Erythropoietin-producing Hepatocellular Receptor A2 Research Articles

Abstract A059: Serum EphA2 proteolytic fragment is a potent biomarker for diagnosing a very early stage of ductal pancreatic carcinoma

Abstract Erythropoietin-producing hepatocellular receptor A2 (EphA2) is a receptor tyrosine kinase, and its expression increases with malignant progression of cancer. Therefore, EphA2 is believed to be a candidate for molecular cancer therapy and many candidate drugs targeting EphA2 have been developed, but their effects are limited. The reason is its complex signaling functions. In fact, EphA2 has a tumor suppressor function regulated by the stimulation of the Ephrin-A ligand, while in the absence of ligand stimulation, it has a prooncogenic function in collaboration with EGFR and its downstream signaling molecules. The opposite functions of EphA2 make it difficult to develop it as a cancer drug target. We have previously demonstrated that a type-1 matrix membrane metalloprotease (MT1-MMP) at cancer cell surface interacted with EphA2 and selectively cleaved its N-terminal ligand binding site, converting it into a prooncogenic signaling transmitter (Koshikawa N et al., Cancer Research, 2015). We also found that the N- terminal fragment of EphA2 (termed EphA2-NF) released by MT1-MMP cleavage increased in sera from patients with gastric cancer and ductal pancreatic carcinoma (PDC) (Koshikawa N et al., Cell Death & Disease, 2017). In this study, we compare the expression of EphA2-NF in serum from a healthy donor (HD: 150 cases), patients with chronic pancreatic disease (87 cases), and PDC (578 cases) by automated chemiluminescence immunoassay with a specific EphA2-NF monoclonal antibody. As a result, PDC cases, including stage I/II PDC, show significantly higher levels of EphA2-NF than HD (Sato et al. Cancer Research Communications, 2023). Furthermore, PDC occurred in the intraductal papillary mucinous neoplasm (IPMN) cases with high levels of EphA2-NF during follow-up observation. Therefore, to verify that the EphA2 proteolytic cleavage occurred in the PDC onset, we analyzed 50 surgical resected tissues from patients with PDC and IPMN to detect the N and C-terminal regions of EphA2 by immunohistochemical staining. Interestingly, PDC, including early stage PDC cases, lacked the N-terminus of EphA2. Furthermore, the deletion of N-terminus of EphA2 was observed in all tested cases pathologically diagnosed as intraductal papillary mucinous adenocarcinoma (IPMC) (Sato S et al., Cancer Research Communications, 2023). These results suggest that serum EphA2-NF has the potential to revolutionize the early detection and management of PDC, providing a valuable tool to diagnose the disease in very early stages when treatment results may be more favorable. Collaborators: Masatoshi Nakagawa, Eisaku Yoshida and Toru Yoshimura (Research and Development, Abbott Japan LLC) Citation Format: Naohiko Koshikawa, Shinya Sato, Kouki Nio, Takeshi Terashima, Makoto Ueno, Taro Yamashita. Serum EphA2 proteolytic fragment is a potent biomarker for diagnosing a very early stage of ductal pancreatic carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A059.

Molecular mechanisms of transmitted endoplasmic reticulum stress mediating immune escape of gastric cancer via PVR overexpression in TAMs

Gastric cancer (GC) is the fourth leading cause of cancer death worldwide. Due to the complex tumor microenvironment (TME), the efficacy of immunotherapy in GC has not met expectations. Malignant changes in the TME induce endoplasmic reticulum stress (ERS). ERS can be transmitted between tumor cells and tumor-associated macrophages (TAMs), promoting tumor immune escape, but the specific mechanism in GC remains unclear. We established a TAM model of transmitted ERS (TERS), and iTRAQ proteomic analysis identified overexpressed proteins. The overexpression of poliovirus receptor (PVR) was screened while flow cytometry and ELISA showed that PVR mediated the immunosuppressive function of TAMs by downregulating the proliferative activity and cytotoxicity of cocultured CD8+ T lymphocytes. With EMSA and dual-luciferase reporter assays, we confirmed that erythropoietin-producing hepatocellular receptor A2 (EphA2) affected PVR expression by increasing the transcriptional activity of activator protein-1 (AP-1). MFC cells were mixed with EphA2 knockdown or control RAW264.7 cells to establish subcutaneous tumor models with or without tunicamycin treatment in vivo. The vivo experiments revealed that ERS promoted subcutaneous xenograft growth, which was reversed by EphA2 knockdown. Clinically, GC patients with high expression of PVR and EphA2 tended to have an immunosuppressive TME, which were determined by immunohistochemical and immunofluorescence analyses. In conclusion, the transcriptional activity of AP-1 is upregulated in ERS-transmitted TAMs through EphA2 to increase PVR expression, which promotes immune escape in GC. Our study provides a new perspective on the role of ERS in tumor immunity.

Targeting Interleukin-13 Receptor α2 and EphA2 in Aggressive Breast Cancer Subtypes with Special References to Chimeric Antigen Receptor T-Cell Therapy.

Breast cancer (BCA) remains the leading cause of cancer-related mortality among women worldwide. This review delves into the therapeutic challenges of BCA, emphasizing the roles of interleukin-13 receptor α2 (IL-13Rα2) and erythropoietin-producing hepatocellular receptor A2 (EphA2) in tumor progression and resistance. Highlighting their overexpression in BCA, particularly in aggressive subtypes, such as Her-2-enriched and triple-negative breast cancer (TNBC), we discuss the potential of these receptors as targets for chimeric antigen receptor T-cell (CAR-T) therapies. We examine the structural and functional roles of IL-13Rα2 and EphA2, their pathological significance in BCA, and the promising therapeutic avenues their targeting presents. With an in-depth analysis of current immunotherapeutic strategies, including the limitations of existing treatments and the potential of dual antigen-targeting CAR T-cell therapies, this review aims to summarize potential future novel, more effective therapeutic interventions for BCA. Through a thorough examination of preclinical and clinical studies, it underlines the urgent need for targeted therapies in combating the high mortality rates associated with Her-2-enriched and TNBC subtypes and discusses the potential role of IL-13Rα2 and EphA2 as promising candidates for the development of CAR T-cell therapies.

Upregulation of EphA2 is associated with apoptosis in response to H2O2 and UV radiation-induced cataracts

In this article, we examine the role of erythropoietin-producing hepatocellular receptor A2 (EphA2) in the apoptosis of lens epithelial cells (LECs) in H2O2 and UV radiation-induced cataracts. We treated SRA01/04 cells with H2O2 or ultraviolet (UV) radiation to create a cataract cell model. We constructed a cataract lens model by exposing mice to UV radiation. We used CCK8 assays, Annexin V-FITC analysis, and immunohistochemical staining to explore proliferation and apoptosis of the cataract model. Thereafter, we used quantitative real-time PCR (qPCR) analysis, Western blot assays, and immunofluorescence to determine gene and protein expression levels. We also employed Crispr/Cas9 gene editing to create an EphA2 knockout in SRA01/04 cells. Results: H2O2 or UV radiation induced SRA01/04 cells showed EphA2 gene upregulation. CCK8 and apoptosis assays showed that EphA2 over-expression (OE) reduced epithelial cell apoptosis, but knockout of EphA2 induced it in response to H2O2 and UV radiation, respectively. Mutation of the EphA2 protein kinase domain (c.2003G > A, p. G668D) had a limited effect on cell apoptosis. In vivo, the EphA2 protein level increased in the lenses of UV-treated mice. Our results showed that EphA2 was upregulated in SRA01/04 cells in response to H2O2 and UV radiation. Mutation of the EphA2 protein kinase domain (c.2003G > A, p. G668D) had a limited effect on H2O2 and UV radiation-induced cell apoptosis. We confirmed this change with an experiment on UV-treated mice. The present study established a novel association between EphA2 and LEC apoptosis.

Memory-like Differentiation, Tumor-Targeting mAbs, and Chimeric Antigen Receptors Enhance Natural Killer Cell Responses to Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell therapy-refractory cancers, but are frequently dysfunctional in patients with HNSCC. Strategies are needed to enhance NK cell responses against HNSCC. We hypothesized that memory-like (ML) NK cell differentiation, tumor targeting with cetuximab, and engineering with an anti-EphA2 (Erythropoietin-producing hepatocellular receptor A2) chimeric antigen receptor (CAR) enhance NK cell responses against HNSCC. We generated ML NK and conventional (c)NK cells from healthy donors, then evaluated their ability to produce IFNγ, TNF, degranulate, and kill HNSCC cell lines and primary HNSCC cells, alone or in combination with cetuximab, in vitro and in vivo using xenograft models. ML and cNK cells were engineered to express anti-EphA2 CAR-CD8A-41BB-CD3z, and functional responses were assessed in vitro against HNSCC cell lines and primary HNSCC tumor cells. Human ML NK cells displayed enhanced IFNγ and TNF production and both short- and long-term killing of HNSCC cell lines and primary targets, compared with cNK cells. These enhanced responses were further improved by cetuximab. Compared with controls, ML NK cells expressing anti-EphA2 CAR had increased IFNγ and cytotoxicity in response to EphA2+ cell lines and primary HNSCC targets. These preclinical findings demonstrate that ML differentiation alone or coupled with either cetuximab-directed targeting or EphA2 CAR engineering were effective against HNSCCs and provide the rationale for investigating these combination approaches in early phase clinical trials for patients with HNSCC.

Novel [ 111 In]In-BnDTPA-EphA2-230-1 Antibody for Single-Photon Emission Computed Tomography Imaging Tracer Targeting of EphA2.

Erythropoietin-producing hepatocellular receptor A2 (EphA2) is overexpressed in cancer cells and causes abnormal cell proliferation. Therefore, it has attracted attention as a target for diagnostic agents. In this study, the EphA2-230-1 monoclonal antibody (EphA2-230-1) was labeled with [111In]In and evaluated as an imaging tracer for single-photon emission computed tomography (SPECT) of EphA2. EphA2-230-1 was conjugated with 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-BnDTPA) and then labeled with [111In]In. [111In]In-BnDTPA-EphA2-230-1 was evaluated in cell-binding, biodistribution, and SPECT/computed tomography (CT) studies. The cellular uptake ratio of [111In]In-BnDTPA-EphA2-230-1 was 14.0 ± 2.1%/mg protein at 4 h in the cell-binding study. In the biodistribution study, a high uptake of [111In]In-BnDTPA-EphA2-230-1 was observed in tumor tissue (14.6 ± 3.2% injected dose/g at 72 h). The superior accumulation of [111In]In-BnDTPA-EphA2-230-1 in tumors was also confirmed using SPECT/CT. Therefore, [111In]In-BnDTPA-EphA2-230-1 has potential as a SPECT imaging tracer for EphA2.

MiR-10b-5p Suppresses the Proliferation and Invasion of Primary Hepatic Carcinoma Cells by Downregulating EphA2.

Objective To analyze the function of miR-10b-5p in suppressing the invasion and proliferation of primary hepatic carcinoma cells by downregulating erythropoietin-producing hepatocellular receptor A2 (EphA2). Material and Methods. Eighty-six hepatic carcinoma (HCC) tissue specimens and 86 corresponding adjacent tissue specimens were collected, and the mRNA expression of miR-10b-5p and Ephrin type-A receptor 2 (EphA2) in the specimens was determined using a reverse transcription-polymerase chain reaction (RT-PCR) assay. Western blot was employed to quantify EphA2, B-cell chronic lymphocytic leukemia/lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-3 in the cells, and CCK8, Transwell assay, and flow cytometry were applied to evaluate the proliferation, invasion, and apoptosis of cells, respectively. Moreover, the dual luciferase reporter assay was utilized for correlation analysis between miR-10b-5p and EphA2. Results miR-10b-5p was lowly expressed in HCC, while EphA2 was highly expressed. Cell experiments revealed that miR-10b-5p overexpression or EphA2 knockdown could reduce cell proliferation, accelerate apoptosis, strongly upregulate Bax and Caspase-3, and downregulate Bcl-2. In contrast, miR-10b-5p knockdown or EphA2 overexpression gave rise to reverse biological phenotypes. Furthermore, dual luciferase reporter assay verified that miR-10b-5p was a target of EphA2, and the rescue experiment implied that transfection of pCMV-EphA2 or Si-EphA2 could reverse EphA2 expression and cell biological functions caused by miR-10b-5p overexpression or knockdown. Conclusions miR-10b-5p reduced HCC cell proliferation but accelerate apoptosis by regulating EphA2, suggesting it has the potential to be a clinical target for HCC.

Radiomics-based MRI for predicting Erythropoietin-producing hepatocellular receptor A2 expression and tumor grade in brain diffuse gliomas.

EphA2 is a key factor underlying invasive propensity of gliomas, and is associated with poor prognosis of tumors. We aimed to develop a radiomics-based imaging index for predicting EphA2 expression in diffuse gliomas, and further estimating its value for grading of tumors. A total of 182 patients with diffuse gliomas were included. All subjects underwent pre-operative MRI and post-operative pathological diagnosis. EphA2 expression of tumors was scored on pathological sections with immunohistochemical staining using monoclonal EphA2 antibody. MRI radiomics features were extracted from three-dimensional contrast-enhanced T1-weighted imaging and diffusion kurtosis imaging. Predictive models were constructed using machine learning-based radiomics features selection and three classifiers for predicting EphA2 expression and tumor grade. Features of best EphA2 expression model were subsequently used to construct another model of tumor grading. For each model, 146 cases (80%) were randomly picked as training and the rest 36 (20%) were testing cohorts. EphA2 expression was further correlated to the radiomics features in both grade models using Spearman's correlation. Logistic regression model presented highest performance for predicting EphA2 expression (AUC: 0.836/0.724 in training/validation set). Tumor gradings model guided by features from EphA2 expression model demonstrated comparable performance (AUC: 0.930/0.983) to that constructed directly using imaging radiomics features (AUC: 0.960/0.977). Two radiomics features which included in both LR-grade models showed strong correlation (P < 0.05) with EphA2 expression. The expression of EphA2 in gliomas could be predicted by radiomics features extracted from diffusion kurtosis MRI, which could also be used to assist tumor grading.

Abstract 691: Antibody conjugated to a bispecific RNA molecule targeting RIG-I and PLK1

Abstract Simultaneous access to several targets has become the subject of intense studies in immuno-oncology. In order to benefit from the synergies provided by the activation of different signaling pathways in immunology and the knockdown of proteins involved in cancer cell survival, we established a bispecific approach. The activation of the innate immune response by delivering agonists of pattern-recognition receptors (PRR) such as RIG-I (retinoic acid-inducible I) represents a promising strategy. RIG-I detects short double-stranded RNA molecules ended by a 5'-di/triphosphate moiety (5'ppp-dsRNA). RIG-I activation promotes type I IFN secretion and cancer-cell selective apoptosis. To obtain a bifunctional molecule, the 5'ppp-dsRNA sequence was designed to silence PLK1 (polo-like kinase 1). Suppressing PLK1 expression with small interfering RNAs (siRNA) leads to cell cycle arrest and retards cancer cell growth. This concept of bifunctional RNAs has been validated by using non-targeted systems.1 To enhance this synergy, we conjugated this 5'ppp-siPLK1 to an antibody for a specific delivery to cancerous cells that overexpress erythropoietin-producing hepatocellular receptor A2 (EphA2) at their surface. Upon binding to EphA2 receptor, the antibody is well internalized, thus making it a good vehicle to deliver the bispecific 5'ppp-siPLK1. After EphA2-positive cells treatment, we observed RIG-I specific activation as well as PLK1 depletion. Both effects were correlated with cellular apoptosis and the mode of action was further confirmed with mechanistic and kinetic studies. Finally, while non-modified unconjugated siRNA has a very short half-life in plasma, we observed an increase in stability for the antibody-5'ppp-siPLK1 conjugates. These data suggest that anti-EphA2 receptor antibody could be used to deliver a bispecific RNA molecule.

Ginsenoside Rg5 inhibits cancer cell migration by inhibiting the nuclear factor-κB and erythropoietin-producing hepatocellular receptor A2 signaling pathways.

The majority of cancer-associated deaths are caused by cancer metastasis, the first step of which is the acquisition of migratory ability by cancer cells. Therefore, the suppression of cancer cell migration represents a potential efficient strategy to inhibit cancer metastasis. Inflammation induces cancer cell migration through the activation of nuclear factor-κB (NF-κB), which is a transcription factor that serves a central role in inflammatory signaling. Recent studies have demonstrated that the phosphorylation of the receptor tyrosine kinase erythropoietin-producing hepatocellular receptor A2 (EphA2) at S897 promotes cancer cell migration. Therefore, a compound with the ability to abolish these two factors may suppress cancer metastasis. In the present study, ginseng saponin ginsenoside Rg5 was found to inhibit the phosphorylation of NF-κB and EphA2. Therefore, this study aimed to elucidate the molecular mechanisms of ginsenoside Rg5 and determine whether it inhibited cancer cell migration. The results demonstrated that ginsenoside Rg5 inhibited the activation of NF-κB by suppressing its upstream kinase transforming growth factor β-activated kinase 1 in TNF-α treated HeLa or A549 cells compared with that in the untreated control group. Furthermore, ginsenoside Rg5 attenuated the expression of EphA2 by lysosomal degradation, which inhibited its phosphorylation. In addition, ginsenoside Rg5 suppressed inflammatory cytokine-induced cancer cell migration. In conclusion, the results of the present study provided a scientific basis for the development of ginsenoside Rg5 as a potential antimetastatic drug.

Identification of key genes and their functions in palbociclib-resistant breast carcinoma by using bioinformatics analysis

Background: Palbociclib resistance is a significant problem in breast carcinoma, and its underlying molecular mechanisms remain poorly understood. This study aims to elucidate the molecular mechanisms of palbociclib resistance and to identify the key genes and pathways mediating progesterone resistance in breast cancer (BC). Methods: Gene dataset GSE117743 was downloaded from the Gene Expression Omnibus (GEO) database, which included 3 palbociclib-resistant and 3 palbociclib-sensitive BC cell lines. Then, we calculated the differentially expressed genes (DEGs) by using R software. Gene ontology and Enriched pathway analysis of genes we identified were analyzed by using the Database for Database of Annotation Visualization and Integrated Discovery (DAVID) and R software. The protein-protein interaction network was performed according to Metascape, String, and Cytoscape software. Results: In total, 447 DEGs were selected, which consisted of 67 upregulated and 380 downregulated genes. According to gene ontology annotation, DEGs were associated with cytoplasm, signal transduction, and protein binding. The research of the Kyoto Encyclopedia of Genes and Genomes (KEGG) demonstrated that genes enriched in certain tumor pathways, including IL-17 signaling pathways and Herpes simplex infection signaling pathways. Also, certain hub genes were highlighted after constructed and analyzed the protein-protein interaction network, including α-2A adrenergic receptor, cytochrome P450 subfamily IIR polypeptide, Cystathionine β-synthase, nucleotide-binding oligomerization domain-containing, erythropoietin-producing hepatocellular receptor A2 and adrenomedullin, which may be related with BC prognosis. A total of 4 of 6 hub genes had a significant relationship with the overall survival (P&lt;0.05). Conclusions: Using microarray and bioinformatics analyses, we identified DEGs and determined a comprehensive gene network of progesterone resistance. We offered several possible mechanisms of progesterone resistance and identified therapeutic and prognostic targets of palbociclib resistance in BC.

Elevated Expression of EPHA2 Is Associated With Poor Prognosis After Radical Prostatectomy in Prostate Cancer.

Therapeutic targeting of receptor protein tyrosine kinases (PTKs) has proven successful in treating cancer. However, reports about PTKs in treating prostate cancer are few. Elevated expression of the erythropoietin-producing hepatocellular receptor A2 (EPHA2) receptor tyrosine kinase, a transmembrane protein, is associated with poor prognosis of certain cancer types when the enzyme is dephosphorylated. This study investigated whether EPHA2 is useful in predicting the biochemical recurrence of prostate cancer. Data from 241 patients who had undergone total prostatectomy between 2007 and 2011 were used. EPHA2 protein expression was categorized as high or low by two pathologists. The relationship was examined between EPHA2 expression level (high vs. low) and clinicopathological factors including biochemical recurrence. Correlations were examined between EPHA2, low-molecular-weight protein tyrosine phosphatase (LMW-PTP), E-cadherin, and Ki-67. EPHA2 expression was high in 121 (50.2%) and low in 120 (49.8%) patients. A log-rank test revealed early biochemical recurrence in the high-expression group. Gleason score, Ki-67 labeling index, and biochemical recurrence were more frequent in the high-expression group. Furthermore, multivariate analyses revealed that high EPHA2 expression was an independent prognostic factor for biochemical recurrence (hazard ratio=3.62, 95% confidence interval=2.39-5.61). Correlations between EPHA2 and both LMW-PTP and Ki-67 labeling index were positive, whereas EPHA2 and E-cadherin were negatively correlated. EPHA2 overexpression is predictive of aggressive prostate cancer behavior. EPHA2 may be a powerful prognostic biomarker for decision-making in postoperative follow-up after total prostatectomy, and regarding the need for palliative treatment. Additionally, it may be an important therapeutic target.

EphA2-to-YAP pathway drives gastric cancer growth and therapy resistance.

Yes-associated protein (YAP) is a transcriptional coactivator that promotes cell proliferation, stem cell maintenance and tissue homeostasis. The YAP activity is primarily regulated through an inhibitory phosphorylation by the serine/threonine kinases of Hippo pathway. Here, we show that receptor tyrosine kinase (RTK) erythropoietin-producing hepatocellular receptor A2 (EphA2) interacts with and phosphorylates YAP protein, leading to stabilization, nuclear translocation and activation of YAP in gastric cancer (GC) cells. EphA2 induces chemotherapy-resistance by increasing YAP stability and nuclear YAP protein. Knockdown of YAP blocks EphA2-induced tumor growth in GC xenograft mouse models. Importantly, the coactivation of EphA2 and YAP is manifested in clinical human GC, and is related to GC recurrence. Thus, our results establish a novel EphA2-to-YAP pathway that drives GC growth, progression and therapy-resistance, targeting this pathway would be an efficient way for the treatment of GC, particularly chemotherapy-resistant GC.

EPHA2 blockade reverses acquired resistance to afatinib induced by EPHA2-mediated MAPK pathway activation in gastric cancer cells and avatar mice.

Afatinib is a pan-HER inhibitor approved for specific types of lung cancer. We explored antitumor activity, predictive biomarkers and the potential mechanisms underlying antitumor effect and acquired resistance of afatinib in gastric cancer (GC) in vitro and in vivo. Five human GC cell lines and eight patient-derived xenograft (PDX) models with clear molecular profiling were used to evaluate the antitumor activity and mechanisms of afatinib. The ErbB family and downstream PI3K/AKT/mTOR and mitogen-activated protein kinase (MAPK) pathways were evaluated before and after afatinib treatment. An afatinib-resistant PDX model was established to explore both the potential mechanisms of drug resistance and reversal strategies. We found that afatinib exerted a strong tumor suppression in EGFR/HER2 highly amplified (copy number >6) or overexpressed (IHC 3+) PDX models and a moderate tumor suppression in EGFR/HER2 moderately expressed (IHC 2+) PDX models. Afatinib selectively inhibited the proliferation of HER2 highly amplified GC cells in a dose-dependent manner in vitro. Afatinib also exerted its antitumor effect by inducing cell apoptosis and cell arrest at G1 phase. Diminished activation of the ErbB family and downstream PI3K/AKT/mTOR and MAPK pathways was also observed. Erythropoietin-producing hepatocellular receptor A2 (EPHA2) upregulation and phosphorylation might be involved in afatinib-acquired resistance, and EPHA2 blockade could restore afatinib sensitivity. GC patients with amplification (copy number >6) or overexpression (IHC 3+) of EGFR/HER2 were most likely to benefit from afatinib treatment and EPHA2 blockade reversed acquired resistance to afatinib treatment, which could provide solid evidences for future clinical trials.

Dose escalation and expansion cohort study for DS-8895a in patients with advanced solid tumors.

99 Background: Erythropoietin-producing hepatocellular receptor A2 (EPHA2) is overexpressed on the cell surface of many tumors and is associated with poor prognosis, suggesting EPHA2 as a target for cancer therapy. DS-8895a is an afucosylated, humanized anti-EPHA2 IgG1 monoclonal antibody with potent cytotoxicity. We report results from a phase I clinical trial to determine safety, tolerability, and pharmacokinetics (PK) of DS-8895a in Japanese patients with advanced solid tumors (NCT02004717). Methods: Step 1 (dose escalation cohort) had patients with advanced solid tumors and comprised of six dose levels (0.1–20 mg/mL, intravenous infusion, every 2 weeks [Q2W]) with a 28-day dose limiting toxicity (DLT) evaluation period. Step 2 (expansion cohort) patients had gastric or esophageal cancer confirmed to be EPHA2 positive by immunohistochemistry. Dose level in Step 2 was determined based on results obtained in Step 1. We evaluated safety, PK, potential biomarkers including circulating NK cells and cytokines, and tumor response. Results: Maximum tolerated dose was not reached in Step 1 (n = 22). DS-8895a was administered at 20 mg/kg Q2W in Step 2 (n = 15). Among 37 patients in the safety analysis set, adverse events (AEs) were reported in 97.3% (64.9% drug-related); 35.1% presented grade ≥ 3 AEs (8.1% drug-related). Dose delay and study discontinuation due to AEs (treatment related: grade 4 platelet decrease, hypoesthesia, hypotension, peripheral coldness, nausea, and vomiting) were observed in one and four patients (20 mg/kg), respectively. Infusion-related reactions occurred in 51.4% of patients resulting in 10 dose interruptions with one discontinuation. Serum inflammatory cytokines were transiently increased 4 h from the end of infusion drug administration. Serum DS-8895a maximum and trough concentrations increased dose-dependently. Biomarkers had no apparent relationship to best overall response. Seven patients in Step 1 achieved stable disease; in Step 2, six patients achieved stable disease and one patient achieved partial response. Conclusions: DS-8895a was safe and well tolerated up to 20 mg/kg. The PK of DS-8895a was dose-dependent as expected. Clinical trial information: NCT02004717.

Dysregulated erythropoietin-producing hepatocellular receptor A2 (EphA2) is involved in tubal pregnancy via regulating cell adhesion of the Fallopian tube epithelial cells

BackgroundTyrosine kinase receptor erythropoietin-producing hepatocellular receptor A2 (EphA2) is abundant in the endometrium and plays a role in the establishment of eutopic implantation. A similar molecular mechanism may exist between uterine implantation and tubal implantation, therefore EphA2 involvement in tubal pregnancy is suspected. Due to the limited availability of human Fallopian tube specimens, EphA2 expression in human Fallopian tube epithelium remains largely unknown.MethodsA total of 31 women with tubal pregnancy and 41 non-pregnant women with benign uterine diseases were enrolled in this study. Immunohistochemistry was used to investigate the expression pattern of EphA2 in the Fallopian tube epithelium of non-pregnant women (n = 29) and women with tubal pregnancy (n = 17). The changes of EphA2 and its activated form, phosphorylated-EphA2 (Pho-EphA2), in the Fallopian tube epithelium from non-pregnant women (n = 12) and women with tubal pregnancy (n = 14) were compared by quantitative RT-PCR and western blot assay.ResultsEphA2 was expressed throughout the Fallopian tube epithelium, including the isthmus, the ampulla and the infundibulum. EphA2 concentration remained unchanged throughout the whole menstrual cycle, irrespective of menstrual phases and tubal regions. EphA2 mRNA in the Fallopian tube epithelium did not differ between normal women and women with tubal pregnancy (P > 0.05). With respect to the protein level, a significantly higher ratio of EphA2 over Pho-EphA2 was shown in women with tubal pregnancy (P < 0.05).ConclusionsEphA2 is widely expressed in human Fallopian tube epithelium in a temporospatial-independent manner. Dysregulated EphA2 and its phosphorylation-dependent regulatory mechanism may unexpectedly enhance the cell adhesion activity of the Fallopian tube epithelial cells, leading to a mis-contact between the Fallopian tube epithelium and the embryo.

EphA2 affects the sensitivity of oxaliplatin by inducing EMT in oxaliplatin-resistant gastric cancer cells.

Erythropoietin-producing hepatocellular receptor A2 (EphA2) is upregulated in gastric cancer tissues and cells, which is accompanied by epithelial–mesenchymal transition (EMT). The current study was designed to establish the oxaliplatin-resistant human gastric cancer cell line SGC-7901/L-OHP, to determine if EMT in these cells could be reversed, and to determine if the susceptibility of these cells to oxaliplatin was affected by silencing EphA2 expression. We found that EphA2 expression levels were upregulated in gastric cancer and associated with chemotherapy sensitivity. EphA2 and the EMT molecular markers N-cadherin and Snail were upregulated in SGC-7901/L-OHP cells, while silencing of EphA2 using small interfering RNA had the opposite effect. Moreover, silencing of EphA2 inhibited cell migration and invasion, and significantly enhanced the sensitivity of oxaliplatin-resistant gastric cancer cells to oxaliplatin. These observations demonstrate that EphA2 affects the sensitivity to oxaliplatin by inducing EMT in oxaliplatin-resistant gastric cancer cells.

Non-canonical Activation of Receptor Tyrosine Kinases in Cancer Progression.

Receptor tyrosine kinases (RTKs) are known to be key regulators of cancer cell proliferation, migration, invasion and metastatic spread. Ligand-binding to the extracellular domain triggers canonical activation of the intracellular tyrosine kinase domain. In contrast, it has become evident that RTKs are also regulated by non-canonical tyrosine kinase-independent mechanisms via phosphorylation of their serine/threonine residues. In this review, I mainly introduce our recent findings on the non-canonical regulation of epidermal growth factor receptor (EGFR), ErbB2 and erythropoietin-producing hepatocellular receptor A2 (EphA2), and discuss the roles of non-canonical activation of RTKs in cancer progression and resistance to targeted cancer agents. Further characterization of non-canonical regulation will contribute to the development of new target cancer therapies.

Emerging and Diverse Functions of the EphA2 Noncanonical Pathway in Cancer Progression.

Erythropoietin-producing hepatocellular receptor A2 (EphA2) receptor tyrosine kinase controls multiple physiological processes to maintain homeostasis in normal cells. In many types of solid tumors, it has been reported that EphA2 is overexpressed and plays a critical role in oncogenic signaling. However, in recent years, the opposing functions of EphA2 have been explained by the canonical and noncanonical signaling pathways. Ligand- and tyrosine kinase-dependent EphA2 activation (the canonical pathway) inhibits cancer cell proliferation and motility. In contrast, ligand- and tyrosine kinase-independent EphA2 signaling (the noncanonical pathway) promotes tumor survival and metastasis and controls acquired drug resistance and maintenance of cancer stem cell-like properties. Evidence has accumulated showing that the EphA2 noncanonical pathway is mainly regulated by inflammatory cytokines and growth factors via phosphorylation at Ser-897 in the intracellular C-tail region via some serine/threonine kinases, including p90 ribosomal S6 kinase. In this review, we focus on the regulation of Ser-897 phosphorylation and its functional importance in tumor malignancy and discuss future therapeutic targeting.

Inhibition of BRAF and BRAF+MEK drives a metastatic switch in melanoma

Recent analyses by our group and others showed that the majority of melanoma patients who fail BRAF inhibitor therapy do so at new disease sites. Using phosphoproteomics we showed that BRAF inhibition mediates a switch to an aggressive/metastatic melanoma phenotype that is driven by ligand-independent erythropoietin-producing hepatocellular receptor A2 (EphA2) signaling.