Background: Bile acids (BAs) affect cellular membranes. Results: BAs stabilize domains in plasma membranes, leading to reorganization of membrane proteins and signaling perturbations. Conclusion: BAs affect cell function by modulating the stability of plasma membrane nanodomains. Significance: These results suggest mechanisms for regulation of functional membrane domains and nonreceptor-mediated BA signaling.

The Ras-extracellular signal-regulated kinase (ERK) cascade is an important signaling module in cells. One regulator of the Ras-ERK cascade is phosphatidic acid (PA) generated by phospholipase D (PLD) and diacylglycerol kinase (DGK). Using a newly developed PA biosensor, PASS (phosphatidic acid biosensor with superior sensitivity), we found that PA was generated sequentially by PLD and DGK in epidermal growth factor (EGF)-stimulated HCC1806 breast cancer cells. Inhibition of PLD2, one of the two PLD members, was sufficient to eliminate most of the PA production, whereas inhibition of DGK decreased PA production only at the later stages of EGF stimulation, suggesting that PLD2 precedes DGK activation. The temporal production of PA by PLD2 is important for the nuclear activation of ERK. While inhibition of both PLD and DGK had no effect on the overall ERK activity, inhibition of PLD2 but not PLD1 or DGK blocked the nuclear ERK activity in several cancer cell lines. The decrease of active ERK in the nucleus inhibited the activation of Elk1, c-fos, and Fra1, the ERK nuclear targets, leading to decreased proliferation of HCC1806 cells. Together, these findings reveal that PA production by PLD2 determines the output of ERK in cancer cell growth factor signaling.

i. Summary/Abstract Phospholipids are important signaling molecules that regulate cell proliferation, death, migration and metabolism. Many phospholipid signaling cascades are altered in breast cancer. To understanding the functions of phospholipid signaling molecules, genetically encoded phospholipid biosensors have been developed to monitor their spatiotemporal dynamics. Compared to other phospholipids, much less is known about the subcellular production and cellular functions of phosphatidic acid (PA), partially due to the lack of a specific and sensitive PA biosensor in the past. This chapter describes the use of a newly developed PA biosensor, PASS, in two applications: regular fluorescent microscopy and fluorescence lifetime imaging microscopy-Förster/fluorescence resonance energy transfer (FLIM-FRET). These protocols can be also used with other phospholipid biosensors.

Metastasis is the major cause of breast cancer death. However, despite considerable progress, the signaling events specifically driving cancer cell invasion and metastasis remain largely unknown. In this study, we show that removal of the gene encoding phospholipase D2 (PLD2), which generates the signaling lipid phosphatidic acid (PA), strongly reduced circulating tumor cells and lung metastases in the MMTV‐Neu breast cancer mouse model, whereas it had no effect on tumor initiation and growth. Consistently, migration, invasion and invadopodia, were greatly reduced in primary mouse tumor cells and human breast cancer cell lines lacking PLD2, suggesting that PLD2 promotes tumor metastasis by regulating the local invasion of tumor cells. A PA‐binding protein screening and biochemical analyses revealed that PA specifically binds to a motor protein, Kinesin‐1, and regulates its association with intracellular vesicles. Inhibition of the PLD2‐PA‐Kinesin‐1 pathway by inhibitors or PLD2 knockout blocked the plasma membrane targeting of MT1‐MMP, a matrix metalloproteinase that is required for cancer cell invasion and invadopodia formation. Taken together, these results indicate that PLD2‐generated PA promotes breast cancer metastasis by regulating membrane trafficking of proteins that are critical for cancer cell invasion, and identify that PLD2 is a potential target for treating metastatic breast cancer.