Inga Mertens-Walker

Evidence for a dual function of EphB4 as tumor promoter and suppressor regulated by the absence or presence of the ephrin-B2 ligand.

Overexpression of the receptor tyrosine kinase EphB4 is common in epithelial cancers and linked to tumor progression by promoting angiogenesis, increasing survival and facilitating invasion and migration. However, other studies have reported loss of EphB4 suggesting a tumor suppressor function in some cancers. These opposing roles may be regulated by (i) the presence of the primary ligand ephrin‐B2 that regulates pathways involved in tumor suppression or (ii) the absence of ephrin‐B2 that allows EphB4 signaling via ligand‐independent pathways that contribute to tumor promotion. To explore this theory, EphB4 was overexpressed in the prostate cancer cell line 22Rv1 and the mammary epithelial cell line MCF‐10A. Overexpressed EphB4 localized to lipid‐rich regions of the plasma membrane and confirmed to be ligand‐responsive as demonstrated by increased phosphorylation of ERK1/2 and internalization. EphB4 overexpressing cells demonstrated enhanced anchorage‐independent growth, migration and invasion, all characteristics associated with an aggressive phenotype, and therefore supporting the hypothesis that overexpressed EphB4 facilitates tumor promotion. Importantly, these effects were reversed in the presence of ephrin‐B2 which led to a reduction in EphB4 protein levels, demonstrating that ligand‐dependent signaling is tumor suppressive. Furthermore, extended ligand stimulation caused a significant decrease in proliferation that correlated with a rise in caspase‐3/7 and ‐8 activities. Together, these results demonstrate that overexpression of EphB4 confers a transformed phenotype in the case of MCF‐10A cells and an increased metastatic phenotype in the case of 22Rv1 cancer cells and that both phenotypes can be restrained by stimulation with ephrin‐B2, in part by reducing EphB4 levels.

Gonadotropin signalling in epithelial ovarian cancer

Ovarian cancer is the most lethal of all gynecologic malignancies, although its aetiology remains poorly understood. A role for the gonadotropins, follicle-stimulating hormone (FSH) and luteinising hormone (LH), has been implicated in a variety of different aspects of ovarian cancer tumorigenesis, including cellular proliferation, migration and invasion. This review focuses on the latest advances in knowledge concerning signalling pathways and functional consequences of gonadotropin action, including changes in protein-, miRNA- and gene expression, in epithelial ovarian cancer cells.

Gonadotropin-induced ovarian cancer cell migration and proliferation require extracellular signal-regulated kinase 1/2 activation regulated by calcium and protein kinase Cδ

The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKCdelta inhibitor rottlerin, and downregulation of PKCdelta was inhibited by small interfering RNA (siRNA), highlighting the importance of PKCdelta in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKCdelta siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKCdelta-dependent manner.

Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: implications for xenograft models of human prostate cancer.

BACKGROUND Ephrin-B2 is the sole physiologically-relevant ligand of the receptor tyrosine kinase EphB4, which is over-expressed in many epithelial cancers, including 66% of prostate cancers, and contributes to cancer cell survival, invasion and migration. Crucially, however, the cancer-promoting EphB4 signalling pathways are independent of interaction with its ligand ephrin-B2, as activation of ligand-dependent signalling causes tumour suppression. Ephrin-B2, however, is often found on the surface of endothelial cells of the tumour vasculature, where it can regulate angiogenesis to support tumour growth. Proteolytic cleavage of endothelial cell ephrin-B2 has previously been suggested as one mechanism whereby the interaction between tumour cell-expressed EphB4 and endothelial cell ephrin-B2 is regulated to support both cancer promotion and angiogenesis. METHODS An in silico approach was used to search accessible surfaces of 3D protein models for cleavage sites for the key prostate cancer serine protease, KLK4, and this identified murine ephrin-B2 as a potential KLK4 substrate. Mouse ephrin-B2 was then confirmed as a KLK4 substrate by in vitro incubation of recombinant mouse ephrin-B2 with active recombinant human KLK4. Cleavage products were visualised by SDS-PAGE, silver staining and Western blot and confirmed by N-terminal sequencing. RESULTS At low molar ratios, KLK4 cleaved murine ephrin-B2 but other prostate-specific KLK family members (KLK2 and KLK3/PSA) were less efficient, suggesting cleavage was KLK4-selective. The primary KLK4 cleavage site in murine ephrin-B2 was verified and shown to correspond to one of the in silico predicted sites between extracellular domain residues arginine 178 and asparagine 179. Surprisingly, the highly homologous human ephrin-B2 was poorly cleaved by KLK4 at these low molar ratios, likely due to the 3 amino acid differences at this primary cleavage site. CONCLUSION These data suggest that in in vivo mouse xenograft models, endogenous mouse ephrin-B2, but not human tumour ephrin-B2, may be a downstream target of cancer cell secreted human KLK4. This is a critical consideration when interpreting data from murine explants of human EphB4+/KLK4+ cancer cells, such as prostate cancer cells, where differential effects may be seen in mouse models as opposed to human clinical situations.

Signaling of receptor tyrosine kinases in the nucleus

Since the discovery of the first receptor tyrosine kinase (RTK) proteins in the late 1970s and early 1980s, many scientists have explored the functions of these important cell signaling molecules. The finding that these proteins are often deregulated or mutated in diseases such as cancers and diabetes, together with their potential as clinical therapeutic targets, has further highlighted the necessity for understanding the signaling functions of these important proteins. The mechanisms of RTK regulation and function have been recently reviewed by Lemmon & Schlessinger (2010) but in this review we instead focus on the results of several recent studies that show receptor tyrosine kinases can function from subcellular localisations, including in particular the nucleus, in addition to their classical plasma membrane location. Nuclear localisation of receptor tyrosine kinases has been demonstrated to be important for normal cell function but is also believed to contribute to the pathogenesis of several human diseases.

EphB4 localises to the nucleus of prostate cancer cells

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.

Inhibiting Eph kinase activity may not be “Eph”ective for cancer treatment

Abstract Several Eph receptor tyrosine kinases (RTKs) are commonly over-expressed in epithelial and mesenchymal cancers and are recognized as promising therapeutic targets. Although normal interaction between Eph receptors and their ephrin ligands stimulates kinase activity and is generally tumor suppressive, significant Eph over-expression allows activation of ligand- and/or kinase-independent signaling pathways that promote oncogenesis. Single-agent kinase inhibitors are widely used to target RTK-driven tumors but acquired and de novo resistance to such agents is a major limitation to effective clinical use. Accumulating evidence suggests that Ephs can be inhibited by “leaky” or low-specificity kinase inhibitors targeted at other RTKs. Such off-target effects may therefore inadvertently promote ligand- and/or kinase-independent oncogenic Eph signaling, thereby providing a new mechanism by which resistance to the RTK inhibitors can emerge. We propose that combining specific, non-leaky kinase inhibitors with tumor-suppressive stimulators of Eph signaling may provide more effective treatment options for overcoming treatment-induced resistance and clinical failure.

Abstract 1085: Nuclear localization and function of EphB4 in prostate cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL EphB4 is a member of the largest group of receptor tyrosine kinases. EphB4 is significantly overexpressed in prostate cancer where it contributes to tumour progression as demonstrated through knock down of EphB4 in both in vitro and in vivo cell models. EphB4s interaction with its preferred ligand ephrin-B2 may lead, in some cases, to tumour suppression, suggesting therefore that ligand-independent effects are oncogenic. EphB4 is usually located to the plasma membrane, but using an in-house specific monoclonal antibody we have identified EphB4 in the nucleus of prostate cancer cell lines and patient tumour samples. Accordingly, we hypothesise that EphB4 can directly regulate gene expression in prostate cancer cells and that this may be at least one mechanism through which the ligand-independent effects on tumour progression may be manifested. Using chromatin immunoprecipitation with the specific EphB4 antibody we were able to precipitate numerous DNA sequences that are currently being validated. Using confocal microscopy we have discovered that EphB4 colocalises with SUMO-1 (small ubiquitin-like modifier-1). SUMOylation is a posttranslational modification by which one or multiple SUMO molecules are attached to certain lysine residues of a target protein and this can result in cytoplasmic-nuclear shuttling. In silico analysis of EphB4 revealed that it contains a highly conserved potential sumoylation site, K616. This is a highly novel and significant finding in the Eph receptor field and may provide a mechanism by which EphB4 localises to the nucleus. Further characterisation of EphB4 sumoylation and nuclear translocation is currently being undertaken. This study provides novel insight into Eph receptor biology and may identify a major new mechanism by which EphB4 can control gene expression and potentially influence cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1085. doi:10.1158/1538-7445.AM2011-1085