Abstract 4025: An old foe with a new function: JAM-A regulates expression of EphB4 and inhibitor of apoptosis proteins in breast cancer

Abstract

Abstract Over-expression of Junctional Adhesion Molecule-A (JAM-A) in breast tumors has been linked with increased risk of metastasis. Since JAM-A is co-expressed with the receptor tyrosine kinase (RTK) HER2 and regulates HER2 protein expression, we hypothesized that JAM-A regulates the expression and function of other RTKs. Using a joint RT-PCR array/western blot approach in three breast cell lines with modified JAM-A expression, we identified EphB4 as a downstream target of JAM-A. Interestingly, EphB4 gene silencing also cross-regulated the protein expression of JAM-A. Immunofluorescence analysis revealed colocalisation of JAM-A and EphB4 at the cell membrane. Since JAM-A contains a PDZ-binding motif and EphB4 contains a PDZ domain, we speculated that this might underlie the basis of an interaction. Accordingly, overexpression of a JAM-A mutant lacking the PDZ domain failed to upregulate EphB4 expression to the same extent as wild type JAM-A. Next we investigated the signalling events downstream of JAM-A and EphB4 and found downregulation of the tight junction protein ZO-1 in breast cells silenced for JAM-A or EphB4. Using a bioinformatics approach, we identified the transcription factor c-FOS as a potential regulator of JAM-A and EphB4 expression. In JAM-A- or EphB4-silenced cells, c-FOS expression was not localized in the nucleus (relative to cells transfected with negative control siRNAs). Furthermore, ERK5 phosphorylation was inhibited in JAM-A- or EphB4-silenced cells, suggesting a mechanism for reduced c-FOS phosphorylation on Serine-32 and its consequent exclusion from the nucleus. In the context of a proposed block on c-FOS transcriptional activity, we examined the genes affected and show for the first time downregulation of inhibitor of apoptosis proteins (IAPs) in JAM-A- or EphB4-silenced cells. In conclusion, our data are consistent with a model whereby JAM-A regulates EphB4 expression in a PDZ-dependent manner and influences apoptosis via a pathway involving ERK5 and c-FOS. Our data suggest that the pharmacological antagonism of JAM-A in JAM-overexpressing breast cancers merits consideration by virtue of its predicted ability to downregulate EphB4 expression and remove a blockade on apoptosis. Citation Format: Sri HariKrishna Vellanki, Rodrigo G. Cruz, Cathy E. Richards, Kieran Brennan, Ann M. Hopkins. An old foe with a new function: JAM-A regulates expression of EphB4 and inhibitor of apoptosis proteins in breast 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 4025.