Alternative phospholipase D/mTOR survival signal in human ... - Nature

Oncogene (2005) 24, 672–679

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Alternative phospholipase D/mTOR survival signal in human breast cancer cells Yuhong Chen1, Vanessa Rodrik1 and David A Foster*,1 1

Department of Biological Sciences, Hunter College of The City University of New York, New York, NY 10021, USA

Cancer cells generate survival signals to suppress default apoptotic programs that protect from cancer. Phosphatidylinositol-3-kinase (PI3K) generates a survival signal that is frequently dysregulated in human cancers. Phospholipase D (PLD) has also been implicated in signals that promote survival. One of the targets of PLD signaling is mTOR (mammalian target of rapamycin), a critical regulator of cell cycle progression and cell growth. We report here that elevated PLD activity in the MDAMB-231 human breast cancer cell line generates an mTOR-dependent survival signal that is independent of PI3K. In contrast, MDA-MB-435S breast cancer cells, which have very low levels of PLD activity, are dependent on PI3K for survival signals. The data presented here identify an alternative survival signal that is dependent on PLD and mTOR and is active in a breast cancer cell line where the PI3K survival pathway is not active. Oncogene (2005) 24, 672–679. doi:10.1038/sj.onc.1208099 Published online 6 December 2004 Keywords: phospholipase D; survival signals; rapamycin; phosphatidylinositol-3-kinase; Akt; breast cancer

Introduction In the progression of a normal cell to a malignant cancer there are a number of obstacles that must be overcome in order for the cancer cells to divide and spread (Hanahan and Weinberg, 2000). One of these obstacles is overcoming default apoptosis programs that kick in when there is inappropriate proliferation signals or damage to the genome. This can be accomplished by the activation of survival signals that suppress apoptosis and allow progression through critical cell cycle checkpoints (Igney and Krammer, 2002). The beststudied survival signals are those generated by elevating the activity of phosphatidylinositol (PI)-3-kinase (PI3K). PI3K generates PI-3,4,5-tris-phosphate (PIP3), which leads to the activation of Akt protein kinase. Akt then phosphorylates and inactivates many substrate proteins that negatively regulate cell cycle progression *Correspondence: DA Foster; E-mail: [email protected] Received 15 October 2003; revised 6 April 2004; accepted 26 April 2004; published online 6 December 2004

and/or facilitate apoptosis (Vivanco and Sawyers, 2002). This survival signal pathway is activated in many cancers, frequently by the loss or inactivation of PTEN, a phosphatase that dephosphorylates PIP3 to PIP2 (Fry, 2001, Vivanco and Sawyers, 2002). One of the downstream targets of this PI3K/Akt pathway is mTOR (mammalian target of rapamycin), a protein kinase that regulates cell cycle progression and cell growth (Kuruvilla and Schreiber, 1999; Schmelzie and Hall, 2000). Rapamycin is a highly specific inhibitor of mTOR (Cafferkey et al., 1993; Brown et al., 1994; Sabatini et al., 1994) and has been widely used to block cell proliferation and is in clinical trials for treatment of breast and other cancers (Hidalgo and Rowinsky, 2000). Rapamycin acts by binding to the immunophilin FK506 binding protein 12, which then binds mTOR and inhibits its function in a poorly understood manner (Chen and Fang, 2002). mTOR has a requirement for phosphatidic acid (PA) for activation and PA interacts with the FK506 binding protein 12-rapamycin-binding domain of mTOR (Fang et al., 2001; Chen and Fang, 2002). It was proposed that rapamycin competes with PA binding to mTOR (Fang et al., 2001). PA is generated in response to mitogenic signals by the phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to PA and choline (Exton, 1999). Consistent with the hypothesis that rapamycin competes with PA for binding to mTOR, breast cancer cells with elevated PLD activity required higher levels of rapamycin to block cell proliferation (Chen et al., 2003). Elevated expression of PLD was also shown to lead to increased phosphorylation of the mTOR substrate ribosomal subunit S6 kinase 1 (S6K1) (Chen et al., 2003). More recently, PLD has been implicated in the activation of mTOR and S6K1 by Cdc42 (Fang et al., 2003) and lysophosphatidic acid (Kam and Exton, 2004). PLD activity is elevated in response to most mitogenic signals and has been reported to prevent cell cycle arrest and apoptosis (Joseph et al., 2002), and to fully transform cells with weakly transformed phenotypes (Lu et al., 2000; Joseph et al., 2001; Min et al., 2001; Ahn et al., 2003). PLD activity and expression has been reported to be elevated in a majority of breast (Noh et al., 2000) and renal (Zhao et al., 2000) cancers that were examined. MDA-MB-231 breast cancer cells have very highly elevated levels of PLD activity relative to other breast cancer cells, and PLD activity was

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implicated in the survival of this breast cancer cell line subjected to apoptotic stress (Zhong et al., 2003). Understanding survival signals activated in human cancers offers great therapeutic opportunities, since suppression of these signals, in principle, will induce cancers to kill themselves. In this report, we have characterized a novel survival signal generated by PLD that may be elevated in cancers where the wellcharacterized PTEN/PI3K/Akt survival pathway has not been activated. Results

used to represent highly malignant and relatively benign cancer cells, respectively (Yu et al., 2002). Consistent with this paradigm, the MCF-7 cells undergo apoptosis when subjected to serum withdrawal, whereas the MDA-MB-231 cells are resistant to this stress (Figure 1). Two additional breast cancer cell lines were also examined for tolerance to serum withdrawal, MDA-MB-435S and MCF-7R. The MCF-7R cells are a fast growing derivative of MCF-7 cells (Chen et al., 2003). As shown in Figure 1, both of these cell lines are resistant to the apoptotic stress of serum withdrawal as indicated by cell viability and the lack of PARP cleavage.

Resistance of breast cancer cell lines to serum withdrawal A hallmark of cancer cells is the ability to generate survival signals that overcome default apoptosis programs (Hanahan and Weinberg, 2000). Many cells undergo apoptosis in the absence of growth factors that stimulate survival signals. The MDA-MB-231 and MCF-7 breast cancer cell lines have frequently been

Survival signals are frequently generated through the PI3K/Akt signaling pathway. We therefore examined Akt phosphorylation in the four breast cancer cell lines examined in Figure 1. As shown in Figure 2a, there were elevated levels of Akt phosphorylation in the MDA-MB-435S cells relative to the MDA-MB-231, MCF-7, and MCF-7R. We previously reported that

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Figure 2 Survival signals in breast cancer cells. (a) The breast cancer cells examined in Figure 1 were examined for the level of phosphorylated Akt by Western blot analysis using an antibody specific for the phosphorylated form of Akt (upper row). Total Akt protein levels (lower panel) were determined using an anti-Akt antibody. The cells were prepared as in Figure 1 and placed in media lacking serum for 16 h. The experiment shown is representative of three independent experiments. (b) PLD activity in the breast cancer cells prepared as in (a) was determined as described previously (Chen et al., 2003). PLD activity was normalized to the PLD activity present in the MDA-MB-435S cells, which was given a value of 1. Error bars represent the standard deviation for three independent experiments Oncogene


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(Joseph et al., 2002; Zhong et al., 2003), we compared the levels of PLD activity in the MDA-MB-231 and MCF-7R cells with that in the MDA-MB-435S cells. As shown in Figure 2b, there were very low levels of PLD activity in the MDA-MB-435S cells relative to the MCF-7R and MDA-MB-231 cells. In fact, there was more PLD activity in the MCF-7 cells than in the MDAMB-435S cells (Figure 2b). These data suggested that alternative survival signals were being generated in the breast cancer cell lines resistant to the apoptotic stress of serum withdrawal. Differential sensitivity of breast cancer cells to inhibition of PI3K Akt phosphorylation is dependent on PI3K, which generates PIP3. PIP3 recruits both Akt and PDK1, the kinase that phosphorylates and activates Akt (Vivanco and Sawyers, 2002). To characterize the dependence of survival signaling upon PI3K and Akt, we examined the effect of the PI3K inhibitor LY294002 on cell survival in the absence of serum. LY294002 reduced cell viability (Figure 3a) and increased PARP cleavage (Figure 3b) in the MDA-MB-435S cells. We also examined the percentage of apoptotic cells using the Annexin V– FITC Apoptosis Detection Kit I (BD Biosciences Pharmingen) and obtained essentially the same pattern observed in Figure 3a (data not shown). Interestingly, both the MDA-MB-231 and MCF-7R cells were largely resistant to LY294002 in the absence of serum. As shown, in Figure 3c, LY294002 strongly reduced Akt phosphorylation in the MDA-MB-435S cells, which have elevated levels of Akt. In the MDA-MB-231 and the MCF-7R cells, LY294002 suppressed Akt phosphorylation slightly from an apparent uninduced level. mTOR, a target of the PI3K/Akt pathway phosphorylates S6K1 upon activation (Vivanco and Sawyers, 2002). We therefore examined whether LY294002

there were elevated levels of PLD activity in the MDAMB-231 and MCF-7R cells (Chen et al., 2003), and since PLD has also been reported to generate survival signals Oncogene

Figure 3 Differential sensitivity of breast cancer cells to inhibition of PI3K. (a) MDA-MB-435, MDA-MB-231, and MCF7R cells were plated as described in Figure 1 for 16 h. At this point, the cells were shifted to media lacking serum in the presence of the indicated concentration of LY294002. After 24 h, the percentage of nonviable cells was determined as in Figure 1. Error bars represent the standard deviation for three independent experiments. (b) MDAMB-435S, MDA-MB-231, and MCF7R cells were prepared as in (a) with the indicated levels of LY294002 and the level of cleaved PARP was determined by Western blot analysis as in Figure 1. Blots were stripped and reprobed with an antibody to b-tubulin to control for loading as in Figure 1. (c) MDA-MB-435S, MDA-MB231, and MCF-7R cells were prepared as in (a) and (b) with the indicated levels of LY294002 and the level Akt phosphorylation was determined as in Figure 2A. (d) MDA-MB-435S, MDA-MB231, and MCF7R cells were prepared as in (a) and (b) with the indicated levels of LY294002 and the level S6K1 phosphorylation was determined using an antibody specific for the phosphorylated form of S6K1 (upper panel). Total S6K1 levels (lower panel) were determined using an anti-S6K1 antibody. (e) MDA-MB-435S cells were prepared as in (a) and (b) with the indicated levels of LY294002 and the level BAD phosphorylation was determined using an antibody specific for the phosphorylated form of BAD (upper panel). Total BAD protein levels (lower panel) were determined using an anti-BAD antibody. The experiments shown in (b–e) are representative of three independent experiments


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inhibited S6K1 phosphorylation, and as shown in Figure 3d, LY294002 inhibited S6K1 phosphorylation in all three cell lines including the MDA-MB-231 and MCF-7R cells that were resistant to this treatment. We also examined the effect of LY294002 upon the phosphorylation state of the Akt substrate BAD (Datta et al., 1997) in the MDA-MB-435S cells, and as shown in Figure 3e, LY294002 also reduced the phosphorylation of the BAD in these cells. These data reveal that in MDA-MB-435S cells where there is elevated Akt phosphorylation, survival in the absence of serum is dependent on PI3K. More significantly, however, breast cancer cell lines with elevated PLD activity were apparently not dependent on the PI3K/Akt for survival in the absence of serum. These data support the hypothesis that alternative survival signals are operational in different breast cancer cells.

cells, which have the highest level of PLD activity. We also examined the effect of rapamycin on S6K1 phosphorylation, and as shown in Figure 4c, rapamycin blocked the phosphorylation of S6K1 in all three cell lines. The higher concentrations of rapamycin required for the inhibition of proliferation (Chen et al., 2003)

Rapamycin-sensitive survival signals in breast cancer cells with elevated PLD activity The data in Figure 3 indicated that survival in the absence of serum of the MDA-MB-231 and MCF-7R cells was independent of the PI3K/Akt pathway. As demonstrated in Figure 2, MDA-MB-231 and the MCF7R cells have elevated PLD activity, which was previously implicated in survival signaling of MDAMB-231 cells subjected to serum withdrawal (Zhong et al., 2003). PA, the metabolic product of PLD, has been reported to activate mTOR (Fang et al., 2001; Chen et al., 2003), which has also been implicated in survival signaling (Schmelzie and Hall, 2000). mTOR is inhibited by rapamycin, which was reported to be competitive with PA for binding mTOR (Fang et al., 2001). Rapamycin induced apoptosis in serum-deprived MDA-MB-231 and MCF-7R cells, but not in MDAMB-435S cells as indicated by reduced cell viability (Figure 4a) increased PARP cleavage (Figure 4b), and Annexin V–FITC staining (not shown). Consistent with rapamycin being competitive with PA (Fang et al., 2001, Chen et al., 2003), higher concentrations of rapamycin were required to induce apoptosis in the MDA-MB-231 Figure 4 Rapamycin-sensitive survival signals in breast cancer cells with elevated PLD activity. (a) MDA-MB-231, MCF7R, and MDA-MB-435S cells were plated as described in Figure 1 for 16 h. At this point, the cells were shifted to media lacking serum in the presence of the indicated concentration of rapamycin. After 24 h, the percentage of nonviable cells was determined as in Figure 1. Error bars represent the standard deviation for three independent experiments. (b) MDA-MB-231, MCF7R, and MDA-MB-435S cells were prepared as in (a) with the indicated levels of rapamycin, and the level of cleaved PARP was determined by Western blot analysis as in Figure 1. Blots were stripped and reprobed with an antibody to b-tubulin to control for loading as in Figure 1. (c) MDA-MB-231, MCF-7R, and MDA-MB-231 cells were prepared as in (a) and (b) with the indicated levels of rapamycin and the level S6K1 phosphorylation and total S6K1 protein levels were determined as in Figure 3. (d) MDA-MB-231 were transiently transfected with parental pcDNA3 expression vector and pcDNA3 vectors containing WT, RR, and RR/KD mTOR genes (Brunn et al., 1997; Fingar et al., 2004). After 24 h, rapamycin was added and cell viability was determined as in (a). The experiments shown in (b–d) are representative of at least two independent experiments Oncogene


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Elevated expression of PLD2 provides a rapamycin-sensitive survival signal in MCF-7 cells If PLD activity provides an mTOR-dependent survival signal, then elevating PLD activity in MCF-7 cells, which undergo apoptosis when deprived of serum, could generate a survival signal that would be sensitive to rapamycin. To test this hypothesis, PLD2 was transiently transfected into MCF-7 cells. PLD2 was used preferentially to PLD1 because cells tolerate PLD2 expression better than PLD1 (Joseph et al., 2001, 2002), and as reported previously (Zhong et al., 2003), both PLD1 and PLD2 were able to generate survival signals in rat fibroblasts. As shown in Figure 5a, cells transfected with PLD2 had a three-fold elevation of PLD activity. As reported previously, elevated PLD activity also leads to increased phosphorylation of S6K1 in MCF-7 cells (Figure 5b). The PLD2-transfected cells were tested for both cell viability (Figure 5c) and PARP cleavage (Figure 5d) upon serum withdrawal in the presence and absence of rapamycin. As expected, PLD2 inhibited apoptosis in the absence but not in the presence of rapamycin. These data demonstrate that PLD2 generates an mTOR-dependent survival signal.

Discussion In this report, we have provided evidence that survival signals generated by PLD are dependent on mTOR and active in breast cancer cells. MDA-MB-231 cells, which have highly elevated levels of PLD activity, have relatively low levels of Akt phosphorylation, indicating that the PI3K/Akt survival pathway is not highly active in these cells. In contrast, MDA-MB-435S cells have high levels of Akt phosphorylation and low levels of PLD activity. Inhibition of mTOR with rapamycin sensitized the MDA-MB-231, but not the MDA-MB435S cells, to the apoptotic stress of serum withdrawal. Oncogene

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caused no further decrease in S6K1 phosphorylation (data not shown). The induction of apoptosis also required more rapamycin than did inhibition of S6K1. To establish that the higher concentration of rapamycin required to induce apoptosis in the MDA-MB-231 cells was still dependent on mTOR, we introduced a rapamycin-resistant mutant of mTOR gene (mTOR-rr) and a rapamycin-resistant (RR) mTOR mutant that was kinase dead (mTOR-rr/KD) (Brunn et al., 1997; Fingar et al., 2004) into these cells and investigated whether the RR mutant could overcome the effects of rapamycin on cell viability and PARP cleavage. The RR mTOR, but not the KD mutant, was able to suppress rapamycininduced increases in cell death (Figure 4d) and PARP cleavage (not shown) in the MDA-MB-231 cells. The data presented in Figure 4 show that the effect of rapamycin upon cell survival was largely restricted to the cells with the elevated PLD activity. These data also demonstrate that the survival signals in the MDA-MB231 cells, which have highly elevated levels of PLD activity, are dependent on mTOR.

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Figure 5 Elevated expression of PLD2 provides a rapamycinsensitive survival signal in MCF-7 cells. (a) MCF-7 cells were plated in 60 mm culture dish at 2 105 cells/dish. After 16 h, the cells were transfected with either pCGN-mPLD2, which expresses wild-type PLD2 (Colley et al., 1997), or the pCGN empty vector. After 30 h, cells were placed in media containing 0.5% bovine calf serum for overnight and the relative PLD activity in the MCF-7 cells transfected with pCGN-mPLD2 relative to those transfected with the empty vector was determined as in Figure 2b. The data were normalized to the PLD activity in the MCF-7 cells transfected with the empty vector, which was given a value of 1. (b) Vectorand PLD2-transfected MCF-7 cells prepared in (a) were examined for the level S6K1 phosphorylation and total S6K1 protein as in Figure 3. (c) MCF-7 cells were prepared and transfected as in (a). At 30 h after transfection, the cells were placed in media with either 0 or 10% serum and rapamycin (5 mM) as indicated and the percentage of nonviable cells was determined 24 h later as in Figure 1. Error bars represent the standard deviation for three independent experiments. (d) PARP cleavage was also determined in the cells used in (c) as in Figure 1. The experiment shown is representative of three independent experiments

In contrast, inhibition of PI3K with LY294002 sensitized the MDA-MB-435S but not the MDA-MB-231 cells to apoptotic stress. These data reveal distinguishable alternative survival signals active in different


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human breast cancer cell lines – the well-established PI3K/Akt pathway and a novel PLD/mTOR pathway. We previously reported that elevated PLD activity provides resistance to rapamycin (Chen et al., 2003). It is therefore ironic that it is breast cancer cells with elevated PLD activity that are killed by rapamycin. We attribute this to the previously reported competitive binding of rapamycin and PA to mTOR (Fang et al., 2001). Thus, while higher concentrations of rapamycin are required to induce apoptosis in the cells with elevated PLD activity, the elevated PLD activity and mTOR activation are required for cell survival and, ironically, higher concentrations of rapamycin are required to block mTOR and the survival signals they mediate in the cells with the higher levels of PLD activity. This subtle point could have important implications for the efficacy of rapamycin in therapeutic strategies, because if PLD activity is elevated, as it is in many tumors, higher concentrations of rapamycin would be required to suppress mTOR. mTOR has also been implicated as one of several targets of the PI3K/Akt survival pathway (Nave et al., 1999; Sekulic et al., 2000). Akt phosphorylates and inactivates tuberous sclerosis complex-1 and -2, which negatively regulate mTOR (Inoki et al., 2002; Manning et al., 2002; Potter et al., 2002). Akt also phosphorylates and inactivates several other proteins that negatively regulate cell cycle progression. These include glycogen synthase kinase-3b (GSK3b), BAD, forkhead family transcription factors and MDM2 (Vivanco and Sawyers, 2002). Phosphorylation of proteins by GSK3b often targets proteins such as cyclin D for degradation by the proteasome (Vivanco and Sawyers, 2002). The impact of Akt upon mTOR is apparently critical since cells with elevated Akt activity are highly sensitive to rapamycin (Aoki et al., 2001; Mills et al., 2001; Neshat et al., 2001; Podsypanina et al., 2001). It is therefore of interest that Akt phosphorylation is apparently low in breast cancer cells with elevated PLD activity. This suggests that in cells with elevated PLD activity and subsequent activation of mTOR, survival signals are generated that circumvent the need for dysregulation of the PTEN/PI3K/Akt pathway. It has been proposed that dysregulation of the protein synthesis machinery through mTOR may generate cell cycle progression signals that contribute to cancer (Ruggero and Pandolfi, 2003). Thus, the ability of PLD to stimulate mTOR (Fang et al., 2001, 2003) may provide the most significant component of PLD-generated survival signals. A model for the alternative survival signals generated by the PLD/mTOR and PI3K/Akt pathways is presented in Figure 6. It may also be of interest that since PLD activity is dependent on PIP2 (Exton, 1999; Foster and Xu, 2003) and since PIP2 is converted to PIP3 by PI3K and the reverse reaction is catalysed by PTEN, these pathways could alternatively be activated by the regulation of PIP2 and PIP3 levels. Thus, activation of PI3K could shut down the PLD pathway by reducing PIP2 levels. Similarly, activating phospholipase C (PLC) could also reduce the levels of PIP2 and thereby suppress both survival pathways (Figure 6).

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Figure 6 Alternative survival signaling through the PLD/mTOR and PI3K/Akt pathways. Alternative survival signals are generated by the activation of the lipid modifying enzymes PLD and PI3K. PI3K generates PIP3, which recruits PDK1 and Akt. PDK1 phosphorylates and activated Akt. Akt then phosphorylates and inactivates several substrate proteins that negatively regulate cell proliferation or stimulate apoptosis. These include GSK3b, BAD, forkhead family transcription factors (FKHR) and MDM2 (reviewed by Vivanco and Sawyers, 2002). Akt also activates mTOR indirectly as discussed in the text, which indicates that there is overlap in the two pathways. PLD generates PA, which leads to the activation of mTOR. mTOR then phosphorylates several substrate proteins that regulate protein synthesis. It has been proposed that regulators of protein synthesis provide survival signals (Ruggero and Pandolfi, 2003). The two pathways are also connected by their different lipid requirements. Akt requires PIP3 and PLD requires PIP2, thus the generation of PIP3 from PIP2 by PI3K would deplete PIP2, whereas PTEN would generate PIP2 from PIP3. It is also possible that PLC could be a critical negative regulator of both survival pathways by removing PIP2 and thus shutting down both pathways

Rapamycin or the rapamycin analogue CCI-779 has been reported to block the growth of tumors that lack functional PTEN and the concomitant elevation of Akt (Neshat et al., 2001; Podsypanina et al., 2001). Similarly, rapamycin blocked the transformation of chicken embryo fibroblasts transformed by Akt (Aoki et al., 2001). The effect of rapamycin on PTEN-deficient cells was cytostatic and resulted in G1 arrest rather than apoptosis, nor was there any apoptosis reported in response to rapamycin in the Akt-transformed cells. However, these experiments were not performed under the low serum conditions used here. Therefore, it is difficult to compare these studies here. However, our studies clearly reveal a differential ability of rapamycin to induce apoptosis in the MDA-MB-435S and MDAMB-231 cells subjected to serum withdrawal. The MDA-MB-435S cells, which have elevated Akt phosphorylation, were less sensitive to rapamycin than the MDA-MB-231 cells, which do not have high levels of Akt phosphorylation. Moreover, the MDA-MB-231 cells required higher levels of rapamycin to induce apoptosis (Chen et al., 2003) – presumably due to competition with the PA generated by PLD (Fang et al., 2001; Chen and Fang, 2002). And this high concentration of rapamycin did not induce significant apoptosis in Oncogene


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the MDA-MB-435S cells. Thus, while rapamycin is cytostatic in cells with elevated PI3K/Akt signals, this treatment does not appear to induce apoptosis. PLD activity has been reported to be elevated in cells transformed by v-Src (Song et al., 1991), v-Ras (Jiang et al., 1995a), and v-Raf (Frankel et al., 1999). PLD activity is also elevated in response to mitogens including platelet-derived growth factor (Plevin et al., 1991), epidermal growth factor (Song et al., 1994), and insulin (Shome et al., 1997). More recently, PLD activity has been shown to cooperate with overexpression of a tyrosine kinase to transform rat fibroblasts (Lu et al., 2000; Joseph et al., 2001) and to overcome cell cycle arrest and apoptosis in cells with high intensity Raf signals (Joseph et al., 2002). In addition, many proteins that regulate PLD activity have also been implicated in regulating cell cycle progression and cell transformation, including RalA (Jiang et al., 1995b), Rho family GTPases (Exton, 1999), and PKC a (Hornia et al., 1999). Thus, evidence supporting a role for PLD in regulating both growth factor- and oncogene-induced cell proliferation is substantial. And, consistent with this hypothesis, elevated expression of PLD1 has been reported to be common in breast cancer tissues (Uchida et al., 1997; Noh et al., 2000). PLD activity has also been reported to be elevated in renal (Zhao et al., 2000) and gastric (Uchida et al., 1999) cancers and a polymorphism of the PLD2 gene was recently reported to be associated with the prevalence of colorectal cancer (Yamada et al., 2003). These reports suggest that elevated PLD activity could be providing a survival signal in these cancers and is therefore likely to be a contributing factor in cancer progression. The ability of PLD to provide a survival signal in breast cancer cells and in cells transformed by v-Src (Zhong et al., 2003) suggests that a critical aspect of PLD signaling is to prevent apoptosis. The data presented here indicate that mTOR-dependent PLD signals provide a novel survival signal in cells where the PI3K/Akt pathway is not activated.

polymerase (PARP) (human specific), Akt, phosphorylated Akt (Ser 473), BAD, and phosphorylated BAD (Ser 136) were obtained from Cell Signaling Technology. Antibody to b-tubulin was obtained from Santa Cruz Biotechnology. [3H]Myristic acid was obtained from New England Nuclear. Precoated silica 60A thin-layer chromatography plates were from Whatman.

Materials and methods

Western blot analysis

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Extraction of proteins from cultured cells and Western blot analysis of extracted proteins was performed using the ECL system (Amersham) as described (Lu et al., 2000; Zhong et al., 2002).

MCF-7, MDA-MB-435S, and MDA-MB-231 cells were obtained from the American Type Culture Collection and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% bovine calf serum. The MCF-7R cells are a fast growing derivative of MCF-7 cells described previously (Chen et al., 2003). Transfections were performed by using LipofectAMINE PLUSt reagent (GIBCO) according to the vendor’s instructions. Transfection efficiency was determined by transfection of pEGFP-C1 (Clonetech), which expresses green fluorescent protein. The percentage of green cells was determined microscopically and was routinely in excess of 90%. Materials Rapamycin was obtained from Sigma-Aldrich and LY294002 was from Calbiochem. Polyclonal antibodies to poly-(ADP-ribose) Oncogene

Plasmids The plasmid expression vector for PLD2 (pCGN-mPLD2) (Colley et al., 1997) was a generous gift from Dr Michael Frohman (SUNY, Stony Brook). pcDNA3-mTOR expression plasmids encoding wild-type (WT), RR (Ser2035Ile), KD (KD; Asp2338Ala), and double RR/KD alleles of rat mTOR (Brunn et al., 1997; Fingar et al., 2004) were kindly provided by Robert Abraham (Burnham Institute, San Diego, C52, USA). PLD assays MCF-7 and MDA-MB-435S cells were plated in 60 mm culture dish at 4 105 cells/dish; MCF-7R cells and MDA-MB-231cells were plated in 60 mm culture dish at 2 105 cells/dish. The next day, cells were made quiescent by shifting into DMEM containing 0.5% bovine calf serum overnight. Cells were then prelabeled for 4 h with [3H]myristate (3 mCi, 40 Ci/mmol) in 3 ml of medium. PLD catalysed transphosphatidylation in the presence of 0.8% 1-BtOH, and the extraction and characterization of lipids by thin-layer chromatography was performed as described (Chen et al., 2003). Cell viability and apoptosis assays Cell viability was determined by trypan blue exclusion. After various treatments, cells were harvested, washed, and treated with trypan blue at a concentration of 0.4% (w/v). After 10 min, trypan blue uptake (dead cells) was determined by counting on a hemocytometer. For determining the percentage of apoptotic cells, cells were stained according to the Annexin V–FITC Apoptosis Detection Kit I (BD Biosciences Pharmingen) and analysed using a FAC-Sort flow cytometer, with acquisition of a total of 10 000 events/sample to ensure adequate mean data.

Acknowledgements We thank Michael Frohman (SUNY, Stony Brook) for the mPLD2 genes used in this study and Bob Abraham (Burnham Institute) for the mTOR constructs. John Blenis is acknowledged for several helpful discussions. This work was supported by grants from the National Cancer Institute (CA46677) and a SCORE grant from the National Institutes of Health (GM60654). Research Centers in Minority Institutions award RR-03037 from the National Center for Research Resources of the National Institutes of Health, which supports infrastructure and instrumentation in the Biological Sciences Department at Hunter College, is also acknowledged.


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