Phospholipase D (PLD) drives cell invasion, tumor growth and metastasis in a human breast cancer xenograph model

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 short hairpin RNA, 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 with 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 (5-fluoro-2-indolyl des-chlorohalopemide and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide). 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, Wiscott-Aldrich Syndrome protein, growth receptor-bound protein 2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows for the first time that PLD2 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.

Figure 1. A stably silenced PLD2 MDA-MB-231 cell line using lentiviral shRNA shows decreased cancer cell invasion

A, Vector map of pKLO-shPLD2. B, Western-blot analysis of cell lysates from MDA-MB-231 shControl or shPLD2 cells, with anti-PLD2 antibody. B inset, Quantification of PLD2 silencing from Western-blot analysis of cell lysates from MDA-MB-231 shControl or shPLD2 cells. C, Cell proliferation of puromycin-resistant, stable MDA-MB-231 pKLO cells expressing shPLD2. D, PLD activity. E–F, Negative effect of PLD silencing on physiological functions (cell invasion and chemotaxis, respectively). Triplicate results are mean ± SEM. The symbols * and # denote statistically significant (p<0.05) differences (increases or decreases, respectively) between samples and controls.

Figure 2. PLD2 silencing of SCID mouse metastastic breast cancer model decreases tumor size

Metastatic breast cancer cells MDA-MB-231-shPLD2 were implanted into the mammary fat pad of immunodeficient 8 week old female SCID mice. Mammary tumor growth and lung metastasis were determined after the duration of the study (at least 5 weeks). A, Primary tumor onset (# days post-injection) for SCID mice injected with MDA-MB-231 pKLO (either shControl or shPLD2) stable cell lines. B, Growth curves of primary tumor volume (mm³). Representative histology images of primary (C–D) and metastatic tumor sections (E–F) and lung sections (G, H, I) detected by hematoxylin-eosin staining of 3–4 different cross sections (7–10 µm- thick) of tissue from at least 3 different mice from each group at 10× magnification, respectively. C, E, G, Histology of SCID mice injected with MDA-MB-231 shControl cells. D, F, H, I, Histology of SCID mice injected with MDA-MB-231 shPLD2 cells. Black and yellow arrowhead denotes presence of pleural carcinoma. I, 2× magnification. G–H, Scale bar = 200 µm. C–F, I, Scale bar = 1 mm.

Figure 3. A MCF-7 Cancer Cell Line Stably Overexpressing Recombinant Human PLD2 shows enhanced cancer cell invasion

A, Simplified scheme of MIEG-PLD2 mRNA. B–D, Immunofluorescence of GFP in puromycin-resistant, stable MCF-7·pMIEG cells overexpressing either GFP vector, PLD1 or PLD2. E, Western-blot analyses of cell lysates. F, Effect of PLD overexpression on cell proliferation. G, Effect of PLD overexpression on PLD activity. H-I, Effect of PLD overexpression on physiological functions (cell invasion and chemotaxis, respectively). Triplicate results are mean ± SEM. * denotes statistically significant (p<0.05) increases respect to controls.

Figure 4. PLD2 overexpression of SCID mouse metastastic breast cancer model increases tumor size

Metastatic breast cancer cells MCF-7·pMIEG were implanted into the mammary fat pad of SCID mice. A, Primary tumor onset (# days post-injection) of SCID mice injected with MCF-7·pMIEG (either GFP, PLD1 or PLD2) stable cell lines. B, Effect of PLD overexpression on growth curves of primary tumor volume (mm³) in the PLD-xenotransplanted SCID mice. C, Increase in the number of metastatic tumors generated in PLD-xenotransplanted SCID mice. The symbols * and # denote statistically significant (p<0.05) differences (increases or decreases, respectively) between samples and controls. D–L, Representative histology images of lung sections (D–F), primary tumor sections (G–I) or metastatic axillary tumor sections (J-L) detected by hematoxylin-eosin staining of 3–4 different cross sections (7–10 µm- thick) of tissue from at least 3 different mice from each group at 10× magnification, respectively. D, G, J, Histology of SCID mice injected with MCF-7·pMIEG GFP cells. E, H, K, Histology of SCID mice injected with MCF-7·pMIEG PLD1 cells. F, I, L, Histology of SCID mice injected with MCF-7·pMIEG PLD2 cells. Black and white arrowhead denotes presence of pleural carcinoma (D); black and yellow arrowheads point at multifocal perivascular metastatic carcinomas in the lung parenchyma (F). D–F, Scale bar = 200 µm. G-L, Scale bar = 1 mm.

Figure 5. Small-molecule inhibitors, FIPI, NOPT and apigenin, inhibit cell invasion of two breast cancer cell lines, which correlates to a decreased PLD2 activity independent of cell proliferation

Exponentially growing cells in culture were used for these experiments. A, Relative difference of cell invasiveness of highly invasive MDA-MB-231 compared to less invasive MCF-7 cells in response to 3 nM EGF. B–D, Effect of small molecule inhibitors on cell invasion. B, FIPI dose response. C, NOPT dose response. D, Apigenin dose response. Triplicate results are mean ± SEM. B inset, FIPI schematic. C inset, NOPT schematic. D inset, Apigenin inset. E, Time dependent effect of FIPI or NOPT on endogenous PLD activity after incubation for 5 min or for 30 min with the inhibitors prior to the enzymatic assay. F, Time dependent effects of small molecule inhibitors (300 nM concentration of each) on MDA-MB-231 cell proliferation. G, Effect of 300 nM concentration of each inhibitor on cell invasion or lipase assay (at 30 min) of MDA-MB-231 cancer cells overexpressing recombinant PLD2-WT. Triplicate results are mean ± SEM. The symbols * and # denote statistically significant (p<0.05) differences (increases or decreases, respectively) between samples and controls.

Figure 6. Small-molecule inhibitors reduce tumor size on SCID mouse metastastic breast cancer model

A, Schematic drawing of Alzet pump implantation in SCID mice. B, Photographic representation of surgical area on SCID mice after Alzet pump implantation. C, Delayed primary tumor onset (# days post-injection) for SCID mice xenotransplanted with MDA-MB-231 cells in the presence of small molecule inhibitors. D, Decreased primary tumor volume (mm³) following dosing of xenotransplanted SCID mice with small molecule inhibitors. E, Reduction in the number of secondary tumors of xenotransplanted SCID mice in the presence of inhibitors. Triplicate results are mean ± SEM. The symbols * and # denote statistically significant (p<0.05) differences (increases or decreases, respectively) between samples and controls. F–O, Representative histology images of lung sections (F–I), primary tumor sections (J–M) or secondary tumor sections (N–O) detected by hematoxylin-eosin staining of 3–4 different cross sections (7–10 µm- thick) of tissue from at least 3 different mice from each group at 10× or 2× magnification, respectively. Black and yellow arrowhead denotes presence of small focal metastatic perivascular lung carcinomas (F); black and white arrow heads point at small 2–20 cell carcinoma emboli in the alveolar walls (H–I). F-I, Scale bar = 200 µm. J–O, Scale bar = 1 mm.

Figure 7. The mechanism that regulates PLD-mediated cell invasion and metastasis involves PA, Rac2 and Grb2

A, PA sensor used for transfection into MDA-MB-231 or MCF-7 cells. B, Immunofluorescence of PA sensor expression. White arrowheads denote PA sensor localized to a membranous surface that contained PA. C, Cell invasion of MCF-7 cells stably expressing lipase-inactive constructs (PLD1-K866R or PLD2-K758R). D, Tumor size of mice injected with MCF-7 cells stably expressing PLD2, a WASp construct or a combination of the two. E, EGF-mediated cell invasion in cells that overexpress PLD2, Grb2 and Rac2 constructs (WT and SH2-binding deficient). F, PLD2, Grb2 and Rac2 protein expression in MDA-MB-231 cells. G, EGF-mediated cell invasion in cells silenced with siRNA for Grb2 and/or PLD2 or with siRNA for Rac2.

Figure 8. Model for the mechanism that regulates PLD-mediated metastasis

A, Cell invasion (MTLn3) or cell migration (HL60 and AML14) following overexpression of signaling molecules in three cancer cell lines other than MDA’s or MCF’s. Astericks represent significant increase (p<0.05). B, Model that summarizes graphically the findings of this study (histological samples are from Figs. 4F and 6F).