activated protein kinase-activated protein kinase 2 mediates resistance to hydrogen peroxide-induced oxidative stress in human hepatobiliary cancer cells

The development and progression of liver cancer are characterized by increased levels of reactive oxygen species (ROS). ROS-induced oxidative stress impairs cell proliferation and ultimately leads to cell death. Although liver cancer cells are especially resistant to oxidative stress, mechanisms of such resistance remain understudied. We identified the MAPK-activated protein kinase 2 (MK2)/heat shock protein 27 (Hsp27) signaling pathway mediating defenses against oxidative stress. In addition to MK2 and Hsp27 overexpression in primary liver tumors compared to adjacent nontumorous tissues, the MK2/Hsp27 pathway is activated by hydrogen peroxide-induced oxidative stress in hepatobiliary cancer cells. MK2 inactivation or inhibition of MK2 or Hsp27 expression increases caspase-3 and PARP cleavage and DNA breaks and therefore cell death. Interestingly, MK2/Hsp27 inhibition decreases antioxidant defenses such as heme oxygenase 1 through downregulation of the transcription factor nuclear factor erythroid-derived 2-like 2. Moreover, MK2/Hsp27 inhibition decreases both phosphorylation of epidermal growth factor receptor (EGFR) and expression of its ligand, heparin-binding EGF-like growth factor. A new identified partner of MK2, the scaffold PDZ protein EBP50, could facilitate these effects through MK2/Hsp27 pathway regulation. These findings demonstrate that the MK2/Hsp27 pathway actively participates in resistance to oxidative stress and may contribute to liver cancer progression.

MicroRNA-132 enhances transition from inflammation to proliferation during wound healing.

Wound healing is a complex process that is characterized by an initial inflammatory phase followed by a proliferative phase. This transition is a critical regulatory point; however, the factors that mediate this process are not fully understood. Here, we evaluated microRNAs (miRs) in skin wound healing and characterized the dynamic change of the miRNome in human skin wounds. miR-132 was highly upregulated during the inflammatory phase of wound repair, predominantly expressed in epidermal keratinocytes, and peaked in the subsequent proliferative phase. TGF-β1 and TGF-β2 induced miR-132 expression in keratinocytes, and transcriptome analysis of these cells revealed that miR-132 regulates a large number of immune response- and cell cycle-related genes. In keratinocytes, miR-132 decreased the production of chemokines and the capability to attract leukocytes by suppressing the NF-κB pathway. Conversely, miR-132 increased activity of the STAT3 and ERK pathways, thereby promoting keratinocyte growth. Silencing of the miR-132 target heparin-binding EGF-like growth factor (HB-EGF) phenocopied miR-132 overexpression in keratinocytes. Using mouse and human ex vivo wound models, we found that miR-132 blockade delayed healing, which was accompanied by severe inflammation and deficient keratinocyte proliferation. Together, our results indicate that miR-132 is a critical regulator of skin wound healing that facilitates the transition from the inflammatory to the proliferative phase.

The Soluble Heparin-Binding EGF-Like Growth Factor Stimulates EGF Receptor Trafficking to the Nucleus.

Most ligands of epidermal growth factor receptor (EGFR) have the ability to induce EGFR translocation into the nucleus, where EGFR acts as an important transcriptional regulator. Soluble form of heparin-binding EGF-like growth factor (sHB-EGF) is one of the ligands for EGFR in many cell types; however, there is no evidence for the ability of sHB-EGF to induce EGFR nuclear importation. Here, we demonstrated that treatment of A431 cells with sHB-EGF resulted in nuclear localization of EGFR and such translocation occurs via retrograde pathway. It was shown by confocal microscopy and co-immunoprecipitation assay that the translocation complex consisted of both ligand and receptor. The chromatin immunoprecipitation assay showed the association of sHB-EGF–EGFR complex with promoter region of cyclin D1 in the cell nucleus and this association was prevented by application of EGFR kinase inhibitor AG-1478. The obtained data suggest that sHB-EGF acts similarly to other EGFR ligands and is capable to induce EGFR nuclear translocation as a part of ligand-receptor complex in a tyrosine phosphorylation-dependent manner.

Abstract 358: Targeting of Heparin Binding EGF-like Growth Factor (HB-EGF) Efficiently Inhibits Atherosclerosis

Objective: Heparin-binding EGF-like growth factor (HB-EGF) is a receptor ligand that regulates inflammatory gene expression in the human aortic endothelial cells. In this study, we examined the effects of targeting HB-EGF using an antisense oligonucleotide (ASO) in the development of atherosclerosis. Methods and Results: Control and HB-EGF ASOs were injected intraperitoneally (i.p.; weekly; Dose: 50 mg/kg/wk) for 12 weeks into LDLR deficient mice fed a fat-enriched diet (21% fat; 0.2% cholesterol). Compared with the control ASO injected group (N=13), the HB-EGF ASO injected group (N=15) had efficiently reduced atherosclerosis in the arch of aorta (p<0.0001). The HB-EGF ASO also significantly reduced plasma cholesterol concentration (p = 0.0397). We observed that the VLDL was consistently reduced in all HB-EGF ASO injected mice tested. In liver tissue, the HB-EGF ASO induced significant downregulation of gene expressions of the sterol synthetic pathway as determined by a microarray and DAVID functional analysis (adjusted p = 5.5 x 10-9)). The HB-EGF ASO also induced lipid droplet accumulation in liver tissues as determined by histological analysis with liver tissue sections. Conclusions: This study indicates that HB-EGF is a key mediator for atherosclerosis and the targeting of HB-EGF is an efficient approach to inhibit lesion formation in the aorta. Downregulation of the sterol synthesis pathway and reduction of VLDL particles in the blood may contribute to the protective effects of HB-EGF ASO.