Knockout of the PHLDA1 gene in breast cancer cells reveals multiple roles for PHLDA1 in cancer phenotypes

Abstract

PHLDA1 or pleckstrin‐homology‐like domain family A, member 1, also known as TDAG51, is a small, PH‐domain containing protein that acts as a potential modulator of apoptosis and cell proliferation in breast carcinomas. However, many contradictory findings have been reported and the mechanisms by which PHLDA1 affects cancer cells are not well defined. We have reported that in estrogen receptor (ER)‐positive breast tumors, high PHLDA1 mRNA expression was found to be associated with a high risk of distant metastasis, while in ER negative breast tumors, high PHLDA1 expression coincided with a more favorable outcome, suggesting that PHLDA1 has opposite effects depending on the ER status of a tumor. We have recently shown that PHLDA1 contributes to the survival and growth of breast cancer stem cells in ER positive mammospheres, where PHLDA1 mRNA and protein are regulated directly by a crosstalk between ER and the inflammatory nuclear factor κB (NFκB) pathway, and indirectly through inhibition of miR‐181a and b, which targets PHLDA1 for degradation. Here, using CRISPR/Cas9 mediated genome editing, we have permanently knocked out PHLDA1 gene from ER positive MCF‐7 and ER negative MDA‐MB‐231 breast cancer cell lines, and characterized their phenotype. While MDA‐MB‐231 cells are still under investigation, here we report that MCF‐7 cells missing the PHLDA1 gene showed decreased cell proliferation, migration, and invasion across a matrigel layer, consistent with a role for PHLDA1 in more aggressive ER+ breast cancers. In contrast, PHLDA1‐knock out cells were less susceptible to apoptosis than control cells, as shown by decreased activity of caspase‐3 and −7, and were rendered more resistant to doxorubicin and paclitaxel‐induced cytotoxicity, suggestive of a pro‐apoptotic role for PHLDA1. This phenotype was reversed by overexpressing PHLDA1 in the PHLDA1 (−/−) cells. We also compared PI3K activation and Akt phosphorylation in parental and knock out cells in order to determine the signaling pathway mediating the effects of PHLDA1. The PH domains, or phosphoinositide binding protein modules, are known to govern the function of two other PHLDA isoforms, PHLDA2 and PHLDA3, but no information on the role of PH domain in the function of PHLDA1 has been described. We created a PH domain deletion mutant (PHLDA1‐ΔPH), and using confocal microscopy assessed its subcellular localization, as well as Akt/PI3K signaling pathway activity, and compared it to the WT PHLDA1. Our study sheds new light on the function of PHLDA1 in breast cancer cells, suggesting a broad role as an anti‐proliferative/anti‐invasive but pro‐apoptotic protein. Moreover, we describe the signaling pathway involving PHLDA1, an understanding of which will potentially define novel therapeutic targets in treatment of breast carcinomas.Support or Funding InformationThis study was supported by the NIH fund 1 R01 CA200669 01 and DOD fund W81XWH‐16‐1‐0044 to JF