Lithocholic Acid Is an Eph-ephrin Ligand Interfering with Eph-kinase Activation

Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or de-regulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumour phenotype. In particular EphA2 upregulation is correlated with tumour stage and progression and the expression of EphA2 in non-trasformed cells induces malignant transformation and confers tumorigenic potential. Based on these evidences our aim was to identify small molecules able to modulate EphA2-ephrinA1 activity through an ELISA-based binding screening. We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 interaction (Ki = 49 µM). Since each ephrin binds many Eph receptors, also LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Structurally related bile acids neither inhibited Eph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibited EphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate and colon adenocarcinoma cell lines (IC50 = 48 and 66 µM, respectively) without affecting cell viability or other receptor tyrosine-kinase (EGFR, VEGFR, IGFR1β, IRKβ) activity. LCA did not inhibit the enzymatic kinase activity of EphA2 at 100 µM (LANCE method) confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells. In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, as ephrin signalling is a key player in the intestinal cell renewal, our work could provide an interesting starting point for further investigations about the role of LCA in the intestinal homeostasis

Disease stage variation in CD4+ and CD8+ T-cell reactivity to the receptor tyrosine kinase EphA2 in patients with renal cell carcinoma.

We have evaluated CD8+ and CD4+ T-cell responses against a new tumor-associated antigen, the receptor tyrosine kinase EphA2, which is broadly expressed in diverse cancer histologies and is frequently overexpressed in advanced stage/metastatic disease. We report herein that EphA2 is overexpressed in renal cell carcinoma (RCC) cell lines and clinical specimens of RCC, and rind that the highest levels of EphA2 are consistently found in the most advanced stages of the disease. We identified and synthesized five putative HLA class I-binding and three class II-binding peptides derived from EphA2 that might serve as targets for immune reactivity. Each peptide induced specific, tumor-reactive CD8+ or CD4+T-cell responses as measured using IFN-gamma enzyme-linked immunospot assays. The EphA2 peptides elicited relatively weak responses from CD8+ T cells derived from normal healthy volunteers or from RCC patients with active disease. In marked contrast, immune reactivity to EphA2-derived epitopes was greatly enhanced in CD8+ T cells that had been isolated from patients who were rendered disease-free, after surgery. Furthermore, enzyme-linked immunospot analyses demonstrated prominent EphA2-restricted T-helper 1-type CD4+ T cell activity in patients with early stage disease, whereas T-helper 2-type and T regulatory-type responses predominated in patients with more advanced forms of RCC. These data suggest that the immune system of cancer patients actively monitors EphA2-derived epitopes, and that the magnitude and character of T-cell responses to EphA2 epitopes may convey much-needed predictive information about disease stage and outcome

Claudin-4 controls the receptor tyrosine kinase EphA2 pro-oncogenic switch through β-catenin.

BackgroundThe EphA2 receptor, which is expressed in many types of cancer, is activated by two different mechanisms. Activation by engagement with one of its ephrin ligands is anti-oncogenic whereas phosphorylation of S897 by AKT increases migration, invasion and metastasis. Down-regulation of claudin-4 (CLDN4) produces a loss of E-cadherin and increased β-catenin signaling and a phenotype similar to that produced by oncogenic activation of EphA2, suggesting that CLDN4 may serve to restrain the pro-oncogenic signaling of EphA2.ResultsWe found that constitutive knockdown of CLDN4 was associated with a 4.5-fold increase in EphA2 mRNA and a 2.5-fold increase in EphA2 protein which was reversible by re-expression of CLDN4. Knockdown of EphA2 blocked the migratory phenotype induced by loss of CLDN4. Knockdown of CLDN4 resulted in a 5.8-fold increase in pEphA(S897), the oncogenic form of the receptor, as well as partial mislocalization of the excess EphA2 to the interior of the cell. Forced expression of E-cadherin did not reduce total EphA2 or pEphA(S897) whereas re-expression of CLDN4 restored localization and reduced EphA2 and pEphA(S897) even in cells not expressing E-cadherin. Transient siRNA-mediated knockdown of EphA2 and β-catenin, and inhibition of PI3K by LY294002, demonstrated that increased pEphA(S897) in the CLDN4 knockdown cells was attributable to an increase in the level of active dephospho-β-catenin upstream of PI3K and AKT.ConclusionsWe conclude that CLDN4 serves to restrain pro-oncogenic signaling from EphA2 by limiting the activity of β-catenin and PI3K and preventing phosphorylation of EphA2 on S897 by AKT. This suggests that interventions directed at enhancing the level or functional activity of CLDN4 may be of therapeutic interest