Targeted therapy for gastric cancer: molecular pathways and ongoing investigations.

Gastric cancer is currently the second leading cause of worldwide cancer mortality. Ongoing collaborative sequencing efforts have highlighted recurrent somatic genomic aberrations in gastric cancer, however, despite advances in characterizing the genomic landscape, there have been few advances in patient outcomes. Prognosis remains poor with a median overall survival of 12 months for advanced disease. The improved survival with trastuzumab, and more recently ramucirumab, underscore the promise of targeted and biologic therapies and the importance of molecular tumor characterization in gastric cancer. Here we review the most frequent actionable alterations in gastric cancer and highlight ongoing clinical investigations attempting to translate biologic understanding into improved clinical outcomes.

Modulation of dorsal root ganglion development by ErbB signaling and the scaffold protein Sorbs3.

The multipotent cells of the vertebrate neural crest (NC) arise at the dorsal aspect of the neural tube, then migrate throughout the developing embryo and differentiate into diverse cell types, including the sensory neurons and glia of the dorsal root ganglia (DRG). As multiple cell types are derived from this lineage, it is ideal for examining mechanisms of fate restriction during development. We have isolated a mutant, ouchless, that specifically fails to develop DRG neurons, although other NC derivatives develop normally. This mutation affects the expression of Sorbs3, a scaffold protein known to interact with proteins involved in focal adhesions and several signaling pathways. ouchless mutants share some phenotypic similarities with mutants in ErbB receptors, EGFR homologs that are implicated in diverse developmental processes and associated with several cancers; and ouchless interacts genetically with an allele of erbb3 in DRG neurogenesis. However, the defect in ouchless DRG neurogenesis is distinct from ErbB loss of function in that it is not associated with a loss of glia. Both ouchless and neurogenin1 heterozygous fish are sensitized to the effects of ErbB chemical inhibitors, which block the development of DRG in a dose-dependent manner. Inhibitors of MEK show similar effects on DRG neurogenesis. We propose a model in which Sorbs3 helps to integrate ErbB signals to promote DRG neurogenesis through the activation of MAPK and upregulation of neurogenin1.

Exogenous GLP-2 and IGF-I induce a differential intestinal response in IGF binding protein-3 and -5 double knockout mice.

Glucagon-like peptide-2 (GLP-2) action is dependent on intestinal expression of IGF-I, and IGF-I action is modulated by IGF binding proteins (IGFBP). Our objective was to evaluate whether the intestinal response to GLP-2 or IGF-I is dependent on expression of IGFBP-3 and -5. Male, adult mice in six treatment groups, three wild-type (WT) and three double IGFBP-3/-5 knockout (KO), received twice daily intraperitoneal injections of GLP-2 (0.5 µg/g body wt), IGF-I (4 µg/g body wt), or PBS (vehicle) for 7 days. IGFBP-3/-5 KO mice showed a phenotype of lower plasma IGF-I concentration, but greater body weight and relative mass of visceral organs, compared with WT mice (P < 0.001). WT mice showed jejunal growth with either IGF-I or GLP-2 treatment. In KO mice, IGF-I did not stimulate jejunal growth, crypt mitosis, sucrase activity, and IGF-I receptor (IGF-IR) expression, suggesting that the intestinotrophic actions of IGF-I are dependent on expression of IGFBP-3 and -5. In KO mice, GLP-2 induced significant increases in jejunal mucosal cellularity, crypt mitosis, villus height, and crypt depth that was associated with increased expression of the ErbB ligand epiregulin and decreased expression of IGF-I and IGF-IR. This suggests that in KO mice, GLP-2 action in jejunal mucosa is independent of the IGF-I system and linked with ErbB ligands. In summary, the intestinotrophic actions of IGF-I, but not GLP-2, in mucosa are dependent on IGFBP-3 and -5. These findings support the role of multiple downstream mediators for the mucosal growth induced by GLP-2.

Activation of the neuregulin/ErbB system during physiological ventricular remodeling in pregnancy.

The neuregulin-1 (NRG1)/ErbB system has emerged as a paracrine endothelium-controlled system in the heart, which preserves left ventricular (LV) performance in pathophysiological conditions. Here, we analyze the activity and function of this system in pregnancy, which imparts a physiological condition of LV hemodynamic overload. NRG1 expression and ErbB receptor activation were studied by Western blot analyses in rats and mice at different stages of pregnancy. LV performance was evaluated by transthoracic echocardiography, and myocardial performance was assessed from twitches of isolated papillary muscles. NRG1/ErbB signaling was inhibited by oral treatment of animals with the dual ErbB1/ErbB2 tyrosine kinase inhibitor lapatinib. Analyses of LV tissue revealed that protein expression of different NRG1 isoforms and levels of phosphorylated ErbB2 and ErbB4 significantly increased after 1-2 wk of pregnancy. Lapatinib prevented phosphorylation of ErbB2 and ERK1/2, but not of ErbB4 and protein kinase B (Akt), revealing that lapatinib only partially inhibited NRG1/ErbB signaling in the LV. Lapatinib did not prevent pregnancy-induced changes in LV mass and did not cause apoptotic cell death or fibrosis in the LV. Nevertheless, lapatinib led to premature maternal death of ∼25% during pregnancy and it accentuated pregnancy-induced LV dilatation, significantly reduced LV fractional shortening, and induced abnormalities of twitch relaxation (but not twitch amplitude) of isolated papillary muscles. This is the first study showing that the NRG1/ErbB system is activated, and plays a modulatory role, during physiological hemodynamic overload associated with pregnancy. Inhibiting this system during physiological overload may cause LV dysfunction in the absence of myocardial cell death.

ErbB receptor signaling in astrocytes: a mediator of neuron-glia communication in the mature central nervous system.

Astrocytes are now recognized as active players in the developing and mature central nervous system. Each astrocyte contacts vascular structures and thousands of synapses within discrete territories. These cells receive a myriad of inputs and generate appropriate responses to regulate the function of brain microdomains. Emerging evidence has implicated receptors of the ErbB tyrosine kinase family in the integration and processing of neuronal inputs by astrocytes: ErbB receptors can be activated by a wide range of neuronal stimuli; they control critical steps of glutamate-glutamine metabolism; and they regulate the biosynthesis and release of various glial-derived neurotrophic factors, gliomediators and gliotransmitters. These key properties of astrocytic ErbB signaling in neuron-glia interactions have significance for the physiology of the mature central nervous system, as exemplified by the central control of reproduction within the hypothalamus, and are also likely to contribute to pathological situations, since both dysregulation of ErbB signaling and glial dysfunction occur in many neurological disorders.

ErbB4 genotype predicts left frontotemporal structural connectivity in human brain.

Diminished left frontotemporal connectivity is among the most frequently reported findings in schizophrenia and there is evidence that altered neuronal myelination may in part account for this deficit. Several investigations have suggested that variations of the genes that encode the Neuregulin 1 (NRG1)-ErbB4 receptor complex are associated with schizophrenia illness. As NRG1--ErbB4 has been implicated in neuronal myelination, we investigated with diffusion tensor imaging (DTI) whether fractional anisotropy (FA)--a putative measure of neuronal myelination--is predicted by a risk haplotype of the ErbB4 gene. The effects of the ErbB4 genotype were investigated in healthy subjects (N=59; mean age: 22.6+/-1.8 years). We also measured reaction time (RT) during a selective attention/working memory paradigm (visual oddball). In the schizophrenia risk genotype group, we found lower FA in the temporal lobe white matter (WM) including frontotemporal fiber tracts, predominantly in the left hemisphere. RT was increased in the risk genotype group and correlated with FA in the affected brain region. As FA is considered to index structural integrity of WM, to which neuronal fiber myelination is contributing, our results suggest that variations of the ErbB4 genotype may confer risk for schizophrenia illness via its impact on left frontotemporal connectivity in human brain. Reliability and validity of the result is suggested by our observation that (1) the FA-genotype association was not only obtained in the entire sample but also in both the split halves and (2) a statistical relationship was found among RT, genotype and FA.

The epidermal growth factor receptor family: biology driving targeted therapeutics.

The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in regulating cell proliferation, survival, differentiation and migration. The ErbB receptors carry out both redundant and restricted functions in mammalian development and in the maintenance of tissues in the adult mammal. Loss of regulation of the ErbB receptors underlies many human diseases, most notably cancer. Our understanding of the function and complex regulation of these receptors has fueled the development of targeted therapeutic agents for human malignancies in the last 15 years. Here we review the biology of ErbB receptors, including their structure, signaling, regulation, and roles in development and disease, then briefly touch on their increasing roles as targets for cancer therapy.

Role of neuregulin-1/ErbB signaling in cardiovascular physiology and disease: implications for therapy of heart failure.

Since the discovery that neuregulin-1 (NRG-1)/ErbB signaling is indispensable in cardiac development, evidence has shown that this system also plays a crucial role in the adult heart. In patients, an inhibitory ErbB2 antibody, trastuzumab, used in the treatment of mammary carcinomas, increases the risk for the development of cardiotoxic cardiomyopathy. Postnatal disruption of NRG-1/ErbB signaling by gene targeting in mice leads to dilated cardiomyopathy. Initially, the search for the mechanisms behind these observations focused mainly on the effects of NRG-1 on cardiomyocyte growth and survival and revealed that NRG-1 has Akt-dependent antiapoptotic effects in cultured cardiomyocytes. In vivo studies, however, did not uniformly reinforce a role for apoptosis in the development of cardiomyopathy induced by impaired NRG-1/ErbB signaling. More recent studies have revealed that NRG-1 is involved in the regulation of cardiac sympathovagal balances by counterbalancing adrenergic stimulation of the adult myocardium and through an obligatory interaction with the muscarinic cholinergic system. NRG-1 is synthesized and released by the endocardial and cardiac microvascular endothelium, dynamically controlled by neurohormonal and biomechanical stimuli. The physiology of the cardiac NRG-1/ErbB system has implications for the treatment of both cancer and heart failure. Clinical studies in breast cancer with novel ErbB inhibitors are currently underway. Novel oncological indications for ErbB inhibition are emerging; cardiovascular side effects need to be carefully monitored. On the other hand, pharmacological activation of ErbB signaling is likely an unrecognized and beneficial effect of currently used drugs in heart failure and a promising therapeutic approach to prevent or reverse myocardial dysfunction.

EGFR kinase possesses a broad specificity for ErbB phosphorylation sites, and ligand increases catalytic-centre activity without affecting substrate binding affinity.

We previously found that EGF (epidermal growth factor) increases the EGFR (EGF receptor) kinase-binding affinity towards the major tyrosine phosphorylation sites in downstream adaptor proteins such as Gab1 (Grb2-associated binding protein 1) and Shc [Src homology 2 (SH2) domain and collagen containing protein], but not that towards EGFR autophosphorylation sites [Fan, Wong, Deb and Johnson (2004) J. Biol. Chem. 279 , 38143-38150]. EGFR activation can also result in transphosphorylation of tyrosine resides in the C-terminal region of the related receptors ErbB2, ErbB3 and ErbB4 in heterodimers which are formed upon ligand stimulation. In the present study, we investigated the specificity of EGFR kinase by comparing the steady state kinetic parameters for peptides derived from all four ErbBs in the absence or presence of EGF. Our results demonstrated that (i) EGFR kinase can efficiently phosphorylate a broad range of diverse peptide sequences representing ErbB sites; (ii) certain ErbB2, ErbB3 and ErbB4 sites had higher specificity constants than any EGFR sequence and (iii) EGF stimulation consistently increases the k(cat) approx. 5-fold, but does not significantly alter the K(m) for any ErbB peptides. Furthermore, peptides containing lysine at position -2 or -3 N-terminal to the target tyrosine were found to be poor EGFR kinase substrates, and substitution of these lysines with glutamine decreased the K(m) and increased the k(cat) for these substrates. We conclude that EGFR kinase-mediated ErbB transphosphorylations are mostly controlled at the level of oligomerization, and not by a preference of the EGFR kinase for phosphorylation sites in any particular ErbB. The results also demonstrated that, unlike phosphorylation sites in select downstream targets, EGF does not regulate the recognition of phosphorylation sites in the C-terminal region of any of the ErbBs.

Negative regulation of ErbB family receptor tyrosine kinases.

Receptors of the EGF receptor or ErbB family of growth factor receptor tyrosine kinases are frequently overexpressed in a variety of solid tumours, and the aberrant activation of their tyrosine kinase activities is thought to contribute to tumour growth and progression. Much effort has been put into developing inhibitors of ErbB receptors, and both antibody and small-molecule approaches have exhibited clinical success. Recently, a number of endogenous negative regulatory proteins have been identified that suppress the signalling activity of ErbB receptors in cells. These include intracellular RING finger E3 ubiquitin ligases such as cbl and Nrdp1 that mediate ErbB receptor degradation, and may include a wide variety of secreted and transmembrane proteins that suppress receptor activation by growth factor ligands. It will be of interest to determine the extent to which tumour cells suppress these pathways to promote their progression, and whether restoration of endogenous receptor-negative regulatory pathways may be exploited for therapeutic benefit.

Coexpression patterns of EGFR, HER2, HER3 and HER4 in non-melanoma skin cancer.

The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR), HER2, HER3 and HER4 are involved in the pathogenesis of multiple human malignant neoplasias. However, their role in the carcinogenesis of basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) remains to be elucidated. In order to further define the role of these RTKs, 56 human skin tissue samples of normal skin, BCC and SCC were studied by conventional and differential and quantitative reverse transcriptase-polymerase chain reaction (rtPCR). EGFR and HER3 were predominantly expressed in the BCCs and SCCs, while HER2 was ubiquitously expressed. HER4 was not expressed in any sample. Since in vitro studies have provided compelling evidence that heterodimer formation of these receptors are associated with different signal transduction processes, coexpression patterns might be decisive for the induction and maintenance of a malignant phenotype. These results confirm this concept: isolated HER2 expression and EGFR/HER2 were predominantly found in normal skin, while HER2/HER3 and the triple expression of EGFR/HER2/HER3 were seen more frequently in the BCCs and SCCs compared with normal skin (50% and 40% compared with 26%, respectively). The activation of HER3, in addition to EGFR and HER2, might therefore be associated with the malignant phenotype. However, due to the small numbers in this study, further confirmation of the patterns is needed.

Heregulin, but not ErbB2 or ErbB3, heterozygous mutant mice exhibit hyperactivity in multiple behavioral tasks.

Genetic redundancy is a problem in gene targeting studies because functionally relevant sister proteins can compensate for the lack of protein product of a targeted gene. A molecular system is chosen in which it is hoped to demonstrate both the lack and presence of compensation after disruption of particular single genes. Mammals may not be able to compensate for the lack of heregulin, a single ligand for multiple ErbB receptors, however, compensation is expected when a single ErbB receptor is knocked out. To investigate this the heregulin-1, ErbB2, or ErbB3 locus was disrupted in a targeted manner and mice heterozygous for the mutation were analyzed. Heregulin and its receptors were shown to be involved in embryonic brain development and, more recently, in plastic changes associated with adult brain function in rodents. Although they have never been shown to play roles in mammalian behavior, it was decided to characterize the mice behaviorally using a battery of simple tests. Heregulin mutant mice exhibited elevated activity levels in the open field, showed improved rotorod performance, and finished T-maze spontaneous alternation task faster compared to control wild type littermates, findings that suggest a consistent hyperactivity across tests. ErbB2 and ErbB3 mutant mice, whose strain origin was identical to that of heregulin mutants, showed no sign of the behavioral alterations. It is suggested that the abnormalities seen in heregulin mutant mice are due to mutation at that locus and the lack of alterations seen in ErbB2 and ErbB3 mutant mice is the result of compensation by unaltered sister receptors.