Elevated PLD activity has been observed in a large number of human cancers and human cancer cells (8
). PLD activity has also been implicated in survival signaling in cancer cells (5
). The PLD metabolite PA has been implicated in the activation of mTOR (7
), which has also been widely implicated in cancer survival signals (13
). However, a role for PLD in the survival signals mediated by mTOR has not been widely accepted. The data provided here reveal that PLD and its metabolite PA are critical for the formation of both mTORC1 and mTORC2 complexes. This study reinforces the concepts that rapamycin suppresses mTOR by interfering with the interaction between mTOR and PA and that mTOR can become more sensitive to rapamycin by reducing PA levels. Data presented here demonstrate that the mTORC2-dependent phosphorylation of Akt at Ser473 requires PLD activity and PA. Akt, like mTOR, is a critical node for cancer survival signals, and phosphorylation of Akt at Ser473 has been strongly correlated with elevated Akt activity (13
). The dependence of Akt phosphorylation at Ser 473 on PLD implicates PA as a critical regulator of Akt-mediated survival signals. The PLD dependence was observed in renal cancer cells, where there is elevated basal Akt phosphorylation, and also in insulin-stimulated increases in Akt phosphorylation in a breast cancer cell line, where there are reduced levels of Akt phosphorylation. Data were also presented demonstrating that the suppression of mTORC1 by PRAS40 is reversed when PLD activity is suppressed. Collectively, these data firmly establish a role for PLD-generated PA in the regulation of both mTORC1 and mTORC2 in human cancer cells and suggest that targeting PLD signaling represents a means for suppressing mTOR-dependent survival signals and enhancing the efficacy of rapamycin-based therapeutic strategies in cancers where PLD and mTOR suppress default apoptotic programs that protect against cancer.
While the specificity of rapamycin for mTOR has been known for some time, the mechanism of action has not been established. Jie Chen's group reported that PA interacted with mTOR in a manner that was competitive with rapamycin but did not have any apparent impact on the kinase activity of mTOR (4
). Our group subsequently demonstrated that elevated PLD activity increased the concentration of rapamycin needed to suppress S6 kinase phosphorylation and cell proliferation (2
). Recent structural studies have revealed that PA interacts with the FRB domain of mTOR and causes structural changes similar to those observed when rapamycin-FKBP12 binds to the FRB domain (35
). Sabatini's group reported recently that rapamycin could prevent the association of mTOR with other components of the mTORC2 complex. Our finding that PLD is required for the stability of complexes between mTOR and Rictor and between mTOR and Raptor is consistent with the observations that rapamycin prevents mTOR2 complex formation and that PA interacts with mTOR in a manner that is competitive with rapamycin (2
). While rapamycin disrupts the interaction between mTOR and Raptor, the presence of a cross-linker in mTOR immunoprecipitations restored the association (17
). In contrast, reducing PA levels with 1-BtOH apparently disrupted the mTOR complexes such that cross-linking did not restore the association between mTOR and either Raptor or Rictor—implying that the lack of PA disrupts the mTOR complexes more than rapamycin does. The major implications of this study are that PLD and PA regulate mTOR signaling by facilitating the formation of mTOR complexes and that rapamycin inhibits mTOR by interfering with the PA-mTOR interaction.
Our previous study demonstrating that elevated PLD activity increased the dose of rapamycin needed to suppress S6 kinase phosphorylation and cell proliferation (2
) revealed something that was confusing. The amount of rapamycin needed to suppress cell proliferation was greater than that needed to suppress S6 kinase phosphorylation. The effect of high-dose rapamycin needed to suppress proliferation and induce apoptosis in MDA-MB-231 cells was reversed when rapamycin-resistant mutant mTOR was used (1
)—indicating that the effect of the high doses of rapamycin involved mTOR. Data provided in Fig. reveal that higher concentrations of 1-BtOH are required to suppress Akt phosphorylation at Ser 473 than are needed to suppress S6 kinase phosphorylation. This suggests that higher concentrations of 1-BtOH are required to suppress mTORC2 than are required to suppress mTORC1. The implications of this finding are that mTORC2 binds PA more strongly than mTORC1 and that lower concentrations of PA in the cell are needed when PA dissociates in order for PA to stay dissociated from mTORC2. This observation also suggests why higher concentrations of rapamycin are needed to compete for binding to mTORC2 than to compete for binding to mTORC1. The data provided in Fig. reveal a differential rapamycin dose response for S6 kinase phosphorylation and Akt phosphorylation at Ser473. The higher sensitivity of S6 kinase phosphorylation to rapamycin is consistent with a requirement for mTORC1, which has an apparently lower affinity for PA than does mTORC2. The weaker association of PA with mTORC1 means that PA and mTORC1 will dissociate more often, and thus, lower concentrations of rapamycin would be needed to replace PA on mTORC1 when there is a dissociation. In contrast, the higher concentration of rapamycin needed to suppress Akt phosphorylation at Ser473 reflects a requirement for only mTORC2, which apparently binds PA more strongly, and therefore dissociations are rare—meaning that very high concentrations of rapamycin would be needed to bind the low concentrations of mTOR obtained when PA dissociates from mTORC2. Thus, the dissociation constant for PA and mTORC2 (Kd2
) would be less than the dissociation constant for mTORC1 and PA (Kd1
). This is depicted in a simplified model for the differential strength of association between mTORC1 and mTORC2 with PA in Fig. . We propose that rapamycin-FKBP12 binds the FRB domain of mTOR only when PA is dissociated from the FRB domain. However, since the association of PA with mTORC2 is stronger than the association with mTORC1, the dissociation of PA from mTORC2 is less frequent and higher concentrations of rapamycin are needed to interact with the rare mTOR released from mTORC2. The rate constants described in the proposed model are only for the interaction between mTOR and PA, and therefore the model represents an oversimplification, since the involvement of Rictor and Raptor, along with other components of the mTORC1 and mTORC2 complexes, has been neglected. However, the model does provide a first approximation of the differential stability of mTORC1 and mTORC2 complexes that is consistent with the observed differential sensitivities to rapamycin and PA.
FIG. 8. Model of the differential effects of rapamycin (Rap) and 1-BtOH on mTORC1 and mTORC2. The constants of mTORC1 and mTORC2 dissociation (KDs) from PA represent the ratios of the rate constants of dissociation (kd) and formation (kf). The data provided here (more ...)
We used two cell lines in this study, 786-O renal cancer cells and MDA-MB-231 breast cancer cells—both of which have high levels of PLD activity (32
). The PLD activity of MDA-MB-231 cells is highly elevated only in the absence of serum, whereas the elevated PLD activity in 786-O cells occurs both in the presence and in the absence of serum. While the significance of this difference is not clear, interestingly, Akt phosphorylation at Ser473 is high in 786-O cells and low in MDA-MB-231 cells. The low level of Akt phosphorylation in MDA-MB-231 cells in the absence of serum, where PLD activity is high (39
), clearly reveals that PLD activity and PA are not sufficient by themselves to activate mTORC2 and cause the phosphorylation of Akt at Ser473. In contrast, the introduction of an exogenous PLD gene did stimulate the phosphorylation of S6 kinase (1
), suggesting that mTORC1 can be activated by elevated levels of PA. Exogenously provided PA has been reported to stimulate S6 kinase, but only in the presence of amino acids (4
) or suppressed TSC2 (31
). Thus, PA stimulates mTORC1 in conjunction with other input. It will be of interest to determine what signals, in addition to those that activate PLD, are necessary for the activation of mTORC2. The data provided here showing that insulin can stimulate Akt phosphorylation in a PLD-dependent manner in MDA-MB-231 cells may provide a lead as to the additional signals needed to activate mTORC2. While insulin increases PLD activity (8
), the already elevated PLD activity in MDA-MB-231 cells makes it likely that another aspect of insulin signaling is required for the activation of mTORC2. Recently, Rosen and colleagues demonstrated that suppression of mTORC1 led to an increase in Akt phosphorylation at Ser473 that was dependent on IGF-1 (20
). A similar observation made by Wan et al. (36
) demonstrated that suppression of S6 kinase increased the phosphorylation of Akt at Ser473. Thus, components of IGF-1 and insulin signaling are likely to be an important component, in addition to PA, for mTORC2 activation.
There are two major classes of PLD isoforms—PLD1 and PLD2. We have found that dominant negative mutant forms of both PLD1 and PLD2 suppress S6 kinase and Akt phosphorylation and that using both mutant forms together is more effective than using either one by itself. This implies that both PLD1 and PLD2 are involved. We have noticed the same phenomenon for receptor endocytosis (26
) and HIFα expression (32
). Thus, we believe that both PLD isoforms are involved. In this regard, there are two recent reports that support this hypothesis—at least for mTORC1. Sung Ho Ryu and colleagues reported that PLD2 associates functionally with Raptor (15
), implicating PLD2 in the activation of mTORC1, and very recently Chen and colleagues reported that PLD1 is a direct target of Rheb (28
), which functions upstream from mTORC1—implicating PLD1 in the activation of mTORC1. While this study used short hairpin RNA for PLD2 to rule out a role for PLD2 in Rheb-induced S6 kinase phosphorylation, it is possible that this procedure did not reduce PLD2 protein levels sufficiently to completely suppress its effect. We have found that PLD2 is especially resistant to downregulation when using siRNA approaches (32
; our unpublished observations). Thus, at present, there is evidence supporting a role for both PLD1 and PLD2 in the activation of mTORC1. The data presented here indicate that both PLD1 and PLD2 are also involved in the activation of mTORC2, although the mechanism remains to be worked out.
Rapamycin and rapamycin derivatives have been widely employed in clinical trials, with mostly disappointing results (25
). A recent clinical study focused on glioblastoma, where there are commonly defects in PTEN (3
). This study indicated that there was cell cycle arrest in response to rapamycin and effects on S6 kinase phosphorylation—implicating mTORC1. As indicated here, mTORC1 is much more sensitive to rapamycin than is mTORC2. However, Akt phosphorylation at Ser473 is dependent on mTORC2, indicating that mTORC2 may more critical in cancer—in that Akt phosphorylates many key substrates critical for cancer cell survival (19
). Thus, targeting mTOR effectively may require strategies that suppress mTORC2. Consistent with this hypothesis, we recently found that mTORC2 is required for the expression of HIF2α in kidney cancer cells (33
Kaelin and colleagues have reported that HIF2α is required for tumor formation by kidney cancer cells (18
). These two studies reinforce the concept that mTORC2 could be an important target of anticancer therapies. As indicated in this study, suppressing PLD activity makes rapamycin effective in suppressing mTORC2 in 786-O cells, which have high levels of PLD activity (32
). It is therefore possible that combining strategies that suppress PLD activity with rapamycin could improve the efficacy of rapamycin. While there are no drugs currently being used to target PLD directly, targeting the intracellular signals that increase PLD activity remains a possibility. We just recently reported that a natural product from Magnolia grandiflora
known as honokiol suppresses PLD activity (12
) and might therefore be used in combination with rapamycin to suppress mTORC2. It will therefore be of interest to determine whether honokiol can improve the efficacy of rapamycin in cancer cells with elevated PLD activity. This study provides a rationale for targeting the signals that regulate PLD activity to increase the efficacy of rapamycin, which has produced mixed results in clinical trials.