Dysregulation of Phospholipase D (PLD) isoforms increases breast cancer cell invasion

Breast cancer remains the second most prevalent cancer among women in the U.S. with metastatic breast cancer having the worst prognosis. A rapidly proliferating tumor under various stressors will promote phenotypic cellular changes, known as epithelial-to-mesenchymal transition (EMT), which allows cells to begin to invade surrounding tissue, enter the circulatory system, and eventually seed a distant metastatic site. The phospholipase D (PLD) enzymes are critical regulators of cell signaling pathways necessary for cell migration. While the importance of PLD enzymes in cancer cell invasion is well known, clinically applicable methods of PLD inhibition are not yet available. The best-studied isoforms are the `classical’ PLD1 and PLD2 membrane lipases that hydrolyze phosphatidylcholine (PC) to free choline and phosphatidic acid (PA). PA acts as a secondary messenger activating numerous pathways leading to cell growth and proliferation, vesicle trafficking, and cell migration. PLD1 and PLD2 are upregulated in several cancers including breast and, furthermore, promote tumorigenesis and disease progression. However, the specific molecular mechanism that regulates overexpression of PLD1 and PLD2 in cancer remains unclear. I investigated post-transcriptional regulation of the classical PLD isoforms by microRNA (miRNA) in cancer cell invasion and in stress (nutrient starvation) leading to EMT. MicroRNA are currently widely investigated as potential therapeutic agents involved in a myriad of diseases. Naturally produced in the body, miRNA are attractive therapeutic molecules based on their small size, surprising stability, and potent regulatory capabilities. A repertoire of four microRNAs present in non-cancerous breast cells that are down-regulated in invasive breast cancer cell were found and demonstration of their regulation of PLD1 or PLD2 mRNA was done by luciferase reporter assay. Exogenous addition of these microRNAs to invasive breast cancer cells reduced cell invasion. Additionally, initial nutrient starvation increased classical PLD protein levels, concomitant with cell invasion. With prolonged starvation, PLD-targeting microRNA were upregulated and functioned in a negative feedback loop to downregulate PLD protein expression. While much is known about the PLD1 and PLD2 isoforms, the other mammalian PLD isoforms (PLD3, PLD4, and PLD6) are vastly less studied. Interestingly, PLD6 has recently been found to be overexpressed in breast cancer tumors, however, further studies investigating the role of PLD6 in cancer progression have not been published. PLD6 localizes to the mitochondrial outer membrane and uses cardiolipin as a substrate to produce PA. This reaction promotes mitochondrial fusion, thereby inhibiting apoptosis, while increasing oxidative phosphorylation, both of which are critical to cancer progression. While PLD1 and PLD2 are ubiquitously expressed, PLD6 is expressed at lower basal levels in most tissues, making PLD6 an attractive therapeutic target, when upregulated in cancer, with potentially less adverse side effects. The protein and gene expression of PLD6 are upregulated in invasive breast cancer cells compared to non-cancerous breast cells. PLD6 subcellular localization changed in response to invasion-promoting factors, nutrient starvation and EGF signaling. Furthermore, this is the first report showing that modulating PLD6 expression influences cell invasion of aggressive breast cancer cells. Overall, I show that increased expression of PLD1, PLD2, and PLD6 in invasive breast cancer cells leads to higher cell invasion. A novel molecular regulation of classical PLD by a repertoire of microRNA was demonstrated. Additionally, PLD6 increases breast cancer cell invasion of certain breast cancer cell lines. This work highlights the importance that targeting PLD family members could have in breast cancer therapeutics

A Phosphatidic Acid (PA) conveyor system of continuous intracellular transport from cell membrane to nucleus maintains EGF receptor homeostasis

The intracellular concentration of the mitogen phosphatidic acid (PA) must be maintained at low levels until the need arises for cell proliferation. How temporal and spatial trafficking of PA affects its target proteins in the different cellular compartments is not fully understood. We report that in cancer cells, PA cycles back and forth from the cellular membrane to the nucleus, affecting the function of epidermal growth factor (EGF), in a process that involves PPARα/LXRα signaling. Upon binding to its ligand, EGF receptor (EGFR)-initiated activation of phospholipase D (PLD) causes a spike in intracellular PA production that forms vesicles transporting EGFR from early endosomes (EEA1 marker) and prolonged internalization in late endosomes and Golgi (RCAS marker). Cells incubated with fluorescent-labeled PA (NBD-PA) show PA in “diffuse” locations throughout the cytoplasm, punctae (small, 0.5 μm) vesicles that co-localize with EGFR. We also report that PPARα/LXRα form heterodimers that bind to new Responsive Elements (RE) in the EGFR promoter. Nuclear PA enhances EGFR expression, a role compatible with the mitogenic ability of the phospholipid. Newly made EGFR is packaged into PA recycling vesicles (Rab11 marker) and transported back to the cytoplasm and plasma membrane. However, a PLD+PA combination impedes binding of PPARα/LXRα to the EGFR promoter. Thus, if PA levels inside the nucleus reach a certain threshold (>100 nM) PA outcompetes the nuclear receptors and transcription is inhibited. This new signaling function of PLD-PA targeting EGFR trafficking and biphasically modulating its transcription, could explain cell proliferation initiation and its maintenance in cancer cells