THE ROLE OF EPHS AND EPHRINS IN MELANOMA METASTASIS AND AS POTENTIAL TARGETS FOR IMMUNOTHERAPY

During development, Eph receptors and their ephrin ligands are expressed in a temporally and spatially restricted manner, governing the migration and positioning of cells, performing critical developmental roles including the formation of the vascular system. Ephs are expressed at far lower levels in adult tissues, and are ectopically overexpressed in a variety of malignancies where they consistently correlate with increased tumourigenesis. In this context, they are proposed to mediate metastasis, the most insidious aspect of cancer. Cytokines within the tumour microenvironment may facilitate tumourigenesis. The ability of cytokines to regulate the expression of Ephs and ephrins suggests there may be an underlying pathway linking these three factors. Further, the aberrant expression of Eph and ephrins in neoplastic tissues, coupled to their absence in adult tissues, makes them ideal candidates for targeted immunotherapy. Real Time PCR was used to determine if cytokines could regulate the expression of Eph and ephrin genes in three melanoma cell lines. Tumour necrosis factor (TNF) decreased EphA3 expression and Interferon-g (IFN-g) increased the expression of ephrin A4, and decreased the expression of ephrin A1 and EphB3. Insulin-like growth factor-1 (IGF) increased the expression of EphA3, EphA4, EphB1, ephrin A3 and ephrin B1. All-trans retinoic acid (RA) increased the expression of EphB2 whereas transforming growth factor-b (TGF-b), fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) did not affect the expression of any Eph or ephrins. The potential role of EphA3, ephrins A1, A5, B2 and B3 in melanoma metastasis was examined in vivo following ectopic over-expression of these proteins in a nonmetastatic melanoma cell line (A02-JLO). The ectopic over-expression of these proteins did not confer to A02-JLO cells the ability to form metastases following subcutaneous or intravenous inoculation into nude mice. However, significant differences were noted in subcutaneous growth. EphA3, ephrin A1, ephrin B2 or ephrin B3 expression significantly attenuated, whereas ephrin A5 expression significantly increased tumour growth relative to parental or vector control cells. In vitro proliferation assays indicated all lines proliferated equally and CD31 immunostaining identified increased vessel density in EphA3, ephrin A1, ephrin B2 or ephrin B3 expressing tumours, suggesting the presence of functionally impaired vessels. The more rapidly growing ephrin A5 expressing tumours displayed similar blood vessel density to parental and vector control cells, however examination of H&E sections identified the presence of patterned networks indicative of a more aggressive tumour phenotype. Further, the lack of correlation between CD31 immunoreactivity and the number of vessel-like structures observed in H&E sections suggests the occurrence of vasculogenic mimicry. Finally, the therapeutic potential of an anti-EphA3 monoclonal antibody (monomeric or dimeric IIIA4) was determined both in vitro and in vivo. Real time-PCR (Q-PCR) of nude mouse tissues and an in vivo tissue distribution study of radiolabeled IIIA4 in an EphA3 positive melanoma xenograft model, confirmed the absence of EphA3 in adult nude mouse tissues, and the ability of IIIA4 to selectively target EphA3 positive melanoma xenografts. Prevention studies identified that IIIA4 dimer, but not IIIA4 monomer, was able to significantly attenuate EphA3 positive melanoma xenograft growth, which coincided with a significant increase in vessel number. In a model of disseminated leukaemia, monomeric IIIA4 and dimeric IIIA4 significantly attenuated leukaemic cell engraftment in the peripheral blood and infiltration into splenic tissue, whereas Mabthera (Rituxan®) could not attenuate either. In vitro assays determined that monomeric or dimeric IIIA4 failed to stimulate significant immune mediated lysis. Taken together, the above data suggests that Eph or ephrin expression alone is not sufficient to mediate melanoma metastasis. The present study identifies for the first time that ephrin A5 expression increases tumour aggressiveness and significantly alters tumour vascularisation. Further, manipulation of Eph or ephrin expression may lead to the formation of function-impaired blood vessels. Importantly, IIIA4 can effectively target EphA3 positive tumours and may be a powerful immunotherapeutic tool following humanisation to boost immunogenicity, or used as a vehicle to deliver cytotoxic drugs with unsurpassed avidity