Changes in phospholipase D isoform activity and expression in multidrug-resistant human cancer cells

Abstract

Multidrug resistance (MDR) is a major cause of failure of cancer chemotherapy and is often associated with elevated expression of drug transporters such as P-glycoprotein (P-gp) in the cancer cells. MDR is, however, accompanied by additional biochemical changes including modifications of membrane composition and properties. We have shown that MDR is associated with a massive up-regulation of caveolin expression and an elevated surface density of caveolae. We report that phospholipase D (PLD), a constituent enzyme of caveolae and detergent-insoluble glycolipid-rich membranes (DIGs), is up-regulated in human MDR cancer cells. Lysates of HT-29-MDR human colon adenocarcinoma cells, MCF-7 AdrR human breast adenocarcinoma cells and the corresponding parental drug-sensitive cells, were fractionated on discontinuous sucrose density gradients. PLD activity was found to be enriched in low density fractions that contain DIGs and caveolar membranes, and the activity in these fractions was 4-to 6-fold higher in the MDR cells compared with the parental drug-sensitive cells. Utilizing specific antibodies to PLD1 and PLD2, the distribution of PLD isoforms along the gradient was determined and the PLD localized in DIGs and caveolar membranes has been identified as PLD2. Northern blot analysis of PLD1 and PLD2 mRNA levels has indicated that PLD2 mRNA is elevated in both HT-29-MDR and MCF-7 AdrR cells. PLD1 mRNA levels were either unchanged or reduced in the MDR cells. Finally, in vivo experiments have confirmed previous results showing that activation of PLD by phorbol esters is markedly potentiated in the MDR cells. We conclude that MDR is accompanied by an increase in PLD2 activity in DIGs and caveolar membranes.

A Novel Phosphatidic Acid-Protein-tyrosine Phosphatase D2 Axis Is Essential for ERBB2 Signaling in Mammary Epithelial Cells

“…Although we did not observe tyrosine phosphorylation of PLD2 upon acute stimulation of chimeric ERBB2, it is possible that there exist other mechanisms of PLD2 activation in the context of ERBB2 signaling. Consistent with a pro-oncogenic role for this enzyme, a high level of PLD activity has been observed in breast cancers (45,46). We also observed that exogenously added phosphatidic acid can rescue the PLD inhibition phenotype, but only when PTPD2 was present.…”

Temporal Production of the Signaling Lipid Phosphatidic Acid by Phospholipase D2 Determines the Output of Extracellular Signal-Regulated Kinase Signaling in Cancer Cells

“…PLD expression and activity are elevated in a variety of human cancer tissues (66)(67)(68)(69)(70)(71) and human cancer cell lines (72)(73)(74)(75)(76). Interestingly, high levels of PLD2 activity also correlate with more aggressive cancer phenotypes (77). Moreover, it has been proposed that PLD2 could be a "driver" in breast tumorigenesis, based on the relatively high rate of somatic mutation of PLD2 found in a sequencing analysis of human breast cancer patients (78).…”

Inhibition of Platelet-derived Growth Factor-induced Cell Growth Signaling by a Short Interfering RNA for EWS-Fli1 via Down-regulation of Phospholipase D2 in Ewing Sarcoma Cells

“…We have observed that ADP-ribosylation factoractivated PLD activity was transiently increased in regenerating liver nuclei and that PLD2 levels were markedly elevated in the nuclei of hepatoma and kidney tumor cells (35,36). There is also evidence that PLD2 levels are markedly elevated in the caveolae of stimulated oncogenic cells and multidrugresistant cancer cells (37,38). Furthermore, overexpression of PLD1 or PLD2 in mouse fibroblasts induces colony formation in soft agar, and both transformants induce undifferentiated sarcoma when transplanted into nude mice (30).…”