Functional role of PHLDA1 down regulation leads to morphological changes and increased proliferation in MCF-7 breast cancer cells

The PHLDA1 (pleckstrin homology-like domain Family A, member 1) belongs to a family of genes that encode phosphatidylinositol (PIP) binding proteins and function as inhibitors of Akt activation. Previously, we have shown that PHLDA1 down-regulation is a strong predictor of poor prognosis for breast cancer (BC) patients. We have also demonstrated that decreased PHLDA1 expression is associated with more aggressive behavior in non-malignant mammary cells and is linked to a more aggressive BC phenotype and a worse prognosis. Additionally, we demonstrated that PHLDA1 expression is regulated by estrogen via ER. Here, we sought to investigate the effects of PHLDA1 knockdown on cell proliferation, migration, and colony formation, and further assess its involvement in the response to endocrine therapies. The MCF-7 cells were transfected using a CRISPR/Cas9 vector (p5pCas(BB)-2A-Puro (PX459) V2.0) that included guide RNAs (gRNAs) designed to silence the PHLDA1 gene, as well as a control vector with scrambled gRNAs. PHLDA1 knockdown clones (MCF-7koPHLDA1) were confirmed by western blot and qRT-PCR. MCF-7koPHLDA1 clones displayed morphological changes, including loss of regular angular and spindle shapes and a more dispersed cell arrangement. We performed the colorimetric MTT reduction assay to evaluate the effect of PHLDA1 knockdown on the proliferation. MCF-7koPHLDA1 has significantly higher proliferation rates than MCF-7 transfected with the vector containing a scramble (MCF-7-SC). In addition, a clonogenic assay followed by crystal violet staining showed that MCF-7koPHLDA1 also exhibited higher colony-forming ability, with a dispersed organization and limited cell-cell contact, compared with MCF-7-SC. Our data suggest that decreased PHLDA1 expression enhances the proliferation and colony-forming ability of MCF7 cells. Additionally, the obtained clones will be assessed for how PHLDA1 expression influences their response to tamoxifen and fulvestrant treatments. Supported by FAPESP.