Effects of BARD1 isoforms on BRCA1 localization and patient survival in non-small lung cancer

Overexpression of BRCA1 mRNA was previously reported to be strongly associated with poor survival in NSCLC patients. However, BRCA1 tumor suppressor functions are linked to its binding to BARD1 (BRCA1-associated RING protein 1), which is required for stability, nuclear localization, and ubiquitin ligase function of the BRCA1 protein. BARD1 is alternatively spliced in breast and ovarian cancers and expression of splice-isoforms was correlated with poor prognosis. BARD1 isoforms do not bind BRCA1 and have oncogenic potential, as their repression leads to growth arrest of cancer cells in vitro. We therefore investigated BARD1 expression in tumors from 100 NSCLC patients on the mRNA and protein levels by RTPCR and immunohistochemistry, respectively. We found previously identified BARD1 mRNA isoforms in in all NSCLC samples and in addition two novel forms κ and π. Importantly, all isoforms were expressed in tumors and morphologically normal peritumor tissues, and only isoform π was specifically upregulated in tumors. This was consistent with immunostaining of tumor sections that showed expression of BARD1 epitopes compatible with isoform and not full-length BARD1 expression. BRCA1 staining was cytoplasmic not correlated with BARD1 staining. Statistical analysis showed that the expression of two BARD1 epitopes, PVC and WFS, compatible with the structure of isoform π, was significantly correlated with decreased patient survival. Thus, overexpression of BARD1 isoforms affects mis-localization and presumably mis-functioning of BRCA1 and might be involved in lung carcinogenesis. In particular, expression of isoform π, or epitopes PVC and WFS, might be involved in progressive tumor stages, and thus might represent a novel prognostic marker for NSCLC.