Oncogenic p95HER2 regulates Na+-HCO3- cotransporter NBCn1 mRNA stability in breast cancer cells via 3'UTR-dependent processes

The Na + -HCO 3 - -cotransporter NBCn1 (SLC4A7) is upregulated in breast cancer, important for tumor growth, and a Single Nucleotide Polymorphism (SNP), rs4973768, in its 39untranslated region (39UTR) correlates with increased breast cancer risk. We previously demonstrated that NBCn1 expression and promoter activity are strongly increased in breast cancer cells expressing a constitutively active oncogenic HER2 receptor (p95HER2). Here, we address the roles of p95HER2 in regulating NBCn1 expression via posttranscriptional mechanisms. p95HER2 expression in MCF-7 cells reduced the rate of NBCn1 mRNA degradation. The NBCn1 39UTR downregulated luciferase reporter expression in control cells and this was reversed by p95HER2, suggesting that p95HER2 counteracts 39UTR-mediated suppression of NBCn1 expression. Truncation analyses identified three NBCn1 39UTR regions of regulatory importance. Mutation of putative miRNA binding sites (miR-374a/b, miR-200b/c, miR-29a/b/c, miR-488) in these regions did not significantly impact 39UTR activity. The NBCn1 39UTR interacted directly with the RNA-binding protein HuR, and HuR knockdown reduced NBCn1 expression. Conversely, ablation of a distal AU-rich Element (ARE) increased 39UTR-driven reporter activity, suggesting complex regulatory roles of these sites. The cancer-associated SNP variant decreased reporter expression in T-47D breast cancer cells, yet not in MCF-7, MDA-MB-231 and SK-BR-3 cells, arguing against a general role in regulating NBCn1 expression. Finally, p95HER2 expression increased total and plasma membrane NBCn1 protein levels and decreased the rate of NBCn1 protein degradation. Collectively, this work is the first to demonstrate 39UTR-mediated NBCn1 regulation, shows that p95HER2 regulates NBCn1 expression at multiple levels, and substantiates the central position of p95HER2-NBCn1 signaling in breast cancer.