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Originally published In Press as doi:10.1074/jbc.M600660200 on April 4, 2006

J. Biol. Chem., Vol. 281, Issue 23, 15862-15868, June 9, 2006
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Phospholipase D Couples Survival and Migration Signals in Stress Response of Human Cancer Cells*

Yang Zheng, Vanessa Rodrik, Alfredo Toschi, Ming Shi, Li Hui, Yingjie Shen, and David A. Foster1

From the Department of Biological Sciences, Hunter College of the City University of New York, New York, New York 10021

Received for publication, January 23, 2006 , and in revised form, March 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
MDA-MB-231 human breast cancer cells belong to a highly invasive metastatic cell line that depends on phospholipase D (PLD) activity for survival when deprived of serum growth factors. In response to the stress of serum withdrawal, there is a rapid and dramatic increase in PLD activity. Concomitant with increased PLD activity, there was an increase in the ability of MDA-MB-231 cells to both migrate and invade MatrigelTM. The ability of MDA-MB-231 cells to both migrate and invade MatrigelTM was dependent on both PLD and mTOR, a downstream target of PLD signals. Serum withdrawal also led to a PLD-dependent increase in the expression of the stress factor, hypoxia-inducible factor-1{alpha}. These data reveal that PLD survival signals not only prevent apoptosis but also stimulate cell migration and invasion, linking the ability to suppress apoptosis with the ability to metastasize.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The conversion of a normal cell to a malignant cancer cell involves multiple genetic alterations that overcome the many protections built into cells that prevent unwanted proliferation (1). Perhaps the most crucial step in progression to malignancy is gaining the ability to migrate or metastasize to distant sites where the growth of multiple tumors ultimately causes the lethal consequences of the cancer. Although there are several cellular properties that correlate with increased metastatic potential, such as increased protease secretion (2), there has never been a clear genetic event that confers metastatic capability. However, it has been suggested that mutations occurring at early stages of tumorigenesis that confer a proliferative advantage may also contribute to the ability to metastasize at later stages of tumor progression (3).

Among the obstacles to be overcome in a developing tumor are default apoptotic programs that cause cells with faulty division signals to undergo apoptosis (1). A cell must generate "survival signals" to suppress these apoptotic programs (4–6). Interestingly, signals that have been shown to suppress apoptosis have also been linked to cell migration, a hallmark of the metastatic phenotype. Both phosphatidylinositol 3-kinase and phospholipase D (PLD),2 which provide survival signals in human cancer cells (7–9), have also been linked with cellular processes that contribute to cell migration (8, 10). This correlation between survival and cell migration suggests that generating a survival signal early in tumorigenesis could also endow the cell with the ability to migrate. This raises the question as to how the migration would be triggered. One possibility is that although a primary tumor mass is forming, survival signals are selected for in cells deprived of blood serum to suppress the apoptosis that would occur in an unvascularized tumor mass. If the survival response of cells also includes increased cell migration, then in addition to suppression of apoptosis, the response would also include migration to sites where growth factors and nutrition could be obtained.

We recently described a survival signal in the highly malignant human breast cancer cell line MDA-MB-231 that involves PLD and one of its downstream targets, mTOR, the mammalian target of rapamycin (11). Under the stress of serum withdrawal, MDA-MB-231 cells undergo apoptosis if either PLD or mTOR was suppressed (11, 12). We report here that depriving MDA-MB-231 and other human cancer cells of serum results in a PLD-dependent survival signal that also promotes cell migration. We propose that the ability to metastasize is part of a "survival program" that suppresses apoptosis and enhances migration of stressed cells to more hospitable conditions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells, Cell Culture Conditions, and Transfection—All human cancer cell lines used in this study were obtained from the American Type Culture Collection and were maintained in Dulbecco's modified Eagle's medium with 10% bovine calf serum. Transfections were performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Transfection efficiency was determined by transfection of pEGFP-C1 (Clontech), which expresses green fluorescent protein. The percentage of green cells was determined microscopically and was routinely in excess of 70%. The generation of MCF-7 cells expressing PLD2 (MCF-7-P2 cells) has been described previously (13). These cells represent a pool of clones selected for G418 resistance as described (13).

Materials—Rapamycin was obtained from Sigma. Antibodies for hypoxia-inducible factor-1{alpha} (HIF1{alpha}), actin, and hemagglutinin were purchased from Santa Cruz Biotechnology. [3H]Myristic acid was obtained from PerkinElmer Life Sciences. Precoated silica 60A thin layer chromatography plates were from Whatman. Plasmid expression vectors for PLD2 (pCGN-mPLD2) and mPLD2-K758R (pCGN-mPLD2-K758R) (14) were the generous gift of Dr. Michael Frohman (SUNY, Stony Brook).

Western Blot Analysis—Extraction of proteins from cultured cells and Western blot analysis of extracted proteins was performed using the ECL system (Amersham Biosciences) as described (15).

Phospholipase D Assays—Cells were plated in 60-mm culture dishes at 1–2 x 105 cells/dish depending on their rate of growth with faster growing cells such as MDA-MB-231 cells being plated at the lower cell density. Two days later, cells were shifted to Dulbecco's modified Eagle's medium containing 0.5% bovine calf serum and kept overnight. Cells were then prelabeled for 4 h with [3H]myristate (3 µCi, 40 Ci/mmol) in 3 ml of medium. PLD catalyzed transphosphatidylation in the presence of 0.8% 1-BtOH, and the extraction and characterization of lipids by thin layer chromatography were performed as described previously (16).


Figure 1
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  		FIGURE 1.
Serum withdrawal increases PLD activity in MDA-MB-231 cells. A, MDA-MB-231 cells were plated in media containing 10% serum for 24 h. At this point, cells were shifted to media containing 0.5% serum for the times indicated. BtOH was added 20 min prior to harvesting of cells, and the transphosphatidylation product phosphatidylbutanol (PtBt) was determined by thin layer chromatography as described under "Experimental Procedures." The levels of phosphatidylbutanol are shown. B, MDA-MB-231 cells were plated in media containing 10% serum as described for A and then shifted to media containing 0.5% serum for 24 h. At this time, the cells were shifted back to media containing 10% serum for the times indicated. Phosphatidylbutanol levels were determined as described for A. Experiments shown are representative of ones repeated at least three times.

 
Migration and Invasion Assays—The assays were carried out using BIOCOATTM cell culture inserts that had polyethylene terephthalate filters (8-µm pore size) on the bottom. For migration assays, inserts were used directly without coating; and for invasion assays, the inserts were coated with MatrigelTM purified from the Engelbreth-Holm-Swarm mouse sarcoma, a tumor rich in extracellular matrix proteins, which closely mimics the basement membrane in vivo. Single cell suspensions in varying serum concentrations were added into the inserts. The inserts were set into 24-well plates that held 0.75 ml/well growth medium with the indicated serum concentration and incubated under normal growth condition for 24 h. Cells that had not penetrated the filters were wiped out with cotton swabs, and cells that had migrated or invaded to the lower surface of the filters were fixed in methanol and then stained with a 0.2% (v/v) solution of crystal violet in 2% (v/v) ethanol. The number of migrated or invaded cells was counted under microscope. The mean of five individual fields in the center of the filter where migration or invasion was the highest was obtained for each well.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Serum Withdrawal Increases PLD Activity in MDA-MB-231 Cells—We reported previously that PLD activity is elevated in MDA-MB-231 human breast cancers cells relative to the PLD activity in MCF-7 cells (12). Importantly, the elevated PLD activity in MDA-MB-231 cells provides an mTOR-dependent survival signal that suppresses apoptosis when these cells are deprived of serum (11, 12). Much of our previous work evaluating PLD activity in breast cancer cell lines was performed under low serum conditions to reduce background PLD activity. We were therefore surprised to find that serum actually suppressed PLD activity in MDA-MB-231 cells. Shown in Fig. 1A is the PLD activity in MDA-MB-231 cells in 10 and 0.5% serum. Surprisingly, there was more than 20-fold higher PLD activity in low (0.5%) serum than in high (10%) serum. Significant increases in PLD activity could be observed by 10 min after serum withdrawal (Fig. 1A). Adding back serum to MDA-MB-231 cells led to the immediate suppression of PLD activity, which could be observed within 10 min (Fig. 1B). These data indicate that the elevated PLD activity observed in MDA-MB-231 cells is a response to the lack of serum growth factors.

Serum Withdrawal Increases Cell Migration and Invasion in MDA-MB-231 Cells—As indicated in Fig. 1, serum withdrawal results in elevation of PLD activity. The absence of serum in culture mimics in part the lack of vascularization in an evolving tumor. To survive under these conditions, cells need to either stimulate vascularization or migrate to a site where oxygen, growth factors, and nutrition can be obtained. To investigate whether the removal of serum enhances cell migration, we examined the ability of MDA-MB-231 cells to migrate in the presence and absence of serum. To examine cell migration, we used the Transwell cell migration assay and found that MDA-MB-231 cells migrate 10 times better in low serum than in high serum (Fig. 2A). We also examined the ability to invade MatrigelTM, a hallmark of metastasis. As shown in Fig. 2B, serum withdrawal also increased the ability of the MDA-MB-231 cells to invade MatrigelTM by about 10-fold. These data reveal a correlation between increased PLD activity in response to serum withdrawal and increased ability to migrate and invade MatrigelTM.


Figure 2
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  		FIGURE 2.
Serum withdrawal increases cell motility and invasiveness of MDA-MB-231 cells. A, MDA-MB-231 cells placed in Transwell chambers with media containing either 0.5 or 10% serum as indicated for 24 h. At this time, cells that had migrated to the lower chamber were counted. Error bars represent the standard deviation from three independent experiments. B, MDA-MB-231 cells were placed in Transwell chambers that had been coated with MatrigelTM in media containing either 0.5 or 10% serum as indicated for 24 h. At this time, cells that had invaded the MatrigelTM and migrated to the lower chamber were counted. Error bars represent the standard deviation from three independent experiments.

 
The Migration and Invasion of MDA-MB-231 Cells is Dependent on PLD—We next examined whether the increased ability to migrate and invade MatrigelTM was dependent upon the increased PLD activity. To suppress PLD activity in the MDA-MB-231 cells, we introduced a catalytically inactive PLD2 (K758R), which we have used previously as a dominant negative mutant that suppresses both PLD activity and survival signals in these cells (11, 12, 16). The introduction of PLD2-K758R into the MDA-MB-231 cells via transient transfection suppressed the PLD activity in these cells to less than 50% of a vector control (Fig. 3A). The dominant negative PLD2 also suppressed both cell migration (Fig. 3B) and the invasion of MatrigelTM (Fig. 3C). For reasons that are not clear, invasion was consistently more sensitive to suppression of PLD activity than migration.

We demonstrated previously that survival signals generated by PLD in MDA-MB-231 cells are dependent upon mTOR (11). We therefore examined the effect of rapamycin, which inhibits mTOR, on the ability of MDA-MB-231 cells to both migrate and invade MatrigelTM. Rapamycin also blocked both cell migration (Fig. 4A) and invasion (Fig. 4B). These data indicate that PLD-induced cell migration and invasion, like PLD-induced survival (11), is through mTOR. The sensitivity to rapamycin also further indicates that cell migration and invasion in low serum are dependent on PLD because PLD activates mTOR in these cells (11).


Figure 3
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  		FIGURE 3.
The migration and invasion of MDA-MB-231 cells is dependent on PLD. A, PLD activity in MDA-MB-231 cells that had been transiently transfected with either a vector control (Con) or with pCGN-PLD2-K758R. 24 h after transfection, cells were placed in media containing 0.5% serum, and 24 h later, PLD activity was determined using the transphosphatidylation reaction. The phosphatidylbutanol bands were determined by densitometry. Error bars represent the standard deviation from three independent experiments. B, the cell motility of MDA-MB-231 cells transiently transfected with either a vector control or with pCGN-PLD2-K758R. 24 h after transfection, cells were placed in Transwell chambers in media containing 0.5% serum. 24 h later, cells in the bottom chamber were counted as described in the legend to Fig. 2. Error bars represent the standard deviation from three independent experiments. C, the ability to invade MatrigelTM of MDA-MB-231 cells transiently transfected with either a vector control or with pCGN-PLD2-K758R. 24 h after transfection, cells were placed in Transwell chambers coated with MatrigelTM in media containing 0.5% serum. 24 h later, cells in the bottom chamber were counted as described in the legend to Fig. 2. D, lysates from cells used in A, B, and C were subjected to Western blot analysis with antibodies raised against the hemagglutinin (HA) tag on PLD2-K758R (14) and actin. Error bars represent the standard deviation from three independent experiments.

 


Figure 4
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  		FIGURE 4.
The migration and invasion of MDA-MB-231 cells is dependent on mTOR. A, MDA-MB-231 cells placed in Transwell chambers in media containing 0.5% serum along with either Me2SO control (Con) or rapamycin (Rap) (15 µM). 24 h later, cells in the bottom chamber were counted as described in the legend to Fig. 2. Error bars represent the standard deviation from three independent experiments. B, MDA-MB-231 cells placed in Transwell chambers coated with MatrigelTM in media containing 0.5% serum along with either Me2SO control or rapamycin (15 µM). 24 h later, cells in the bottom chamber were counted as described in the legend to Fig. 2. Error bars represent the standard deviation from three independent experiments.

 
Elevated PLD Activity in MCF-7 Cells Enhances Cell Migration—Many studies have compared MDA-MB-231 cells with MCF-7 cells to distinguish between relatively benign phenotypes (MCF-7) and aggressive malignant phenotypes (MDA-MB-231) (17, 18). MDA-MB-231 cells have a much more malignant phenotype than MCF-7 cells with regard to cell motility (17, 18). We reported previously that MDA-MB-231 cells have as much as 50-fold higher levels of PLD activity than MCF-7 cells (11, 12, 19). We therefore wished to examine the impact of elevated PLD activity on cell migration in MCF-7 cells. As observed previously, the MDA-MB-231 cells have substantially elevated PLD activity relative to the MCF-7 cells and also have a substantially increased ability to migrate (Fig. 5, A and B). MCF-7 cells were stably transfected with PLD2, and pools of clones (MCF-7-P2 cells) were examined for PLD activity and for the ability to migrate. As shown in Fig. 5, C and D, the MCF-7-P2 cells had substantially elevated levels of PLD activity relative to vector control MCF-7v cells and had increased ability to migrate. Although the ability of the MCF-7-P2 cells to migrate was substantially increased relative to the MCF-7v cells, these cells did not migrate nearly as efficiently as the MDA-MB-231 cells, indicating that there are greater differences between MDA-MB-231 cells and MCF-7 cells than different PLD activity levels. However, because elevated PLD activity confers a survival signal in MCF-7 cells (11), the data provided here further link survival and migration signals in human breast cancer cells.

Serum Withdrawal from MDA-MB-231 Cells Increases Expression of HIF1{alpha}—In response to the stress of hypoxia, cells increase expression of HIF1{alpha} (20, 21). HIF1{alpha} is a component of the HIF1 transcription factor that stimulates transcription of several genes that promote angiogenesis such as vascular endothelial growth factor and the vascular endothelial growth factor receptor (20). Elevated HIF1{alpha} expression in cancer cells has been correlated with the "Warburg Effect" whereby there is increased aerobic glycolysis during normoxia (22, 23). Because of the link between oxidative stress and the stress of serum withdrawal, we examined the effect of serum withdrawal on HIF1{alpha} expression in the MDA-MB-231 cells. As shown in Fig. 6, there was a substantially higher level of HIF1{alpha} in cells in 0.5% serum than in cells maintained in 10% serum. The dependence of HIF1{alpha} on PLD activity was assessed using the "alcohol trap" assay (16) whereby primary but not tertiary alcohols are preferentially utilized over water in the hydrolysis of phosphatidylcholine to a corresponding inert phosphatidylalcohol rather than phosphatidic acid. As shown in Fig. 6, the elevated HIF1{alpha} seen in low serum was suppressed by primary but not tertiary butanol, indicating a dependence upon PLD activity. Consistent with PLD dependence, the dominant negative PLD2-K758R mutant also suppressed the increase in HIF1{alpha} expression induced by serum withdrawal (Fig. 6). The data in Fig. 6 indicate that the elevated PLD activity in MDA-MB-231 cells contributes to elevated expression of the stress factor HIF1{alpha}.


Figure 5
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  		FIGURE 5.
Elevated PLD activity in MCF-7 cells enhances cell migration and invasion. A, MDA-MB-231 and MCF-7 cells were plated in media containing 10% serum for 24 h. At that point, cells were shifted to media containing 0.5% serum. 24 h later, the PLD activity was determined as described in the legend to Fig. 3. Error bars represent the standard deviation from three independent experiments. B, MDA-MB-231 and MCF-7 cells placed in Transwell chambers in media containing 0.5% serum. 24 h later, cells in the bottom chamber were counted as described in the legend to Fig. 2. Error bars represent the standard deviation from three independent experiments. C, MCF-7 and MCF-7 cells stably transfected with PLD2 (52) were plated in media containing 10% serum for 24 h. At that point, cells were shifted to media containing 0.5% serum. 24 h later, the PLD activity was determined as described in the legend to Fig. 3. Error bars represent the standard deviation from three independent experiments. D, MCF-7 and MCF-7 cells stably transfected with PLD2 were placed in Transwell chambers in media containing 0.5% serum. 24 h later, cells in the bottom chamber were counted as described in the legend to Fig. 2. Error bars represent the standard deviation from three independent experiments.

 


Figure 6
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  		FIGURE 6.
Serum withdrawal elevates HIF1{alpha} in MDA-MB-231 cells. MDA-MB-231 cells (231) were plated in media containing 10% serum. 24 h later, the cells were placed in 0.5% serum as indicated. The cells were treated with the indicated alcohol, either 1-butanol (1-Bt) or tertiary-butanol (t-Bt), 4 h prior to harvest. Alternatively, MDA-MB-231 cells stably transfected with either vector control (231v) or pCGN-PLD2-K758R were placed in media containing 0.5% serum for 24 h. All cells were harvested, and the level of HIF1{alpha} was determined by Western blot analysis. Blots were reprobed for actin as a loading control. The experiment is representative of one repeated three times.

 
The Effect of Serum Withdrawal Increases Cell Migration in Other Cancer Cell Lines—To determine whether the effect of serum withdrawal on MDA-MB-231 cells was a general response of cancer cell or restricted to the MDA-MB-231 cells, we examined the effect of serum withdrawal upon migration, invasion, and PLD activity in a panel of human cancer cell lines with very different properties. The cell lines examined included the breast cancer cell lines MCF-7, MDA-MB-468, MDA-MB-435s, BT-549, T47D, and SK-BR3. We also examined T24 bladder carcinoma and Calu-1 lung carcinoma cells, which have highly elevated levels of PLD activity. Cells were grouped according to their PLD activity as indicated in Fig. 7A. Cells could be put into three categories. The first category included those with highly elevated levels of PLD activity, which included the T24 and Calu-1 cells in addition to the MDA-MB-231 cells. It was very apparent that the PLD activity in this group of cells was substantially higher in 0.5% serum than in 10% serum. In the second category were cell lines that had detectably elevated PLD activity in the presence of serum but did not have increased PLD activity when serum was reduced. These cell lines included BT-549, MDA-MB-468, and MCF-7 (Fig. 7A). In the third category, several cancer cell lines had very low levels of PLD activity in both low and high serum, including MDA-MB-435s, T47D, and SK-BR3 (Fig. 7A). Thus, there appears a pattern: 1) where PLD activity is elevated as a stress response (MDA-MB-231, T24, and Calu-1); 2) where PLD is elevated but not in response to stress (BT-549, MDA-MB-468, and MCF-7); and 3) where there is no elevated PLD activity (MDA-MB-435s, T47D, and SK-BR3).

We next evaluated the ability of these cancer cell lines to migrate and invade MatrigelTM. Five of the cell lines exhibited strong migration and invasive behavior. Interestingly, all five of these cell lines migrated (Fig. 7B) and invaded MatrigelTM (Fig. 7C) far more efficiently in 0.5% serum than in 10% serum. The five cell lines with enhanced migration and invasive behavior included not only all three cell lines where PLD activity was elevated in low serum (MDA-MB-231, T24, and Calu-1) but also included the MDA-MB-435s and BT-549 cells (Fig. 7, B and C). The MDA-MB-435s cells had very little PLD activity, and the BT-549 cells had elevated PLD activity but no significant increases in PLD activity in low serum. Thus, although there was a correlation between a stress-induced increase in PLD activity and increased cell migration, there were also cases where there was a stress-induced migration that was apparently independent of PLD activity. In all cases, the ability to migrate and invade MatrigelTM was enhanced under the stress of serum withdrawal, indicating that the migration and invasive properties of these cancer cells was a stress response.


Figure 7
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  		FIGURE 7.
The effect of serum withdrawal on PLD activity, cell migration, and invasion in other cancer cell lines. A, the indicated human cancer cell lines were plated in media containing 10% serum for 24 h. At that point, cells were shifted to media containing either 10 or 0.5% serum as indicated. 24 h later, the PLD activity was determined as described in the legend to Fig. 3. Error bars represent the standard deviation from at least two independent experiments. B, cells were placed in Transwell chambers in media containing either 10 or 0.5% serum as indicated. 24 h later, cells in the bottom chamber were counted as described in the legend to Fig. 2. Error bars represent the standard deviation from at least two independent experiments. C, cells were placed in Transwell chambers coated with MatrigelTM in media containing either 0.5 or 10% serum as indicated. Cells that had invaded the MatrigelTM and migrated to the lower chamber were counted 24 h later. Error bars represent the standard deviation from at least two independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In an emerging tumor, cells have to be able to survive in the absence of serum growth factors prior to vascularization. In this report, we have provided evidence that in response to the stress of serum withdrawal, there is an increase in PLD activity in MDA-MB-231 and other human cancer cells. The elevated PLD activity, which suppresses apoptosis in MDA-MB-231 cells under these conditions (11, 12), also stimulates increased cell migration and invasion. In addition, there was a PLD-dependent increase in the expression of HIF1{alpha}, which is elevated in response to oxidative stress and promotes vascularization (21). These data indicate that in response to the stress of serum withdrawal, there is the activation of a PLD-dependent survival program in MDA-MB-231 cells that suppresses apoptosis and enhances cell migration and invasion. Elevated PLD activity was also observed in T24 bladder carcinoma and Calu-1 lung carcinoma cells upon serum withdrawal, and there was also increased migration and invasion in response to serum withdrawal. The implication is that the genetic alterations that suppress default apoptotic pathways early in tumorigenesis are the same genetic alterations that ultimately facilitate cell migration and metastasis.


Figure 8
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  		FIGURE 8.
Model for enhanced survival and migration signals in a developing tumor. In a developing tumor mass (A), cells inside the mass (green cells) were subjected to hypoxia, nutrient, and growth factor deprivation (B). It is proposed that cells that elevate their PLD activity (red cells) in response to this stress will not only survive (C) but also gain the ability to migrate (D). Cells that find a site where vasculature can provide nutrients, oxygen, and growth factors will form secondary metastatic tumors (E).

 
It is not unreasonable to postulate that survival and cell motility are linked. In the absence of blood serum, if the cell is to survive, it needs to first suppress default apoptotic signals, but ultimately it needs to provide a means for obtaining needed factors provided by blood serum, including oxygen, nutrients, and growth factors. This involves bringing serum to the dividing cells either by stimulating the generation of blood vessels or, alternatively, by inducing migration to sites where blood vessels already exist. This is shown schematically in Fig. 8, where in an emerging tumor mass lacking vascularization there is a selection for cells with suppressed apoptosis. This could involve the activation of PLD or perhaps another survival pathway such as the phosphatidylinositol 3-kinase pathway (24). In this model, the activation of survival signals also stimulates cells to migrate to sites where serum growth factors are available.

At the core of stress responses may be mTOR, which regulates a variety of stress responses including those for nutrients, oxygen, and growth factor deprivation (25, 26). In this regard, the finding that mTOR has a phosphatidic acid requirement (27) likely represents a distinct mechanism for responding to stressful conditions. The PLD activity elevated in response to serum withdrawal in MDA-MB-231 cells generates an mTOR-dependent survival signal that suppresses apoptosis (11) and as reported here stimulates cell migration in an mTOR-dependent manner. mTOR is also targeted by survival signals generated by phosphatidylinositol 3-kinase (24) and has been the subject of much discussion on the targeting of mTOR in the treatment of cancer (5, 28). However, clinical trials with rapamycin derivatives have been largely disappointing (28). One important complication in targeting mTOR in cancer cells is the observation that elevated PLD activity leads to rapamycin resistance (19), presumably because of a competition between phosphatidic acid and rapamycin for mTOR (27). Therefore, although targeting mTOR in cancer makes sense because of its role in survival and migration signals, the effectiveness of treatment may depend upon reducing PLD activity to reduce the level of phosphatidic acid and consequently the level of rapamycin needed to block mTOR. This point underscores the importance of knowing whether PLD activity is elevated in a given tumor. In this regard, it is possible that anti-angiogenesis strategies, by limiting the supply of serum growth factors, could actually elevate PLD activity in tumors and increase the dose of rapamycin needed to suppress mTOR. Thus, suppressing PLD activity could be important for targeting mTOR with rapamycin derivatives.

It is not yet clear how PLD activity is elevated in MDA-MB-231 cells. There is elevated expression of PLD1 in these cells (12); however the increased expression does not account for the high level of activity seen in these cells. Moreover, the increase seen in response to serum withdrawal occurs before significant protein synthesis could occur. Although little is known about what activates PLD activity in MDA-MB-231 cells, it is of interest that the regulators of PLD1, RalA and Arf6 (8, 29–31), are both implicated in the regulation of cell migration. RalA was recently reported to stimulate the metastasis of transformed cells (32, 33) and was shown previously to activate metalloproteases (34). Other studies also link PLD activity with protease secretion (35–37). Similarly, Arf6 is shown to enhance cell migration (38, 39). The formation of stress fibers is linked to cell migration, and PLD activity is also implicated in stress fiber formation (40, 41). These studies make it clear that factors that regulate PLD activity and the responses to PLD signals are involved with cell migration and invasion and provide support for the coupling of survival signals and the processes that drive metastasis.

PLD activity is reported to be elevated in a wide variety of cancers including breast (42, 43), renal (44), gastric (45), and colon (46) cancer. As indicated here, PLD activity is elevated in T24 bladder carcinoma cells, which express an activated H-Ras gene (47) and in Calu-1 lung carcinoma cells, which express an elevated K-Ras gene (48). PLD is also shown to contribute to the transformation of cells in culture (15, 49), and PLD activity is elevated in response to both oncogenic and mitogenic stimuli (8, 9). Thus, it is becoming apparent that PLD likely plays a significant role in tumorigenesis. The data provided here indicate that in addition to providing survival signals thought to be critical in the early stages of tumorigenesis to suppress default apoptotic programs, PLD also stimulates cell migration in response to some of the stresses that occur in an unvascularized tumor. The ability of PLD to suppress apoptosis and enhance cell migration links survival signaling with metastasis and suggests that metastatic potential may be acquired earlier in tumorigenesis than generally thought. If metastasis is also part of a survival program acquiring the capability to metastasize early might explain, at least in part, the recent controversy over the benefits of mammography in reducing breast cancer mortality (50). Gøtzsche and Olsen (51) have provided evidence that mammography does not significantly reduce breast cancer mortality. Although this report has been widely criticized, it is remarkable that one could even make a case for no impact on mortality. If metastasis is a late event in tumorigenesis, then the impact of early detection should have an enormous effect on mortality. Whether there is an effect or not, it is clear that the impact of mammography is not as great as expected. This could be explained by the coupling of survival signaling and metastasis. In tumors where the processes are linked, metastasis may occur much earlier, and earlier detection may not have much of an impact on mortality. Therefore it is critical to know the genetic defects in a cancer cell that may define whether it has metastatic potential linked with survival mechanisms.


   FOOTNOTES
 
* This work was supported by NCI, National Institutes of Health (NIH) Grant CA46677, NIH SCORE Grant GM60654, and Research Centers in Minority Institutions Award RR-03037 from the National Center for Research Resources, NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biological Sciences, Hunter College of the City University of New York, 695 Park Ave., New York, NY 10021. Tel.: 212-772-4075; Fax: 212-772-5227; E-mail: foster{at}genectr.hunter.cuny.edu.

2 The abbreviations used are: PLD, phospholipase D; HIF1{alpha}, hypoxia-inducible factor-1{alpha}; mTOR, mammalian target of rapamycin. Back


   ACKNOWLEDGMENTS
 
We thank M. Frohman (SUNY, Stony Brook) for the PLD2 genes used to generate the inducible PLD expression vectors.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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