Subfamily Of Receptor Tyrosine Kinases Research Articles

Abstract 2638: Galectin-3 activates the receptor tyrosine kinase Tyro3 and stimulates ERK signaling, cell survival and migration in human head and neck cancer cells

Abstract The beta-galactoside-binding protein galectin-3 (Gal-3) has been shown to influence the tumorigenic behaviour of cancer cells, and elevated serum levels of Gal-3 have been linked to tumor metastasis in several different cancers. Previously, Gal-3 was shown to be a ligand MerTK, a member of the TAM (Tyro3, Axl, MerTK) subfamily of receptor tyrosine kinases (RTKs) - these RTKs are implicated in tumorigenesis and/or enabling tumor chemoresistance in many different cancers. However, it is not known whether Gal-3 is also a ligand for the other two TAM RTKs, nor what signaling pathways it activates to regulate cancer cell functions. This study is aimed at determining the ligand properties of Gal-3 against Tyro3 and Axl RTKs in human cancer cells and the oncogenic signal pathways linked to them. The cultured human head and neck squamous cell carcinoma (HNSCC) cell line SCC-25 was determined by western blot and qPCR to express both Axl and Tyro3, but not MerTK. Recombinant Gal-3 and the TAM ligands Gas6 and ProS1 were added to serum-starved cells over a time-period of minutes and cells lysed to determine activation/phosphorylation of RTKs and intracellular signal mediators by western blotting. Longer incubations were performed to measure cell viability (MTS assay) and cell migration rate (scratch assay). All experiments were carried out a minimum three times. The known TAM ligands Gas6 and ProS1 stimulated TAM RTKs differentially in SCC-25 cells: Gas6 activated Axl strongly within minutes, with ProS1 having a weak effect. In contrast, ProS1 was the strongest ligand for Tyro3, activating it by fourfold, followed by Gal-3 at threefold and Gas6 at twofold. All three exogenous proteins also rapidly stimulated ERK1/2 phosphorylation by two-fold, whereas only Gas6 and Gal-3 stimulated Akt phosphorylation. All three proteins significantly maintained cell survival during acute induction of apoptosis by staurosporine. However, only Gas6 conferred a survival effect over long-term serum starvation-induced cell death. In addition, Gal-3 significantly stimulated cell migration during Axl blockade with the small molecule Axl inhibitor BGB324, further suggesting a role for Tyro3 in this effect. These results have shown that Gal-3 is a ligand for Tyro3 RTK, and that Gal-3 promotes cell survival and migration in HNSCC cells, coupled with activation of both the related signal pathways ERK and Akt. The data therefore reveal a new mechanism of Tyro3 activation; however, it remains to be determined whether Gal-3 interacts with the glycosyl chain on the RTK or else via other interfaces. Finally, cancer therapeutic strategies targeting Gal-3 action and/or cell surface glycosylation may be viable approaches in further research. Citation Format: Sassan Hafizi, Nour Al Kafri. Galectin-3 activates the receptor tyrosine kinase Tyro3 and stimulates ERK signaling, cell survival and migration in human head and neck cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2638.

PF466 POTENTIAL COMBINATIONAL THERAPY OF LEUKEMIC STEM CELLS USING BONE MORPHOGENETIC PROTEIN AND TYROSINE KINASE INHIBITORS

Background:Chronic myeloid leukemia (CML) disease outcomes have improved since the introduction of tyrosine kinase inhibitors (TKIs), however a proportion of patients will have TKI resistance and TKIs are not curative in the majority of patients. Low‐levels of Bcr‐Abl transcripts persist and a major clinical aim is to understand mechanism underlying disease persistence and the role of leukemic stem cells (LSCs) in this. To date it has proved difficult to both understand how LSCs dominate and alter the niche, and to target LSCs with current therapies. Previous studies have highlighted the importance of the bone morphogenetic protein (BMP) pathway in CML LSCs with altered stem cell fate, persistence and the response to BMP antagonists affecting cell behavior with altered cell cycle, apoptosis and expansion (Laperrousaz, B. et al, Blood, 2013; Grockowiak, E. et al, Blood, 2017).Aims:To investigate the effect of BMP pathway inhibition in combination with TKIs in primary CML samples to improve insights and identify new potential therapeutic approaches.Methods:Primary CML CD34+cells were treated with Imatinib (IM) or Saracatinib (SC), a Src/Abl dual inhibitor, alone or in combination with BMP inhibitor Dorsomorphin (DOR). Additionally, cell cultures were supplemented with soluble BMP4 or co‐cultured with the human stromal cell line HS‐5. Apoptosis, cell cycle and proliferation were investigated by flow cytometry. RNA was harvested, cDNA generated and pre‐amplified prior to the performance of multiplex Real‐Time PCR (Fluidigm) on 89 lineage‐specific, cell cycle, proliferation, and survival genes. Furthermore, we investigated tyrosine kinase activity in cell lysates of primary CML patient samples post single or dual treatment of SC and DOR using a chip‐based microarray assay from PamGene containing 196 peptides. Kinase phosphorylation activities measured on peptides were further evaluated by relating them to kinases that are able to phosphorylate a particular site on the peptide sequence.Results:Combinatorial treatments targeting the BMP pathway together with TKIs display a synergistic mode of action. Increases in late apoptosis (p < 0.0002), altered cell cycle (G2‐M, p = 0.0058), fewer cell divisions, and reduced numbers of CD34+cells were observed in dual treatments. BMP4 supplementation revealed similar differences whereas stroma co‐cultures displayed a protective effect. Furthermore, multiplex quantitative PCR has revealed significant deregulation of genes involved in cell cycle,apoptosis and differentiation, such as CDKN1a, CDKN2b, BCL6, GATA1 and SMAD5 showing differential expression in relation to response. Upstream kinase analysis using the PamGene platform, revealed kinases involved in cell growth, development, differentiation, apoptosis and cell‐cell adhesion. Amongst the top 20 potential differentially expressed kinases after SC treatment, were the expected members of the Src family kinases. DOR treatment revealed an up regulation of the largest known subfamily of receptor tyrosine kinases, the Ephrin receptors and Met, a single pass tyrosine kinase receptor that had already been identified as a potential therapy target in CML for IM resistant cells.Summary/Conclusion:Our results highlight the potential of a combined approach of targeting the BMP pathway with BMP antagonists or small inhibitors together with TKIs, which could open up new therapeutic possibilities. Future experiments will assess the potential of combinatorial treatment in a 3D bone marrow model, comprising magnetically levitated Mesenchymal Stem Cell spheroids embedded in medical‐grade collagen type I.

Ephrin B receptor tyrosine kinase ligands modulate the germinal center reaction and control humoral immune responses to malaria

Abstract Malaria remains a global health priority causing half a million deaths annually. Protective antibody responses that can control the Plasmodium parasites that cause malaria are an essential component of naturally acquired immunity and develop after years of continuous exposure to Plasmodium parasites. The protective humoral response is short-lived and tends to wane in the absence of parasite re-infections. The cellular and molecular mechanisms that prevent the development of long-lived humoral immunity against malaria remain poorly understood. Recently, ephrin B1, a ligand for the Eph B receptor tyrosine kinase subfamily, was shown to be involved in T follicular helper (Tfh) cell recruitment, retention and contact-dependent interaction with germinal center (GC) B cells, key processes in the production of efficacious antibody responses. We hypothesize that EphB/EphrinB signaling pathway is required for the development of Plasmodium humoral responses. Using a non-lethal P. yoelii XNL infection model, we observed an upregulation in the expression of Ephrin B on both GC B cell and Tfh cells at the peak of the infection in wild type mice. Selective deficiency of Ephrin B1/B2 on B cells (CD19cre+ Ephrin B1/B2fl/fl mice) led to lethality in some mice infected with an otherwise non-lethal P. yoelii XNL infection. On the other hand mice that lack the expression of Ephrin B1/B2 on T cells (CD4cre+ Eprhin B1/B2fl/fl mice) were able to control P. yoelii XNL infection. Our data show a requirement for Ephrin B1/B2 signaling on B cells for an effective contact-dependent interaction with Tfh cells and for optimal antibody responses during Plasmodium infection.

Receptor Tyrosine Kinase-Targeted Cancer Therapy.

In the past two decades, several molecular targeted inhibitors have been developed and evaluated clinically to improve the survival of patients with cancer. Molecular targeted inhibitors inhibit the activities of pathogenic tyrosine kinases. Particularly, aberrant receptor tyrosine kinase (RTK) activation is a potential therapeutic target. An increased understanding of genetics, cellular biology and structural biology has led to the development of numerous important therapeutics. Pathogenic RTK mutations, deletions, translocations and amplification/over-expressions have been identified and are currently being examined for their roles in cancers. Therapies targeting RTKs are categorized as small-molecule inhibitors and monoclonal antibodies. Studies are underway to explore abnormalities in 20 types of RTK subfamilies in patients with cancer or other diseases. In this review, we describe representative RTKs important for developing cancer therapeutics and predicting or evaluated resistance mechanisms.

Overexpression-mediated activation of MET in the Golgi promotes HER3/ERBB3 phosphorylation.

Ligand-dependent oligomerization of receptor tyrosine kinases (RTKs) results in their activation through highly specific conformational changes in the extracellular and intracellular receptor domains. These conformational changes are unique for each RTK sub-family, limiting cross-activation between unrelated RTKs. The proto-oncogene MET receptor tyrosine kinase overcomes these structural constraints and phosphorylates unrelated RTKs in numerous cancer cell lines. The molecular basis for these interactions is unknown. We investigated the mechanism by which MET phosphorylates the human epidermal growth factor receptor-3 (HER3 or ERBB3), a catalytically impaired RTK whose phosphorylation by MET has been described as an essential component of drug resistance to inhibitors targeting EGFR and HER2. We find that in untransformed cells, HER3 is not phosphorylated by MET in response to ligand stimulation, but rather to increasing levels of MET expression, which results in MET activation in a ligand-independent manner. Phosphorylation of HER3 by its canonical dimerization partners, EGFR and HER2, is achieved by engaging an allosteric site on the HER3 kinase domain, but this site is not required when HER3 is phosphorylated by MET. We also observe that HER3 preferentially interacts with MET during its maturation along the secretory pathway, before MET is post-translationally processed by cleavage within its extracellular domain. This results in accumulation of phosphorylated HER3 in the Golgi apparatus. We further show that in addition to HER3, MET phosphorylates other RTKs in the Golgi, suggesting that this mechanism is not limited to HER3 phosphorylation. These data demonstrate a link between MET overexpression and its aberrant activation in the Golgi endomembranes and suggest that non-canonical interactions between MET and unrelated RTKs occur during maturation of receptors. Our study highlights a novel aspect of MET signaling in cancer that would not be accessible to inhibition by therapeutic antibodies.

EphA8 is a Prognostic Factor for Oral Tongue Squamous Cell Carcinoma.

BackgroundOral tongue squamous cell carcinoma (OTSCC) is the most common malignancy of the oral cavity. Here we explore the potential effects of EphA8, which is one of the receptors in Ephs subfamily of RTKs (receptor tyrosine kinases), in the progression and prognosis of OTSCC.Material/MethodsA total of 119 OTSCC patients were enrolled in this retrospective study. Immunohistochemistry (IHC) staining and quantitative polymerase chain reaction (Q-PCR) were utilized to examine the expression of EphA8 in OTSSC tissues and adjacent non-tumor tissues. The relationship between EphA8 expression and the clinicopathological features of OTSCC patients were analyzed by chi-square. Survival analysis was carried out with Kaplan-Meier curve and the related log-rank test. Multivariate analysis was then undertaken to assess the prognosis factor by utilizing the Cox proportional hazard regression model. In addition, MTT assay and Matrigel invasion assay were performed to examine the effects of EphA8 on the proliferation and invasion capacities of human oral squamous carcinoma cells (SCC-25) and human tongue squamous cell carcinoma cells (H357).ResultsQ-PCR and IHC staining revealed that EphA8 was highly expressed in OTSCC tissues, especially in advanced stage OTSCC tissues. Kaplan-Meier curve showed that high EphA8 expression was significantly associated with poor prognosis, similar to age, smoking habit, drinking habit, tumor size, and TNM stage. Multivariate analysis indicated that EphA8 expression could serve as an independent prognostic factor in OTSCC. In vitro experiments revealed that overexpression of EphA8 might promote the progression of OTSCC via enhancing the invasion capacity but not proliferation capacity of tumor cells.ConclusionsEphA8 was highly expressed in OTSCC tissues and was significantly associated with poor prognosis of OTSCC.

PET Imaging of Receptor Tyrosine Kinases in Cancer.

Overexpression and/or mutations of the receptor tyrosine kinase (RTK) subfamilies, such as epidermal growth factor receptors (EGFR) and vascular endothelial growth factor receptors (VEGFR), are closely associated with tumor cell growth, differentiation, proliferation, apoptosis, and cellular invasiveness. Monoclonal antibodies (mAb) and tyrosine kinase inhibitors (TKI) specifically inhibiting these RTKs have shown remarkable success in improving patient survival in many cancer types. However, poor response and even drug resistance inevitably occur. In this setting, the ability to detect and visualize RTKs with noninvasive diagnostic tools will greatly refine clinical treatment strategies for cancer patients, facilitate precise response prediction, and improve drug development. Positron emission tomography (PET) agents using targeted radioactively labeled antibodies have been developed to visualize tumor RTKs and are changing clinical decisions for certain cancer types. In the present review, we primarily focus on PET imaging of RTKs using radiolabeled antibodies with an emphasis on the clinical applications of these immunoPET probes. Mol Cancer Ther; 17(8); 1625-36. ©2018 AACR.

Abstract 2488: Functional characterization of nuclear TYRO3 in colorectal cancer

Abstract Colorectal cancer is one of most common death-related cancers worldwide. Aberrant expression of oncogenes is regarded as a predominant factor in colorectal cancer development. TYRO3 belongs to TAM (TYRO3, AXL, and MER) family, which is a subfamily of receptor tyrosine kinases. Our previous study discovered TYRO3, an oncogene overexpressed in colorectal cancer, promotes cell proliferation, metastasis, and tumor progression. Herein, we further identified that TYRO3 is present in nucleus of colon cancer cells using immunofluorescence staining in conjunction with confocal microscopy. By staining with three different antibodies recognizing N-terminal, central region, and C-terminal of TYRO3, respectively, we identified that central and C-terminal of TYRO3 is the fragment to be translocated to the nucleus. Transfection of exogenous Myc-TYRO3-GFP fusion protein further confirmed this finding. By bioinformatic tool prediction, we found there is a nuclear localization signal (NLS) and a nuclear export signal (NES) in TYRO3. To evaluate the function of nuclear TYRO3, we mutated NLS of TYRO3 and performed several functional studies. Results of immunofluorescent staining showed that after NLS mutation, TYRO3 cannot be translocated to nucleus, which was accompanied by distorted morphology. Furthermore, cancer cells transfected with NLS-mutated TYRO3 increased activated caspase-3 and number of apoptotic cells, suggesting nuclear localization of TYRO3 is essential for cell survival. Clinical data further support that levels of nuclear TYRO3 are a poor prognostic marker that is positively associated with colon cancer treatment failure and malignancy. These data support that nuclear TYRO3 is important for colon cancer cell survival and is a valuable biomarker for prognosis. Citation Format: Shaw-Jenq Tsai, Chun-Wei Chien, Sih-Yu Chen, Shao-Chieh Lin, Bo-Wen Lin, Jenq-Chang Lee. Functional characterization of nuclear TYRO3 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2488.

Abstract 746: Development of novel formats of anti-AXL ADCs

Abstract AXL is a member of the Tyro3- AXL -Mer (TAM) receptor tyrosine kinase subfamily which has been associated with several cellular functions including growth, migration, aggregation, and anti-inflammation (Li Y et al., 2009) via activation through its ligand, Gas6. Originally identified as an oncogene in chronic myelogenous leukemia, expression of Axl has been reported to be upregulated in a variety of cancers including breast, gastric, prostate, ovarian, and lung. AXL expression has been shown to be negatively associated with patient survival and implicated in epithelial-to-mesenchymal transition, a process closely linked with invasive motility and metastasis of malignant cells. Several recent studies have reported that AXL is overexpressed during resistance to chemotherapy and other molecularly targeted therapies, as well as during hypoxia (Schoumacher M et al 2017). Consequently, Axl is promising therapeutic target for development into ADCs for the first or second line treatment of cancer. At the National Research Council (NRC) of Canada, we have selected mouse monoclonal (mAbs) or llama derived single domain antibodies (sdAbs) that are selective for the extracellular domain of human Axl and which have been shown to be internalized into Axl expressing cells for antibody drug conjugate (ADC) development using a surrogate ADC screen. These antibodies were also characterized based on their binding domains and ability to compete with Gas6, the extracellular ligand for Axl. A panel of Axl-specific ADCs was subsequently directly conjugated to maytansine, the microtubule disrupting agent through a noncleavable linker. Axl-DM1 ADCs were shown to efficiently induce cytotoxicity in vitro, in Axl expressing cells including breast (MDA-MB 231), lung (NCI-H292) and ovarian (SKOV3) cell lines with sub nM potencies achieved with both sdAb and conventional mAb-based ADCs. When tested in vivo using ovarian (SKOV3) and breast cancer (MDA-MB 231) xenograft models, Axl-DM1 ADCs were shown to have potent anti-tumor activity. Biparatopic antibodies (i.e. those which target two non-overlapping epitopes) directed against HER2 have been previously shown to induce target clustering and promote robust internalization, lysosomal trafficking, and degradation (LI JY et al 2016). Since these properties are important in the mechanism of action of ADCs, we have further tested anti-Axl antibodies in various configurations and have identified potent biparatopic antibodies for further development into ADCs. Citation Format: Maria L. Jaramillo, Myriam Banville, Alma Robert, Luc Meury, Maurizio Acchione, Anne Marcil, Christine Gadoury, Cunle Wu, Yves Fortin, Kevin Henry, Joseph Schrag, The-Minh Tu, Binbing (Erica) Ling, Jacqueline Slinn, Maria Moreno. Development of novel formats of anti-AXL ADCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 746.

Structural and clinical consequences of activation loop mutations in class III receptor tyrosine kinases.

Mutations within the activation loop of members of the class III receptor tyrosine kinase (RTK) subfamily, which includes KIT, PDGFRA, and FLT3, have been observed in multiple human tumor types. These mutations confer constitutive activation as well as resistance to the type II tyrosine kinase inhibitors (TKI) that are currently clinically available, such as imatinib and sunitinib. It is now understood that activation loop mutations in class III RTKs shift the activation state equilibrium away from inactive states, to which type II TKIs bind, to the active state by destabilizing the inactive conformation. Recently, type I TKIs, which can bind to active kinase conformations, have been developed with specificity for class III RTK members. Some type I TKIs, such as crenolanib and avapritinib (BLU-285), have entered clinical studies for patients with activation loop mutations in KIT, PDGFRA, or FLT3. Preliminary results suggest that these type I TKIs show activity in these patient populations that previously lacked effective treatments. This article reviews the inactive and active structures of KIT, PDGFRA, and FLT3, how the mutations seen in human cancers affect kinase structure, and the clinical implications of these mutations in terms of type I vs. type II TKI binding.

EphrinB/EphB signaling contributes to spinal nociceptive processing via calpain‑1 and caspase‑3.

Previous studies have indicated that an important subfamily of receptor tyrosine kinases, ephrins and their receptors, are important in pain signaling, particularly in spinal nociceptive processing. In the present study, the role of the ephrin/Eph signaling pathway was confirmed, and it was shown that this signaling was also involved in spinal nociceptive processing through the actions of calpain-1 and caspase-3. First, the ephrinB ligands, ephrinB1-Fc or ephrinB2-Fc, were introduced into experimental mice via intrathecal injection, and it was found that this injection induced marked time- and dose-dependent mechanical allodynia and thermal hyperalgesia, accompanied by increased levels of calpain-1 and caspase-3 in the spinal cord. MDL28170, an inhibitor of calpain-1, reversed the behavioral effects and ameliorated the increases in calpain-1 and caspase-3. Second, it was found that the administration of EphB1 between L5 and L6 in mice inhibited the mechanical allodynia and thermal hyperalgesia induced by chronic constrictive injury. In addition, to demonstrate the cell phenotypes responsible for the increased levels of calpain-1 and caspase-3 in the spinal cord following injection with ephrinB2-Fc, double immunofluorescent labeling was performed, which indicated that calpain-1 and caspase-3 were localized in neurons, but not in astrocytes or microglial cells. In conclusion, the present study suggested that ephrinB/EphB signaling contributes to spinal nociceptive processing via the actions of calpain-1 and caspase-3.

Structure-Based Discovery of a Series of 5H-Pyrrolo[2,3-b]pyrazine FGFR Kinase Inhibitors.

Fibroblast growth factor receptors (FGFRs), a subfamily of receptor tyrosine kinases, are aberrant in various cancer types, and considered to be promising targets for cancer therapy. We started with a weak-active compound that was identified from our internal hepatocyte growth factor receptor (also called c-Met) inhibitor project, and optimized it with the guidance of a co-crystal structure of compound 8 with FGFR1. Through rational design, synthesis, and the biological evaluation of a series of 5H-pyrrolo[2,3-b]pyrazine derivatives, we discovered several potent FGFR kinase inhibitors. Among them, compound 13 displayed high selectivity and favorable metabolic properties, demonstrating a promising lead for further development.

Soluble TAM receptor tyrosine kinases in rheumatoid arthritis: correlation with disease activity and bone destruction.

The TAM receptor tyrosine kinases (TAM RTK) are a subfamily of receptor tyrosine kinases, the role of which in autoimmune diseases such as systemic lupus erythematosus has been well explored, while their functions in rheumatoid arthritis (RA) remain largely unknown. In this study, we investigated the role of soluble TAM receptor tyrosine kinases (sAxl/sMer/sTyro3) in patients with RA. A total of 306 RA patients, 100 osteoarthritis (OA) patients and 120 healthy controls (HCs) were enrolled into this study. The serum concentrations of sAxl/sMer/sTyro3 were measured by enzyme-linked immunosorbent assay (ELISA), then the associations between sAxl/sMer/sTyro3 levels and clinical features of RA patients were analysed. We also investigated whether sTyro3 could promote osteoclast differentiation in vitro in RA patients. The results showed that compared with healthy controls (HCs), sTyro3 levels in the serum of RA patients were elevated remarkably and sMer levels were decreased significantly, whereas there was no difference between HCs and RA patients on sAxl levels. The sTyro3 levels were correlated weakly but positively with white blood cells (WBC), immunoglobulin (Ig)M, rheumatoid factor (RF), swollen joint counts, tender joint counts, total sharp scores and joint erosion scores. Conversely, there were no significant correlations between sMer levels and the above indices. Moreover, RA patients with high disease activity also showed higher sTyro3 levels. In-vitro osteoclast differentiation assay showed further that tartrate-resistant acid phosphatase (TRAP)+ osteoclasts were increased significantly in the presence of sTyro3. Collectively, our study indicated that serum sTyro3 levels were elevated in RA patients and correlated positively with disease activity and bone destruction, which may serve as an important participant in RA pathogenesis.

SUMOylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the ErbB4 receptor tyrosine kinase

Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.

Regulation of the prometastatic neuregulin-MMP13 axis by SRC family kinases: therapeutic implications.

Metastatic dissemination of tumor cells is responsible for the fatal outcome of breast cancer. Therefore, understanding the mechanisms involved in dissemination is essential for the development of new therapeutic strategies to prevent metastasis. One mechanism involved in metastatic dissemination of breast cancer cells is dependent on control of the production of matrix metalloproteinases by the neuregulins (NRGs). The NRGs are polypeptide factors that act by binding to the ErbB/HER subfamily of receptor tyrosine kinases. NRG‐mediated activation of HER receptors causes an increase in the production of metalloprotease 13 (MMP13, also termed collagenase‐3), which facilitates metastatic dissemination of breast tumors. In this context, we aimed to explore whether the clinically approved tyrosine kinase inhibitor dasatinib was able to neutralize this mechanism of metastatic dissemination. Here, we show that dasatinib restricted NRG‐induced MMP13 upregulation, both in vitro and in vivo, and in vivo metastatic dissemination of breast cancer cells. Chemical proteomics studies showed that the main cellular targets of dasatinib were SRC family kinases (SFKs). Moreover, genetic studies showed that knockdown of SRC or YES strongly inhibited NRG‐induced MMP13 upregulation in vitro. Mechanistically, dasatinib treatment or knockdown of SRC also inhibited ERK1/2 kinases in vitro, which were required for NRG‐induced MMP13 upregulation. These results open the possibility of clinically exploring the antitumoral action of dasatinib in those tumors in which the NRG–MMP13 signaling axis may play a relevant role in the control of tumor cell dissemination.

Role of the venus kinase receptor in the female reproductive physiology of the desert locust, Schistocerca gregaria

Venus kinase receptors (VKR) are a subfamily of invertebrate receptor tyrosine kinases, which have only recently been discovered. They contain an intracellular tyrosine kinase domain and an extracellular Venus FlyTrap domain. VKRs have been functionally and pharmacologically characterized in only two invertebrate species, namely the human parasite Schistosoma mansoni and the mosquito Aedes aegypti, where they play a crucial role in oogenesis. Here, we report the characterization of a VKR in the desert locust, Schistocerca gregaria. We performed an in-depth profiling study of the SgVKR transcript levels in different tissues throughout the female adult stage. Using the RNA interference technique, the possible role of SgVKR was investigated. SgVKR knockdown had significant effects on ovarian ecdysteroid levels and on the size of oocytes during the vitellogenic stage. SgVKR is probably involved in the complex cross-talk between several important pathways regulating female reproductive physiology. Contrary to A. aegypti and S. mansoni, we cannot conclude that this receptor is essential for reproduction, since silencing SgVKR did not affect fecundity or fertility. Considering the evolutionary distance between A. aegypti and S. gregaria, as well as the differences in regulation of their female reproductive physiology, this article constitutes a valuable asset in better understanding VKRs.

EphA2 gene targeting using neutral liposomal small interfering RNA (EPHARNA) delivery: A phase I clinical trial.

TPS2604 Background: EphA2 is a member of the largest subfamily of receptor tyrosine kinases, with over 14 receptors and 8 ligands. EphA2 overexpression is common in many human cancers, including lung, breast, prostate, colorectal, pancreatic, melanoma, esophageal and endometrial cancers. EphA2 can function as an oncoprotein when introduced into cells with low expression. In addition, downregulation of constitutive expression reduces tumorigenicity in breast, endometrial, ovarian and pancreatic cancers in vitro and in vivo models. EphA2 is a desirable target because of its selective expression in cancer (vs. adult normal tissue), and its important role in promoting tumor growth and metastasis. It has kinase-dependent and independent functions, making it an ideal target for RNAi-based targeting. We have previously reported that EphA2 siRNA incorporated in DOPC nanoliposomes (EPHARNA) was highly effective in reducing EphA2 protein levels after a single dose. In addition, three weeks of treatment with EPHARNA (150 μg/kg twice weekly) in an orthotopic mouse model of ovarian cancer (HeyA8 or SKOV3ip1) significantly reduced tumor growth compared with non-silencing siRNA, and demonstrated synergistic anti-tumor activity when combined with conventional chemotherapy. EPHARNA underwent GLP development in 2 animal models (murine and primate) at M.D. Anderson to support the IND (#72924). The first-in-human trial (NCT01591356) is ongoing and recruiting study subjects. Methods: Adult Patients > 18 years of age with histologic proof of advanced recurrent solid tumors, who are not candidates for known regimens or protocol treatments of higher efficacy or priority. All patients (dose escalation and dose expansion phases) must be willing to undergo pre- and post-treatment biopsies. For dose expansion phase, patients must have EphA2 overexpression by IHC evaluation. Enrollment is ongoing for the dose escalation with the plan for dose expansion. A total of 16 patients have been enrolled and treated in the dose escalation phase. Clinical trial information: NCT01591356.

Review of the endothelial pathogenic mechanism of TIE2-related venous malformation

Venous malformation (VM) is a type of disease involving vascular morphogenesis in humans. Clinically, VM can be sporadic or inherited. TIE2, also known as TEK or HYK, is a member of the receptor tyrosine kinase subfamily and is highly conserved among species. In 1996, an arginine-to-tryptophan substitution at position 849 (R849W) in TIE2 was found to induce hereditary VM. Additional alterations in TIE2 involved in the pathogenesis of inherited or sporadic VM have since been reported. The relevant key literature was selectively reviewed, including case reports, reviews, research studies, and meta-analyses. TIE2 can be thought of as the basis for VM, with a potential role in determining locations, through intracorporal endothelium-specific distribution and expression from the embryonic phase. It has a sophisticated protein structure, and various point mutations destroy its function and physiologic processes by obviously different activation mechanisms, of which some inhibit dephosphorylation and others maintain phosphorylation. Extracellularly, whereas angiopoietins (ANGs) are ligands of TIE2, the chaotic balance between ANG1 and ANG2 in VM is related to their effects on switching between the cell-cell/cell-extracellular matrix contact conditions and vascular quiescence/angiogenesis state, resulting in corrupted contacts. Intracellularly, among diverse cellular pathways, phosphatidylinositol 4,5-bisphosphate 3-kinase/AKT serine-threonine kinase, mitogen-activated protein kinase, and Dok-related protein are irreplaceable keys underlying changes in endothelial morphology and behavioral biology in VM. For example, R849W and L914F (a leucine-to-phenylalanine substitution at position 914), the most important and frequent TIE2 mutations associated with VM, share similar phenotypes but differ with respect to signaling pathways, heredity, and triggering factors. Based on this comprehensive analysis, we propose an avalanche theory, in which mutant TIE2 is a trigger and pathogenic core, the intercellular network is a tool, altered extracellular matrix and contacts are the final foothold, and fragile contacts are the result. Precise classification according to TIE2 mutation type in VM, especially the mutation site, is important for future targeted therapies.

IGF-1 Mediates EphrinB1 Activation in Regulating Tertiary Dentin Formation

Eph receptors belong to a subfamily of receptor tyrosine kinases that are activated by membrane-spanning ligands called ephrins. Previously, we demonstrated that the ephrinB1-EphB2 interaction regulates odontogenic/osteogenic differentiation from dental pulp cells (DPCs) in vitro. The goal of this study was to identify the molecular mechanisms regulated by the EphB2/ephrinB1 system that govern tertiary dentin formation in vitro and in vivo. During tooth development, ephrinB1, and EphB2 were expressed in preodontoblast and odontoblasts at postnatal day 4. EphrinB1 was continuously expressed in odontoblasts and odontoblastic processes until the completion of tooth eruption. In addition, ephrinB1 was expressed in odontoblastic processes 2 wk following tooth injury without pulp exposure, whereas EphB2 was expressed in the center of pulp niches but not odontoblasts. In a model of tooth injury with pulp exposure, ephrinB1 was strongly expressed in odontoblasts 4 wk postinjury. In vitro studies with human and mouse DPCs treated with calcium hydroxide (CH) or mineral trioxide aggregate (MTA) showed an increased expression of insulin-like growth factor 1 (IGF-1). Experiments using several inhibitors of IGF-1 receptor signaling revealed that inhibiting the Ras/Raf-1/MAPK pathway inhibited EphB2 expression, and inhibiting the PI3K/Akt/mTOR pathway specifically inhibited ephrinB1 gene expression. Tooth injury in mice with odontoblast-specific IGF-1 receptor ablation exhibited a reduced tertiary dentin volume, mineral density, and ephrinB1 expression 4 wk following injury. We conclude that the IGF-1/ephrinB1 axis plays significant roles in the early stages of tooth injury. Further research is needed to fully understand the potential of targeting ephrinB1 as a regenerative pulp therapy.

EphB3 protein is associated with histological grade and FIGO stage in ovarian serous carcinomas.

Eph (Erythropoietin-producing human hepatocellular carcinoma cell) is the largest subfamily of receptor tyrosine kinases. Eph receptors and their ephrin ligands are involved in embryonic development and physiological processes. Aberrant expression of Eph/ephrin may contribute to a variety of diseases including cancer. EphB3 is a member of Eph receptors and has been found to play roles in carcinogenesis of some types of human cancer. But, its expression and clinical significance in ovarian serous carcinoma have not been well investigated and are unknown. In this study, a set of ovarian tissues including normal fallopian tube, serous borderline tumor, and serous carcinoma were subjected to immunohistochemistry using a specific polyclonal antibody for EphB3. The relationship between EphB3 expression and clinicopathological parameters was statistically analyzed. EphB3 was strongly expressed in all fallopian tube specimens (19/19, 100%). EphB3 was negatively or weekly expressed in 1 of 17 (5.8%) in borderline tumors and 26 of 50 (52.0%) in serous carcinomas, moderately expressed in 7 of 17 (41.2%) in borderline tumors and 14 of 50 (28%) in serous carcinomas, and strongly expressed in 9 17 (52.9%) in borderline tumors and 10 of 50 (20%) in serous carcinomas. EphB3 expression is significantly reduced in serous carcinomas compared with normal fallopian tubes and borderline tumors (p < 0.001). EphB3 expression is negatively associated with histological grade (p < 0.001, rs = -0.613) and FIGO stage (p = 0.001, rs = -0.464) of serous carcinomas. Our results show EphB3 protein lost in ovarian serous carcinoma and is associated with tumor grade and FIGO stage, which indicate EphB3 protein may play a role in carcinogenesis of ovarian serous carcinoma and may be used as a molecular marker for prognosis.