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Glucocorticoid Receptor Activation Inhibits p53-induced Apoptosis of MCF10Amyc Cells via Induction of Protein Kinase Cϵ*

Open AccessPublished:July 06, 2012DOI:https://doi.org/10.1074/jbc.M112.393256
      Glucocorticoid receptor (GR) is a ligand-dependent transcription factor that can promote apoptosis or survival in a cell-specific manner. Activated GR has been reported to inhibit apoptosis in mammary epithelial cells and breast cancer cells by increasing pro-survival gene expression. In this study, activated GR inhibited p53-dependent apoptosis in MCF10A cells and human mammary epithelial cells that overexpress the MYC oncogene. Specifically, GR agonists hydrocortisone or dexamethasone inhibited p53-dependent apoptosis induced by cisplatin, ionizing radiation, or the MDM2 antagonist Nutlin-3. In contrast, the GR antagonist RU486 sensitized the cells to apoptosis by these agents. Apoptosis inhibition was associated with maintenance of mitochondrial membrane potential, diminished caspase-3 and -7 activation, and increased expression at both the mRNA and protein level of the anti-apoptotic PKC family member PKCϵ. Knockdown of PKCϵ via siRNA targeting reversed the protective effect of dexamethasone and restored apoptosis sensitivity. These data provide evidence that activated GR can inhibit p53-dependent apoptosis through induction of the anti-apoptotic factor PKCϵ.

      Introduction

      The tumor suppressor protein p53 is a key regulator of multiple cellular processes, and evidence suggests that apoptosis is critical for its tumor suppressor function (
      • Zilfou J.T.
      • Lowe S.W.
      Tumor-suppressive functions of p53.
      ). The main function of p53 is to maintain genetic stability in response to various oncogenic challenges, such as DNA damage or inappropriate oncogene signaling. p53 carries out this function by inducing cell cycle arrest, apoptosis, or senescence. As such, p53 is often referred to as the “guardian of the genome.” p53 is negatively regulated by MDM2 through different mechanisms in coordination with MDMX (MDM4). Upon overexpression, MDM2 binds the transcription domain of p53 and blocks its ability to activate gene transcription (
      • Chen J.
      • Marechal V.
      • Levine A.J.
      Mapping of the p53 and mdm-2 interaction domains.
      ,
      • Oliner J.D.
      • Pietenpol J.A.
      • Thiagalingam S.
      • Gyuris J.
      • Kinzler K.W.
      • Vogelstein B.
      Oncoprotein MDM2 conceals the activation domain of tumor suppressor p53.
      ,
      • Huang L.
      • Yan Z.
      • Liao X.
      • Li Y.
      • Yang J.
      • Wang Z.G.
      • Zuo Y.
      • Kawai H.
      • Shadfan M.
      • Ganapathy S.
      • Yuan Z.M.
      The p53 inhibitors MDM2/MDMX complex is required for control of p53 activity in vivo.
      ). MDM2 also functions as an E3 ubiquitin ligase, mediating the ubiquitination and proteasome degradation of p53 (
      • Huang L.
      • Yan Z.
      • Liao X.
      • Li Y.
      • Yang J.
      • Wang Z.G.
      • Zuo Y.
      • Kawai H.
      • Shadfan M.
      • Ganapathy S.
      • Yuan Z.M.
      The p53 inhibitors MDM2/MDMX complex is required for control of p53 activity in vivo.
      ,
      • Haupt Y.
      • Maya R.
      • Kazaz A.
      • Oren M.
      Mdm2 promotes the rapid degradation of p53.
      ,
      • Kubbutat M.H.
      • Jones S.N.
      • Vousden K.H.
      Regulation of p53 stability by Mdm2.
      ). The type of response that follows p53 activation depends on a number of factors. Importantly, oncogenic transformation can cause a switch in the cell's response to p53 activation from growth arrest to programmed cell death (
      • Lowe S.W.
      • Ruley H.E.
      • Jacks T.
      • Housman D.E.
      p53-dependent apoptosis modulates the cytotoxicity of anticancer agents.
      ). As a result, transformed tumor cells may be more prone to undergo apoptosis following p53 activation than corresponding normal cells. Inactivation of p53 not only promotes tumorigenesis and cancer progression but also confers cancer cells with an ability to evade death induced by therapeutic agents (
      • O'Connor P.M.
      • Jackman J.
      • Bae I.
      • Myers T.G.
      • Fan S.
      • Mutoh M.
      • Scudiero D.A.
      • Monks A.
      • Sausville E.A.
      • Weinstein J.N.
      • Friend S.
      • Fornace Jr., A.J.
      • Kohn K.W.
      Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents.
      ).
      Breast cancer is the leading cause of cancer-related death among women worldwide. In 2011, according to the American Cancer Society, more than 230,000 new cases of breast cancer and ∼40,000 breast cancer-related deaths in women have been registered in the United States alone (American Cancer Society Breast Cancer Facts and Figures). A major factor contributing to the development of breast cancer is inactivation of p53 (
      • Gasco M.
      • Shami S.
      • Crook T.
      The p53 pathway in breast cancer.
      ). Thus, inactivating point mutations in the P53 gene are observed in 20–30% of breast cancers (
      • Soussi T.
      The p53 tumor suppressor gene. From molecular biology to clinical investigation.
      ,
      • Tennis M.
      • Krishnan S.
      • Bonner M.
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      • Moysich K.
      • Swede H.
      • McCann S.
      • Hall P.
      • Shields P.G.
      • Freudenheim J.L.
      p53 mutation analysis in breast tumors by a DNA microarray method.
      ). Furthermore, wild-type p53 is often inactivated through alternative mechanisms in cancers where the P53 gene is not mutated. Mechanisms for wild-type p53 inactivation in breast cancer include overexpression of MDM2, silencing of p14Arf (which causes hyper-activation of MDM2), and cytoplasmic sequestration (
      • Vogelstein B.
      • Lane D.
      • Levine A.J.
      Surfing the p53 network.
      ,
      • Moll U.M.
      • Riou G.
      • Levine A.J.
      Two distinct mechanisms alter p53 in breast cancer. Mutation and nuclear exclusion.
      ). Many cytotoxic drugs (e.g. cisplatin) and irradiation can damage DNA and can activate wild-type p53 (
      • Bragado P.
      • Armesilla A.
      • Silva A.
      • Porras A.
      Apoptosis by cisplatin requires p53-mediated p38α MAPK activation through ROS generation.
      ,
      • Impicciatore G.
      • Sancilio S.
      • Miscia S.
      • Di Pietro R.
      Nutlins and ionizing radiation in cancer therapy.
      ). In several reports, p53 wild-type cancer cells respond better to DNA-damaging therapeutics than p53-mutated or p53-null cancer cells due to activation of wild-type p53 growth-inhibitory pathways (
      • Wu G.S.
      • El-Deiry W.S.
      Apoptotic death of tumor cells correlates with chemosensitivity, independent of p53 or bcl-2.
      ,
      • Wu G.S.
      • Ding Z.
      Caspase 9 is required for p53-dependent apoptosis and chemosensitivity in a human ovarian cancer cell line.
      ).
      Conzen and co-workers (
      • Wu W.
      • Chaudhuri S.
      • Brickley D.R.
      • Pang D.
      • Karrison T.
      • Conzen S.D.
      Microarray analysis reveals glucocorticoid-regulated survival genes that are associated with inhibition of apoptosis in breast epithelial cells.
      ,
      • Wu W.
      • Pew T.
      • Zou M.
      • Pang D.
      • Conzen S.D.
      Glucocorticoid receptor-induced MAPK phosphatase-1 (MPK-1) expression inhibits paclitaxel-associated MAPK activation and contributes to breast cancer cell survival.
      ,
      • Moran T.J.
      • Gray S.
      • Mikosz C.A.
      • Conzen S.D.
      The glucocorticoid receptor mediates a survival signal in human mammary epithelial cells.
      ,
      • Mikosz C.A.
      • Brickley D.R.
      • Sharkey M.S.
      • Moran T.W.
      • Conzen S.D.
      Glucocorticoid receptor-mediated protection from apoptosis is associated with induction of the serine/threonine survival kinase gene, sgk-1.
      ) have reported that activation of the glucocorticoid receptor (GR)
      The abbreviations used are: GR
      glucocorticoid receptor
      HMEC
      human mammary epithelial cell
      Dex
      dexamethasone
      Cis
      cisplatin
      IR
      ionizing radiation
      Gy
      gray
      DAG
      diacylglycerol
      PE
      phorbol ester.
      inhibits apoptosis and promotes survival of breast cancer and breast epithelial cells by increasing expression of pro-survival genes. Recent studies indicate that GR activation is associated with poor prognosis in estrogen receptor-negative breast cancer (
      • Pan D.
      • Kocherginsky M.
      • Conzen S.D.
      Activation of the glucocorticoid receptor is associated with poor prognosis in estrogen receptor-negative breast cancer.
      ). Glucocorticoid synthesis is enhanced following stressful conditions leading and acting mostly through GR to regulate inflammatory and immune responses, as well as cellular proliferation and apoptosis. Most glucocorticoid-mediated effects result from the ability of activated GR to act as a transcription factor, either through a DNA binding-dependent mechanism or through cross-talk and/or interference with other transcription factors such as activator protein-1 (AP-1) (
      • Jonat C.
      • Rahmsdorf H.J.
      • Park K.K.
      • Cato A.C.
      • Gebel S.
      • Ponta H.
      • Herrlich P.
      Antitumor promotion and anti-inflammation. Down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone.
      ), signal transducers and activators of transcription-5 (Stat-5) (
      • Wyszomierski S.L.
      • Yeh J.
      • Rosen J.M.
      Glucocorticoid receptor/signal transducer and activator of transcription 5 (STAT5) interactions enhance STAT5 activation by prolonging STAT5 DNA binding and tyrosine phosphorylation.
      ), and nuclear factor-κB (NF-κB) (
      • Brostjan C.
      • Anrather J.
      • Csizmadia V.
      • Stroka D.
      • Soares M.
      • Bach F.H.
      • Winkler H.
      Glucocorticoid-mediated repression of NFκB activity in endothelial cells does not involve induction of IκBα synthesis.
      ). The accumulated evidence shows that GR activation has a dual and cell type-specific role in cell death regulation. GR is able to induce apoptosis in lymphocytes, leukemia, lymphoma, and multiple myeloma cells (
      • Distelhorst C.W.
      Recent insights into the mechanism of glucocorticosteroid-induced apoptosis.
      ,
      • Greenstein S.
      • Ghias K.
      • Krett N.L.
      • Rosen S.T.
      Mechanisms of glucocorticoid-mediated apoptosis in hematological malignancies.
      ). However, in other cell types such as hepatocytes, vascular endothelial cells, osteoclasts, and particularly in mammary epithelial cells, GR can inhibit apoptosis induced by a variety of signaling events (
      • Wu W.
      • Chaudhuri S.
      • Brickley D.R.
      • Pang D.
      • Karrison T.
      • Conzen S.D.
      Microarray analysis reveals glucocorticoid-regulated survival genes that are associated with inhibition of apoptosis in breast epithelial cells.
      ). Several groups have observed that glucocorticoids can inhibit chemotherapy-induced apoptosis in vitro (
      • Wu W.
      • Chaudhuri S.
      • Brickley D.R.
      • Pang D.
      • Karrison T.
      • Conzen S.D.
      Microarray analysis reveals glucocorticoid-regulated survival genes that are associated with inhibition of apoptosis in breast epithelial cells.
      ,
      • Wu W.
      • Pew T.
      • Zou M.
      • Pang D.
      • Conzen S.D.
      Glucocorticoid receptor-induced MAPK phosphatase-1 (MPK-1) expression inhibits paclitaxel-associated MAPK activation and contributes to breast cancer cell survival.
      ) and in vivo (
      • Herr I.
      • Ucur E.
      • Herzer K.
      • Okouoyo S.
      • Ridder R.
      • Krammer P.H.
      • von Knebel Doeberitz M.
      • Debatin K.M.
      Glucocorticoid cotreatment induces apoptosis resistance toward cancer therapy in carcinomas.
      ).
      We wished to gain insight into the mechanisms by which GR activation inhibits apoptosis and promotes survival in breast epithelial cells. To this end, we addressed the involvement of protein kinase Cϵ (PKCϵ) as a potential mediator, because overexpression of PKCϵ is found in various cancers, including breast cancer, and is considered an important marker of negative disease outcome (
      • Pan Q.
      • Bao L.W.
      • Kleer C.G.
      • Sabel M.S.
      • Griffith K.A.
      • Teknos T.N.
      • Merajver S.D.
      Protein kinase Cϵ is a predictive biomarker of aggressive breast cancer and a validated target for RNA interference anticancer therapy.
      ). PKC is a family of serine/threonine kinases involved in several processes, including proliferation, differentiation, survival, apoptosis, and migration (
      • Aziz M.H.
      • Sundling K.E.
      • Dreckschmidt N.E.
      • Verma A.K.
      Protein kinase Cϵ inhibits UVR-induced expression of FADD, an adaptor protein, linked to both Fas- and TNFR1-mediated apoptosis.
      ,
      • Aziz M.H.
      • Hafeez B.B.
      • Sand J.M.
      • Pierce D.B.
      • Aziz S.W.
      • Dreckschmidt N.E.
      • Verma A.K.
      Protein kinase Cvarϵ mediates Stat3Ser-727 phosphorylation, Stat3-regulated gene expression, and cell invasion in various human cancer cell lines through integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2).
      ,
      • Bourguignon L.Y.
      • Spevak C.C.
      • Wong G.
      • Xia W.
      • Gilad E.
      Hyaluronan-CD44 interaction with protein kinase Cϵ promotes oncogenic signaling by the stem cell marker Nanog and the production of microRNA-21, leading to down-regulation of the tumor suppressor protein PDCD4, anti-apoptosis, and chemotherapy resistance in breast tumor cells.
      ). Based on the structure of the regulatory domain, PKC isoforms are divided into three subgroups as follows: classical (PKCα, -βI, -βII, and -γ), novel (PKCδ, -ϵ, -η, and -θ), and atypical (PKCζ and -ι/λ). Classical and novel PKCs contain a diacylglycerol (DAG)-binding C1 domain and are therefore regulated by activation of pathways that lead to DAG generation. Atypical PKCs are DAG-insensitive and regulated in a different manner (
      • Aziz M.H.
      • Hafeez B.B.
      • Sand J.M.
      • Pierce D.B.
      • Aziz S.W.
      • Dreckschmidt N.E.
      • Verma A.K.
      Protein kinase Cvarϵ mediates Stat3Ser-727 phosphorylation, Stat3-regulated gene expression, and cell invasion in various human cancer cell lines through integration with MAPK cascade (RAF-1, MEK1/2, and ERK1/2).
      ). Previous studies have implicated the DAG-sensitive classical and novel PKC isoforms in promoting malignant features of breast cancer cells. In addition, earlier studies reported that the synthetic GR agonist dexamethasone (Dex) could increase PKCϵ expression (
      • Dwivedi Y.
      • Pandey G.N.
      Administration of dexamethasone up-regulates protein kinase C activity and the expression of γ and κ protein kinase C isozymes in the rat brain.
      ,
      • Maddali K.K.
      • Korzick D.H.
      • Turk J.R.
      • Bowles D.K.
      Isoform-specific modulation of coronary artery PKC by glucocorticoids.
      ). However, the potential involvement of PKCϵ in GR-regulated inhibition of apoptosis has not been explored.
      Here, we demonstrate that the GR agonists Dex and hydrocortisone can protect MCF10Amyc and HMEC+myc mammary epithelial cells from p53-induced apoptosis. Activation of GR by hydrocortisone/Dex and attenuation of p53-induced apoptosis is associated with increased expression of PKCϵ mRNA and protein, maintenance of mitochondrial membrane potential, and diminished caspase-3/7 activation. In contrast, the GR antagonist RU486 suppressed the anti-apoptotic effect of GR and enhanced apoptosis in MCF10Amyc cells. Finally, siRNA-mediated knockdown of PKCϵ reversed the protective effect of Dex, rendering the cells susceptible to apoptosis. These data suggest that PKCϵ plays an important role in the signaling pathway activated by Dex during GR-induced inhibition of apoptosis.

      DISCUSSION

      Wild-type p53 and GR have opposing effects on the response of breast cancer cells to chemotherapeutic drugs. P53 promotes growth arrest or death in breast cancer cells exposed to chemotherapeutic drugs (
      • Gasco M.
      • Shami S.
      • Crook T.
      The p53 pathway in breast cancer.
      ), whereas GR promotes survival (
      • Wu W.
      • Chaudhuri S.
      • Brickley D.R.
      • Pang D.
      • Karrison T.
      • Conzen S.D.
      Microarray analysis reveals glucocorticoid-regulated survival genes that are associated with inhibition of apoptosis in breast epithelial cells.
      ,
      • Moran T.J.
      • Gray S.
      • Mikosz C.A.
      • Conzen S.D.
      The glucocorticoid receptor mediates a survival signal in human mammary epithelial cells.
      ). PKCϵ is a novel PKC isoform with anti-apoptotic activity that is expressed, through an unknown mechanism, at high levels in multiple cancer types (
      • Gorin M.A.
      • Pan Q.
      Protein kinase Cϵ. An oncogene and emerging tumor biomarker.
      ). In breast cancer, increased PKCϵ is linked with high histologic grade and poor disease-free survival (
      • Pan Q.
      • Bao L.W.
      • Kleer C.G.
      • Sabel M.S.
      • Griffith K.A.
      • Teknos T.N.
      • Merajver S.D.
      Protein kinase Cϵ is a predictive biomarker of aggressive breast cancer and a validated target for RNA interference anticancer therapy.
      ). In this study, the GR agonists Dex and hydrocortisone promoted PKCϵ expression in MCF10A and HMECs that overexpress the MYC oncogene. Importantly, this effect was reversed by the GR antagonist RU486, confirming increased PKCϵ expression resulted from activation of GR. Moreover, Dex protected the cells from apoptosis that was at least partly p53-dependent and induced by Cis, IR, or the p53 activator Nutlin. Knockdown of PKCϵ abrogated the protective effect of Dex and sensitized the cells to Cis, IR, and Nutlin. Together, these data demonstrate that GR activation can inhibit therapy- and p53-dependent apoptosis and that this occurs through increased expression of PKCϵ.
      Wasylyk and co-workers (
      • Sengupta S.
      • Vonesch J.L.
      • Waltzinger C.
      • Zheng H.
      • Wasylyk B.
      Negative cross-talk between p53 and the glucocorticoid receptor and its role in neuroblastoma cells.
      ,
      • Sengupta S.
      • Wasylyk B.
      Ligand-dependent interaction of the glucocorticoid receptor with p53 enhances their degradation by Hdm2.
      ) have demonstrated negative cross-talk between GR and p53. In their studies, activation of endogenous GR by Dex treatment inhibited p53-induced cell cycle arrest in stressed cells, whereas activation of endogenous p53 by DNA damage inhibited GR transcriptional activity. Mechanistically, they found that GR could form a ligand-dependent (Dex-dependent) complex with p53. MDM2 was recruited to this GR-p53 complex via p53 and promoted the ubiquitin-dependent degradation of both p53 and GR, thus inhibiting both proteins. Their model suggested that activated GR binds and inhibits p53 by promoting its MDM2-mediated degradation. This model is unlikely to explain GR protection against p53-dependent apoptosis in our system for two reasons. First, p53 was induced comparably by Nutlin, Cis, and IR in the presence or absence of Dex (FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6, FIGURE 7). Thus, degradation was not limiting the accumulation of p53 when GR was activated. Second, Dex inhibited the apoptosis induced by Nutlin, even though Nutlin disrupts binding between p53 and MDM2. This suggests activated GR can inhibit p53-induced apoptosis in a manner that does not involve MDM2 interaction with p53. Rather, our results demonstrate Dex treatment increases levels of the anti-apoptotic PKC family member PKCϵ. This increase is at both the mRNA and protein level. The GR antagonist RU486 blocks the increase in PKCϵ expression. Our results are consistent with those of Dwivedi and Pandey (
      • Dwivedi Y.
      • Pandey G.N.
      Administration of dexamethasone up-regulates protein kinase C activity and the expression of γ and κ protein kinase C isozymes in the rat brain.
      ) and Maddali et al. (
      • Maddali K.K.
      • Korzick D.H.
      • Turk J.R.
      • Bowles D.K.
      Isoform-specific modulation of coronary artery PKC by glucocorticoids.
      ), who reported that administration of Dex coincided with increased PKCϵ expression in coronary arteries and in the rat brain. We speculate PKCϵ is either a direct or indirect transcription target of GR that is required for apoptosis inhibition.
      How does PKCϵ induced by Dex treatment inhibit Nutlin-, Cis-, and IR-induced apoptosis? siRNA knockdown studies showed apoptosis in response to all three agents is, in part, p53-dependent in MCF10Amyc cells (Fig. 11). Interestingly, p53 transcriptional activity was apparently unaffected by Dex under conditions in which Dex induced PKCϵ expression and inhibited apoptosis. This is based on the fact that multiple p53 target genes (P21, MDM2, PUMA, and Bax) were induced comparably by Nutlin treatment in the presence or absence of Dex (Fig. 3). Thus, it is unlikely PKCϵ blocks apoptosis by inhibiting p53 transcriptional activity. p53-responsive factors PUMA and Bax promote the formation of pores in the mitochondrial membrane that causes loss of membrane potential and release of factors that activate caspases and drive apoptosis. We found that mitochondrial membrane potential was maintained by Dex treatment in cells exposed to Nutlin, and caspase-3/7 activation was diminished (Fig. 2, B and C). Based on this, we speculate PKCϵ blocks apoptosis by somehow maintaining mitochondrial membrane potential and blocking caspase activation. In this regard, it is worth pointing out that PKCϵ has been reported to localize in cardiac mitochondria, associate with mitochondrial ATPase and MAPKs, and promote phosphorylation and inhibition of the pro-apoptotic factor Bad (
      • Baines C.P.
      • Zhang J.
      • Wang G.W.
      • Zheng Y.T.
      • Xiu J.X.
      • Cardwell E.M.
      • Bolli R.
      • Ping P.
      Mitochondrial PKCϵ and MAPK form signaling modules in the murine heart. Enhanced mitochondrial PKCϵ-MAPK interactions and differential MAPK activation in PKCϵ-induced cardioprotection.
      ,
      • Jabůrek M.
      • Costa A.D.
      • Burton J.R.
      • Costa C.L.
      • Garlid K.D.
      Mitochondrial PKC epsilon and mitochondrial ATP-sensitive K+ channel copurify and coreconstitute to form a functioning signaling module in proteoliposomes.
      ). These activities of PKCϵ are believed to confer protection from ischemic/reperfusion injury in the heart (
      • Cohen M.V.
      • Baines C.P.
      • Downey J.M.
      Ischemic preconditioning. From adenosine receptor to KATP channel.
      ,
      • Baines C.P.
      • Pass J.M.
      • Ping P.
      Protein kinases and kinase-modulated effectors in the late phase of ischemic preconditioning.
      ). However, it is unknown whether mitochondrial PKCϵ would maintain mitochondrial membrane integrity and block apoptosis in cardiac cells exposed to chemotherapeutic drugs or Nutlin. Likewise, it is unknown whether or the extent to which PKCϵ may localize in mitochondria in our experiments. Interestingly, pro-apoptotic Noxa expression was decreased in Dex-treated cells, although it was not significantly up-regulated by Nutlin/p53 in our studies (Fig. 3B). It is unclear whether decreased Noxa expression may contribute to apoptosis protection by Dex.
      An interesting finding from our studies is that Nutlin-induced apoptosis in MCF10Amyc cells but not MCF10A parental cells or MCF10NeoT cells. Furthermore, siRNA-mediated Myc knockdown diminished Nutlin-induced apoptosis in MCF10Amyc cells, and HMEC+myc cells were susceptible to apoptosis by Nutlin although control HMECs were not (Fig. 8). These data suggest MYC expression can render MCF10A cells susceptible to p53-dependent apoptosis. Previous studies reported MYC is directly recruited to the p21 gene promoter by the DNA-binding protein Miz-1 and inhibits p21 expression, favoring the initiation of apoptosis (
      • Seoane J.
      • Le H.V.
      • Massagué J.
      Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage.
      ). We do not believe MYC sensitizes MCF10A cells through this mechanism in our studies, because p21 was highly expressed in MCF10Amyc cells that were treated with Nutlin and targeted for apoptosis (Fig. 3). It is also interesting that MCF10ANeoT cells are transformed, but remain resistant to apoptosis (Fig. 7). This suggests sensitivity to Nutlin is a consequence of increased MYC expression and does not result from cellular transformation alone. Finally, it is interesting that Dex treatment caused a more pronounced induction of PKCϵ expression in MCF10-Amyc cells compared with either MCF10A or MCF10ANeoT (Fig. 7B). How MYC may affect PKCϵ expression and sensitivity to apoptosis remains to be determined.
      Conzen and co-workers (
      • Wu W.
      • Chaudhuri S.
      • Brickley D.R.
      • Pang D.
      • Karrison T.
      • Conzen S.D.
      Microarray analysis reveals glucocorticoid-regulated survival genes that are associated with inhibition of apoptosis in breast epithelial cells.
      ,
      • Wu W.
      • Pew T.
      • Zou M.
      • Pang D.
      • Conzen S.D.
      Glucocorticoid receptor-induced MAPK phosphatase-1 (MPK-1) expression inhibits paclitaxel-associated MAPK activation and contributes to breast cancer cell survival.
      ,
      • Mikosz C.A.
      • Brickley D.R.
      • Sharkey M.S.
      • Moran T.W.
      • Conzen S.D.
      Glucocorticoid receptor-mediated protection from apoptosis is associated with induction of the serine/threonine survival kinase gene, sgk-1.
      ) previously identified GR target genes associated with survival and apoptosis inhibition in MCF10Amyc cells and different breast cancer cell lines. Their studies included gene expression analysis to identify GR targets induced at early time points after Dex treatment (30 min after Dex). They reported two GR target genes, SGK1 and MKP1, conferred survival in MCF10Amyc cells, and inhibited apoptosis following either serum deprivation or paclitaxel treatment (
      • Wu W.
      • Chaudhuri S.
      • Brickley D.R.
      • Pang D.
      • Karrison T.
      • Conzen S.D.
      Microarray analysis reveals glucocorticoid-regulated survival genes that are associated with inhibition of apoptosis in breast epithelial cells.
      ,
      • Wu W.
      • Pew T.
      • Zou M.
      • Pang D.
      • Conzen S.D.
      Glucocorticoid receptor-induced MAPK phosphatase-1 (MPK-1) expression inhibits paclitaxel-associated MAPK activation and contributes to breast cancer cell survival.
      ,
      • Mikosz C.A.
      • Brickley D.R.
      • Sharkey M.S.
      • Moran T.W.
      • Conzen S.D.
      Glucocorticoid receptor-mediated protection from apoptosis is associated with induction of the serine/threonine survival kinase gene, sgk-1.
      ). Our results identify PKCϵ as another factor whose increased expression after Dex treatment confers protection against apoptosis. PKCϵ expression increases in response to GR agonists (Dex and hydrocortisone) and decreases in response to GR antagonists (RU486), suggesting PKCϵ is a direct or indirect target of GR. The results suggest GR agonists can promote expression of multiple factors (SGK1, MKP1, and PKCϵ) that confer apoptosis protection.

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