Induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA in mouse skin organ culture was blocked by two pan-ErbB receptor tyrosine kinase (RTK) inhibitors but not by genetic ablation of ErbB1, suggesting involvement of multiple ErbB species in skin physiology. Human skin, cultured normal keratinocytes, and A431 skin carcinoma cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4. Skin and A431 cells expressed more ErbB3 than did keratinocytes. Despite strong expression of ErbB2 and ErbB3, heregulin was inactive in stimulating tyrosine phosphorylation in A431 cells. In contrast, it was highly active in MDA-MB-453 breast carcinoma cells. ErbB2 displayed punctate cytoplasmic staining in A431 and keratinocytes, compared to strong cell surface staining in MDA-MB-453. In skin, ErbB2 was cytoplasmic in basal keratinocytes, assuming a cell surface pattern in the upper suprabasal layers. In contrast, ErbB1 retained a cell surface distribution in all epidermal layers. Keratinocyte proliferation in culture was found to be ErbB1-RTK-dependent, using a selective inhibitor. These results suggest that in skin keratinocytes, ErbB2 transduces ligand-dependent differentiation signals, whereas ErbB1 transduces ligand-dependent proliferation/survival signals. Intracellular sequestration of ErbB2 may contribute to the malignant phenotype of A431 cells, by allowing them to respond to ErbB1-dependent growth/survival signals, while evading ErbB2-dependent differentiation signals.

ErbB4, a type I transmembrane receptor tyrosine kinase, is a member of the epidermal growth factor receptor family. Its cleavage releases an intracellular C-terminal domain (ICD), which can be either degraded following ubiqitination or translocated to the nucleus and regulate gene expression. There are 2 ErbB4 ICD isoforms: CYT-1 and CYT-2. We and others have previously reported that following cleavage, CYT-2 selectively translocates to the nucleus. In the current study we found that following cleavage, the intracellular levels of CYT-1 ICD decreased rapidly, while levels of CYT-2 ICD remained relatively stable. CYT-1 ICD degradation could be prevented by administration of either the proteasome inhibitor lactacystin or the lysosome inhibitor chloroquine, indicating both proteasomal and lysosomal degradation. Further studies implicated Nedd4, an E3 ubiquitin ligase, as a mediator of CYT-1 ubiquitination and degradation. The interaction of Nedd4 with CYT-1 was shown by coimmnunoprecipitation, an in vitro direct binding assay, and an in vitro ubiquitination assay. Three PPxY or PY motifs present in the CYT-1 C terminus are necessary for binding by Nedd4 WW domains, because impaired interactions are seen in mutation of any of the PY motifs. Nedd4-CYT-1 binding was associated with increased CYT-1 ubiquitination following proteasome inhibitor treatment. Impaired Nedd4 binding to CYT-1 by PY motif mutations led to increased CYT-1 ICD stability, whereas only one of the PY motif mutations (Y1056A), which disrupts the binding sites for both a WW domain and an SH2 domain of PI3 kinase, demonstrated enhanced nuclear translocation following HB-EGF treatment. These studies indicate that Nedd4 mediates ErbB4 CYT-1 ICD ubiquitination and degradation, and the prevention of both WW binding and PI3 kinase activity are required for ErbB4 nuclear translocation.

OBJECTIVE

Patients with recurrent prostate cancer are commonly treated with androgen withdrawal therapy (AWT); however, almost all patients eventually progress to castration resistant prostate cancer (CRPC), indicating failure of AWT to eliminate androgen-sensitive prostate cancer. The overall goal of these studies is to determine whether dual inhibition of the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and HER2 would prolong the effectiveness of this treatment in prostate cancer.

METHODS

We used androgen-dependent LNCaP cells and its CRPC sublines LNCaP-AI and C4-2. Additional data were collected in pRNS-1-1 cells stably expressing a mutant androgen receptor (AR-T877A), and in nude mice harboring CWR22 tumors. Studies utilized EGFR inhibitors erlotinib and AG1478, and HER2 inhibitors trastuzumab and AG879.

RESULTS

Dual EGFR/HER2 inhibition induced apoptosis selectively in androgen-sensitive prostate cancer cells undergoing AWT, but not in the presence of androgens, or in CRPC cells. We show that AWT alone failed to induce significant apoptosis in androgen-dependent cells, due to AWT-induced increase in HER2 and ErbB3, which promoted survival by increasing Akt phosphorylation. AWT-induced ErbB3 stabilized the AR and stimulated PSA, while it was inactivated only by inhibition of both its dimerization partners EGFR and HER2 (prostate cancer cells do not express ErbB4); but not the inhibition of any one receptor alone, explaining the success of dual EGFR/HER2 inhibition in sensitizing androgen-dependent cells to AWT. The effectiveness of the inhibitors in suppressing growth correlated with its ability to prevent Akt phosphorylation.

CONCLUSIONS

These studies indicate that dual EGFR/HER2 inhibition, administered together with AWT, sensitize prostate cancer cells to apoptosis during AWT.

The neuregulins comprise a subfamily of epidermal growth factor (EGF)-like growth factors that elicit diverse cellular responses by activating members of the ErbB family of receptor tyrosine kinases. Although neuregulin-1 and neuregulin-2 are both binding ligands for the ErbB3 and ErbB4 receptors, they exhibit distinct biological activities depending on cellular context. In MDA-MB-468 human mammary tumor cells, neuregulin-2beta (NRG2beta) inhibits cell growth, whereas neuregulin-1beta (NRG1beta) does not. In these cells, NRG2beta appears to preferentially act through the EGF receptor, stimulating receptor tyrosine phosphorylation and the recruitment of phospholipase C-gamma, Cbl, SHP2, and Shc to that receptor. NRG1beta preferentially acts through ErbB3 in these cells by stimulating the tyrosine phosphorylation and recruitment of phosphatidylinositol 3-kinase and Shc to that receptor. In MDA-MB-453 cells, both NRG1beta and NRG2beta stimulate the tyrosine phosphorylation of the ErbB2 and ErbB3 receptors to similar extents, but only NRG1beta potently stimulates morphological changes consistent with their differentiation. The profiles of SH2 domain-containing proteins that are efficiently recruited to activated receptors differ for the two factors. These observations indicate that despite their overlapping receptor specificity, the neuregulins exhibit distinct biological and biochemical properties. Since both of these cell lines express only two of the known ErbB receptors, our results imply that EGF-like ligands might elicit differential signaling within the context of a single receptor heterodimer.

Protease-mediated signaling is an important modulator of the nervous system. However, identifying the specific signaling substrates of such proteases is limited by the rapidity with which intermediate substrate forms are cleaved and released. Here, a screening method to detect noncleaved enzyme-bound forms was developed and used to identify a novel neuropsin/neuregulin-1 (NRG-1) proteolytic signaling system, which is specifically localized in the microdomain of synaptic cleft, in the mouse hippocampus. The extracellular protease, neuropsin, cleaved mature NRG-1 (comprising the extracellular domain of the NRG-1) at three newly identified sites to remove the heparin-binding domain of NRG-1. This released the ligand moiety from the matrix-glycosaminoglycan pool and enabled it to trigger the phosphorylation of NRG-1 receptor, p185 (ErbB4). Proteolysis of mature NRG-1 by neuropsin led to colocalization of the processed NRG-1 with ErbB4 in parvalbumin-positive hippocampal interneurons and consequent phosphorylation of tyrosine residues of proteins in the cells. Moreover, neuropsin knock-out mice exhibited impairments in Schaffer collateral early phase long-term potentiation, and application of the recombinant NRG-1 lacking heparin-binding activity reversed the effects through the activation of ErbB4 and GABA(A) receptors. Thus, ErbB4 signaling induced by neuropsin-dependent processing of NRG-1 contributes to the modulation of synaptic plasticity via regulation of GABAergic transmission. This signaling system may be involved in human cognition and mental disorders, such as schizophrenia and bipolar disorder, by its dysfunction.

The topological relationships between erbB receptors and ligands of the epidermal growth factor family were characterized by immunocytochemistry in normal and psoriatic epidermis and in proliferating and differentiating human keratinocytes in culture. Spatial colocalization of receptors and ligands was assessed by dual immunostaining. Expression of epidermal growth factor receptor (EGFr), erbB2, and erbB3, but not erbB4, was detected throughout the epidermis, although labeling for erbB2 and erbB3 was accentuated in the upper spinous layers, and EGFr was more strongly labeled in basal cells. Of the tested growth factors, heparin-binding epidermal growth factor (HB-EGF) was diffusely expressed throughout normal and psoriatic epidermis and sparsely colocalized with EGFr in all viable epidermal layers, with increased colocalization in psoriatic epidermis. In contrast, betacellulin and heregulin/neu differentiation factor (NDF) alpha were largely restricted in their distribution to the upper spinous and granular layers. Betacellulin was downregulated in psoriatic keratinocytes. Although heregulin/NDF-beta was undetectable in normal epidermis, it was upregulated in psoriasis. Betacellulin and heregulin/NDF-alpha strikingly colocalized with EGFr and erbB3 receptors in the granular layer and in a declining gradient from the granular zone to the basal layer, respectively. Similar patterns were observed in cultured keratinocytes under proliferative conditions and upon differentiation in high-calcium medium. These morphological data collectively suggest divergent functions for members of the growth factor family, and in particular, we propose that betacellulin and heregulin/NDF-alpha are involved in epidermal morphogenesis and/or in maintenance of the differentiated phenotype.

HER-2 (also called ErbB2 or Neu) tyrosine kinase, one of the four members of ErbB receptor family (ErbB1, i.e., EGFR ErbB2, ErbB3 and ErbB4), plays a critical role in the control of diverse cellular functions involved in differentiation, proliferation, migration and cell survival via multiple signal transduction pathways. Overexpression of HER-2, observed in HER-2-positive breast cancer patients, is believed to cause the tumor resistance to an array of anti-cancer agents and poor prognosis. Although HER-2 antibodies have shown growth inhibitory effects, more efficient molecular targets against HER-2-mediated tumor resistance need to be developed. The molecular mechanisms underlying HER-2-mediated tumor resistance, especially the connections between HER-2 and therapy-resistant signaling networks, need to be further investigated. NF-kappaB, a key stress transcription factor that can initiate a pro-survival network, was found to be activated in many cancer cells overexpressing HER-2 and to be responsible for the radiation resistance in HER-2 transfected breast cancer cells. Recent findings in literature and data from this laboratory suggest a possible co-operation between HER-2 and NF-KB in signaling tumor resistance to radiotherapy. This review will discuss the mechanisms of HER-2 mediated NF-kappaB signaling pathway and potential target for therapeutic intervention.

To investigate the expression of human papillomavirus type 16 (HPV-16) E5 protein in squamous neoplastic changes in the uterine cervix, the specific E5 antibody was generated and used to identify the expression of E5 protein in 40 cases of HPV-16-positive tissues and 5 previously identified HPV-negative normal cervical tissues. The results revealed that E5 protein was primarily expressed in the lower third of the epithelium in low-grade squamous intraepithelial lesions (SILs) and throughout the whole epithelium in high-grade SILs. In invasive squamous carcinoma, 60% of HPV-16-infected cancers which contained the episomal viral genome had the E5 gene, and could express E5 protein which was located throughout the whole epithelium. Previously, we documented the expression of type I growth factor receptors [ERBB1/EGFR (epidermal growth factor receptor), ERBB2, ERBB3 and ERBB4] in the full range of cervical neoplasias by immunohistochemistry assay. Hence, in this study, we extensively analyzed the correlation between the expression of E5 protein and the expression of type I growth factor receptors. Among 40 HPV-16- infected cervical neoplasias, we found that the expression of E5 protein was significantly correlated with either the expression of the ERBB1 or the ERBB4 receptor.

Sporadic and neurofibromatosis type 2-associated schwannomas contain a glial growth factor (GGF)-like activity that has been hypothesized to promote neoplastic Schwann cell mitogenesis. It is not known whether this GGF-like activity is neuregulin-1 (NRG-1), an epidermal growth factor (EGF)-related molecule that regulates the proliferation, survival, and differentiation of developing Schwann cells, the related factor NRG-2, or another NRG/EGF ligand. We report that neoplastic Schwann cells within schwannomas overexpress multiple alpha and beta transmembrane precursors from the class II and class III NRG-1 subfamilies. NRG-2 alpha and beta transcripts are similarly overexpressed in some tumors. Of the other 8 known NRG/EGF ligands, only heparin-binding EGF, epiregulin, and TGFalpha are detectable in schwannomas. Neoplastic Schwann cells almost uniformly express erbB2 and erbB3, 2 membrane receptor tyrosine kinases mediating NRG-1 and NRG-2 action. Expression of the NRG receptor erbB4 and EGF receptor is also evident in schwannomas, but is more limited, occurring in only a subset of these tumors. ErbB2, the preferred dimerization partner for all erbB kinases, is constitutively phosphorylated in schwannomas. These observations suggest that autocrine, paracrine, and/or juxtacrine NRG-1/NRG-2 signaling promotes schwannoma pathogenesis and that this signaling pathway may be an important therapeutic target in schwannomas.

The expression of all four ErbB receptors was compared by immunohistochemistry, using receptor-specific polyclonal antisera, in 32 invasive, 11 in situ carcinomas, six benign lesions, and 22 samples of histologically normal mucosa adjacent to specimens of carcinoma originating from oral cavity epithelium. Among invasive and in situ carcinoma, EGFR expression was the most prevalent (in 29/32 and 8/11 cases, respectively) followed by ErbB2 (17/32 and 2/11) and ErbB4 (9/32 and 1/10), while ErbB3 was only detected in invasive tumours (12/32). Specific patterns included invasive tumours with expression of EGFR (8/32) or ErbB4 (1/32) alone, as well as different receptor combinations (EGFR+ErbB2, EGFR+ErbB4, EGFR+ErbB2+ErbB3, EGFR+ErbB2+ErbB4, and all four receptors). Simultaneous expression of three or four ErbB receptors correlated with tumour invasion (p=2.2x10(-4)) and localized in the intermediate epithelial cell layer of well and moderately differentiated tumours. No other significant correlation with clinico-pathological features was noticed. Some benign lesions and histologically normal mucosa adjacent to carcinomas showed weak immunostaining of EGFR (10/28), ErbB2 (4/28) or ErbB4 (3/28). By comparison, overexpression, as indicated by increased staining intensity, was observed in invasive tumours for EGFR (18/32), ErbB2 (8/32), ErbB4 (3/32), and ErbB3 (3/32). Statistical evaluation demonstrated a significant association of EGFR or ErbB2 overexpression with invasive carcinoma when compared with benign lesions and apparently normal epithelium (p=5.2x10(-7) and p=5x10(-3), respectively). Tumour-specific overexpression of ErbB receptors and their co-expression, most frequently involving EGFR and ErbB2, in the same cell layer of neoplastic epithelium, implicate receptor heterodimers in the pathogenesis of oral squamous carcinoma.

Cognitive deficits in individuals with schizophrenia (SCZ) are considered core symptoms of this disorder, and can manifest at the prodromal stage. Antipsychotics ameliorate positive symptoms but only modestly improve cognitive symptoms. The lack of treatments that improve cognitive abilities currently represents a major obstacle in developing more effective therapeutic strategies for this debilitating disorder. While D4 receptor (D4R)-specific antagonists are ineffective in the treatment of positive symptoms, animal studies suggest that D4R drugs can improve cognitive deficits. Moreover, recent work from our group suggests that D4Rs synergize with the neuregulin/ErbB4 signaling pathway, genetically identified as risk factors for SCZ, in parvalbumin (PV)-expressing interneurons to modulate gamma oscillations. These high-frequency network oscillations correlate with attention and increase during cognitive tasks in healthy subjects, and this correlation is attenuated in affected individuals. This finding, along with other observations indicating impaired GABAergic function, has led to the idea that abnormal neural activity in the prefrontal cortex (PFC) in individuals with SCZ reflects a perturbation in the balance of excitation and inhibition. Here we review the current state of knowledge of D4R functions in the PFC and hippocampus, two major brain areas implicated in SCZ. Special emphasis is given to studies focusing on the potential role of D4Rs in modulating GABAergic transmission and to an emerging concept of a close synergistic relationship between dopamine/D4R and neuregulin/ErbB4 signaling pathways that tunes the activity of PV interneurons to regulate gamma frequency network oscillations and potentially cognitive processes.

Inhibitory neurotransmission in amygdala is important for fear learning and memory. However, mechanisms that control the inhibitory activity in amygdala are not well understood. We provide evidence that neuregulin 1 (NRG1) and its receptor ErbB4 tyrosine kinase are critical for maintaining GABAergic activity in amygdala. Neutralizing endogenous NRG1, inhibition, or genetic ablation of ErbB4, which was expressed in a majority of palvalbumin (PV)+ neurons in amygdala, reduced GABAergic transmission and inhibited tone-cued fear conditioning. Specific ablation of ErbB4 in PV+ neurons reduced eIPSC/eEPSC ratios and impaired fear conditioning. Notably, expression of ErbB4 in amygdala was sufficient to diminish synaptic dysfunction and fear conditioning deficits in PV-ErbB4-/- mice. These observations indicated that NRG1 signaling maintains high GABAergic activity in amygdala and, thus, regulates fear memory. Considering that both NRG1 and ErbB4 are susceptibility genes of schizophrenia, our study sheds light on potential pathophysiological mechanisms of this disorder.

Approximately 25% of breast cancers overexpress and depend on the receptor tyrosine kinase ERBB2, one of 4 ERBB family members. Targeted therapies directed against ERBB2 have been developed and used clinically, but many patients continue to develop resistance to such therapies. Although much effort has been focused on elucidating the mechanisms of acquired resistance to ERBB2-targeted therapies, the involvement of ERBB4 remains elusive and controversial. We demonstrate that genetic ablation of ERBB4, but not ERBB1-3, led to apoptosis in lapatinib-resistant cells, suggesting that the efficacy of pan-ERBB inhibitors was, at least in part, mediated by the inhibition of ERBB4. Moreover, ERBB4 was upregulated at the protein level in ERBB2+ breast cancer cell lines selected for acquired lapatinib resistance in vitro and in MMTV-Neu mice following prolonged lapatinib treatment. Knockdown of ERBB4 caused a decrease in AKT phosphorylation in resistant cells but not in sensitive cells, suggesting that ERBB4 activated the PI3K/AKT pathway in lapatinib-resistant cells. Importantly, ERBB4 knockdown triggered apoptosis not only in lapatinib-resistant cells but also in trastuzumab-resistant cells. Our results suggest that although ERBB4 is dispensable for naïve ERBB2+ breast cancer cells, it may play a key role in the survival of ERBB2+ cancer cells after they develop resistance to ERBB2 inhibitors, lapatinib and trastuzumab.

Epidermal growth factor receptor (EGFR) and its family members, ErbB2, ErbB3 and ErbB4, are receptor tyrosine kinases which send signals into the cell to regulate many critical processes including development, tissue homeostasis, and tumorigenesis. Central to the signaling of these receptors is their intracellular kinase domain, which is activated by ligand-induced dimerization of the receptor and phosphorylates several tyrosine residues in the C-terminal tail. The phosphorylated tail then recruits other signaling molecules and relays the signal to downstream pathways. A model of the autoinhibition, activation and feedback inhibition mechanisms for the ErbB kinase domain has emerged from a number of recent structural studies. Meanwhile, recent clinical studies have revealed the relationship between specific ErbB kinase mutations and the responsiveness to kinase inhibitor drugs. We will review these regulation mechanisms of the ErbB kinase domain, and discuss the binding specificity of kinase inhibitors and the effects of kinase domain mutations found in cancer patients from a structural perspective.

Both epidemiological and experimental studies indicate that ethanol is a tumor promoter and may promote metastasis of breast cancer. However, the molecular mechanisms underlying ethanol-mediated tumor promotion remain unknown. Overexpression of ErbB proteins in breast cancer patients is generally associated with poor prognosis. The ErbB proteins are a family of receptor kinases that include four closely related members: epidermal growth factor receptor (EGFR/ErbB1), ErbB2/neu, ErbB3, and ErbB4. Particularly, ErbB2 plays a pivotal role in ErbB-mediated activities. Here we demonstrated that amplification of ErbB2 expression sensitized a specific cellular response to ethanol. Human breast cancer cells or mammary epithelial cells with a high expression of ErbB2 exhibited an enhanced response to ethanol-stimulated cell invasion in vitro. Ethanol also stimulated cell proliferation; however, this stimulation was independent of ErbB2 levels. Ethanol triggered divergent intracellular signaling among cells expressing different ErbB2 levels. In the cells overexpressing ErbB2, ethanol was more effective in the activation of c-Jun NH2 terminal protein kinases (JNKs) and p38 mitogen-activated protein kinase (p38 MAPK) as well as the induction of reactive oxygen species (ROS) than the cells with normal ErbB2 expression. Blockage of either JNKs or p38 MAPK activation eliminated ethanol-mediated cell invasion. In contrast, the reduction of hydrogen peroxide concentration by catalase exposure had little effect on ethanol-induced cell invasion. These results indicated that ethanol-induced cell invasion was primarily mediated by JNKs and p38 MAPK, whereas the involvement of ROS formation might be minimal. Our study suggests that overexpression of ErbB2 may augment ethanol-elicited signaling and promote ethanol-stimulated tumor metastasis.

Neuregulin-1 (Nrg1) is genetically linked to schizophrenia, a disease caused by neurodevelopmental imbalance in dopaminergic function. The Nrg1 receptor ErbB4 is abundantly expressed on midbrain dopaminergic neurons. Nrg1 has been shown to penetrate blood-brain barrier, and peripherally administered Nrg1 activates ErbB4 and leads to a persistent hyperdopaminergic state in neonatal mice. These data prompted us to study the effect of peripheral administration of Nrg1 in the context of Parkinson's disease, a neurodegenerative disorder affecting the dopaminergic system in the adult brain. We observed that systemic injections of the extracellular domain of Nrg1β(1) (Nrg1β(1)-ECD) increased dopamine levels in the substantia nigra and striatum of adult mice. Nrg1β(1)-ECD injections also significantly protected the mouse nigrostriatal dopaminergic system morphologically and functionally against 6-hydroxydopamine-induced toxicity in vivo. Moreover, Nrg1β(1)-ECD also protected human dopaminergic neurons in vitro against 6-hydroxydopamine. In conclusion, we have identified Nrg1β(1)-ECD as a neurotrophic factor for adult mouse and human midbrain dopaminergic neurons with peripheral administratability, warranting further investigation as therapeutic option for Parkinson's disease patients.

The resorption, formation and maintenance of bone are coordinated by the action of several hormones, growth factors and transcription factors. Recent experiments based on genetically modified mouse models, gene microarrays and pharmacological intervention indicate that the epidermal growth factor receptor (EGFR) system plays important roles in skeletal biology and pathology. This network, including a family of seven growth factors - the EGFR ligands - and the related tyrosine kinase receptors EGFR (ERBB1), ERBB2, ERBB3 and ERBB4, regulates aspects such as proliferation and differentiation of osteoblasts, chondrocytes and osteoclasts, parathyroid hormone-mediated bone formation and cancer metastases in bone. Here, we summarize and discuss the role of the EGFR and its ligands in skeletal biology and pathology.

Presenilins (PS) provide the catalytic activity for gamma-secretase, which cleaves physiologically relevant substrates including Notch, ErbB4, and APP. Recent genetic studies indicated that the contribution of PS1 to mouse development includes gamma-secretase-independent functions that cannot be easily explained by any of the demonstrated or hypothesized functions of this protein. To begin a nonbiased analysis of PS1 activity unencumbered by the dominant effect stemming from loss of Notch function, we characterized PS functions in the early land plant Physcomitrella patens, which lacks Notch, ErbB4, and APP. Removal of P. patens PS resulted in phenotypic abnormalities. Further assays performed to delineate the defective pathways in PS-deficient P. patens implicated improper function of the cytoskeletal network. Importantly, this characterization of a nonmetazoan PS uncovered a previously undescribed, evolutionarily conserved function (human PS1 can rescue the growth and light responses) that is gamma-secretase-independent (mutants with substitutions of the catalytic aspartyl residues retain the activity). Introduction of PpPS into PS-deficient mouse embryonic fibroblasts rescues normal growth rates, demonstrating that at least some metazoan functions of PS are evolutionarily conserved.

We recently reported a significant relationship between tumor cell expression of the ERBB4 receptor, the most recently described member of the epidermal growth factor receptor family, and aggressive tumor phenotype in childhood medulloblastoma. Two alternative juxtamembrane (JM) isoforms of the ERBB4 receptor have been described. Termed JMa and JMb, these variants possess different receptor processing and ligand-binding characteristics. In the current study, we employed an RT-PCR and sequencing strategy to determine the pattern of ERBB4 JM isoform expression in a large (n = 78) series of pediatric medulloblastomas. JMa and JMb transcript expression was detected in 53% and 28% of tumor samples, respectively. In addition, two novel ERBB4 JM isoforms, which we have termed JMc and JMd, were isolated from 10% and 36% of tumors, respectively. Sequence analysis revealed the JMc transcript to contain a deletion of the entire JM region. In contrast, JMd includes an extended coding region, retaining both the JMa and JMb sequences. Neither of these novel isoforms was detected in normal human adult cerebellum, but expression of JMd was observed in developing fetal cerebellum, suggesting that this later isoform may represent an ERBB4 transcript restricted to primitive neuroectoderm-derived tissue. To confirm that the four ERBB4 JM isoforms arise by alternative RNA splicing, we sequenced the intron-exon junctions of the human ERBB4 gene within the JM region. This demonstrated the four ERBB4 JM variants to be encoded by two short exons containing the JMb and JMa sequences positioned in the order 5' to 3' and separated by a 121 bp intron.

Neu differentiation factor (NDF)/heregulin activates ErbB2 via heterodimerization with the NDF receptors ErbB3 and ErbB4. Cells which express normal levels of these receptors are often growth stimulated by NDF, whereas SKBR3, and other ErbB2-overexpressing breast tumour cells are growth inhibited. We demonstrate here that in SKBR3 cells, NDF induces G1 progression but also causes a G2 delay from day 1 and apoptosis from days 2-3. G1 progression was associated with ErbB2 transactivation of ErbB3 and subsequent stimulation of the phosphatidylinositol 3-kinase (PI3K) pathway whereas apoptosis was dependent on p38 MAPK. Inhibition of ERK1/ERK2 had no effect on cell cycle progression or apoptosis. Activation of ErbB3 and PI3K was also seen with betacellulin (BTC) but not epidermal growth factor (EGF) and correlated with the growth effects of these ligands. All three ligands induced short-term activation of p38 MAPK in a c-Src-dependent manner. However, only NDF caused a second, c-Src-independent increase in p38 MAPK activity which was required for apoptosis.

Cancer is a heterogeneous disease often requiring a complexity of alterations to drive a normal cell to a malignancy and ultimately to a metastatic state. Certain genetic perturbations have been implicated for initiation and progression. However, to a great extent, underlying mechanisms often remain elusive. These genetic perturbations are most likely reflected by the altered expression of sets of genes or pathways, rather than individual genes, thus creating a need for models of deregulation of pathways to help provide an understanding of the mechanisms of tumorigenesis. We introduce an integrative hierarchical analysis of tumor progression that discovers which a priori defined pathways are relevant either throughout or in particular steps of progression. Pathway interaction networks are inferred for these relevant pathways over the steps in progression. This is followed by the refinement of the relevant pathways to those genes most differentially expressed in particular disease stages. The final analysis infers a gene interaction network for these refined pathways. We apply this approach to model progression in prostate cancer and melanoma, resulting in a deeper understanding of the mechanisms of tumorigenesis. Our analysis supports previous findings for the deregulation of several pathways involved in cell cycle control and proliferation in both cancer types. A novel finding of our analysis is a connection between ErbB4 and primary prostate cancer.

The ligands of the epidermal growth factor family and their receptors, the ErbB proteins, have been linked to the development of different types of cancer. Particular attention has focused on ErbB2, whose activation may occur by receptor overexpression or by ligand-induced oligomerization with other ErbB receptors. Whether these two modes of ErbB2 activation cause the same biological responses is unknown. Here, we uncovered important differences in the signaling, proliferation rates, and the response to anti-ErbB2 antibodies when comparing MCF7 cells expressing the ligand neuregulin, to MCF7 cells overexpressing ErbB2. Expression of neuregulin caused higher proliferation than ErbB2 overexpression. Transmembrane neuregulin expression was accompanied by constitutive activation of ErbB2, ErbB3, and ErbB4 receptors. ErbB2 overexpression caused tyrosine phosphorylation of ErbB2, whereas ErbB3 and ErbB4 were only slightly tyrosine phosphorylated. Autocrine transmembrane neuregulin also caused constitutive activation of several signaling pathways, such as the Erk1/2, Erk5, and Akt routes, which have been linked to breast cancer cell proliferation. Interestingly, expression of neuregulin increased p21 levels and this was required for the proliferation of MCF7 cells. Treatment with the anti-ErbB2 receptor antibody Herceptin had an inhibitory effect on proliferation only in cells expressing neuregulin but not on cells overexpressing ErbB2, and its inhibitory activity was accompanied by a decrease in p21. These results suggest that Herceptin may also be of help in the treatment of tumors in which neuregulin feeds the tumoral tissue.

BACKGROUND

Emerging evidence shows that ErbB2 signaling has a critical role in cardiomyocyte physiology, based mainly on findings that blocking ErbB2 for cancer therapy is toxic to cardiac cells. However, consequences of high levels of ErbB2 activity in the heart have not been previously explored.

RESULTS

We investigated consequences of cardiac-restricted over-expression of ErbB2 in two novel lines of transgenic mice. Both lines develop striking concentric cardiac hypertrophy, without heart failure or decreased life span. ErbB2 transgenic mice display electrocardiographic characteristics similar to those found in patients with Hypertrophic Cardiomyopathy, with susceptibility to adrenergic-induced arrhythmias. The hypertrophic hearts, which are 2-3 times larger than those of control littermates, express increased atrial natriuretic peptide and β-myosin heavy chain mRNA, consistent with a hypertrophic phenotype. Cardiomyocytes in these hearts are significantly larger than wild type cardiomyocytes, with enlarged nuclei and distinctive myocardial disarray. Interestingly, the over-expression of ErbB2 induces a concurrent up-regulation of multiple proteins associated with this signaling pathway, including EGFR, ErbB3, ErbB4, PI3K subunits p110 and p85, bcl-2 and multiple protective heat shock proteins. Additionally, ErbB2 up-regulation leads to an anti-apoptotic shift in the ratio of bcl-xS/xL in the heart. Finally, ErbB2 over-expression results in increased activation of the translation machinery involving S6, 4E-BP1 and eIF4E. The dependence of this hypertrophic phenotype on ErbB family signaling is confirmed by reduction in heart mass and cardiomyocyte size, and inactivation of pro-hypertrophic signaling in transgenic animals treated with the ErbB1/2 inhibitor, lapatinib.

CONCLUSIONS

These studies are the first to demonstrate that increased ErbB2 over-expression in the heart can activate protective signaling pathways and induce a phenotype consistent with Hypertrophic Cardiomyopathy. Furthermore, our work suggests that in the situation where ErbB2 signaling contributes to cardiac hypertrophy, inhibition of this pathway may reverse this process.

Activation of EGF receptors is closely involved in vascular proliferative diseases. The signaling mechanisms of EGF ligands, including betacellulin (BTC) and amphiregulin (AR), are poorly understood. We examined how BTC and AR induced DNA synthesis activity in primary cultures of human thoracic aortic smooth muscle cells (HTASMCs). BTC induced phosphorylation of all four EGF receptors present on HTASMCs: ErbB1, ErbB2, ErbB3, and ErbB4. BTC rapidly induced the phosphorylation of Akt, GSK3alpha/beta, and two FoxO factors, FKHR and AFX, in a dose- and time-dependent manner. BTC increased nuclear beta-catenin accumulation. BTC increased cyclin D1 mRNA, cyclin D1 protein, and DNA synthesis activity. Pretreatment with the phosphatidylinositol 3'-kinase (PI 3'-kinase) inhibitor wortmannin suppressed BTC-induced cyclin D1 mRNA and protein and DNA synthesis activity. In contrast, AR, a specific ErbB1 ligand, induced sustained ERK1/2 and Elk1 phosphorylation, increased cyclin D1 mRNA and protein, and increased DNA synthesis activity. AR did not produce any changes in Akt phosphorylation. Pretreatment with PD98059 suppressed AR-induced cyclin D1 mRNA and protein. Thus, the PI 3'-kinase/Akt/GSK/FoxO/beta-catenin pathway could be the major signaling cascade for BTC-induced upregulation of cyclin D1 protein, whereas a sustained ERK/Elk1 activation could be the major signaling cascade for AR-induced upregulation of cyclin D1 protein in HTASMCs. Moreover, immunohistochemical staining revealed that that BTC, ErbB1, and ErbB4 are upregulated in the plaques of human atherosclerotic coronary arteries. Taken together, BTC and AR could be potent growth factors in proliferative vascular diseases.