Abstract
Breast cancer is one of the most common malignancies in human females in the world. One protein that has elevated enzymatic lipase activity in breast cancers in vitro is phospholipase D (PLD), which is also involved in cell migration. We demonstrate that the PLD2 isoform, which was analyzed directly in the tumors, is crucial for cell invasion that contributes critically to the growth and development of breast tumors and lung metastases in vivo. We used three complementary strategies in a SCID mouse model and also addressed the underlying molecular mechanism. First, the PLD2 gene was silenced in highly metastatic, aggressive breast cancer cells (MDA-MB-231) with lentivirus-based shRNA, which were xenotransplanted in SCID mice. The resulting mouse primary mammary tumors were reduced in size (65%, p<0.05) and their onset delayed when compared to control tumors. Second, we stably overexpressed PLD2 in low-invasive breast cancer cells (MCF-7) with a biscistronic MIEG retroviral vector and observed that these cells were converted into a highly aggressive phenotype, as primary tumors that formed following xenotransplantation were larger, grew faster and developed lung metastases more readily. Third, we implanted osmotic pumps into SCID xenotransplanted mice that delivered two different small-molecule inhibitors of PLD activity (FIPI and NOPT). These inhibitors led to significant (>70%, p<0.05) inhibition of primary tumor growth, metastatic axillary tumors and lung metastases. In order to define the underlying mechanism, we determined that the machinery of PLD-induced cell invasion is mediated by phosphatidic acid (PA), WASp, Grb2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows that PLD has a central role in the development, metastasis and level of aggressiveness of breast cancer, raising the possibility that PLD2 could be used as a new therapeutic target.
Highlights
Breast cancer is globally the most common malignancy affecting greater than one million human females per year in 145 countries surveyed.[1,2] Breast cancer metastasis, which is the primary cause of death in patients, is a complex process that involves cell proliferation, invasion through basement membrane and vessel walls, diapedesis into capillaries or lymphatic vessels and further establishment of new colonies in other tissues.[3,4,5] Cell invasion and metastasis need the interaction of a developmental regulatory program, called epithelial–mesenchymal transition, for transformed epithelial cells to acquire several malignant attributes that enable them to systematically invade adjacent tissues.[6]
Knocking down phospholipase D (PLD) deters invasion of highly aggressive cancer cells It is known that MDA-MB-231 cells are highly invasive and metastatic cancer cells, and some reasons could be due to high PLD activity found in these cells and PLD’s involvement in cancer cell survival.[37]
Considering this earlier finding, we explored the mechanistic importance of Wiscott–Aldrich Syndrome protein (WASp) to our in vivo SCID mouse model using transient overexpression of WASp into MCF-7 cells that stably overexpressed PLD2, which were xenotransplanted into SCID mice
Summary
Breast cancer is globally the most common malignancy affecting greater than one million human females per year in 145 countries surveyed.[1,2] Breast cancer metastasis, which is the primary cause of death in patients, is a complex process that involves cell proliferation, invasion through basement membrane and vessel walls, diapedesis into capillaries or lymphatic vessels and further establishment of new colonies in other tissues.[3,4,5] Cell invasion and metastasis need the interaction of a developmental regulatory program, called epithelial–mesenchymal transition, for transformed epithelial cells to acquire several malignant attributes that enable them to systematically invade adjacent tissues.[6]. Elevation of either PLD or especially the PLD2 isoform has the potential to transform both murine and rat fibroblasts.[25,26,27] The potential exists for stimulation of PLD activity to directly contribute to cell proliferation, which further compounds the formation of a fully malignant phenotype.[28,29,30]
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