The receptor tyrosine kinase EphB4 is overexpressed in ovarian cancer, provides survival signals and predicts poor outcome

EphB4 is a member of the largest family of transmembrane receptor tyrosine kinases and plays critical roles in axonal pathfinding and blood vessel maturation. We wanted to determine the biological role of EphB4 in ovarian cancer. We studied the expression of EphB4 in seven normal ovarian specimens and 85 invasive ovarian carcinomas by immunohistochemistry. EphB4 expression was largely absent in normal ovarian surface epithelium, but was expressed in 86% of ovarian cancers. EphB4 expression was significantly associated with advanced stage of disease and the presence of ascites. Overexpression of EphB4 predicted poor survival in both univariate and multivariate analyses. We also studied the biological significance of EphB4 expression in ovarian tumour cells lines in vitro and in vivo. All five malignant ovarian tumour cell lines tested expressed higher levels of EphB4 compared with the two benign cell lines. Treatment of malignant, but not benign, ovarian tumour cell lines with progesterone, but not oestrogen, led to a 90% reduction in EphB4 levels that was associated with 50% reduction in cell survival. Inhibition of EphB4 expression by specific siRNA or antisense oligonucleotides significantly inhibited tumour cell viability by inducing apoptosis via activation of caspase-8, and also inhibited tumour cell invasion and migration. Furthermore, EphB4 antisense significantly inhibited growth of ovarian tumour xenografts and tumour microvasculature in vivo. Inhibition of EphB4 may hence have prognostic and therapeutic utility in ovarian carcinoma.     

Expression of receptor tyrosine kinase EphB4 and its ligand ephrin-B2 is associated with malignant potential in endometrial cancer

The protein kinases includes many oncogenes and growth-factor receptors, as well as genes that are involved in cell cycle regulation. EphB4 receptors are a subfamily of receptor tyrosine kinases that are activated by ephrin-B2 ligands and are thought to play an important role in the development and oncogenesis of various tissues. However, very little experimental evidence exists to support this hypothesis. To elucidate the involvement of EphB4 and ephrin-B2 in endometrial carcinogenesis, we used fluorescent immunohistochemistry to analyze serial frozen sections of 20 endometrial carcinomas and 20 normal endometria for EphB4 and ephrin-B2 protein expression. We analyzed the relationship between the patient's characteristics and the percentages of EphB4- and ephrin-B2-stained cells. EphB4 expression was significantly associated with histological grade (p < 0.001) and certain clinical stages. Ephrin-B2 Expression was significantly associated with the presence of invasion to > 1/2 myometrium (p = 0.002). Our results demonstrate that increased EphB4 and ephrin-B2 expression may reflect or induce in endometrial carcinomas increased potential for growth and tumorigenicity. Furthermore, these results suggest that EphB4 receptor kinase may modulate the biological behavior of endometrial carcinomas through autocrine and/or paracrine activation, which is caused by ephrin-B2 ligands that are expressed in the same or neighbouring cells by immunohistochemistry.     

The receptor tyrosine kinase Tie1 is expressed and activated in epithelial tumour cell lines

The receptor tyrosine kinase Tie1 is expressed primarily in vascular endothelial cells. The receptor has also been detected in epithelial tumours in breast, thyroid and gastric cancers and in tumour cell lines where it appears as a 45 kDa truncated receptor fragment. In this study, we show that in addition to truncated Tie1, breast and colon tumour cell lines express a full-length Tie1 holoreceptor. In contrast to the situation in endothelial cells, Tie1 truncation is not activated by phorbol esters and generation of truncated Tie1 does not occur via a metalloprotease-inhibitor sensitive mechanism. Examination of the phosphorylation status of Tie1 revealed both the holoreceptor and truncated receptor to be constitutively activated in MCF-7 cells. These data indicate that Tie1 expressed in epithelial tumour cell lines is present in holoreceptor and truncated forms, and in MCF-7 cells both forms are constitutively phosphorylated and competent to signal. Our findings suggest therefore that anti-angiogenic strategies targeting the angiopoietin/Tie system in tumour microvasculature could also have additional direct effects on the tumour epithelial cells within those tumours in which there is also extravascular expression of the Tie1 receptor tyrosine kinase.     

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

Background  

The EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported.     

EphB4 provides survival advantage to squamous cell carcinoma of the head and neck

The receptor tyrosine kinase EphB4 and its ligand EphrinB2 play critical roles in blood vessel maturation, and are frequently overexpressed in a wide variety of cancers. We studied the aberrant expression and biological role of EphB4 in head and neck squamous cell carcinoma (HNSCC). We tested the effect of EphB4-specific siRNA and antisense oligonucleotides (AS-ODN) on cell growth, migration and invasion, and the effect of EphB4 AS-ODN on tumor growth in vivo. All HNSCC tumor samples express EphB4 and levels of expression correlate directly with higher stage and lymph node metastasis. Six of 7 (86%) HNSCC cell lines express EphB4, which is induced either by EGFR activation or by EPHB4 gene amplification. EphrinB2 was expressed in 65% tumors and 5 of 7 (71%) cell lines. EphB4 provides survival advantage to tumor cells in that EphB4 siRNA and AS-ODN significantly inhibit tumor cell viability, induce apoptosis, activate caspase-8, and sensitize cells to TRAIL-induced cell death. Furthermore, EphB4-specific AS-ODN significantly inhibits the growth of HNSCC tumor xenografts in vivo. Expression of EphB4 in HNSCC tumor cells confers survival and invasive properties, and thereby provides a strong rationale for targeting EphB4 as novel therapy for HNSCC.     

受体酪氨酸激酶EphB6在胃癌中的表达及其临床意义#br# #br#

目的 探讨受体酪氨酸激酶EphB6在胃癌组织中的表达及与临床病理特征的关系。方法 采用免疫组织化学技术检测152例胃癌和癌旁组织中EphB6蛋白的表达,并分析其表达与胃癌临床病理特征的关系。结果 152例胃癌组织中, EphB6低表达66例（43.4％）,中等强度表达54例（35.5％）,高表达32例（21.1％）;EphB6在癌旁组织中呈高表达。EphB6蛋白表达与分化程度、淋巴结转移及TNM分期有关（P＜0.05）,而与年龄、性别、肿瘤部位和浸润深度均无关（P＞0.05）。结论EphB6在胃癌组织中表达下调,且与淋巴结转移有关,其可能是预测胃癌转移的潜在指标。     

FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines.

The fms-like tyrosine kinase 4 (FLT4) complementary DNA was cloned from a human HEL erythroleukemia cell library by polymerase chain reaction-amplification. We previously reported a partial sequence of FLT4 and showed that the FLT4 gene maps to chromosomal region 5q33-qter (O. Aprelikova, K. Pajusola, J. Partanen, E. Armstrong, R. Alitalo, S. Bailey, J. McMahon, J. Wasmuth, K. Huebner, and K. Alitalo, Cancer Res., 52: 746-748, 1992). Here we present the full-length sequence of the predicted FLT4 protein. The extracellular domain of FLT4 consists of 7 immunoglobulin-like loops, including 12 potential glycosylation sites. On the basis of structural similarities FLT4 and the previously known FLT1 and kinase insert domain-containing receptor tyrosine kinase/fetal liver kinase 1 (KDR/FLK1) receptors constitute a subfamily of class III tyrosine kinases. FLT4 was expressed as 5.8- and 4.5-kilobase mRNAs which were found to differ in their 3' sequences and to be differentially expressed in the HEL and DAMI leukemia cells. Interestingly, a Wilms' tumor cell line, a retinoblastoma cell line, and a nondifferentiated teratocarcinoma cell line expressed FLT4, whereas differentiated teratocarcinoma cells were negative. Most fetal tissues also expressed the FLT4 mRNA, with spleen, brain intermediate zone, and lung showing the highest levels. In in situ hybridization the FLT4 autoradiographic grains decorated bronchial epithelial cells of fetal lung. No evidence was obtained for the expression of FLT4 in the endothelial cells of blood vessels.     

Activation of the receptor protein tyrosine kinase EphB4 in endometrial hyperplasia and endometrial carcinoma.

BACKGROUND: Members of the Eph family of tyrosine kinases have been implicated in embryonic pattern formation and vascular development; however, little is known about their role in the adult organism. We have observed estrogen-dependent EphB4 expression in the normal breast suggesting its implication in the hormone-controlled homeostasis of this organ. Since the endometrium is a similarly hormone dependent organ and endometrial carcinoma is thought to result from estrogenic stimulation, we have investigated EphB4 expression in normal human endometrium and during its carcinogenesis. PATIENTS AND METHODS: EphB4 expression was analyzed immunohistochemically in 26 normal endometrium specimens, 15 hyperplasias and 102 endometrioid adenocarcinomas and correlated with clinical and prognostic tumor characteristics. RESULTS: In normal endometrial tissue no EphB4 protein was detected. Strikingly, we observed a drastic increase (P <0.0001) in the number of EphB4 protein-expressing glandular epithelial cells in the majority of hyperplasias and carcinomas. Moreover, we found a statistically highly significant positive correlation between EphB4 expression and post-menopausal stage of the patient (P = 0.007). CONCLUSIONS: These findings indicate that in the endometrium, EphB4 is an early indicator of malignant development and, thus, EphB4 may represent a potent tool for diagnosis and therapeutic intervention.     

Loss of EphB4 receptor tyrosine kinase protein expression during carcinogenesis of the human breast

Members of the Eph family of receptor tyrosine kinase have been implicated in cell-cell communication and tissue integrity during embryogenesis. We have previously demonstrated cell type specific and hormone dependent EphB4 expression in the mouse mammary parenchyma suggesting involvement in the homeostasis of this organ. Since disruption of tissue organization is crucial for metastatic dissemination, we have investigated the expression of EphB4 during carcinogenesis of the human breast. Immunohistochemical analysis of 24 normal human breast samples and 124 consecutive breast carcinomas was correlated with tumor characteristics (stage, histology, grade, lymph node involvement) and the expression of ER, PR, Ki-67, p53 and HER2. In normal breast tissue, the EphB4 protein was expressed exclusively in parenchymal cells. Strikingly, a drastic reduction in the number of EphB4 protein expressing cells was observed in almost all invasive carcinomas analyzed, irrespective of the tumor type (p<0.0001). Furthermore, we found a highly significant correlation between EphB4 positivity and low histological grading of the tumor cells (p=0.002) suggesting that in breast cancer, EphB4 expression is not compatible with tumor progression. This raises the possibility that EphB4 could represent a potent tool for therapeutic intervention.     

The RON receptor tyrosine kinase mediates oncogenic phenotypes in pancreatic cancer cells and is increasingly expressed during pancreatic cancer progression

Pancreatic cancer is an aggressive disease characterized by rapid growth and early metastasis. The recepteur d'origine nantais (RON) receptor tyrosine kinase is overexpressed and/or constitutively active in several epithelial cancers, but its role in pancreatic cancer is unknown. In this study, we have characterized RON expression in both murine and human pancreatic cancer. Immunoblotting indicates that RON is expressed in pancreatic intraepithelial neoplasia (PanIN), primary, and metastatic cell lines both in the human and mouse. Immunostaining revealed that 93% of high-grade PanIN, 79% of primary, and 83% of metastatic lesions from human pancreatic tissue samples expressed RON, with minimal expression in normal ducts and low-grade PanIN (6% and 18%, respectively). Moreover, we show a dose-dependent effect of hepatocyte growth factor-like protein (HGFL), the RON-specific ligand, on pancreatic cancer cell migration and invasion, which was reversed by RON inhibition. Although stimulation with HGFL had no effect on proliferation, concurrent RON receptor blockade and gemcitabine treatment increased apoptosis of RON-expressing pancreatic cancer cells versus gemcitabine treatment alone. Finally, HGFL stimulation of pancreatic cancer cells resulted in increased expression of phospho-mitogen-activated protein kinase and phospho-Akt. Taken together, these findings suggest that RON receptor signaling may contribute to pancreatic carcinogenesis, and that further investigation is warranted to assess the potential of RON-directed therapies in this deadly disease.     

Anti-tumour effects of antibodies targeting the extracellular cysteine-rich region of the receptor tyrosine kinase EphB4

EphB4 is a membrane-bound receptor tyrosine kinase (RTK) commonly over-produced by many epithelial cancers but with low to no expression in most normal adult tissues. EphB4 over-production promotes ligand-independent signaling pathways that increase cancer cell viability and stimulate migration and invasion. Several studies have shown that normal ligand-dependent signaling is tumour suppressive and therefore novel therapeutics which block the tumour promoting ligand-independent signaling and/or stimulate tumour suppressive ligand-dependent signaling will find application in the treatment of cancer. An EphB4-specific polyclonal antibody, targeting a region of 200 amino acids in the extracellular portion of EphB4, showed potent in vitro anti-cancer effects measured by an increase in apoptosis and a decrease in anchorage independent growth. Peptide exclusion was used to identify the epitope targeted by this antibody within the cysteine-rich region of the EphB4 protein, a sequence defined as a potential ligand interacting interface. Addition of antibody to cancer cells resulted in phosphorylation and subsequent degradation of the EphB4 protein, suggesting a mechanism that is ligand mimetic and tumour suppressive. A monoclonal antibody which specifically targets this identified extracellular epitope of EphB4 significantly reduced breast cancer xenograft growth in vivo confirming that EphB4 is a useful target for ligand-mimicking antibody-based anti-cancer therapies.     

Paradoxes of the EphB4 receptor in cancer

Recent findings have started to uncover the intriguing roles of the Eph family of receptor tyrosine kinases in normal epithelial cells and during oncogenic transformation. This review focuses on EphB4, an Eph receptor that has both tumor-suppressing and tumor-promoting activities in breast cancer. Understanding the multifaceted role of EphB4 in tumorigenesis may allow the development of new anticancer therapies.     

OCT-4, an embryonic stem cell marker, is highly expressed in bladder cancer

OCT-4 (also known as POU5F1) is a key regulator of self-renewal in embryonic stem cells. Regarding the new cancer stem cell concept, the expression of such genes is potentially correlated with tumorigenesis and can affect some aspects of tumor behavior, such as tumor recurrence or resistance to therapies. Although OCT-4 has been introduced as a molecular marker for germ cell tumors, little is known about its expression in somatic cancers. Here, we have investigated the potential expression of OCT-4 in bladder cancer. We used semiquantitative RT-PCR to examine the expression of OCT-4 in 32 tumors, 13 apparently nontumor tissues taken from the margin of tumors and 9 normal urothelial tissues. The expression of OCT-4 at protein level was further determined by Western blotting and immunohistochemical (IHC) analysis. OCT-4 expression was detected in almost all examined tumors (31/32), but at much lower level (p<0.001) in some nonneoplastic samples (6/22). A significantly strong correlation of 0.6 has been observed between OCT-4 expression and the presence of tumors (p<0.001). Western blot analysis further confirmed the expression of OCT-4 in tumor biopsies. According to IHC results, OCT-4 is primarily localized in the nuclei of tumor cells, with no or low immunoreactivity in nontumor cells. Our study demonstrated, for the first time, the expression of OCT-4 in bladder cancer and a further clue to the involvement of embryonic genes in carcinogenesis.     

Epidermal growth factor receptor tyrosine kinase inhibitors for non-small cell lung cancer

First-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib and erlotinib, have proven to be highly effective agents for advanced non-small cell lung cancer (NSCLC) in patients harboring an activating EGFR mutation such as the exon 19 deletion mutation and L858R. Although those reversible small molecular targeted agents provide a significant response and survival benefit, all responders eventually acquire resistance. Second-generation EGFR-targeting agents, such as irreversible EGFR/HER2 tyrosine kinase inhibitors and pan-HER TKIs, may improve survival further and be useful for patients who acquired resistance to first-generation EGFR-TKIs. This review discusses novel therapeutic strategies for EGFR-mutated advanced NSCLC using first- and second-generation EGFR-TKIs.     

Expression of the receptor tyrosine kinase Axl promotes ocular melanoma cell survival

Metastatic tumor cells originating from cancers of a variety of tissues such as breast, skin, and prostate may remain dormant for long periods of time. In the case of uveal melanoma, the principal malignancy of the eye, complete removal of the primary tumor by enucleation can nonetheless be followed by metastatic tumor growth in distant organs months, years, or even decades later. This suggests that tumor cells have already spread to secondary sites at the time of treatment and remain dormant as micrometastases. Identifying factors that govern long-lived survival of metastatic tumor cells is therefore key to decreasing mortality associated with this and other diseases. While investigating factors differentially expressed in melanoma cells and normal melanocytes, we identified the receptor tyrosine kinase Axl and found up-regulation of Axl in uveal melanomas and melanoma cell lines by RNase protection, Western analysis, and immunohistochemistry. Axl has been shown to mediate cell growth and survival through its ligand Gas6 in non-transformed cells. To test whether stimulation of Axl can enhance survival of uveal melanoma cells, we assessed the degree of mitogenesis and cell survival by bromodeoxyuridine incorporation and trypan blue exclusion, respectively, upon stimulation of Mel 290 uveal melanoma cells with Gas6 in vitro. We show that Gas6 mediates mitogenesis and cell survival in Mel 290 cells. We further demonstrate that these effects occur specifically through the Axl receptor by modulating the expression of Axl with an antisense construct. cDNA microarray analysis of 12,687 genes then revealed that Gas6 stimulation of Axl in Mel 290 cells results primarily in the down-regulation of Cyr61, a member of the CCN protein family involved in tumor progression. These data show that the Axl pathway mediates increased survival of uveal melanoma cells, potentially advantageous during cancer dormancy, and that Axl may function in part through regulation of Cyr61.     

受体酪氨酸激酶EphB6在卵巢浆液性癌中的表达及其意义

目的 探讨受体酪氨酸激酶EphB6在卵巢浆液性癌中的表达及其与临床病理特征的关系。方法 采用免疫组织化学染色检测EphB6蛋白在55例卵巢浆液性癌、24例卵巢良性肿瘤、37例卵巢交界性肿瘤和20例正常输卵管组织蜡块中的表达情况,并分析EphB6蛋白表达与卵巢癌临床病理特征及预后之间的相关性。结果 EphB6蛋白在100.0％（20／20）正常输卵管、100.0％（24／24）卵巢良性肿瘤、78.4％（29／37）卵巢交界性肿瘤和18.2％（10／55）卵巢浆液性癌组织中高表达（P＜0.001）。EphB6蛋白表达水平与肿瘤分化程度、TNM分期及Ki 67表达均有关（P＜0.05）。EphB6高表达组和EphB6中、低表达组的中位生存期（OS）分别为103个月和69个月（95％CI：26～105个月）,差异有统计学意义（P=0.046）。结论 EphB6将有可能成为一种新的鉴别卵巢肿瘤高、低级别的标志物和预测预后的新指标。     

受体酪氨酸激酶EphA1mRNA在膀胱癌中的表达及临床意义

背景与目的:EphA1基因是受体酪氨酸激酶家族的成员之一,其过表达可能在恶性肿瘤的发生、发展中起重要的作用.本研究旨在探讨受体酪氨酸激酶EphA1 mRNA在膀胱痛中的表达及其临床意义.方法:采用实时荧光定量PCR法检测50例膀胱痛组织和15例相应癌旁组织中EphA1 mRNA的表达,分析基因表达的差异与临床病理特征间的关系.结果:EphA1 mRNA在膀胱痛中的表达量为106.86±30.40,高于癌旁组织的表达量10.37±2.69,差异有显著性统计学意义(P<0.001);EphA1 mRNA在高级别膀胱癌中的表达量为210.94±50.51,高于低级别膀胱癌组的52.64±16.01(P(0.001),肌层浸润性膀胱痛EphA1 mRNA的表达量为174.69±43.57,显著高于非肌层浸润性膀胱癌的63.14±17.60(P=0.001).EphA1 mRNA的表达与性别、年龄、肿瘤大小、数日无显著相关性(P>0.05).结论:EphA1 mRNA的表达在肿瘤组织中明显高于正常组织,且与病理分级、临床分期呈正相关,因此EphA1基因的过表达可能促进膀胱癌的进展.     

Axl receptor tyrosine kinase is up-regulated in metformin resistant prostate cancer cells

Recent epidemiological studies showed that metformin, a widely used anti-diabetic drug might prevent certain cancers. Metformin also has an anti-proliferative effect in preclinical studies of both hematologic malignancies as well as solid cancers and clinical studies testing metformin as an anti-cancer drug are in progress. However, all cancer types do not respond to metformin with the same effectiveness or acquire resistance. To understand the mechanism of acquired resistance and possibly its mechanism of action as an anti-proliferative agent, we developed metformin resistant LNCaP prostate cancer cells. Metformin resistant LNCaP cells had an increased proliferation rate, increased migration and invasion ability as compared to the parental cells, and expressed markers of epithelial-mesenchymal transition (EMT). A detailed gene expression microarray comparing the resistant cells to the wild type cells revealed that Edil2, Ereg, Axl, Anax2, CD44 and Anax3 were the top up-regulated genes and calbindin 2 and TPTE (transmembrane phosphatase with tensin homology) and IGF1R were down regulated. We focused on Axl, a receptor tyrosine kinase that has been shown to be up regulated in several drug resistance cancers. Here, we show that the metformin resistant cell line as well as castrate resistant cell lines that over express Axl were more resistant to metformin, as well as to taxotere compared to androgen sensitive LNCaP and CWR22 cells that do not overexpress Axl. Forced overexpression of Axl in LNCaP cells decreased metformin and taxotere sensitivity and knockdown of Axl in resistant cells increased sensitivity to these drugs. Inhibition of Axl activity by R428, a small molecule Axl kinase inhibitor, sensitized metformin resistant cells that overexpressed Axl to metformin. Inhibitors of Axl may enhance tumor responses to metformin and other chemotherapy in cancers that over express Axl.     

The RON receptor tyrosine kinase promotes MSP-independent cell spreading and survival in breast epithelial cells

The recepteur d'origine nantais (RON) is a receptor tyrosine kinase (RTK) in the scatter factor family, which includes the c-Met receptor. RON exhibits increased expression in a significant number of human breast cancer tissues as well as in many established breast cancer cell lines. Recent studies have indicated that in addition to ligand-dependent signaling events, RON also promotes signals in the absence of its only known ligand, MSP, when expressed in epithelial cells. In this study, we found that when expressed in MCF-10A breast epithelial cells, RON exhibits both MSP-dependent and MSP-independent signaling, which lead to distinct biological outcomes. In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors. However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells. Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases. These data suggest that RON has two alternative modes of signaling that can contribute to oncogenic behavior in normal breast epithelial cells.