Alfredo Toschi

Regulation of mTORC1 and mTORC2 Complex Assembly by Phosphatidic Acid: Competition with Rapamycin

ABSTRACT mTOR, the mammalian target of rapamycin, is a critical node for control of cell growth and survival and has widely been implicated in cancer survival signals. mTOR exists in two complexes: mTORC1 and mTORC2. Phospholipase D (PLD) and its metabolite phosphatidic acid (PA) have been implicated in the regulation of mTOR; however, their role has been controversial. We report here that suppression of PLD prevents phosphorylation of the mTORC1 substrate S6 kinase (S6K) at Thr389 and the mTORC2 substrate Akt at Ser473. Suppression of PLD also blocked insulin-stimulated Akt phosphorylation at Ser473 and the mTORC2-dependent phosphorylation of PRAS40. Importantly, PA was required for the association of mTOR with Raptor to form mTORC1 and that of mTOR with Rictor to form mTORC2. The effect of PA was competitive with rapamycin—with much higher concentrations of rapamycin needed to compete with the PA-mTORC2 interaction than with PA-mTORC1. Suppressing PA production substantially increased the sensitivity of mTORC2 to rapamycin. Data provided here demonstrate a PA requirement for the stabilization of both mTORC1 and mTORC2 complexes and reveal a mechanism for the inhibitory effect of rapamycin on mTOR. This study also suggests that by suppressing PLD activity, mTORC2 could be targeted therapeutically with rapamycin.

Differential Dependence of Hypoxia-inducible Factors 1α and 2α on mTORC1 and mTORC2

Constitutive expression of hypoxia-inducible factor (HIF) has been implicated in several proliferative disorders. Constitutive expression of HIF1α and HIF2α has been linked to a number of human cancers, especially renal cell carcinoma (RCC), in which HIF2α expression is the more important contributor. Expression of HIF1α is dependent on the mammalian target of rapamycin (mTOR) and is sensitive to rapamycin. In contrast, there have been no reports linking HIF2α expression with mTOR. mTOR exists in two complexes, mTORC1 and mTORC2, which are differentially sensitive to rapamycin. We report here that although there are clear differences in the sensitivity of HIF1α and HIF2α to rapamycin, both HIF1α and HIF2α expression is dependent on mTOR. HIF1α expression was dependent on both Raptor (a constituent of mTORC1) and Rictor (a constitutive of mTORC2). In contrast, HIF2α was dependent only on the mTORC2 constituent Rictor. These data indicate that although HIF1α is dependent on both mTORC1 and mTORC2, HIF2α is dependent only on mTORC2. We also examined the dependence of HIFα expression on the mTORC2 substrate Akt, which exists as three different isoforms, Akt1, Akt2, and Akt3. Interestingly, the expression of HIF2α was dependent on Akt2, whereas that of HIF1α was dependent on Akt3. Because HIF2α is apparently more critical in RCC, this study underscores the importance of targeting mTORC2 and perhaps Akt2 signaling in RCC and other proliferative disorders in which HIF2α has been implicated.

Differential dependence of HIF1α and HIF2α on mTORC1 and mTORC2

Constitutive expression of hypoxia-inducible factor (HIF) has been implicated in several proliferative disorders. Constitutive expression of HIF1α and HIF2α has been linked to a number of human cancers, especially renal cell carcinoma (RCC), in which HIF2α expression is the more important contributor. Expression of HIF1α is dependent on the mammalian target of rapamycin (mTOR) and is sensitive to rapamycin. In contrast, there have been no reports linking HIF2α expression with mTOR. mTOR exists in two complexes, mTORC1 and mTORC2, which are differentially sensitive to rapamycin. We report here that although there are clear differences in the sensitivity of HIF1α and HIF2α to rapamycin, both HIF1α and HIF2α expression is dependent on mTOR. HIF1α expression was dependent on both Raptor (a constituent of mTORC1) and Rictor (a constitutive of mTORC2). In contrast, HIF2α was dependent only on the mTORC2 constituent Rictor. These data indicate that although HIF1α is dependent on both mTORC1 and mTORC2, HIF2α is dependent only on mTORC2. We also examined the dependence of HIFα expression on the mTORC2 substrate Akt, which exists as three different isoforms, Akt1, Akt2, and Akt3. Interestingly, the expression of HIF2α was dependent on Akt2, whereas that of HIF1α was dependent on Akt3. Because HIF2α is apparently more critical in RCC, this study underscores the importance of targeting mTORC2 and perhaps Akt2 signaling in RCC and other proliferative disorders in which HIF2α has been implicated.

Phospholipase D Couples Survival and Migration Signals in Stress Response of Human Cancer Cells

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 Matrigel™. The ability of MDA-MB-231 cells to both migrate and invade Matrigel™ 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-1α. 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.

Targeting mTOR with rapamycin: One dose does not fit all

A puzzling aspect of rapamycin-based therapeutic strategies is the wide disparity in the doses needed to suppress mTOR under different circumstances. A recent study revealing mechanistically how rapamycin suppresses mTOR provides two explanations for the differential sensitivities to rapamycin. First, mTOR exists as two functionally distinct complexes (mTORC1 and mTORC2), and while rapamycin suppresses both, it does so at very different concentrations. Whereas mTORC1 is suppressed by concentrations of rapamycin in the low nM range, mTORC2 generally requires low μM concentrations. Second, the efficacy of rapamycin is dependent on the level of phosphatidic acid (PA), which is required for the assembly of both mTORC1 and mTORC2 complexes. Rapamycin interacts with mTOR in a manner that is competitive with PA. Therefore, elevated levels of PA, which is common in cancer cells, increases the level of rapamycin needed to suppress both mTORC1 and mTORC2. A practical outcome of the recent study is that if PA levels are suppressed, mTORC2 becomes sensitive to concentrations of rapamycin that can be achieved clinically. Since mTORC2 is likely more critical for survival signals in cancer cells, the recent findings suggest new strategies for enhancing the efficacy of rapamycin-based therapeutic approaches in cancer cells.

Honokiol Suppresses Survival Signals Mediated by Ras-Dependent Phospholipase D Activity in Human Cancer Cells

Purpose: Elevated phospholipase D (PLD) activity provides a survival signal in several human cancer cell lines and suppresses apoptosis when cells are subjected to the stress of serum withdrawal. Thus, targeting PLD survival signals has potential to suppress survival in cancer cells that depend on PLD for survival. Honokiol is a compound that suppresses tumor growth in mouse models. The purpose of this study was to investigate the effect of honokiol on PLD survival signals and the Ras dependence of these signals. Experimental Design: The effect of honokiol upon PLD activity was examined in human cancer cell lines where PLD activity provides a survival signal. The dependence of PLD survival signals on Ras was investigated, as was the effect of honokiol on Ras activation. Results: We report here that honokiol suppresses PLD activity in human cancer cells where PLD has been shown to suppress apoptosis. PLD activity is commonly elevated in response to the stress of serum withdrawal, and, importantly, the stress-induced increase in PLD activity is selectively suppressed by honokiol. The stress-induced increase in PLD activity was accompanied by increased Ras activation, and the stress-induced increase in PLD activity in MDA-MB-231 breast cancer cells was dependent on a Ras. The PLD activity was also dependent on the GTPases RalA and ADP ribosylation factor. Importantly, honokiol suppressed Ras activation. Conclusion: The data provided here indicate that honokiol may be a valuable therapeutic reagent for targeting a large number of human cancers that depend on Ras and PLD for their survival.

HIFα expression in VHL-deficient renal cancer cells is dependent on phospholipase D

Loss of the von Hippel-Lindau (VHL) tumor suppressor gene contributes to proliferative disorders including renal cell carcinoma. The consequence of VHL loss is increased levels of hypoxia-inducible factor-α (HIFα), which is targeted for proteolytic degradation by the VHL gene product pVHL. HIF is a transcription factor that increases the expression of factors critical for tumorigenesis in renal cell carcinoma. We report here another regulatory component of HIFα expression in renal cancer cells. Phospholipase D (PLD), which is commonly elevated in renal and other cancers, is required for elevated levels of both HIF1α and HIF2α in VHL-deficient renal cancer cells. The induction of both HIF1α and HIF2α by hypoxic mimetic conditions was also dependent on PLD in renal cancer cells with restored pVHL expression. The effect of PLD activity upon HIFα expression was at the level of translation. PLD activity also provides a survival signal that suppresses apoptosis induced by serum deprivation in the renal cancer cells. Suppression of HIF2α has been shown to reverse tumorigenesis with renal cancer cells. The finding here that HIF2α expression is dependent on PLD in renal cancer cells suggests that targeting PLD signals may represent an alternative therapeutic strategy for targeting HIF2α in renal cancers where HIF2α is critical for tumorigenesis and elevated PLD activity is common.

Fabrication of Metal Nanoparticles Using Toroidal Plasmid DNA as a Sacrificial Mold

A new method for synthesizing gold, nickel, and cobalt metal nanoparticles at room temperature from metal salts employing plasmid DNA in a toroidal topology as a sacrificial mold is presented. The diameter of the toroidal DNA drives the formation and size of the nanoparticle, and UV light initiates the oxidation of the DNA and concomitant reduction of the DNA bound metal ions. The nanoparticles were characterized by atomic force microscopy (AFM), transmission electron microscopy (TEM), and electron diffraction (ED).

Suppression TGF-β Signaling by Phospholipase D

MDA-MB-231 human breast cancer cells have a survival signal generated by phospholipase D (PLD) that involves the activation of mTOR and MAP kinase. TGF-β signals that block cell cycle progression in G1 are suppressed in MDA-MB-231 cells. We report here that the elevated PLD activity in MDA-MB-231 cells suppresses TGF-β signaling. Suppression of PLD activity or PLD expression resulted in increased phosphorylation of Smad2 and Smad3 on Ser 465/467 – sites on Smads that get phosphorylated by the TGF-β receptor and positively regulate TGF-β signaling. The effect of PLD suppression on Smad2/3 phosphorylation was dependent on the presence of TGF-β. Suppression of PLD also suppressed phosphorylation of Smad2 on Ser 245/250/255 – sites that are phosphorylated by MAP kinase and negatively regulate TGF-β signaling. Suppression of PLD also led to increased expression of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1, the expression of which is stimulated in response to TGF-β. Consistent with the elevated expression of CDK inhibitors, suppression of PLD also suppressed phosphorylation of the CDK substrate pRb. Similar effects were also seen in PANC-1 human pancreatic cancer cells. The data presented here indicate that the suppressed TGF-β signaling in MDA-MB-231 and perhaps many other human cancer cells is due to elevated PLD activity and mediated by mTOR and MAP kinase. These results indicate that the survival signals generated by PLD involve the suppression TGF-β signals that promote G1 arrest.