The heparan sulfate (HS) mimic/heparanase inhibitor roneparstat (SST0001) shows antitumor activity in preclinical sarcoma models. We hypothesized that this 100% N-acetylated and glycol-split heparin could interfere with the functions of several receptor tyrosine kinases (RTK) coexpressed in sarcomas and activated by heparin-binding growth factors. Using a phospho-proteomic approach, we investigated the drug effects on RTK activation in human cell lines representative of different sarcoma subtypes. Inhibition of FGF, IGF, ERBB and PDGF receptors by the drug was biochemically and functionally validated. Roneparstat counteracted the autocrine loop induced by the COL1A1/PDGFB fusion oncogene, expressed in a human dermatofibrosarcoma protuberans primary culture and in NIH3T3COL1A1/PDGFB transfectants, inhibiting cell anchorage-independent growth and invasion. In addition, roneparstat inhibited the activation of cell surface PDGFR and PDGFR-associated FAK, likely contributing to the reversion of NIH3T3COL1A1/PDGFB cell transformed and pro-invasive phenotype. Biochemical and histological/immunohistochemical ex vivo analyses confirmed a reduced activation of ERBB4, EGFR, INSR, IGF1R, associated with apoptosis induction and angiogenesis inhibition in a drug-treated Ewing's sarcoma family tumor xenograft. The combination of roneparstat with irinotecan significantly improved the antitumor effect against A204 rhabdoid xenografts resulting in a high rate of complete responses and cures. These findings reveal that roneparstat exerts a multi-target inhibition of RTKs relevant in the pathobiology of different sarcoma subtypes. These effects, likely cooperating with heparanase inhibition, contribute to the antitumor efficacy of the drug. The study supports heparanase/HS axis targeting as a valuable approach in combination therapies of different sarcoma subtypes providing a preclinical rationale for clinical investigation.

The cytoplasmic protein-tyrosine kinase BTK plays an essential role for differentiation and survival of B-lineage cells and, hence, represents a suitable drug target. The number of BTK inhibitors (BTKis) in the clinic has increased considerably and currently amounts to at least 22. First-in-class was ibrutinib, an irreversible binder forming a covalent bond to a cysteine in the catalytic region of the kinase, for which we have identified 228 active trials listed at ClinicalTrials.gov. Next-generation inhibitors, acalabrutinib and zanubrutinib, are approved both in the United States and in Europe, and zanubrutinib also in China, while tirabrutinib is currently only registered in Japan. In most cases, these compounds have been used for the treatment of B-lymphocyte tumors. However, an increasing number of trials instead addresses autoimmunity and inflammation in multiple sclerosis, rheumatoid arthritis, pemphigus and systemic lupus erythematosus with the use of either irreversibly binding inhibitors, e.g., evobrutinib and tolebrutinib, or reversibly binding inhibitors, like fenebrutinib. Adverse effects (AEs) have predominantly implicated inhibition of other kinases with a BTKi-binding cysteine in their catalytic domain. Analysis of the reported AEs suggests that ibrutinib-associated atrial fibrillation is caused by binding to ERBB2/HER2 and ERBB4/HER4. However, the binding pattern of BTKis to various additional kinases does not correlate with the common assumption that skin manifestations and diarrhoeas are off-target effects related to EGF receptor inhibition. Moreover, dermatological toxicities, diarrhoea, bleedings and invasive fungal infections often develop early after BTKi treatment initiation and subsequently subside. Conversely, cardiovascular AEs, like hypertension and various forms of heart disease, often persist.

Keywords: X-linked agammaglobulinemia; acalabrutinib; atrial fibrillation; diarrhoea; ibrutinib; infection; rash; zanubrutinib.

Tyrosine phosphorylation is important in signaling pathways underlying tumorigenesis. We performed a mutational analysis of the protein tyrosine kinase (PTK) gene family in cutaneous metastatic melanoma. We identified 30 somatic mutations affecting the kinase domains of 19 PTKs and subsequently evaluated the entire coding regions of the genes encoding these 19 PTKs for somatic mutations in 79 melanoma samples. We found ERBB4 mutations in 19% of individuals with melanoma and found mutations in two other kinases (FLT1 and PTK2B) in 10% of individuals with melanomas. We examined seven missense mutations in the most commonly altered PTK gene, ERBB4, and found that they resulted in increased kinase activity and transformation ability. Melanoma cells expressing mutant ERBB4 had reduced cell growth after shRNA-mediated knockdown of ERBB4 or treatment with the ERBB inhibitor lapatinib. These studies could lead to personalized therapeutics specifically targeting the kinases that are mutationally altered in individual melanomas.

The role of ErbB4 and ErbB2 in the heart of adult mammals is well established. The heart also expresses ErbB1 (the epidermal growth factor (EGF) receptor), but this receptor has received less attention. We studied the effect of EGF on the response of isolated mouse heart to low-flow ischemia and reperfusion. Reducing perfusate flow to 10% for 30 min resulted in an increase in anaerobic metabolism and the leakage of lactate dehydrogenase during reperfusion. In addition, left ventricle +dP/dt and developed pressure were depressed (20-25%) during reperfusion. The addition of EGF 5 min before and throughout the ischemic period prevented the increase in anaerobic metabolism and the leakage of intracellular lactate dehydrogenase during reperfusion. EGF improved both +dP/dt and developed pressure during ischemia and prevented the decrease in dP/dt during reperfusion. To determine whether the effect of EGF on cell integrity depends on its effect on contractility, we studied nonbeating isolated myocytes. In these cells, anoxia and reoxygenation reduced cell viability by nearly 25%. EGF prevented such a decrease. Our results indicate that, like ErbB4 and ErbB2, ErbB1 also has an important role in the heart of adult animals.

Poorly differentiated thyroid carcinoma (PDTC) is a rare malignancy with higher mortality than well-differentiated thyroid carcinoma. The histological diagnosis can be difficult as well as the therapy. Improved diagnosis and new targeted therapies require knowledge of DNA sequence changes in cancer-relevant genes. The TruSeq Amplicon Cancer Panel was used to screen cancer genomes from 25 PDTC patients for somatic single-nucleotide variants in 48 genes known to represent mutational hotspots. A total of 4490 variants were found in 23 tissue samples of PDTC. Ninety-eight percent (4392) of these variants did not meet the inclusion criteria, while 98 potentially pathogenic or pathogenic variants remained after filtering. These variants were distributed over 33 genes and were all present in a heterozygous state. Five tissue samples harboured not a single variant. Predominantly, variants in P53 (43% of tissue samples) were identified, while less frequently, variants in APC, ERBB4, FLT3, KIT, SMAD4 and BRAF (each in 17% of tissue samples) as well as ATM, EGFR and FBXW7 (each in 13% of tissue samples) were observed. This study identified new potential genetic targets for further research in PDTC. Of particular interest are four observed ERBB4 (alias HER4) variants, which have not been connected to this type of thyroid carcinoma so far. In addition, APC and SMAD4 mutations have not been reported in this subtype of cancer either. In contrast to other reports, we did not find CTNNB1 variants.

Metaplastic breast carcinoma (MBC) is a rare and aggressive histologic subtype of breast cancer comprising approximately 0.5% to 5.0% of all invasive breast cancers with a poor prognosis and limited therapeutic options.

We investigated MBC at our institution to evaluate outcomes and investigate the molecular profile of our cohort to determine the presence of mutations for which there are targeted therapies.

We found our cohort to consist mainly of the matrix-producing variant (72%) with 48% having the stereotypical estrogen receptor-negative/progesterone receptor-negative/human epidermal growth factor receptor-2-negative phenotype. While the overall survival of our cohort was an average of 1679 days (4.6 years), we had a surprising number of patients with second primaries (40%) and distant metastases (40%), yet few recurrences (12%). Molecular analysis of the tumors indicated that one gene mutation, CSFIR, was significantly associated with outcome (P = .021); however, the cohort was defined by frequent mutations in ERBB4 (36%), PIK3CA (48%), and FLT3 (60%), for which there are now targeted therapies.

While surgery is the appropriate first step in the management of this aggressive malignancy, the collection of data pertaining to the use of targeted agents, although anecdotal, may provide clues to better treatment for these patients.

Neuregulin (NRG) is an EGF-related growth factor that binds to the tyrosine kinase receptors ErbB3 and ErbB4, thus inducing tissue development and muscle glucose utilization during contraction. Here, we analyzed whether NRG has systemic effects regulating glycemia in control and type 2 diabetic rats. To this end, recombinant NRG (rNRG) was injected into Zucker diabetic fatty (ZDF) rats and their respective lean littermates 15 min before a glucose tolerance test (GTT) was performed. rNRG enhanced glucose tolerance without promoting the activation of the insulin receptor (IR) or insulin receptor substrates (IRS) in muscle and liver. However, in control rats, rNRG induced the phosphorylation of protein kinase B (PKB) and glycogen synthase kinase-3 (GSK-3) in liver but not in muscle. In liver, rNRG increased ErbB3 tyrosine phosphorylation and its binding to phosphatidylinositol 3-kinase (PI3K), thus indicating that rNRG activates the ErbB3/PI3K/PKB signaling pathway. rNRG increased glycogen content in liver but not in muscle. rNRG also increased the content of fructose-2,6-bisphosphate (Fru-2,6-P2), an activator of hepatic glycolysis, and lactate in liver but not in muscle. Increases in lactate were abrogated by wortmannin, a PI3K inhibitor, in incubated hepatocytes. The liver of ZDF rats showed a reduced content of ErbB3 receptors, entailing a minor stimulation of the rNRG-induced PKB/GSK-3 cascade and resulting in unaltered hepatic glycogen content. Nonetheless, rNRG increased hepatic Fru-2,6-P2 and augmented lactate both in liver and in plasma of diabetic rats. As a whole, rNRG improved response to the GTT in both control and diabetic rats by enhancing hepatic glucose utilization.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motoneurons, which is preceded by loss of neuromuscular connections in a "dying back" process. Neuregulin-1 (Nrg1) is a neurotrophic factor essential for the development and maintenance of neuromuscular junctions, and Nrg1 receptor ErbB4 loss-of-function mutations have been reported as causative for ALS. Our main goal was to investigate the role of Nrg1 type I (Nrg1-I) in SOD1(G93A) mice muscles. We overexpressed Nrg1-I by means of an adeno-associated viral (AAV) vector, and investigated its effect by means of neurophysiological techniques assessing neuromuscular function, as well as molecular approaches (RT-PCR, western blot, immunohistochemistry, ELISA) to determine the mechanisms underlying Nrg1-I action. AAV-Nrg1-I intramuscular administration promoted motor axon collateral sprouting by acting on terminal Schwann cells, preventing denervation of the injected muscles through Akt and ERK1/2 pathways. We further used a model of muscle partial denervation by transecting the L4 spinal nerve. AAV-Nrg1-I intramuscular injection enhanced muscle reinnervation by collateral sprouting, whereas administration of lapatinib (ErbB receptor inhibitor) completely blocked it. We demonstrated that Nrg1-I plays a crucial role in the collateral reinnervation process, opening a new window for developing novel ALS therapies for functional recovery rather than preservation.

Peritoneal carcinomatosis accompanied by malignant ascites is a major cause of death of advanced gastric cancer (GC). To comprehensively characterize the underlying genomic events involved in GC peritoneal carcinomatosis, we analyzed whole-exome sequences of normal gastric tissues, primary tumors, and malignant ascites from eight GC patients. We identified a unique mutational signature biased toward C-to-A substitutions in malignant ascites. In contrast, the patients who received treatment of adjuvant chemotherapy showed a high rate of C-to-T substitutions along with hypermutation in malignant ascites. Comparative analysis revealed several candidate mutations for GC peritoneal carcinomatosis: recurrent mutations in COL4A6, INTS2, and PTPN13; mutations in druggable genes including TEP1, PRKCD, BRAF, ERBB4, PIK3CA, HDAC9, FYN, FASN, BIRC2, FLT3, ROCK1, CD22, and PIK3C2B; and mutations in metastasis-associated genes including TNFSF12, L1CAM, DIAPH3, ROCK1, TGFBR1, MYO9B, NR4A1, and RHOA. Notably, gene ontology analysis revealed the significant enrichment of mutations in the Rho-ROCK signaling pathway-associated biological processes in malignant ascites. At least four of the eight patients acquired somatic mutations in the Rho-ROCK pathway components, suggesting the possible relevance of this pathway to GC peritoneal carcinomatosis. These results provide a genome-wide molecular understanding of GC peritoneal carcinomatosis and its clinical implications, thereby facilitating the development of effective therapeutics.

Adolescence is a vulnerable period characterized by major cognitive changes. The mechanisms underlying the emergence of new cognitive functions are poorly understood. We find that a long-term depression of inhibitory transmission (iLTD) from parvalbumin-expressing (PV+) interneurons in the hippocampal area Cornu Ammonis 2 (CA2) is absent in young mice but emerges at the end of adolescence. We demonstrate that the maturation of both the perineuronal net (PNN) and signaling through ErbB4 is required for this plasticity. Furthermore, we demonstrate that social recognition memory displays the same age dependence as iLTD and is impaired by targeted degradation of the PNN or iLTD blockade in area CA2. Our data reveal an unusual developmental rule for plasticity at the PV+ interneuron transmission in area CA2 and indicate that this plasticity is involved in the emergence of higher cognitive function, such as social memory formation, in late adolescence.

OBJECTIVE

To identify molecular prognostic factors and potentially actionable mutations in ampulla of Vater cancer (AVC).

BACKGROUND

The largely variable outcomes of AVCs make clinical decisions difficult regarding the need of postsurgical therapy, which is based on morphological and immunohistochemical classification that do not adequately consider the varying degrees of heterogeneity present in many AVCs. No approved targeted therapies for AVC exist, but some show promising results requiring better molecular characterization to identify potential responders.

METHODS

We assessed 80 AVCs for the prognostic value of mutations of kirsten rat sarcoma (KRAS), neuroblastoma RAS (NRAS), B rapidly accelerated fibrosarcoma (BRAF), TP53, and 4 membrane erythroblastosis oncogene B (ERBB) receptor tyrosine kinases (EGFR-ERBB1, HER2-ERBB2, HER3-ERBB3, HER4-ERBB4) amenable to pharmacological inhibition. Moreover, we evaluated mutations in 16 key components of rat sarcoma (RAS), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), protein 53 (P53), transforming growth factor beta (TGF-β), and wingless/integrated (WNT) pathways, recently associated to AVC by whole-exome sequencing.

RESULTS

TP53 and KRAS were mutated in 41% and 35% of cases, respectively, and emerged as independent prognostic factors together with tumor stage and regardless of the histotype (TP53: P = 0.0006; KRAS: P = 0.0018; stage IIB: P = 0.0117; stage III-IV: P = 0.0020). ERBB, WNT and PI3K pathway genes were mutated in 37.5% of cases.

CONCLUSIONS

KRAS and TP53 mutations are negative predictors of survival in AVCs, regardless of histotype. Potentially actionable mutations in ERBB, WNT, and PI3K signaling pathway genes are present in 37.5% of all cases. These might be amenable to target therapy using available drugs like Everolimus in PI3K-mutated cases or compounds under active screening against ERBB and WNT signaling.

Members of the CD44 family of transmembrane glycoproteins control cell signaling pathways from numerous cell surface receptors, including receptor tyrosine kinases (RTKs). The decisive factor (ligand, RTKs or both) that controls the recruitment of specific CD44 isoforms is still unknown. We investigated this question by using the EGFR signaling pathway, in which one receptor can be activated by a broad range of ligands. By means of siRNA-mediated downregulation of CD44 expression and blocking experiments, we identified CD44v6 as a co-receptor for EGF- and ER-induced ErbB1 activation and for NRG1-induced ErbB3 and ErbB4 activation. In contrast, TGFα is independent of all CD44 isoforms, even though it addresses the same receptor pairs as EGF. Moreover, the heparin-sulfated CD44v3 isoform is required for HB-EGF-induced EGFR signaling. These data suggest that specific CD44 isoforms are recruited in a ligand-dependent manner as co-receptors in the EGFR signaling pathways and that the specificity is determined by the ligand and not by the receptors themselves. The in vivo relevance of this interplay between CD44 isoforms and EGFR ligands is underlined by the decreased metastatic spreading of mammary carcinomas in mice treated with a CD44v6-specific peptide. Most importantly, we found a clear correlation between the presence of CD44v6/ErbB1 complexes in breast cancer patients and lymph node metastases.

Neuregulin-1 (NRG1) signaling through the tyrosine kinase receptors erbB2 and erbB4 is required for cardiac morphogenesis, and it plays an essential role in maintaining the myocardial architecture during adulthood. The tyrosine kinase receptor erbB2 was first linked to the amplification and overexpression of erbb2 gene in a subtype of breast tumor cells, which is indicative of highly proliferative cells and likely a poor prognosis following conventional chemotherapy. The development of targeted therapies to block the survival of erbB2-positive cancer cells revealed that impaired NRG1 signaling through erbB2/erbB4 heterodimers combined with anthracycline chemotherapy may lead to dilated cardiomyopathy in a subpopulation of treated patients. The ventricular-specific deletion of either erbb2 or erbb4 manifested dilated cardiomyopathy, which is aggravated by the administration of doxorubicin. Based on the exacerbated toxicity displayed by the combined treatment, it is expected that the relevant pathways would be affected in a synergistic manner. This review examines the NRG1 activities that were monitored in different model systems, focusing on the emerging pathways and molecular targets, which may aid in understanding the acquired dilated cardiomyopathy that occurs under the conditions of NRG1-deficient signaling.

Aberrant increases in NMDA receptor (NMDAR) signaling contributes to central nervous system sensitization and chronic pain by activating neuronal nitric oxide synthase (nNOS) and generating nitric oxide (NO). Because the scaffolding protein postsynaptic density 95kDA (PSD95) tethers nNOS to NMDARs, the PSD95-nNOS complex represents a therapeutic target. Small molecule inhibitors IC87201 (EC5O: 23.94 μM) and ZL006 (EC50: 12.88 μM) directly inhibited binding of purified PSD95 and nNOS proteins in AlphaScreen without altering binding of PSD95 to ErbB4. Both PSD95-nNOS inhibitors suppressed glutamate-induced cell death with efficacy comparable to MK-801. IC87201 and ZL006 preferentially suppressed phase 2A pain behavior in the formalin test and suppressed allodynia induced by intraplantar complete Freund's adjuvant administration. IC87201 and ZL006 suppressed mechanical and cold allodynia induced by the chemotherapeutic agent paclitaxel (ED50s: 2.47 and 0.93 mg/kg i.p. for IC87201 and ZL006, respectively). Efficacy of PSD95-nNOS disruptors was similar to MK-801. Motor ataxic effects were induced by MK-801 but not by ZL006 or IC87201. Finally, MK-801 produced hyperalgesia in the tail-flick test whereas IC87201 and ZL006 did not alter basal nociceptive thresholds. Our studies establish the utility of using AlphaScreen and purified protein pairs to establish and quantify disruption of protein-protein interactions. Our results demonstrate previously unrecognized antinociceptive efficacy of ZL006 and establish, using two small molecules, a broad application for PSD95-nNOS inhibitors in treating neuropathic and inflammatory pain. Collectively, our results demonstrate that disrupting PSD95-nNOS protein-protein interactions is effective in attenuating pathological pain without producing unwanted side effects (i.e. motor ataxia) associated with NMDAR antagonists.

In the present study, we investigate the expression profile of the epidermal growth factor receptor family, which comprises EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia (LP). The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus (OLP) and normal oral mucosa (NOM) from 14 healthy donors by real-time polymerase chain reaction (PCR) and immunohistochemistry. Synchronous mRNA coexpression of ErbB1, ErbB2, ErbB3 and ErbB4 was detected in LP lesions. Out of the receptors, only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues. These were strongly expressed by epithelial keratinocytes in LP lesions, as shown by immunohistochemistry. Regarding the ligands, the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues. Therefore, enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP.

The NRG1 growth factor and ERBB4 receptor have been identified as leading schizophrenia risk genes. Although NRG1 and ERBB4 have been shown to modulate neuronal functions involved in schizophrenia, including both GABAergic and glutamatergic synapses, the exact molecular mechanisms remain poorly understood. Here we investigated ERBB4 intracellular domain, 4ICD, transactivator function in rat hippocampal cultures by inhibiting γ-secretase mediated ERBB4 regulated intramembrane proteolysis (RIP). NRG1 stimulation resulted in a dramatic increase in the number of hippocampal cells displaying nuclear 4ICD which was abolished in cultures pretreated with the γ-secretase inhibitor compound E (CE). To identify NRG1-4ICD transactivated genes we compared global gene expression profiles of hippocampal cultures stimulated with NRG1 in the absence or presence of CE. In concordance with the contribution of NRG1-ERBB4 signaling to dendritic spine maturation and schizophrenia, global gene expression analysis followed by Ingenuity Pathway Analysis of the dataset identified NRG1-4ICD regulated genes significantly represented in semaphorin signaling and actin cytoskeletal plasticity and multiple genes with confirmed roles in dendritic spine morphogenesis. Using the power of global gene expression analysis our data provides a proof-of-concept supporting a role for non-canonical NRG1-4ICD signaling in the regulation of gene expression contributing to normal and schizophrenic neuronal function.

Among the 518 protein kinases encoded by the human kinome, several of them act as oncoproteins in human cancers. Like other eukaryotic genes, oncogenes encoding protein kinases are frequently subjected to alternative splicing in coding as well as noncoding sequences. In the present paper, we will illustrate how alternative splicing can significantly impact on the physiological functions of oncogenic protein kinases, as demonstrated by mouse genetic model studies. This includes examples of membrane-bound tyrosine kinases receptors (FGFR2, Ret, TrkB, ErbB4, and VEGFR) as well as cytosolic protein kinases (B-Raf). We will further discuss how regular alternative splicing events of these kinases are in some instances implicated in oncogenic processes during tumor progression (FGFR, TrkB, ErbB2, Abl, and AuroraA). Finally, we will present typical examples of aberrant splicing responsible for the deregulation of oncogenic kinases activity in cancers (AuroraB, Jak2, Kit, Met, and Ron).

Neuregulin-1 signaling molecules have various roles in the nervous system, including effects on neuronal migration and differentiation. In mouse cerebellar granule neurons the effects of neuregulin-1 (type I) are mediated by a homo-dimeric ErbB4 receptor, which can interact with the PSD-95 protein through a C-terminal motif. To determine whether this interaction is critical for signaling we introduced into granule cells a decoy peptide representing the C-terminal 20 amino acids of the mouse ErbB4 receptor. Exposure of unloaded cells to neuregulin-1 did not alter neurite density but increased average neurite length by 50%. In contrast, granule cell cultures loaded with the decoy peptide were unresponsive to the ligand. We conclude that the association of ErbB4 with PSD-95 is important for neuregulin-induced neuronal differentiation.

Identifying novel neuroprotectants that can halt or even reverse the effects of stroke is of interest to both clinicians and scientists. Neuregulin 1 (NRG1) is an effective neuroprotectant, but its molecular mechanisms are largely unclear. In this study, NRG1 rescued cortical neurons from oxygen-glucose deprivation (OGD) model, but the effect was blocked by neutralizing NRG1 and ErbB4 inhibition. In addition, γ-Aminobutyric acid (GABA) receptor agonists had no synergistic effect with NRG1, and the neuroprotective effect of NRG1 against OGD was partly blocked by GABA receptor antagonists. Importantly, NRG1 neuroprotection against brain ischemia was abolished in the mice with specific deletion of ErbB4 in parvalbumin (PV)-positive interneurons. In summary, NRG1 protects against ischemic brain injury via ErbB4 receptors by enhancing GABAergic transmission.

Endothelin-1 (ET-1) regulates contractility and growth of the mammalian heart by binding endothelin receptor type A (ET(A)) and endothelin receptor type B (ET(B)) G-protein-coupled receptors. To identify growth signaling pathways associated with ET-1 receptors in adult myocardium, a combined immunoprecipitation/proteomic analysis was performed. Signaling proteins believed to function downstream of ET(A) such as Galpha(q), phospholipase C-beta1, protein kinase C (PKC) epsilon, and PKCdelta were identified in immunoprecipitates of ET(A) by matrix-assisted laser desorption ionization/time of flight mass spectrometry. Also prominent were the growth factor receptor tyrosine kinases erbB2 and erbB4 and their downstream growth signaling effectors phosphoinositide-3 kinase (PI3 kinase), Akt, Raf-1, mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (Erk). Western blot analysis confirmed coimmunoprecipitation of erbB2/4, PI3 kinase, and Akt with ET(A), and confocal microscopy revealed their colocalization in cardiac transverse tubules (T-tubules). The erbB4 receptor ligand neuregulin-1beta (NRG1beta) promoted erbB2/4 tryosine phosphorylation and Akt serine phosphorylation in ventricular myocytes, whereas treatment with ET-1 did not. This observation argues against ET-1 growth signaling occurring via erbB2/4 transactivation in adult myocardium. ET-1 did, however, stimulate Erk1/2 phosphorylation and substantially blunted several NRG1beta-mediated actions, including erbB2/4 phosphorylation, serine phosphorylation of Akt, and negative inotropy. This inhibitory cross-talk between ET(A) and erbB2/4-Akt pathways was mimicked by a phorbol ester and blocked by pharmacological inhibition of PKC or MEK/Erk. The proteomic analysis and subsequent investigation of receptor cross-talk indicate that growth signaling between ET(A) and erbB pathways is fundamentally different in adult versus neonatal cardiac myocytes. The results may be relevant to cardiomyopathies associated with 1) prolonged exposure to ET-1; 2) degeneration of T-tubules; and 3) therapies targeted at erbB2 inhibition.

OBJECTIVE

Epidermal growth factor receptor (EGFR) and other members of the ErbB family of receptor tyrosine kinases (RTK) mediate autocrine growth regulation in a wide spectrum of human tumor cells. We have previously demonstrated that in stably transfected mammary carcinoma cells a dominant negative (DN) mutant of EGFR, EGFR-CD533 is a potent inhibitor of EGFR and its cytoprotective signaling after exposure to ionizing radiation. In the present study, we further investigate the capacity of a genetic approach, using replication-incompetent adenovirus (Ad)-mediated transfer of EGFR-CD533 (Ad-EGFR-CD533), to enhance the radiosensitivity in vitro of four cell lines representative of three major cancer phenotypes.

METHODS

The cell lines MDA-MB-231 and T-47D mammary carcinoma, A-431 squamous carcinoma, and U-373 MG malignant glioma cells were used. The ErbB expression profiles and the EGFR tyrosine phosphorylation (Tyr-P) levels following irradiation were quantified by Western blotting. The relative radiosensitivities of tumor cells were assessed by standard colony formation assays after infection with control vector (Ad-LacZ) or Ad-EGFR-CD533.

RESULTS

The expression profiles demonstrated varying levels of EGFR, ErbB2, ErbB3, and ErbB4 expression. The overexpression of EGFR-CD533 after infection with Ad-EGFR-CD533 completely inhibited the radiation-induced stimulation of EGFR Tyr-P relative to the immediate 2.4- to 3.1-fold increases in EGFR Tyr-P in control infected cells (Ad-LacZ). Ad-EGFR-CD533-infected cells demonstrated significant (p < 0.001) radiosensitization over a range of radiation doses (1-8 Gy), yielding dose-enhancement ratios (DER) between 1.4 and 1.7. This radiosensitization was maintained under conditions of repeated radiation exposures, using 3 x 2 Gy, yielding DERs of 1.6 and 1.7 for MDA-MB-231 and U-373 cells, respectively.

CONCLUSIONS

Overexpression of EGFR-CD533 significantly sensitizes human carcinoma and glioma cells to single and repeated radiation exposures irrespective of their ErbB expression levels. Therefore, transduction of human tumor cells with EGFR-CD533 holds promise as a gene therapeutic approach for the radiosensitization of neoplastic cells that are growth-regulated by EGFR or other ErbB receptors.

The molecular genetic events involved in the etiology of human granulosa cell (GC) tumors, which represent approximately 7% of all malignant ovarian neoplasms, are unknown. Amplification and/or overexpression of the ERBB genes are a feature of many cancer types, and overexpression of erbB2 correlates with poor prognosis in epithelial ovarian cancer. In the present study, we used immunohistochemistry to determine the level and frequency of expression of different erbB receptors in GC tumors. Ten of 12 tumors expressed erbB4 at moderate to high levels in >50% of cancer cells, whereas erbB2 (6 of 12) and erbB3 (2 of 12) were expressed less frequently. Western blot experiments showed that the only available GC tumor cell line, COV434, also expressed erbB receptors. Heregulin (HRG)-beta2, a ligand for erbB3 and erbB4 receptors, stimulated tyrosine phosphorylation of the erbB receptors, which was accompanied by activation of Erk1 and Erk2, two mitogen-activated protein kinases with a functional role in mitogenesis. Importantly, HRG increased cell proliferation in COV434 cells, and treatment with HRG/PE40, a ligand toxin shown previously to be cytotoxic against human breast cancer cells overexpressing erbB receptors, led to a dramatic and irreversible decrease in cell number. These results indicate that erbB receptor signaling pathways may be critical in the control of GC tumor cell proliferation and that HRG/PE40 is a potential therapeutic agent for the treatment of GC tumors.

Glucocorticoids stimulate foetal surfactant synthesis. Therefore, they are used in impending pre-term birth. One mechanism of action on surfactant synthesis is through the induction of neuregulin (NRG) secretion by foetal lung fibroblasts. The direct effects on signalling pathways, and specifically on erbB receptors in foetal type II cell surfactant synthesis, are less well understood. The present authors studied the effect of known promoters of foetal surfactant synthesis (namely dexamethasone and mature (i.e. NRG-containing) fibroblast-conditioned medium (FCM)) on erbB receptor activation, protein content and dimerisation patterns in foetal mouse lung type II cells. Dexamethasone inhibited surfactant synthesis in immature type II cells at day (d)16 of gestation, while the mature FCM had stimulatory effects. Both treatments directly stimulated surfactant synthesis in more mature (d17) cells. At this gestational day, dexamethasone had only a small effect on phosphorylation, but it stimulated the protein levels of all four erbB receptors. Dexamethasone effects were distinct from those of mature FCM, which stimulated both protein content and phosphorylation of all erbB receptors and of the signalling intermediate phospholipase Cgamma. Dexamethasone modulated erbB receptor dimerisation patterns, such that erbB2 became the main dimerisation partner for erbB4. In conclusion, dexamethasone signalling involves erbB receptors in foetal type II cells, in a manner similar to, but distinct from, neuregulin-containing fibroblast-conditioned medium signalling.

The growth factor heregulin (HRG), a ligand of ErbB3 and ErbB4 receptors, contributes to breast cancer development and the promotion of metastatic disease, and its expression in breast tumors has been associated with poor clinical outcome and resistance to therapy. In this study, we found that breast cancer cells exposed to sustained HRG treatment show markedly enhanced Rac1 activation and migratory activity in response to the CXCR4 ligand SDF-1/CXCL12, effects mediated by P-Rex1, a Rac-guanine nucleotide exchange factor (GEF) aberrantly expressed in breast cancer. Notably, HRG treatment upregulates surface expression levels of CXCR4, a G protein-coupled receptor (GPCR) implicated in breast cancer metastasis and an indicator of poor prognosis in breast cancer patients. A detailed mechanistic analysis revealed that CXCR4 upregulation and sensitization of the Rac response/motility by HRG are mediated by the transcription factor hypoxia-inducible factor 1α (HIF-1α) via ErbB3 and independently of ErbB4. HRG caused prominent induction in the nuclear expression of HIF-1α, which transcriptionally activates the CXCR4 gene via binding to a responsive element located in positions -1376 to -1372 in the CXCR4 promoter, as revealed by mutagenesis analysis and chromatin immunoprecipitation (ChIP). Our results uncovered a novel function for ErbB3 in enhancing breast cancer cell motility and sensitization of the P-Rex1/Rac1 pathway through HIF-1α-mediated transcriptional induction of CXCR4.