PHLDA1 Expression is Controlled by an Estrogen Receptor (ER)-NFκB-miR-181 Regulatory Loop and is Essential for Formation of ER+ Mammospheres

Crosstalk between estrogen receptor (ER) and the inflammatory nuclear factor κB (NFκB) pathway in ER+ breast cancers may contribute to a more aggressive phenotype. PHLDA1 (Pleckstrin Homology-Like Domain, Family A, member 1), one target gene of ER-NFκB crosstalk, has been implicated in cell survival and stem cell properties. 17β-estradiol (E2), acting through ERα, and pro-inflammatory cytokines, acting through NFκB, increase the nascent transcript and PHLDA1 mRNA stability, indicating both transcriptional and post-transcriptional control of PHLDA1 expression. We show that PHLDA1 is a direct target of miR-181 and that mature miR-181a and b, as well as their host gene, are synergistically down-regulated by E2 and TNFα, also in an ER and NFκB-dependent manner. Thus, ER and NFκB work together to up-regulate PHLDA1 directly through enhanced transcription and indirectly through repression of miR-181a and b. Previous studies have suggested that PHLDA1 may be a stem cell marker in the human intestine that contributes to tumorigenesis. Our findings that PHLDA1 is up-regulated in mammospheres (MS) of ER+ breast cancer cells and that PHLDA1 knockdown impairs both MS formation and the expansion of aldehyde dehydrogenase (ALDH)-positive population, suggest that PHLDA1 may play a similar role in breast cancer cells. Up-regulation of PHLDA1 in MS is largely dependent on the NFκB pathway, with down-regulated miR-181 expression a contributing factor. Over-expression of miR-181 phenocopied PHLDA1 knockdown and significantly impaired MS formation, which was reversed, in part, by protection of the PHLDA1 3′UTR or overexpression of PHLDA1 lacking the 3′UTR. Furthermore, we find that elevated PHLDA1 expression is associated with a higher risk of distant metastasis in ER+ breast cancer patients. Altogether, these data suggest that high PHLDA1 expression is controlled through an ER-NFκB-miR-181 regulatory axis and may contribute to a poor clinical outcome in patients with ER+ breast tumors by enhancing stem-like properties in these tumors.