Upregulated phospholipase D2 expression and activity is related to the metastatic properties of melanoma
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- Published online on: March 9, 2022 https://doi.org/10.3892/ol.2022.13260
- Article Number: 140
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Copyright: © Perez‑Valle et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Phospholipase D (PLD) is a phospholipase that participates in the catabolism of glycerol-based phospholipids. There are six different isoforms of the phospholipase D subclass (PLD1-6) and PLD1 and PLD2 are the best-studied isoforms. The lipase activity of both these enzymes involves the preferential hydrolysis of phosphatidylcholines (PCs), to release phosphatidic acid (PA) and choline (Cho), crucial lipid mediators of cell signaling pathways involved in cell proliferation, growth and survival (1).
Cho is an essential nutrient for humans and it has a wide range of vital physiological roles (2,3), although higher levels of Cho have been associated with an increased risk of melanoma and other cancer types (3–5). However, as only small amounts of this molecule may be synthesized de novo in humans, it must be obtained from other sources, such as through the hydrolysis of the most abundant phospholipid, PC, by the lipase activity of PLDs (3). PA is the other lipid mediator produced by the lipase activity of PLDs, which may subsequently be converted into essential second messengers such as lyso-PA by phospholipase A2 (PLA2) or diacylglycerol (DAG) by PA-phosphohydrolase. These products significantly expand the range of the effects in which these enzymes may participate.
Aside from the lipase activity of PLD2, a unique feature of this isoform is that it also acts as a guanine nucleotide exchange factor (GEF) (6,7). Certain small GTPases regulate PLD2 activity through a complex regulatory program of phosphorylation and dephosphorylation switching involving S6K, Grb2, SoS, WASp and Rac2 (7). The GEF activity of PLD2 has been associated with the migration of cancer cells, hypothesizing that PLD2 constitutively activates Rac2, a GTPase known to participate in cancer cell metastasis (8). Furthermore, the GEF activity of PLD2 regulates the PA released by hydrolysis of PC, adding a further layer of complexity to these regulatory circuits (6,9,10).
It is known that both the expression and activity of PLD1 and PLD2 are upregulated in several cancer types (1,10–18), altering numerous signaling pathways involved in tumor progression and metastasis. Indeed, it has been suggested that PLD2 has a pivotal role in tumor cells, primarily in metastatic dissemination (16,19). In fact, PLD2 activity promotes proliferation and invasiveness of lymphomas (20) and breast cancer cells (12,21), as well as representing a marker of poor prognosis in colorectal and renal neoplasms (14–16). PLD2 has also been observed to be overexpressed in melanoma along with enhanced lipase activity (22,23), although there is no clear evidence of its specific role in this pathology.
Melanoma is a cancer that arises from the transformation of melanocytes and it mainly occurs in the skin. It is the seventh most common tumor type in Europe and although the incidence of most cancers is declining, the number of melanoma cases is expected to continue to rise in western populations. While early diagnosis guarantees a 5-year overall survival of 99% for most melanomas, this rate falls to 25% in melanomas with distant metastases (24). Although numerous treatment options have been approved for metastatic tumors in the past decade (25), there is still no effective therapy for advanced melanoma. Thus, the aim of the present study was to obtain novel insight into the contribution of PLD2 in the development and progression of melanoma, assessing its value as a potential novel biomarker or therapeutic target for melanoma. It was indicated that an increase in the expression of PLD2 and its lipase activity contribute to increased proliferation, migration and invasion of melanoma cells.
Materials and methods
Commercial cell lines
The HEMn-LP, HEMn-MP and HEMn-DP melanocyte cell lines were purchased from Thermo Fisher Scientific, Inc., while the A375, G361, Sk-Mel-28, HT144, Hs294t, A2058, Sk-Mel-2, Sk-Mel-3, WM-266-4 and VMM1 melanoma cell lines were obtained from the American Type Culture Collection (ATCC). The Mel-Ho, Colo-800 and RPMI-7951 melanoma cell lines were acquired from Innoprot S.L.
The cell lines used in the present study may be classified into three different groups according to their origin: Normal melanocytes (HEMn-LP, HEMn-MP and HEMn-DP), primary melanomas (A375, G361, Sk-Mel-28, ME4405 and Mel-Ho) and metastatic melanomas (HT144, Hs294t, A2058, Sk-Mel-2, Sk-Mel-3, WM-266-4, VMM1, RPMI-7951 and Colo-800). Table SI provides a brief description of these cell lines.
The Mel-Ho cell line was authenticated by short-tandem repeat DNA profiling analysis (TH01: 7; D21S11: 29; D5S818: 12; D13S317: 11; D7S820: 10,11; D16S539: 11; CSF1P0: 12; AMEL: X; VWA: 14,18; TPOX: 8,10. The numbers after the loci name indicate the number of repeats of the repeat unit for that locus in the analysed cells' genome).
Cell culture
Melanocyte cell lines were cultured in medium 254 supplemented with human melanocyte growth supplement (Thermo Fisher Scientific, Inc.). The A375, ME4405, Hs294t, RPMI-7951, A2058 and HT144 lines were maintained in DMEM culture medium (MilliporeSigma), while the Sk-Mel-28, WM-266-4 and Sk-Mel-2 lines were cultured in Eagle's Minimum Essential Medium (ATCC). RPMI 1640-GlutaMAX™ (Thermo Fisher Scientific, Inc.) was used to culture the Mel-Ho, Colo-800 and VMM1 cell lines, and McCoy's 5A was used for G361 and Sk-Mel-3 cells (Thermo Fisher Scientific, Inc.). All the culture media used for melanoma cell lines were supplemented with 10% FBS (v/v; Cytiva), 100 UI/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific, Inc.). The cells were cultured at 37°C in a humidified atmosphere with 5% CO2. All of the cultured cell lines tested negative for mycoplasma (Venor® GeM One Step Test Kit; Minerva Biolabs).
Western blot analysis
Cell protein extracts were obtained from cells incubated for 15 min in RIPA buffer (Thermo Fisher Scientific, Inc.) containing protease inhibitor cocktail-3 and a phosphatase inhibitor (MilliporeSigma), and then centrifuged for 5 min at 15,000 × g at 4°C. The proteins were quantified with a BCA assay (EMD Millipore) and equal amounts (40 µg) of each protein extract were denatured for 30 min at 37°C (22) and separated by 7.5% SDS-PAGE electrophoresis under reducing conditions. The proteins were then wet-transferred to a nitrocellulose membrane (Cytiva) that was probed overnight at 4°C with primary antibodies against PLA2, PLC, PLD1, PLD2 and tubulin. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. The details of the antibodies are provided in Table I. The bands were visualized by enhanced chemiluminescence and digital images of the bands were obtained with a charge-couple device camera-based imager, the Syngene G box imaging system (Syngene).
Immunofluorescence staining
Cells were seeded on round coverslips, incubated overnight, fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were incubated overnight at 4°C with anti-human PLD2 primary antibody (1:200 dilution; Abgent), washed and then incubated for 2 h at room temperature in the dark with an Alexa-Fluor 594-conjugated secondary antibody (1:5,000 dilution; cat. no. ab150080; Abcam). Finally, the cells were incubated with Hoechst-33342 (Thermo Fisher Scientific, Inc.) for 5 min and the coverslips were mounted with Fluoromount-G (MilliporeSigma) on a microscope slide. The cells were visualized by generating 20 z-stacks on a Leica SP8 confocal fluorescence microscope (Leica Microsystems GmbH) at an original magnification of ×63.
PLD lipase activity
PLD activity in melanoma cells was assessed using a protocol described previously (26). First, liposomes were prepared from a short chain PC (8:0/8:0) (3.5 mM; Avanti Polar Lipids) in HEPES (pH 7.8; 45 mM), phosphatidylinositol 4,5-biphosphate (1 µM; Avanti Polar Lipids, Inc.) and 1.0 µCi [3H]-n-butanol (American Radiolabeled Chemicals Inc.). Pellets of the cell lines studied were resuspended in lysis buffer [5 mM HEPES (pH 7.8), 100 µM Na orthovanadate, 0.4% Triton X-100, 2 µg/ml aprotinin and 5 µg/ml leupeptin], sonicated on ice and the protein content of the sonicates was measured with a BCA assay. The liposomes were incubated for 20 min on a shaker with 50 µg of the cell sonicates at 30°C to achieve transphosphatidylation of PLD. This reaction was stopped by adding ice-cold chloroform/methanol (1:2) and the lipids in the samples were isolated and resolved by thin-layer chromatography. The amount of [3H]-phosphatidylbutanol (PBut) that co-migrated with the PBut standards was measured in a scintillation counter (PerkinElmer, Inc.).
PLD2 overexpression
Melanoma cells were seeded in 6-well plates and grown until 60–70% confluence. They were then transfected by incubation for 48 h with a transfection mixture of 1 µg of plasmid DNA (cat. no. RC202042; Origene Technologies, Inc.), TransIT®−2020 transfection reagent (Mirus Bio, LLC) and Opti-MEM™ (Thermo Fisher Scientific, Inc.). Control cells were transfected with empty plasmid (pc-DNA3.1-myc; Origene Technologies, Inc.) following the same protocol and the overexpression efficiency was confirmed using western blot analysis.
PLD2 silencing
When the melanoma cells cultured in 6-well plates reached 60–70% confluence, the culture medium was replaced with silencing mixture containing 50 nM small interfering (si)RNA (Ambion; Thermo Fisher Scientific, Inc.), Opti-MEM™ and Lipofectamine RNAiMax (Thermo Fisher Scientific, Inc.), and the cells were incubated for 48 h prior to performing the experiments. For control transfections, a non-targeting siRNA molecule suitable for negative controls (cat. no. AM4611; Ambion; Thermo Fisher Scientific, Inc.) was used following the same procedure and the correct silencing of the cells was confirmed using western blot analysis.
Cell proliferation assay
The relative proliferation capacity of cells after PLD2 silencing and overexpression was measured as the number of viable cells in that condition after 24 h in culture, relative to their control. Viable cells were quantified by XTT, which is based on dehydrogenase enzymes that reduce the tetrazolium salt to a highly coloured formazan dye. The amount of the formazan produced is proportional to the number of viable cells. It was confirmed that the treatments did not affect dehydrogenase enzyme activity compared to the results obtained by XTT and those calculated by trypan blue, so XTT is valid for measuring cell viability in the present study. Melanoma cells were grown overnight in 96-well plates in complete culture medium and then the medium was discarded and low-FBS medium (0.5%) was added. After 2 h in the incubator at 37°C, the medium was replaced with complete culture medium and 24 h later, XTT reagent (MilliporeSigma) was added to the cells. The cell absorbance in each experimental condition was measured at 450 nm in a Synergy HT spectrophotometer (BioTek Instruments, Inc.) and the proliferation rate of the cells was expressed as a ratio relative to their corresponding transfection controls.
Cell migration and invasion assay
Cell migration was assessed using Boyden chambers with 8-µm pores (Corning, Inc.). Melanoma cells were incubated for 2 h at 37°C in low-FBS medium (0.5%) and 400 µl of 5×104 cells/ml were seeded in the upper chambers in medium containing 0.5% FBS. For invasion, 400 µl of 7.5×105 cells/ml in 0.5% FBS medium were seeded in the upper chambers of Matrigel®-coated Boyden chambers (Corning, Inc.). Complete culture medium was added to the bottom well and after 20 h of incubation at 37°C, the non-migrated/invaded cells were removed by aspiration and wiping the upper side of the membrane with a cotton bud, while the migrated/invaded cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Inc.) and stained with 0.2% crystal violet at room temperature for 8 min. Finally, the filter was mounted and observed under a microscope at a magnification of ×20. Images of six different fields were acquired and the cells in each field were counted. The migratory/invasive capacity of each cell line was calculated as a ratio relative to their corresponding transfection control.
Statistical analysis
Values are expressed as the mean ± standard error of the mean of at least three experiments and the statistical significance of the differences between the means was calculated using one-way ANOVA and Bonferroni's post-hoc correction for the comparison of more than two data sets, or Dunnett's multiple-comparison test post-hoc correction for comparison of multiple data sets with their control as indicated in each experiment (GraphPad Prism 5.01; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
Results
PLD2 protein expression is upregulated in melanoma cell lines
As several phospholipases are overexpressed in a variety of cancers (1,27) and they participate in their malignant transition, the presence of this family of enzymes was assessed in melanoma. The expression of PLD2, PLD1, PLA2 and PLC was observed to vary in western blots of 17 cell lines, including skin melanocytes, as well as in primary and metastatic melanoma cell lines. In particular, while PLD2 was weakly expressed in skin melanocytes, it was expressed more strongly in melanoma cells (Figs. 1A and B and S1), particularly metastatic cells, although this increase in expression was cell line-specific. Conversely, no clear variation was observed in PLD1 protein expression between skin melanocytes and melanoma cells (Figs. 1A and C and S2). In fact, the expression of this protein in the HEMn-MP cell line was similar to that in all the primary melanomas except Mel-Ho. PLA2 was expressed more strongly by skin melanocytes than by primary melanomas (Fig. S3), although HT144, Colo-800 and RPMI-7951 metastatic melanomas had the highest levels of PLA2, a trait that was cell line-specific as it was not common to all the metastatic melanomas. PLC was expressed relatively homogeneously in the cell lines studied and no marked differences were observed (Fig. S3). The enhanced presence of PLD2 in melanoma cells was specific to this isoform, since the PLD1, PLA2 and PLC proteins remained relatively constant in melanocytes and melanoma cells.
Immunofluorescence analyses confirmed that PLD2 expression was upregulated in the malignant cell lines studied (Fig. 2). PLD2 was expressed more intensely in primary melanomas (A375, Mel-Ho) and metastatic melanomas (Hs294t, Colo-800), while its expression was faint in skin melanocytes (HEMn-LP, HEMn-MP). By contrast, immunofluorescence analysis of the other phospholipases (PLA2, PLC and PLD1) did not indicate any consistent differences between the cell lines studied (data not shown). Indeed, both the western blot and immunofluorescence data corroborated the enhanced expression of PLD2 in primary and metastatic melanomas relative to normal skin melanocytes.
Enhanced PLD lipase activity in melanoma cells
A comparative analysis of PLD activity was performed in melanocytes, primary melanomas and metastatic melanomas (Fig. 3). The results indicated that melanoma cells exhibited enhanced PLD activity and that this was associated with melanoma progression. Indeed, metastatic melanoma cells had significantly more lipase activity, while primary melanoma cell lines also had higher activity than skin melanocytes. However, the activity in the HEMn-LP melanocyte cell line was comparable to that in primary melanomas and therefore, the differences were not statistically significant between both groups. These results confirmed that, overall, the lipase activity of PLD enzymes is increased in melanoma cells and raise the question of whether they have a role in melanoma, particularly in the metastatic process.
PLD2 overexpression and silencing in melanoma cell lines
After demonstrating that aggressive melanoma cells have stronger PLD2 expression and activity than skin melanocytes, modifications to the amounts of this enzyme were performed to shed light on the implication of PLD2 in specific carcinogenic events. The effects of transient overexpression and silencing of PLD2 were examined in primary melanoma cells (A375, Mel-Ho) or metastatic melanoma cells (Colo-800), as confirmed in western blots (Figs. 4A and S4). Furthermore, these molecular alterations were also manifested through enzymatic activity, as lipase activity was altered in accordance with protein expression in all cases, although only in the A375 line, the lipase activity exhibited statistically significant differences compared to the transfection controls. As indicated in Fig. 4B, PLD2 overexpression was associated with an increase in lipase activity when the ratio relative to the transfection control was considered: A375 cells, 1.87; Mel-Ho, 1.54; and Colo-800, 1.4. By contrast, PLD2-silenced cells exhibited weaker lipase activity: A375, 0.52; Mel-Ho, 0.82; and Colo-800, 0.68.
PLD2 enhances the proliferation, migration and invasion of melanoma cells
The functional implications of the modifications to PLD2 expression were analyzed by studying the differences in proliferation, migration and invasion of transformed cells. Although these changes were cell line-specific, there was a clear trend towards an increase in proliferation, migration and invasion of PLD2-overexpressing cells, while these processes were downregulated in PLD2-silenced cells (Figs. 5 and 6). Indeed, cell proliferation increased markedly in A375 and Colo-800 cells overexpressing PLD2 but only slightly in MelHo cells. Conversely, in cells that downregulated PLD2, there was a consistent reduction in proliferation: A375, 0.55 (P<0,001); Mel-Ho, 0.8; and Colo-800, 0.79 (Fig. 5).
Melanoma cell migration and invasion were then assessed and PLD2-overexpressing primary melanoma cells exhibited moderate increments in cell migration: A375, 1.39; and Mel-Ho, 1.31. This effect was markedly enhanced in metastatic cells (Colo-800, 2.13), whereas the results were more similar among the silenced cell lines: A375, 0.66; Mel-Ho, 0.53; and Colo-800, 0.54 (Fig. 6A and B; images are provided in Fig. S5). Finally, increases in cell invasion in association with PLD2 overexpression were observed in primary melanoma cells (A375, 1.68; Mel-Ho, 1.71) relative to metastatic cells (Colo-800, 1.43). However, in the silenced cells, the results were more similar among the three cell lines, although primary melanoma cells were still more strongly affected: A375, 0.48; Mel-Ho, 0.53; and Colo-800, 0.60 (Fig. 6C and D; images are provided in Fig. S6). In conclusion, the present results suggested that higher levels of PLD2 increased the proliferation, migration and invasion of melanoma cells, while silencing of this enzyme decreased those activities.
Discussion
Phospholipases metabolize phospholipids, generating bioactive molecules that influence cell fate by regulating distinct cell activities, including events that favor tumorigenesis and metastasis (11–13,28). Deregulation of the expression and activity of phospholipases has been implicated in different diseases, including cancer. Indeed, the overexpression and enhanced activity of PLA2 serves as a diagnostic marker of breast cancer and is correlated with the tumor stage (29). Similarly, the present analysis of the PLD2 protein demonstrated that it accumulates at higher levels in metastatic melanoma than in normal skin melanocytes, as indicated by the PLD2 expression analyzed by western blot and immunofluorescence. However, the immunohistochemical analysis performed was limited, as neither the subcellular localization of PLD2 nor the changes in PLD2 expression in the transfected cells was examined. The results prompted us to analyze the activity of PLD, based on the notion that not only is PLD expression enhanced in cancer tissues but its activity is also increased (10,30). PLD activity was enhanced particularly in murine melanoma cells, in association with an acidic pH of the tumor microenvironment (31), consistent with the enhanced PLD activity observed previously in melanoma cells relative to skin melanocytes (22). This trait was verified in the present study, as significantly stronger PLD activity was observed in malignant cells. Furthermore, the present study was the first, to the best of our knowledge, to indicate that PLD activity in metastatic melanoma cells was substantially higher than in primary melanoma cells.
One role of PLD2 is the hydrolysis of PC present in cell membranes to release free Cho and a PA molecule. The molecules liberated are known precursors and second messengers that support a tumorigenic phenotype. Indeed, increased Cho levels have been identified in cancer cells (3–5). Cho is essential for the synthesis of PC and sphingomyelin lipids, important structural and signaling lipids involved in generating new cell membranes and reprogramming the metabolism of cancer cells, thereby supporting their malignant transformation. However, the effect of PLD activation is principally related to PA, a ubiquitous second messenger that interacts with important signaling molecules such as Akt and mTOR (13,23,32), both in physiological and pathological processes (1,33). These signaling pathways are constitutively activated in melanoma, promoting cell proliferation, migration, survival, angiogenesis, metabolism and chemoresistance. PLD-produced PA favors the phosphorylation of these interactors in melanoma cells with downregulated liver kinase B1 (23), thereby producing pro-tumorigenic effects (1,33–40). In addition, PA may also be dephosphorylated to yield DAG or hydrolyzed to yield Lyso-PA, both of which are potent signaling molecules known to favor carcinogenesis (32,41). In particular, Lyso-PA supports the migration of melanoma cells (42).
For the first time, to the best of our knowledge, the present study demonstrated the influence of PLD2 expression and activity on distinct carcinogenic events by overexpressing and silencing this enzyme in primary and metastatic melanoma cells. The results demonstrated that augmenting the activity and expression of PLD2 promoted a pro-tumorigenic phenotype in melanoma, driving the proliferation, invasion and migration of these cells, particularly in metastatic cells. Conversely, downregulation of PLD2 inhibited these activities. Similar results have been described in lymphoma (20) and breast cancer cells (12,19), and it was determined that PLD2 promoted metastatic activity by phosphorylating FAK and PAK, and by activating mTOR, JAK3 and Akt (20,43). Furthermore, PLD2 is present in the exosomes secreted by cancer cells, favoring the metastatic spread of prostate cells (44) and the crosstalk of colon cancer cells with their microenvironment (45). Thus, PLD has been associated with cancer progression and metastasis in several tumor types (10–12,17,21,28,44–48), to which melanoma may now be added. Concerning this point, the survival of melanoma patients with high and low PLD2 expression from The Cancer Genome Atlas PanCancer panel data were analysed using the cBioPortal platform (49,50). Although the results of this analysis should be interpreted with caution, they indicate a significant difference in overall survival between the two groups, where higher PLD2 expression (z score >1) is associated with shorter survival (Fig. S7). These data support the experimental results of the present study and reinforce the idea that, in melanoma, high PLD2 expression may be associated with malignant progression.
The association between PLD activity detected and the functional processes studied in transfected cells is not perfect, since the magnitude of this relationship differs between cell lines and the type of process. For instance, in Mel-Ho and Colo-800 cells overexpressing PLD2, similar levels of PLD activity result in quite different rates of proliferation and migration. The variability observed among them may be due to an activity of PLD2 other than its lipase activity. In fact, the GEF activity of PLD2 is important for cell motility and other functions (1,9,10). Indeed, PLD2 is a GEF for the small GTPases Rho, Rac-2 and Arf6, which fulfills an essential role in the actin rearrangements and filopodia formation required for cell motility and migration (6,16,51–53). These activities are consistent with the present results, as it was observed that the migratory and invasive capacities of melanoma cells increase when PLD2 is overexpressed, particularly in metastatic melanoma. In addition, the PLD activity determined indicated the presence of enzymatic activity of both PLD1 and PLD2, while in the functional study, only the protein levels of PLD2 were modified. On the other hand, the fact that the products of PLD2 activity may be converted into essential second messengers means that the range of effects of the enzyme is broad and its effect on biological activities such as those studied may be cell line-dependent.
Pharmacological inhibition or genetic downregulation of PLD2 has been proposed as a potential treatment option for certain cancer types. In breast cancer in particular, previous studies have indicated that the use of a PLD2-specific inhibitor markedly reduced the invasive capacity of cultured cell lines (21) and it promoted cancer cell apoptosis, while overexpression of PLD2 provided resistance to chemotherapeutic agents (54). Silencing of PLD2 or its pharmacological inhibition in a mouse model repressed tumor progression, whereas overexpression of this enzyme translated into an increase in tumorigenic potential (12). In lymphoma, catalytic inactivation of PLD2 reduced the formation of lung metastases (20) and in colorectal cancer cells, the pharmacological inhibition and silencing of PLD2 resulted in cancer cell autophagy (55).
In conclusion, PLD2 contributes to different aspects of cancer development including the production of survival signals (10,46,56–59), proliferation (60–63), resistance to apoptosis (10,64), angiogenesis (10,65), deregulated cellular energetics (10), as well as matrix substrate degradation, invadopodia formation, tumor-cell migration, invasion and metastatic dissemination (10,19,31,43,46,47,66,67). However, in melanoma, a specific role for PLD2 has yet to be described in tumorigenesis. The present study was the first to report increased PLD activity in metastatic melanoma cells relative to primary melanoma cells, as well as the influence of changes in PLD2 levels and activity in several properties of metastatic melanoma. In the light of the data on PLD2 from other cancers and the present in vitro results in melanoma, it is suggested that PLD2 may be a promising therapeutic target and biomarker for melanoma tumors, which merits further study.
Supplementary Material
Supporting Data
Supporting Data
Acknowledgements
The authors would like to thank Dr Julian Gomez-Cambronero from the Department of Biochemistry and Molecular Biology of Wright State University (Dayton, OH, USA) for his support with the PLD lipase activity measurements and PLD2 transfection in cultured cells.
Funding
This study was supported by grants from the University of the Basque Country/EHU (grant no. GIU17/066) and Ministerio de Economía y Competividad MINECO-ONCOFINDER of the Spanish Government (grant no. RTC.2015-3693-1).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Authors' contribution
APV performed the experiments described in the study, collected the data and drafted the manuscript. MDB and AA guided the design and execution of the study and revised the manuscript. BO provided advice on lipid biology and function and analyzed the results of certain experiments. KNS participated in the execution of PLD activity assays and PLD2 transfection assays. EA and GBG obtained the financial support and performed parts of the statistical analysis. APV and AA confirm the authenticity of all the raw data. All authors read, reviewed and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors have no competing interests to declare.
Glossary
Abbreviations
Abbreviations:
|
Cho |
choline |
|
DAG |
diacylglycerol |
|
GEF |
guanine nucleotide exchange factor |
|
PA |
phosphatidic acid |
|
PBut |
phosphatidylbutanol |
|
PC |
phosphatidylcholine |
|
PLD |
phospholipase D |
References
|
Bowling FZ, Frohman MA and Airola MV: Structure and regulation of human phospholipase D. Adv Biol. 79:1007832021.PubMed/NCBI | |
|
Zeisel SH and da Costa KA: Choline: An essential nutrient for public health. Nutr Rev. 67:615–623. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Onono FO and Morris AJ: Phospholipase d and choline metabolism. In: Lipid signaling in human diseases. Gomez-Cambronero J and Frohman MA: 259. Springer International Publishing; Cham: pp. 205–218. 2019 | |
|
Kosmopoulou M, Giannopoulou AF, Iliou A, Benaki D, Panagiotakis A, Velentzas AD, Konstantakou EG, Papassideri IS, Mikros E, Stravopodis DJ and Gikas E: Human melanoma-cell metabolic profiling: Identification of novel biomarkers indicating metastasis. Int J Mol Sci. 21:24362020. View Article : Google Scholar : PubMed/NCBI | |
|
Glunde K, Bhujwalla ZM and Ronen SM: Choline metabolism in malignant transformation. Nat Rev Cancer. 11:835–848. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Mahankali M, Henkels KM and Gomez-Cambronero J: A GEF-to-phospholipase molecular switch caused by phosphatidic acid, Rac and JAK tyrosine kinase that explains leukocyte cell migration. J Cell Sci. 126:1416–1428. 2013.PubMed/NCBI | |
|
Gomez-Cambronero J, Morris AJ and Henkels KM: PLD protein-protein interactions with signaling molecules and modulation by PA. Methods Enzymol. 583:327–357. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Henkels KM, Mahankali M and Gomez-Cambronero J: Increased cell growth due to a new lipase-GEF (Phospholipase D2) fastly acting on Ras. Cell Signal. 25:198–205. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Gomez-Cambronero J: Phospholipase D in cell signaling: From a myriad of cell functions to cancer growth and metastasis. J Biol Chem. 289:22557–22566. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Gomez-Cambronero J: Phosphatidic acid, phospholipase D and tumorigenesis. Adv Biol Regul. 54:197–206. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Brown HA, Thomas PG and Lindsley CW: Targeting phospholipase D in cancer, infection and neurodegenerative disorders. Nat Rev Drug Discov. 16:351–367. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Henkels KM, Boivin GP, Dudley ES, Berberich SJ and Gomez-Cambronero J: Phospholipase D (PLD) drives cell invasion, tumor growth and metastasis in a human breast cancer xenograph model. Oncogene. 32:5551–5562. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Yao Y, Wang X, Li H, Fan J, Qian X, Li H and Xu Y: Phospholipase D as a key modulator of cancer progression. Biol Rev Camb Philos Soc. 95:911–935. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Oshimoto H, Okamura S, Yoshida M and Mori M: Increased activity and expression of phospholipase D2 in human colorectal cancer. Oncol Res. 14:31–37. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Saito M, Iwadate M, Higashimoto M, Ono K, Takebayashi Y and Takenoshita S: Expression of phospholipase D2 in human colorectal carcinoma. Oncol Rep. 18:1329–1334. 2007.PubMed/NCBI | |
|
Kandori S, Kojima T, Matsuoka T, Yoshino T, Sugiyama A, Nakamura E, Shimazui T, Funakoshi Y, Kanaho Y and Nishiyama H: Phospholipase D2 promotes disease progression of renal cell carcinoma through the induction of angiogenin. Cancer Sci. 109:1865–1875. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
McDermott MI, Wang Y, Wakelam MJO and Bankaitis VA: Mammalian phospholipase D: Function, and therapeutics. Prog Lipid Res. 78:1010182020. View Article : Google Scholar : PubMed/NCBI | |
|
Noble AR, Hogg K, Suman R, Berney DM, Bourgoin S, Maitland NJ and Rumsby MG: Phospholipase D2 in prostate cancer: Protein expression changes with Gleason score. Br J Cancer. 121:1016–1026. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Henkels KM, Farkaly T, Mahankali M, Segall JE and Gomez-Cambronero J: Cell invasion of highly metastatic MTLn3 cancer cells is dependent on phospholipase D2 (PLD2) and janus kinase 3 (JAK3). J Mol Biol. 408:850–862. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Knoepp SM, Chahal MS, Xie Y, Zhang Z, Brauner DJ, Hallman MA, Robinson SA, Han S, Imai M, Tomlinson S and Meier KE: Effects of active and inactive phospholipase D2 on signal transduction, adhesion, migration, invasion, and metastasis in EL4 lymphoma cells. Mol Pharmacol. 74:574–584. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Scott SA, Selvy PE, Buck JR, Cho HP, Criswell TL, Thomas AL, Armstrong MD, Arteaga CL, Lindsley CW and Brown HA: Design of isoform-selective phospholipase D inhibitors that modulate cancer cell invasiveness. Nat Chem Biol. 5:108–117. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Riebeling C, Müller C and Geilen C: Expression and regulation of phospholipase D isoenzymes in human melanoma cells and primary melanocytes. Melanoma Res. 13:555–562. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Dogliotti G, Kullmann L, Dhumale P, Thiele C, Panichkina O, Mendl G, Houben R, Haferkamp S, Püschel AW and Krahn MP: Membrane-binding and activation of LKB1 by phosphatidic acid is essential for development and tumour suppression. Nat Commun. 8:157472017. View Article : Google Scholar : PubMed/NCBI | |
|
American Cancer Society, . Cancer Facts & Figures 2020. American Cancer Society; Atlanta, GA: 2020 | |
|
Guterres AN, Herlyn M and Villanueva J: Melanoma. eLS. John Wiley & Sons; Hoboken, NJ: pp. 1–10. 2018 | |
|
Gomez-Cambronero J, Horwitz J and Sha'afi RI: Measurements of phospholipases A2, C, and D (PLA2, PLC, and PLD): In vitro microassays, analysis of enzyme isoforms, and intact-cell assays. Methods Mol Biol. 218:155–176. 2003.PubMed/NCBI | |
|
Henderson F, Johnston HR, Badrock AP, Jones EA, Forster D, Nagaraju RT, Evangelou C, Kamarashev J, Green M, Fairclough M, et al: Enhanced fatty acid scavenging and glycerophospholipid metabolism accompany melanocyte neoplasia progression in zebrafish. Cancer Res. 79:2136–2151. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Park JB, Lee CS, Jang JH, Ghim J, Kim YJ, You S, Hwang D, Suh PG and Ryu SH: Phospholipase signalling networks in cancer. Nat Rev Cancer. 12:782–792. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Qu J, Zhao X, Wang J, Liu C, Sun Y, Cai H and Liu J: Plasma phospholipase A2 activity may serve as a novel diagnostic biomarker for the diagnosis of breast cancer. Oncol Lett. 15:5236–5242. 2018.PubMed/NCBI | |
|
Gomez-Cambronero J, Fite K and Miller TE: How miRs and mRNA deadenylases could post-transcriptionally regulate expression of tumor-promoting protein PLD. Adv Biol Regul. 68:107–119. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Kato Y, Lambert CA, Colige AC, Mineur P, Noël A, Frankenne F, Foidart JM, Baba M, Hata RI, Miyazaki K and Tsukuda M: Acidic extracellular pH induces matrix metalloproteinase-9 expression in mouse metastatic melanoma cells through the phospholipase D-mitogen-activated Protein Kinase Signaling. J Biol Chem. 280:10938–10944. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Jenkins GM and Frohman MA: Phospholipase D: A lipid centric review. Cell Mol Life Sci. 62:2305–2316. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Gomez-Cambronero J: New concepts in phospholipase D signaling in inflammation and cancer. ScientificWorldJournal. 10:1356–1369. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Yoon MS, Rosenberger CL, Wu C, Truong N, Sweedler JV and Chen J: Rapid mitogenic regulation of the mTORC1 inhibitor, DEPTOR, by phosphatidic acid. Mol Cell. 58:549–556. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Yin H, Gui Y, Du G, Frohman MA and Zheng XL: Dependence of Phospholipase D1 multi-monoubiquitination on its enzymatic activity and palmitoylation. J Biol Chem. 285:13580–13588. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Jang YH and Min DS: The hydrophobic amino acids involved in the interdomain association of phospholipase D1 regulate the shuttling of phospholipase D1 from vesicular organelles into the nucleus. Exp Mol Med. 44:571–577. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Donaldson JG: Phospholipase D in endocytosis and endosomal recycling pathways. Biochim Biophys Acta. 1791:845–849. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Dall'Armi C, Hurtado-Lorenzo A, Tian H, Morel E, Nezu A, Chan RB, Yu WH, Robinson KS, Yeku O, Small SA, et al: The phospholipase D1 pathway modulates macroautophagy. Nat Commun. 1:1422010. View Article : Google Scholar : PubMed/NCBI | |
|
Singh NK, Hansen DE III, Kundumani-Sridharan V and Rao GN: Both Kdr and Flt1 play a vital role in hypoxia-induced Src-PLD1-PKCγ-cPLA2 activation and retinal neovascularization. Blood. 121:1911–1923. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Liu Y, Su Y and Wang X: Phosphatidic Acid-Mediated Signaling. Lipid-mediated Protein Signaling. Capelluto DGS: Springer Netherlands; Dordrecht: pp. 159–176. 2013, View Article : Google Scholar | |
|
Frohman MA: The phospholipase D superfamily as therapeutic targets. Trends Pharmacol Sci. 36:137–144. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Lee S and Lynch KR: Brown recluse spider (Loxosceles reclusa) venom phospholipase D (PLD) generates lysophosphatidic acid (LPA). Biochem J. 391:317–323. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Wang Z, Zhang F, He J, Wu P, Tay LWR, Cai M, Nian W, Weng Y, Qin L, Chang JT, et al: Binding of PLD2-generated phosphatidic acid to KIF5B promotes MT1-MMP surface trafficking and lung metastasis of mouse breast cancer cells. Dev Cell. 43:186–197.e7. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Borel M, Lollo G, Magne D, Buchet R, Brizuela L and Mebarek S: Prostate cancer-derived exosomes promote osteoblast differentiation and activity through phospholipase D2. Biochim Biophys Acta Mol Basis Dis. 1866:1659192020. View Article : Google Scholar : PubMed/NCBI | |
|
Muñoz-Galván S, Lucena-Cacace A, Perez M, Otero-Albiol D, Gomez-Cambronero J and Carnero A: Tumor cell-secreted PLD increases tumor stemness by senescence-mediated communication with microenvironment. Oncogene. 38:1309–1323. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Zheng Y, Rodrik V, Toschi A, Shi M, Hui L, Shen Y and Foster DA: Phospholipase D couples survival and migration signals in stress response of human cancer cells. J Biol Chem. 281:15862–15868. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Williger BT, Ho WT and Exton JH: Phospholipase D mediates matrix metalloproteinase-9 secretion in phorbol ester-stimulated human fibrosarcoma cells. J Biol Chem. 274:735–738. 1999. View Article : Google Scholar : PubMed/NCBI | |
|
Noble AR, Maitland NJ, Berney DM and Rumsby MG: Phospholipase D inhibitors reduce human prostate cancer cell proliferation and colony formation. Br J Cancer. 118:189–199. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, Jacobsen A, Byrne CJ, Heuer ML, Larsson E, et al: The cBio cancer genomics portal: An open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2:401–404. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, Sun Y, Jacobsen A, Sinha R, Larsson E, et al: Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 6:pl12013. View Article : Google Scholar : PubMed/NCBI | |
|
Powner DJ and Wakelam MJO: The regulation of phospholipase D by inositol phospholipids and small GTPases. FEBS Lett. 531:62–64. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Santy LC and Casanova JE: Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J Cell Biol. 154:599–610. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Chae YC, Kim JH, Kim KL, Kim HW, Lee HY, Heo WD, Meyer T, Suh PG and Ryu SH: Phospholipase D activity regulates integrin-mediated cell spreading and migration by inducing gtp-rac translocation to the plasma membrane. Mol Biol Cell. 19:3111–3123. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Hwang WC, Kang DW, Kang Y, Jang Y, Kim JA and Min DS: Inhibition of phospholipase D2 augments histone deacetylase inhibitor-induced cell death in breast cancer cells. Biol Res. 53:342020. View Article : Google Scholar : PubMed/NCBI | |
|
Hwang WC, Kim MK, Song JH, Choi KY and Min DS: Inhibition of phospholipase D2 induces autophagy in colorectal cancer cells. Exp Mol Med. 46:e1242014. View Article : Google Scholar : PubMed/NCBI | |
|
Rodrik V, Zheng Y, Harrow F, Chen Y and Foster DA: Survival signals generated by estrogen and phospholipase D in MCF-7 breast cancer cells are dependent on myc. Mol Cell Biol. 25:7917–7925. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Hui L, Rodrik V, Pielak RM, Knirr S, Zheng Y and Foster DA: MTOR-dependent suppression of protein phosphatase 2a is critical for phospholipase D survival signals in human breast cancer cells. J Biol Chem. 280:35829–35835. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Hui L, Zheng Y, Yan Y, Bargonetti J and Foster DA: Mutant p53 in MDA-MB-231 breast cancer cells is stabilized by elevated phospholipase D activity and contributes to survival signals generated by phospholipase D. Oncogene. 25:7305–7310. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Chen Y, Rodrik V and Foster DA: Alternative phospholipase D/mTOR survival signal in human breast cancer cells. Oncogene. 24:672–679. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Moolenaar WH, Kruijer W, Tilly BC, Verlaan I, Bierman AJ and de Laat SW: Growth factor-like action of phosphatidic acid. Nature. 323:171–173. 1986. View Article : Google Scholar : PubMed/NCBI | |
|
Rizzo MA, Shome K, Vasudevan C, Stolz DB, Sung TC, Frohman MA, Watkins SC and Romero G: Phospholipase D and its product, phosphatidic acid, mediate agonist-dependent Raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway. J Biol Chem. 274:1131–1139. 1999. View Article : Google Scholar : PubMed/NCBI | |
|
Bruntz RC, Lindsley CW and Brown HA: Phospholipase D signaling pathways and phosphatidic acid as therapeutic targets in cancer. Pharmacol Rev. 66:1033–1079. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Zhao C, Du G, Skowronek K, Frohman MA and Bar-Sagi D: Phospholipase D2-generated phosphatidic acid couples EGFR stimulation to Ras activation by Sos. Nat Cell Biol. 9:707–712. 2007. View Article : Google Scholar | |
|
Chen Y, Zheng Y and Foster DA: Phospholipase D confers rapamycin resistance in human breast cancer cells. Oncogene. 22:3937–3942. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Ghim J, Moon JS, Lee CS, Lee J, Song P, Lee A, Jang JH, Kim D, Yoon JH, Koh YJ, et al: Endothelial deletion of phospholipase d2 reduces hypoxic response and pathological angiogenesis. Arterioscler Thromb Vasc Biol. 34:1697–1703. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Park MH, Ahn BH, Hong YK and Min DS: Overexpression of phospholipase D enhances matrix metalloproteinase-2 expression and glioma cell invasion via protein kinase C and protein kinase A/NF-κB/Sp1-mediated signaling pathways. Carcinogenesis. 30:356–365. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Kang DW, Park MH, Lee YJ, Kim HS, Lindsley CW, Brown HA and Min DS: Autoregulation of phospholipase D activity is coupled to selective induction of phospholipase D1 expression to promote invasion of breast cancer cells. Int J Cancer. 128:805–816. 2011. View Article : Google Scholar : PubMed/NCBI |
