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.     

Phosphatidic acid signaling to mTOR: Signals for the survival of human cancer cells

During the past decade elevated phospholipase D (PLD) activity has been reported in virtually all cancers where it has been examined. PLD catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger phosphatidic acid (PA). While many targets of PA signaling have been identified, the most critical target of PA in cancer cells is likely to be mTOR — the mammalian target of rapamycin. mTOR has been widely implicated in signals that suppress apoptotic programs in cancer cells — frequently referred to as survival signals. mTOR exists as two multi-component complexes known as mTORC1 and mTORC2. Recent data has revealed that PA is required for the stability of both mTORC1 and mTORC2 complexes — and therefore also required for the kinase activity of both mTORC1 and mTORC2. PA interacts with mTOR in a manner that is competitive with rapamycin, and as a consequence, elevated PLD activity confers rapamycin resistance — a point that has been largely overlooked in clinical trials involving rapamycin-based strategies. The earliest genetic changes occurring in an emerging tumor are generally ones that suppress default apoptotic programs that likely represent the first line of defense of cancer. Targeting survival signals in human cancers represents a rational anti-cancer therapeutic strategy. Therefore, understanding the signals that regulate PA levels and how PA impacts upon mTOR could be important for developing strategies to de-repress the survival signals that suppress apoptosis. This review summarizes the role of PA in regulating the mTOR-mediated signals that promote cancer cell survival.     

p27kip1 protein levels reflect a nexus of oncogenic signaling during cell transformation

SV40 small t-antigen (ST) collaborates with SV40 large T-antigen (LT) and activated rasv12 to promote transformation in a variety of immortalized human cells. A number of oncogenes or the disruption of the general serine-threonine phosphatase protein phosphatase 2A (PP2A) can replace ST in this paradigm. However, the relationship between these oncogenes and PP2A activity is not clear. To address this, we queried the connectivity of these molecules in silico. We found that p27 was connected to each of those oncogenes that could substitute for ST. We further determined that p27 loss can substitute for the expression of ST during transformation of both rodent and human cells. Conversely, knock-in cells expressing the degradation-resistant S10A and T187A mutants of p27 were resistant to the transforming activities of ST. This suggests that p27 is an important target of the tumor-suppressive effects of PP2A and likely an important target of the multitude of cellular oncoproteins that emulate the transforming function of ST.     

Phospholipase D stabilizes HDM2 through an mTORC2/SGK1 pathway

Phosphatidic acid (PA), the primary metabolite of the phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine, has been shown to act as a tumor promoting second messenger in many cancer cell lines. A key target of PA is the mammalian target of rapamycin (mTOR), a serine-threonine kinase that has been widely implicated in cancer cell survival signals. In agreement with its ability to relay survival signals, it has been reported that both PLD and mTOR are required for the stabilization of the p53 E3 ubiquitin ligase human double minute 2 (HDM2) protein. Thus, by stabilizing HDM2, PLD and mTOR are able to counter the pro-apoptotic signaling mediated by p53 and promote survival. mTOR exists in at least two distinct complexes—mTORC1 and mTORC2—that are both dependent on PLD-generated PA. Although PLD and its metabolite PA are clearly implicated in the transduction of survival signals to mTOR, it is not yet apparent which of the two mTOR complexes is critical for the stabilization of HDM2. We report here that the PLD/mTOR-dependent stabilization of HDM2 involves mTORC2 and the AGC family kinase serum- and glucocorticoid-inducible kinase 1 (SGK1). This study reveals that mTORC2 is a critical target of PLD-mediated survival signals and identifies SGK1 as a downstream target of mTORC2 for the stabilization of HDM2.     

Pathologic progression of mammary carcinomas in a C3(1)/SV40 T/t-antigen transgenic rat model of human triple-negative and Her2-positive breast cancer

The C3(1) component of the rat prostate steroid binding protein has been used to target expression of the SV40 T/t-antigen to the mammary epithelium of mice resulting in pre-neoplastic lesions that progress to invasive and metastatic cancer with molecular features of human basal-type breast cancer. However, there are major differences in the histologic architecture of the stromal and epithelial elements between the mouse and human mammary glands. The rat mammary gland is more enriched with epithelial and stromal components than the mouse and more closely resembles the cellular composition of the human gland. Additionally, existing rat models of mammary cancer are typically estrogen receptor positive and hormone responsive, unlike most genetically engineered mouse mammary cancer models. In an attempt to develop a mammary cancer model that might more closely resemble the pathology of human breast cancer, we generated a novel C3(1)/SV40 T/t-antigen transgenic rat model that developed progressive mammary lesions leading to highly invasive adenocarcinomas. However, aggressive tumor development prevented the establishment of transgenic lines. Characterization of the tumors revealed that they were primarily estrogen receptor and progesterone receptor negative, and either her2/neu positive or negative, resembling human triple-negative or Her2 positive breast cancer. Tumors expressed the basal marker K14, as well as the luminal marker K18, and were negative for smooth muscle actin. The triple negative phenotype has not been previously reported in a rat mammary cancer model. Further development of a C3(1)SV40 T/t-antigen based model could establish valuable transgenic rat lines that develop basal-type mammary tumors.     

Viral and cellular oncogene expression during progressive malignant transformation of SV40 transformed human fibroblasts.

In vitro investigation of the multistep neoplastic progression which occurs during transformation of human cells has been hindered by resistance of human cells to both immortalization and tumorigenicity (Mut. Res. 199; 273, 1988). Previously our laboratory established a cell line, HSF4-T12, by transfection of normal human foreskin fibroblasts with the plasmid pSV3-neo which contains the early genes of simian virus 40 (SV40). A multistep progression in karyotypic alterations and transformed phenotype occurred resulting in a neoplastic cell line that was immortal, transformed, and tumorigenic. We have examined changes in the SV40 proteins, large T (T-antigen) and small t (t-antigen) antigens, and in the cellular protein, p53, during progressive transformation of these cells. Total viral protein expression relative to total cellular protein increased following immortalization of HSF4-T12 as did the ratio of T-antigen to t-antigen. Interestingly, no significant change in DNA content accompanied immortalization. However, during the progressive in vitro transformation of HSF4-T12 which occurred primarily post-immortalization, DNA index increased to 1.6 but only small additional increases in T-antigen expression were seen. No consistent or critical role for t-antigen in development of the tumorigenic phenotype was found in this system.     

Reciprocal Regulation of AMP-activated Protein Kinase and Phospholipase D

AMP-activated protein kinase (AMPK), a critical sensor of energy sufficiency, acts as central metabolic switch in cell metabolism. Once activated by low energy status, AMPK phosphorylates key regulatory substrates and turns off anabolic biosynthetic pathways. In contrast, the mammalian/mechanistic target of rapamycin (mTOR) is active when there are sufficient nutrients for anabolic reactions. A critical factor regulating mTOR is phosphatidic acid (PA), a central metabolite of membrane lipid biosynthesis and the product of the phospholipase D (PLD)-catalyzed hydrolysis of phosphatidylcholine. PLD is a downstream target of the GTPase Rheb, which is turned off in response to AMPK via the tuberous sclerosis complex. Although many studies have linked AMPK with mTOR, very little is known about the connection between AMPK and PLD. In this report, we provide evidence for reciprocal regulation of PLD by AMPK and regulation of AMPK by PLD and PA. Suppression of AMPK activity led to an increase in PLD activity, and conversely, activation of AMPK suppressed PLD activity. Suppression of PLD activity resulted in elevated AMPK activity. Exogenously supplied PA abolished the inhibitory effects of elevated AMPK activity on mTOR signaling. In contrast, exogenously supplied PA could not overcome the effect AMPK activation if either mTOR or Raptor was suppressed, indicating that the inhibitory effects of PLD and PA on AMPK activity are mediated by mTOR. These data suggest a reciprocal feedback mechanism involving AMPK and the PLD/mTOR signaling node in cancer cells with therapeutic implications.     

PDK4 Protein Promotes Tumorigenesis through Activation of cAMP-response Element-binding Protein (CREB)-Ras Homolog Enriched in Brain (RHEB)-mTORC1 Signaling Cascade

Mechanistic target of rapamycin (mTOR) integrates multiple extracellular and intracellular signals to regulate cell growth and survival. Hyperactivation of mTOR has been observed in various cancers. Regulation of mTOR activity is thus of importance in physiological processes and tumor development. Here, we present pyruvate dehydrogenase kinase 4 (PDK4) as a novel regulator of mTORC1 signaling. mTORC1 activity was augmented with PDK4 overexpression and reduced by PDK4 suppression in various cell lines. Furthermore, PDK4 bound to cAMP-response element-binding protein (CREB) and prevented its degradation. The enhanced CREB consequently transactivated the expression of Ras homolog enriched in brain (RHEB), a direct key activator of mTORC1, independent of AMP-activated protein kinase or tuberous sclerosis complex protein 2. PDK4 potentiated the mTORC1 effectors hypoxia-inducible factor 1α and pyruvate kinase isozymes M2 and promoted aerobic glycolysis (Warburg effect). Knockdown of PDK4 suppressed the tumor development of cancer cells with activated mTORC1. The abundance of PDK4 dictated the responsiveness of cells to the mTOR inhibitor, rapamycin. Combinatory suppression of mTOR and PDK4 exerted synergistic inhibition on cancer cell proliferation. Therefore, PDK4 promotes tumorigenesis through activation of the CREB-RHEB-mTORC1 signaling cascade.     

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-1alpha. 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.     

Simian virus 40 small-t protein is required for loss of actin cable networks in rat cells.

The ability of the two early simian virus 40 (SV40) coded proteins, the large and small T-antigens, to abortively induce the disappearance of cytoplasmic actin-containing networks in cultured cells has been studied in rat embryo fibroblasts after microinjection of intact SV40 DNA, DNA fragments from the early region of SV40, and a purified SV40 large T-antigen related protein (the D2 hybrid protein) isolated from cells infected with the adenovirus-SV40 hybrid virus Ad2+D2. Injection of either the 107,000-dalton D2 hybrid protein or SV40 DNA from the deletion mutant dl 884 SV40, which lacks part of the region (0.54 to 0.59) encoding small t-antigen, failed to cause any detectable change in the structure of actin cables in recipient cells over a period of 72 h. By contrast, injection of wild-type SV40 DNA or a DNA fragment containing the entire region coding for a small-t antigen leads to the disruption of actin cable networks within 24 h of injection. It appears likely that the SV40 small-t protein is necessary for the abortive loss of actin cables in injected cells. Epidermal growth factor also causes loss of actin cables in rat embryo fibroblasts or Rat 1 cells (an established rat embryo line), but only after exposure of the cells to epidermal growth factor in the culture medium and not after injection of epidermal growth factor into the cells.     

Suppression of cancer cell migration and invasion by protein phosphatase 2A through dephosphorylation of mu- and m-calpains

The mu- and m-calpains are major members of the calpain family that play an essential role in regulating cell motility. We have recently discovered that nicotine-activated protein kinase C iota enhances calpain phosphorylation in association with enhanced calpain activity and accelerated migration and invasion of human lung cancer cells. Here we found that specific disruption of protein phosphatase 2A (PP2A) activity by expression of SV40 small tumor antigen up-regulates phosphorylation of mu- and m-calpains whereas C2-ceramide, a potent PP2A activator, reduces nicotine-induced calpain phosphorylation, suggesting that PP2A may function as a physiological calpain phosphatase. PP2A co-localizes and interacts with mu- and m-calpains. Purified, active PP2A directly dephosphorylates mu- and m-calpains in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppresses nicotine-stimulated phosphorylation of mu- and m-calpains, which is associated with inhibition of calpain activity, wound healing, cell migration, and invasion. By contrast, depletion of PP2A/C by RNA interference enhances calpain phosphorylation, calpain activity, cell migration, and invasion. Importantly, C2-ceramide-induced suppression of calpain phosphorylation results in decreased secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential clinic relevance in controlling metastasis of lung cancer. These findings reveal a novel role for PP2A as a physiological calpain phosphatase that not only directly dephosphorylates but also inactivates mu- and m-calpains, leading to suppression of migration and invasion of human lung cancer cells.     

Phospholipase D elevates the level of MDM2 and suppresses DNA damage-induced increases in p53

Phospholipase D (PLD) has been reported to generate survival signals that prevent apoptosis induced by serum withdrawal. We have now found that elevated expression of PLD also suppresses DNA damage-induced apoptosis. Since DNA damage-induced apoptosis is often mediated by p53, we examined the effect of elevated PLD expression on the regulation of p53 stabilization. We report here that PLD suppresses DNA damage-induced increases in p53 stabilization in cells where PLD has been shown to provide a survival signal. Elevated expression of PLD also led to increased expression of the p53 E3 ubiquitin ligase MDM2 and increased turnover of p53. PLD1-stimulated increases in MDM2 expression and suppression of p53 activation were blocked by inhibition of mTOR and the mitogen-activated protein kinase pathway. Although PLD did not activate the phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway activate the basal levels of PI3K activity were partially required for PLD1-induced increases in MDM2. These data provide evidence that survival signals generated by PLD involve suppression of the p53 response pathway.     

Simian virus 40 small tumor antigen induces deregulation of the actin cytoskeleton and tight junctions in kidney epithelial cells

There is increasing evidence that the transforming DNA tumor virus simian virus 40 (SV40) is associated with human malignancies. SV40 small tumor antigen (small t) interacts with endogenous serine/threonine protein phosphatase 2A (PP2A) and is required for the transforming activity of SV40 in epithelial cells of the lung and kidney. Here, we show that expression of SV40 small t in epithelial MDCK cells induces acute morphological changes and multilayering. Significantly, it also causes severe defects in the biogenesis and barrier properties of tight junctions (TJs) but does not prevent formation of adherens junctions. Small t-induced TJ defects are associated with a loss of PP2A from areas of cell-cell contact; altered distribution and reduced amounts of the TJ proteins ZO-1, occludin, and claudin-1; and marked disorganization of the actin cytoskeleton. Small t-mediated F-actin rearrangements encompass increased Rac-induced membrane ruffling and lamellipodia, Cdc42-initiated filopodia, and loss of Rho-dependent stress fibers. Indeed, these F-actin changes coincide with elevated levels of Rac1 and Cdc42 and decreased amounts of RhoA in small t-expressing cells. Notably, these cellular effects of small t are dependent on its interaction with endogenous PP2A. Thus, our findings provide the first evidence that, in polarized epithelial cells, expression of small t alone is sufficient to induce deregulation of Rho GTPases, F-actin, and intercellular adhesion, through interaction with endogenous PP2A. Because defects in the actin cytoskeleton and TJ disruption have been linked to loss of cell polarity and tumor invasiveness, their deregulation by PP2A and small t likely contributes to the role of SV40 in epithelial cell transformation.     

A novel role for protein phosphatase 2A in the dopaminergic regulation of Na,K-ATPase

Stimulation of dopaminergic type 1 (D(1)) receptors increases lung edema clearance by regulating Na,K-ATPase function in the alveolar epithelium. We studied the role of serine/threonine protein phosphatases in the Na,K-ATPase regulation by D(1) agonists in A549 cells. We found that low doses of the type 1/2A protein phosphatase inhibitor okadaic acid as well as SV40 small t antigen transiently transfected into A549 cells prevented the D(1) agonist-induced increase in Na,K-ATPase activity and translocation from intracellular pools to the plasma membrane. This was associated with a rapid and transient increase in protein phosphatase 2A activity. We conclude that D(1) stimulation regulates Na,K-ATPase activity by promoting recruitment of Na,K-ATPases from intracellular pools into the basolateral membranes of A549 cells via a type 2A protein phosphatase.     

Protein phosphatase 2A enhances the proapoptotic function of Bax through dephosphorylation

Bax is a major proapoptotic member of the Bcl2 family that is required for apoptotic cell death. We have recently discovered that Bax phosphorylation at serine 184 induced by nicotine through activation of protein kinase AKT abolishes its proapoptotic function in human lung cancer cells. Here we found that either treatment of cells with the protein phosphatase 2A (PP2A) inhibitor okadaic acid or specific disruption of PP2A activity by expression of SV40 small tumor antigen enhanced Bax phosphorylation, whereas C(2)-ceramide, a potent PP2A activator, reduced nicotine-induced Bax phosphorylation, suggesting that PP2A may function as a physiological Bax phosphatase. PP2A co-localized and interacted with Bax. Purified, active PP2A directly dephosphorylated Bax in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppressed nicotine-stimulated Bax phosphorylation in association with increased apoptotic cell death. By contrast, depletion of PP2A/C by RNA interference enhanced Bax phosphorylation and prolonged cell survival. Mechanistically C(2)-ceramide-induced Bax dephosphorylation caused a conformational change by exposure of the 6A7 epitope (amino acids 13-19) that is normally hidden at its N terminus that promoted the insertion of Bax into mitochondrial membranes and formation of Bax oligomers leading to cytochrome c release and apoptosis. In addition, PP2A directly disrupted the Bcl2/Bax association to liberate Bax from the heterodimer complex. Thus, PP2A may function as a physiological Bax regulatory phosphatase that not only dephosphorylates Bax but also activates its proapoptotic function.     

Inhibition of PP2A, but not PP5, mediates p53 activation by low levels of okadaic acid in rat liver epithelial cells

The microbial toxin okadaic acid (OA) specifically inhibits PPP-type ser/thr protein phosphatases. OA is an established tumor promoter with numerous cellular effects that include p53-mediated cell cycle arrest. In T51B rat liver epithelial cells, a model useful for tumor promotion studies, p53 activation is induced by tumor-promoting (low nanomolar) concentrations of OA. Two phosphatases sensitive to these concentrations of OA, PP2A and protein phosphatase 5 (PP5), have been implicated as negative regulators of p53. In this study we examined the respective roles of these phosphatases in p53 activation in non-neoplastic T51B cells. Increases in p53 activity were deduced from levels of p21 (cip1) and/or the rat orthologue of mdm2, two p53-regulated gene products whose induction was blocked by siRNA-mediated knockdown of p53. As observed with 10 nM OA, both phospho-ser15-p53 levels and p53 activity were increased by 10 microM fostriecin or SV40 small t-antigen. Both of these treatments selectively inhibit PP2A but not PP5. siRNA-mediated knockdown of PP2A, but not PP5, also increased p53 activity. Finally, adenoviral-mediated over-expression of an OA-resistant form of PP5 did not prevent increased phospho-ser15-p53, p53 protein, or p53 activity caused by 10 nM OA. Together these results indicate that PP5 blockade is not responsible for OA-induced p53 activation and G1 arrest in T51B cells. In contrast, specific blockade of PP2A mimics p53-related responses to OA in T51B cells, suggesting that PP2A is the target for this response to OA.     

LMW-E/CDK2 deregulates acinar morphogenesis, induces tumorigenesis, and associates with the activated b-Raf-ERK1/2-mTOR pathway in breast cancer patients

Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2-associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E-expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing G1/S cell cycle arrest. LMW-E requires CDK2-associated kinase activity to induce mammary tumor formation by disrupting acinar development. The b-Raf-ERK1/2-mTOR signaling pathway is aberrantly activated in breast cancer and can be suppressed by combination treatment with roscovitine plus either rapamycin or sorafenib.