a Discipline of Oncology, Department of Radiology and Oncology , Faculty of Medicine, University of São Paulo , São Paulo , Brazil.
b Laboratory of Molecular Genetics , Center for Translational Research in Oncology, Cancer Institute of São Paulo , São Paulo , Brazil.
Cell Adh Migr. 2018 Jan 2;12(1):37-46. doi: 10.1080/19336918.2017.1313382. Epub 2017 Jun 29.
PHLDA1 (pleckstrin homology-like domain, family A, member 1) is a multifunctional protein that plays distinct roles in several biological processes including cell death and therefore its altered expression has been identified in different types of cancer. Progressively loss of PHLDA1 was found in primary and metastatic melanoma while its overexpression was reported in intestinal and pancreatic tumors. Previous work from our group showed that negative expression of PHLDA1 protein was a strong predictor of poor prognosis for breast cancer disease. However, the function of PHLDA1 in mammary epithelial cells and the tumorigenic process of the breast is unclear. To dissect PHLDA1 role in human breast epithelial cells, we generated a clone of MCF10A cells with stable knockdown of PHLDA1 and performed functional studies. To achieve reduced PHLDA1 expression we used shRNA plasmid transfection and then changes in cell morphology and biological behavior were assessed. We found that PHLDA1 downregulation induced marked morphological alterations in MCF10A cells, such as changes in cell-to-cell adhesion pattern and cytoskeleton reorganization. Regarding cell behavior, MCF10A cells with reduced expression of PHLDA1 showed higher proliferative rate and migration ability in comparison with control cells. We also found that MCF10A cells with PHLDA1 knockdown acquired invasive properties, as evaluated by transwell Matrigel invasion assay and showed enhanced colony-forming ability and irregular growth in low attachment condition. Altogether, our results indicate that PHLDA1 downregulation in MCF10A cells leads to morphological changes and a more aggressive behavior.
PHLDA1（pleckstrin homology-like domain, family A, member 1）是一种多功能蛋白，在包括细胞死亡在内的多种生物学过程中发挥着不同的作用，因此在不同类型的癌症中发现其表达发生了改变。在原发性和转移性黑色素瘤中逐渐发现 PHLDA1 的缺失，而在肠和胰腺肿瘤中则报道其过表达。我们小组的先前工作表明，PHLDA1 蛋白的阴性表达是乳腺癌预后不良的强有力预测指标。然而，PHLDA1 在乳腺上皮细胞中的功能及其在乳腺癌中的致瘤过程尚不清楚。为了剖析 PHLDA1 在人乳腺上皮细胞中的作用，我们生成了 MCF10A 细胞的一个稳定敲低 PHLDA1 的克隆，并进行了功能研究。为了实现 PHLDA1 表达的降低，我们使用 shRNA 质粒转染，然后评估细胞形态和生物学行为的变化。我们发现，PHLDA1 的下调诱导 MCF10A 细胞发生明显的形态改变，例如细胞间粘附模式和细胞骨架重组的改变。关于细胞行为，与对照细胞相比，表达 PHLDA1 降低的 MCF10A 细胞显示出更高的增殖率和迁移能力。我们还发现，用 shRNA 转染敲低 PHLDA1 的 MCF10A 细胞获得了侵袭性，如通过 Transwell Matrigel 侵袭测定评估的那样，并显示出增强的集落形成能力和在低附着条件下的不规则生长。总之，我们的结果表明，MCF10A 细胞中 PHLDA1 的下调导致形态变化和更具侵袭性的行为。
