An ERBB3/ERBB2 oncogenic unit plays a key role in NRG1 signaling and melanoma cell growth and survival

We recently identified neuregulin‐1 (NRG1) as a novel target of Notch1 required in Notch‐dependent melanoma growth. ERBB3 and ERBB4, tyrosine kinase receptors specifically activated by NRG1, have been shown to be either elevated in melanoma cell lines and tumors or to be mutated in 20% of melanomas, respectively. While these data support key roles of NRG1 and its receptors in the pathogenesis of melanoma, whether ERBB3 and ERBB4 display redundant or exclusive functions is not known. Here, we show that ERBB3 and ERBB4 inhibition results in distinct outcomes. ERBB3 inhibition ablates the cellular responses to NRG1, results in AKT inactivation and leads to cell growth arrest and apoptotic cell death. In contrast, ERBB4 knockdown mildly affects cell growth, has no effects on cell survival and, importantly, does not alter the responses to NRG1. Finally, we identified ERBB2 as a key coreceptor in NRG1‐dependent ERBB3 signaling. ERBB2 forms a complex with ERBB3, and its inhibition recapitulates the phenotypes observed upon ERBB3 ablation. We propose that an NRG1‐ERBB3‐ERBB2 signaling unit operates in melanoma cells where it promotes growth and survival.     

Expression analysis and molecular targeting of cyclin-dependent kinases in advanced melanoma: Functional analysis and molecular targeting of cyclin-dependent kinase family members in advanced melanoma

A major focus of melanoma research continues to be the search for genes/proteins that may be suitable targets for molecular therapy of primary and metastatic melanoma. In line with this effort, the objective of the study presented herein was to determine whether interfering with cell cycle progression and in particular, the expression and function of select cyclin-dependent kinases, would impair the biological features of advanced melanoma. We provide data, which document that unlike nevi and melanoma in situ, primary and metastatic melanomas express high levels of CDK2, CDK1, and CDK5. Furthermore, we present the results of in vitro and preclinical in vivo studies, which demonstrate that treatment with a small-molecule cyclin-dependent kinase inhibitor that selectively blocks the function of CDK2, CDK5, CDK1 and CDK9, leads not only to inhibition of melanoma cell proliferation and apoptosis of melanoma cells, but also impairs the growth of human melanoma xenografts.Key words: melanoma, cyclin-dependent kinase expression, small-molecule inhibitor treatment, proliferation, cell cycle progression, apoptosis, tumor xenograft studies     

Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies.     

Constitutive aberrant endogenous interleukin-1 facilitates inflammation and growth in human melanoma

Interleukin (IL)-1-mediated inflammation is proposed to contribute to the development and progression of some cancers. IL-1 family member proteins are known to be expressed constitutively in many melanoma tumor cells, and we hypothesize that these support molecular pathways of inflammation and facilitate tumor growth. To investigate the expression of IL-1α and IL-1β in melanoma patients, and their association with disease progression, immunohistochemical staining was carried out on tissues from 170 patients including benign nevi, primary melanomas, and metastatic melanomas. IL-1β levels were low (or zero) in benign nevi and higher in primary and metastatic melanomas (P < 0.0001). IL-1α was expressed in about 73% of nevi and 55% of metastatic melanomas, with levels significantly higher in primary tumors (P < 0.0001); most (98%) primary melanoma samples were positive for IL-1α. In vitro studies with seven human melanoma cell lines showed that five cell lines expressed IL-1α and IL-1β proteins and mRNA. We identified for the first time several important downstream signaling pathways affected by endogenous IL-1, including reactive oxygen and nitrogen species, COX-2, and phosphorylated NF-κB inhibitor (IκB) and stress-activated protein kinase/c-jun-NH(2)-kinase; all of which were decreased by siRNA to IL-1s. Downregulation of IL-1α, IL-1β, or MyD88 substantially increased p21 and p53 levels. Treatment with IL-1 receptor type I neutralizing antibody or IL-1 pathway-specific siRNAs led to growth arrest in IL-1-positive melanoma cells. Furthermore, blocking the IL-1 pathway increased autophagy in IL-1-positive melanoma cells. These results indicate that the endogenous IL-1 system is functional in most human melanoma and interrupting its signaling inhibits the growth of IL-1-positive melanoma cells.     

Expression analysis and molecular targeting of cyclin-dependent kinases in advanced melanoma

A major focus of melanoma research continues to be the search for genes/proteins that may be suitable targets for molecular therapy of primary and metastatic melanoma. In line with this effort, the objective of the study presented herein was to determine whether interfering with cell cycle progression and in particular, the expression and function of select cyclin-dependent kinases, would impair the biological features of advanced melanoma. We provide data, which document that unlike nevi and melanoma in situ, primary and metastatic melanomas express high levels of CDK2, CDK1, and CDK5. Furthermore, we present the results of in vitro and preclinical in vivo studies, which demonstrate that treatment with a small-molecule cyclin-dependent kinase inhibitor that selectively blocks the function of CDK2, CDK5, CDK1, and CDK9, leads not only to inhibition of melanoma cell proliferation and apoptosis of melanoma cells, but also impairs the growth of human melanoma xenografts.     

Decreased expression of Apaf-1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease-activating factor-1 (Apaf-1) is a cell death effector that acts with cytochrome c and caspase-9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf-1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf-1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf-1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf-1 expression was observed when comparing nevi and melanomas (chi-square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf-1 (chi-square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf-1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf-1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf-1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

Decreased expression of Apaf‐1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease‐activating factor‐1 (Apaf‐1) is a cell death effector that acts with cytochrome c and caspase‐9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf‐1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf‐1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf‐1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf‐1 expression was observed when comparing nevi and melanomas (chi‐square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf‐1 (chi‐square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf‐1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf‐1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf‐1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

Neuregulin Mediates F-actin-driven Cell Migration through Inhibition of Protein Kinase D1 via Rac1 Protein

Neuregulin (NRG; heregulin) is overexpressed in ∼30% of breast cancers and mediates various processes involved in tumor progression, including tumor cell migration and invasion. Here, we show that NRG mediates its effects on tumor cell migration via PKD1. Downstream of RhoA, PKD1 can prevent directed cell migration through phosphorylation of its substrate SSH1L. NRG exerts its inhibitory effects on PKD1 through Rac1/NADPH oxidase, leading to decreased PKD1 activation loop phosphorylation and decreased activity toward SSH1L. The consequence of PKD1 inhibition by NRG is decreased binding of 14-3-3 to SSH1L, localization of SSH1L to F-actin at the leading edge, and increased cofilin activity, resulting in increased reorganization of the actin cytoskeleton and cell motility. Our data provide a mechanism through which the Rho GTPase Rac1 cross-talks with PKD1 signaling pathways to facilitate directed cell migration.     

Association of NRG3 and ERBB4 gene polymorphism with nicotine dependence in Turkish population

Background

Nicotine dependence (ND) is characterized by regular smoking, anxiety, irritation, difficulty concentrating, impatience, restlessness, tremor, dizziness, hunger, nicotine demand, and the individual’s reluctance to quit despite knowing the health risks of smoking. Recently, it has been reported that the Neuregulin 3 (NRG3)/Erb-B2 receptor tyrosine kinase 4 (ERBB4) signaling pathway plays a role in ND. NRG3, which is activated after nicotine intake, binds to ERBB4 and causes GABA release. GABA reduces anxiety and tension, which are one of the nicotine withdrawal symptoms. Therefore we aimed to investigate the relationship between NRG3 and ERBB4 gene polymorphisms and ND.

Materials and methods

The study population was comprised of patients with ND (n?=?200) and healthy non-smoker control subjects (n?=?200) who were matched for age, sex, and compared for comorbidity factors such as alcohol, smoking, duration, and education (age range 18–60). Genotypes were detected by Real-Time PCR using TaqMan technology. The Fagerström Nicotine Dependence Test (FTND) score was 5 and above for the patient group and 0 for the control group. DNA was obtained from whole peripheral blood and six polymorphisms of Neuregulin 3 (NRG3) (rs1836724, rs7562566, and rs10048757) and Erb-B2 Receptor Tyrosine Kinase 4 (ERBB4) (rs1764072, rs6584400, and rs10883934) genes were analyzed by real-time PCR method.

Results

Our findings show that the six selected SNPs are not significantly associated with ND in the Turkish population and no correlation with dependence levels (p?>?0.05).

Conclusion

Although our findings do not show a relationship between ND and these polymorphisms, it is the first study to investigate these single nucleotide polymorphisms (SNPs) for the first time in ND and to find some genotypes in the Turkish population when compared to other populations. Also, our findings are important in terms of their contribution to the literature and forensic genetics.

     

Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal-regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with alpha4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.     

Macroscopic spectral imaging and gene expression analysis of the early stages of melanoma

BACKGROUND: The stages of melanocytic progression are defined as atypical (dysplastic) nevus, melanoma in situ, melanoma in the radial growth phase (RGP), melanoma in the vertical growth phase (VGP), and melanoma in the metastatic growth phase (MGP). Melanoma in situ and RGP melanoma often develop in contiguous association with atypical nevi. This frequently poses a problem with respect to their early detection. Furthermore, unlike cells obtained from VGP and MGP melanomas, cells derived from melanoma in situ and RGP melanoma do not proliferate in vitro. Thus, compared to the late stages of the disease, less information is available regarding genes expressed in the early stages. MATERIALS AND METHODS: To determine whether spectral imaging, a recently developed optical imaging technique, can detect melanoma in situ and RGP melanoma arising in melanoma precursor lesions, atypical nevi in patients with a clinical history of melanoma were subjected to noninvasive macroscopic spectral imaging. To determine at what stage in the progression pathway of melanoma genes having important biological functions in VGP and MGP melanomas are activated and expressed, lesions of melanoma in situ were analyzed by immunohistochemistry and in situ hybridization for expression of some of these known molecular and immunologic markers. RESULTS: The present study demonstrates the capability of noninvasive spectral imaging to detect melanoma in situ and RGP melanoma that arise in contiguous association with atypical nevi. Furthermore, the study provides evidence that genes and antigens expressed in VGP and MGP melanoma are also expressed in melanoma in situ. CONCLUSIONS: Because of the dark and variegated pigmentation of atypical nevi, melanoma in situ and RGP melanoma that arise in these melanoma precursor lesions are often difficult to recognize and thus frequently go unnoticed. The application of new optical screening techniques for early detection of melanoma and the identification of genes expressed in the early stages of melanoma development are two important avenues in the pursuit of melanoma prevention. The investigations presented here document that macroscopic spectral imaging has the potential to detect melanoma in its early stage of development and that genes essential for the proliferation and cell adhesion of VGP and MGP melanoma are already expressed in melanoma in situ.     

Down-Regulation of Neuregulin1/ErbB4 Signaling in the Hippocampus Is Critical for Learning and Memory

Hippocampal function is important for learning and memory, and dysfunction of the hippocampus has been linked to the pathophysiology of neuropsychiatric diseases such as schizophrenia. Neuregulin1 (NRG1) and ErbB4, two susceptibility genes for schizophrenia, reportedly modulate long-term potentiation (LTP) at hippocampal Schaffer collateral (SC)-CA1 synapses. However, little is known regarding the contribution of hippocampal NRG1/ErbB4 signaling to learning and memory function. Here, quantitative real-time PCR and Western blotting were used to assess the mRNA and protein levels of NRG1 and ErbB4. Pharmacological and genetic approaches were used to manipulate NRG1/ErbB4 signaling, following which learning and memory behaviors were evaluated using the Morris water maze, Y-maze test, and the novel object recognition test. Spatial learning was found to reduce hippocampal NRG1 and ErbB4 expression. The blockade of NRG1/ErbB4 signaling in hippocampal CA1, either by neutralizing endogenous NRG1 or inhibiting/ablating ErbB4 receptor activity, enhanced hippocampus-dependent spatial learning, spatial working memory, and novel object recognition memory. Accordingly, administration of exogenous NRG1 impaired those functions. More importantly, the specific ablation of ErbB4 in parvalbumin interneurons also improved learning and memory performance. The manipulation of NRG1/ErbB4 signaling in the present study revealed that NRG1/ErbB4 activity in the hippocampus is critical for learning and memory. These findings might provide novel insights on the pathophysiological mechanisms of schizophrenia and a new target for the treatment of Alzheimer’s disease, which is characterized by a progressive decline in cognitive function.     

The mucin Muc4 potentiates neuregulin signaling by increasing the cell-surface populations of ErbB2 and ErbB3

Mucins provide a protective barrier for epithelial surfaces, and their overexpression in tumors has been implicated in malignancy. We have previously demonstrated that Muc4, a transmembrane mucin that promotes tumor growth and metastasis, physically interacts with the ErbB2 receptor tyrosine kinase and augments receptor tyrosine phosphorylation in response to the neuregulin-1beta (NRG1beta) growth factor. In the present study we demonstrate that Muc4 expression in A375 human melanoma cells, as well as MCF7 and T47D human breast cancer cells, enhances NRG1beta signaling through the phosphatidylinositol 3-kinase pathway. In examining the mechanism underlying Muc4-potentiated ErbB2 signaling, we found that Muc4 expression markedly augments NRG1beta binding to A375 cells without altering the total quantity of receptors expressed by the cells. Cell-surface protein biotinylation experiments and immunofluorescence studies suggest that Muc4 induces the relocalization of the ErbB2 and ErbB3 receptors from intracellular compartments to the plasma membrane. Moreover, Muc4 interferes with the accumulation of surface receptors within internal compartments following NRG1beta treatment by suppressing the efficiency of receptor internalization. These observations suggest that transmembrane mucins can modulate receptor tyrosine kinase signaling by influencing receptor localization and trafficking and contribute to our understanding of the mechanisms by which mucins contribute to tumor growth and progression.     

Identification and cloning of genes displaying elevated expression as a consequence of metastatic progression in human melanoma cells by rapid subtraction hybridization

Although extensively investigated, the complete repertoire of genes associated with and causative of metastasis remain largely unknown. We developed an efficient approach for identifying differentially expressed genes that involves rapid subtraction hybridization (RaSH) of cDNA clones prepared from two cell populations, a driver and a tester. This RaSH approach has previously documented high sensitivity and effectiveness in identifying genes that are differentially expressed as a function of induction of terminal differentiation in human melanoma cells, resistance or sensitivity to human immunodeficiency virus-1 (HIV-1) infection of human T cells and perturbation in gene expression in normal human fetal astrocytes infected with HIV-1 or treated with HIV-1 gp120 viral envelope glycoprotein or tumor necrosis factor-alpha (TNF-alpha). In the present study, RaSH has been applied to a metastatic melanoma model, which mimics the early events of metastasis in humans, comprising weakly metastatic vs. immunosuppressed newborn rat-selected highly metastatic variants. This has now resulted in the identification of eight genes displaying elevated expression in the high metastatic variants vs. normal immortal melanocytes or weakly metastatic parental clones. These include six known genes, 67-kDa laminin receptor (67LR), endothelin receptor B (ENDRB), Na+/K+-ATPase, Ku antigen, interleukin-receptor-associated kinase-1 (IRAK-1) and ribosomal protein RPLA, which may contribute to the complex process of melanoma metastasis. Additionally, two unknown genes (not reported in current databases) that may also impact on the metastatic phenotype have also been identified. These studies provide additional support of the use of the RaSH approach, in this application in the context of closely related variant cell lines with different metastatic potential, for effective differential gene identification and elucidate eight previously unrecognized genes whose role in melanoma progression to metastatic competence can now be scrutinized.     

Schizophrenia susceptibility pathway neuregulin 1-ErbB4 suppresses Src upregulation of NMDA receptors

Hypofunction of the N-methyl D-aspartate subtype of glutamate receptor (NMDAR) is hypothesized to be a mechanism underlying cognitive dysfunction in individuals with schizophrenia. For the schizophrenia-linked genes NRG1 and ERBB4, NMDAR hypofunction is thus considered a key detrimental consequence of the excessive NRG1-ErbB4 signaling found in people with schizophrenia. However, we show here that neuregulin 1β-ErbB4 (NRG1β-ErbB4) signaling does not cause general hypofunction of NMDARs. Rather, we find that, in the hippocampus and prefrontal cortex, NRG1β-ErbB4 signaling suppresses the enhancement of synaptic NMDAR currents by the nonreceptor tyrosine kinase Src. NRG1β-ErbB4 signaling prevented induction of long-term potentiation at hippocampal Schaffer collateral-CA1 synapses and suppressed Src-dependent enhancement of NMDAR responses during theta-burst stimulation. Moreover, NRG1β-ErbB4 signaling prevented theta burst-induced phosphorylation of GluN2B by inhibiting Src kinase activity. We propose that NRG1-ErbB4 signaling participates in cognitive dysfunction in schizophrenia by aberrantly suppressing Src-mediated enhancement of synaptic NMDAR function.