Notch Activation Induces Akt Signaling Via an Autocrine Loop to Prevent

Published OnlineFirst June 2, 2009; DOI: 10.1158/0008-5472.CAN-08-3478

Research Article

Notch Activation Induces Akt Signaling via an Autocrine Loop to Prevent Apoptosis in Breast Epithelial Cells Olivier Meurette, Spyros Stylianou, Rebecca Rock, Giovanna M. Collu, Andrew P. Gilmore, and Keith Brennan

Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

Abstract transcript levels for Notch1 and the ligand Jagged1 correlate with The Notch pathway is aberrantly activated in a wide range of poor prognosis (9) and high NICD levels are associated with an in- cancers, including breast carcinoma, and is required to main- creased risk of recurrence of ductal carcinomas in situ (DCIS) by 5 tain the transformed phenotype of many of these tumors. years (13). Finally, increased Notch signaling is required to main- Notch signaling contributes to the transformed phenotype, tain the transformed phenotype of breast cancer cell lines and the in part, by preventing apoptosis in response to many different growth and survival of primary tumor cells (8, 10). stimuli. However, it is unclear how Notch activation can lead The contribution of Notch signaling to the transformed pheno- to a general suppression of apoptosis. We show here that type of cancer cells appears to be linked to the suppression of Notch signaling induced an autocrine signaling loop that ac- apoptosis (4, 5). We have shown that Notch activation in non- tivates Akt in breast epithelial cells. This activation of Akt was transformed breast epithelial cells induces a general resistance necessary for Notch-induced protection against apoptosis in to apoptotic stimuli (10). Furthermore, inhibiting Notch signaling the nontransformed breast epithelial cell line MCF10A. More- in cancer cells where it is constitutively active induces apoptosis – over, inhibiting Notch signaling in breast cancer cells induced or sensitization to apoptosis (14 16). Notch signaling has been a decrease in Akt activity and an increase in sensitivity to ap- shown to regulate apoptosis in different cancer cell lines through optosis. Finally, the inhibition of ASK1 by Akt was responsible several different mechanisms, including the inhibition of p53, for the protection from apoptosis induced by DNA damage, as Foxo3a, or c-Jun NH2-terminal kinase (JNK) function, as well as the activation of Akt (17–21). A common aspect to all of these it prevented c-Jun NH2-terminal kinase-mediated phosphorylation and activation of p53. [Cancer Res 2009;69(12):5015–22] mechanisms is that they can be linked to the activation of Akt (22); for example, Foxo3a can be phosphorylated and inhibited by Akt (23). Also, activation of Akt has been shown to induce Introduction resistance to a wide range of apoptotic stimuli (22). Consequent- Becoming self-sufficient for growth signals is a key feature of ly, the general apoptotic resistance seen when Notch signaling is transformed cells (1). This ability to grow independently of signals activated could be due to the induction of Akt function. from other cell types can be acquired by either losing or lowering Here we show that Akt activation downstream of Notch signal- the requirement for ligands to activate growth factor receptors ing was necessary for resistance to apoptosis in the normal breast present on the cancer cell (1) or by activating an autocrine signal- epithelial cell line MCF10A. On the other hand, inhibiting Notch ing loop through the secretion of the appropriate growth factors signaling in breast cancer cells decreased Akt phosphorylation (2, 3). and increased sensitivity to apoptosis. In addition, we show that The Notch signaling pathway regulates cell fate decisions, pro- Akt prevented apoptosis in response to DNA damage by inhibiting liferation, and death. The Notch genes encode transmembrane re- ASK1. This inhibition of ASK1 led to a failure to induce JNK signal- ceptors that are cleaved on binding of their ligands, leading to the ing and the subsequent activation of p53. Finally, we show that release of the intracellular domain [Notch intracellular domain Notch-induced activation of Akt is due to the release of an auto- (NICD)], which translocates to the nucleus where it functions as crine factor into the culture medium. a transcriptional coactivator. Notch signaling has been linked to many different human cancers, and in several cases, it has been shown to have a causal role in tumor development (4, 5). In T-cell Materials and Methods Antibodies and Western blotting. Rabbitantibodiesthatrecognize acute lymphoblastic leukemia, activating mutations in Notch1 have 183 185 473 been identified in >50% of all cases (6, 7). Notch signaling is also phospho-Thr /Tyr JNK, phospho-Ser Akt, total Akt, total glycogen synthase kinase (GSK)-3β, phospho-Ser83 ASK1, total ASK1, phospho-Thr81 aberrantly activated in breast cancer (8–12) and changes in the p53, phospho-Ser9 GSK-3β, SHIP2, phospho-Ser73 c-Jun, cleaved caspase-3, pathway may prove to be useful prognostic markers. Elevated and phospho-Ser166 MDM2 were from Cell Signaling Technology. Mouse antibodies that recognize p53, Noxa, FAK, MDM2, and PTEN were from Cell Sig- naling Technology, Abcam, BD Pharmingen, Santa Cruz Biotechnology, and Calbiochem, respectively. The goat antibody that recognizes INPP4B was from Santa Cruz Biotechnology. The mouse monoclonal antibody that recognizes Note: Current address of S. Stylianou: Trojantec Ltd., The Bank of Cyprus Oncology tubulin has been described previously (24). All horseradish peroxidase- Centre, 32 Acropoleos Avenue, 2006 Nicosia, Cyprus. O. Meurette and S. Stylianou contributed equally to this work. conjugated secondary antibodies were from Jackson ImmunoResearch. Requests for reprints: Keith Brennan or Olivier Meurette, Wellcome Trust Centre Western blotting was carried out as described previously (10). for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Oxford Plasmid constructs. cDNAs encoding dominant-negative forms of Road, Manchester M13 9PT, United Kingdom. Phone: 44-161-275-1517; Fax: 44-161-275- Mastermind-like (dnMAML; ref. 25) and Akt (26), constitutively active 1505; E-mail: [email protected] or [email protected]. ©2009 American Association for Cancer Research. forms of JNK (27) and Akt (28), and an NH2-terminally truncated form doi:10.1158/0008-5472.CAN-08-3478 of ASK1 (lacking amino acids 1-649; ΔN-ASK1; ref. 29) were obtained from www.aacrjournals.org 5015 Cancer Res 2009; 69: (12). June 15, 2009

Figure 1. Notch-induced Akt activation was necessary for resistance to apoptosis in MCF10A cells. A, Akt signaling was activated in MCF10A/RBP-Jκ and MCF10A/NICD cells. Whole cell lysates from MCF10A cells, MCF10A cells carrying the empty pcDNA3 (Vector), MCF10A/RBP-Jκ and MCF10A/NICD were analyzed by Western blotting for phosphorylation of Akt and its downstream targets. B, inhibiting Akt signaling sensitized MCF10A/RBP-Jκ and MCF10A/NICD cells to DNA damage-induced apoptosis. Whole cell lysates from MCF10A, MCF10A/RBP-Jκ, and MCF10A/NICD cells treated with 10 μmol/L SH6 and 100 μmol/L melphalan for 8 h as indicated were analyzed by Western blotting for Akt phosphorylation, Noxa expression, JNK phosphorylation, p53 accumulation, and caspase-3 cleavage. C, inhibiting Akt induced apoptosis in MCF10A cells regardless of RBP-Jκ–dependent Notch signaling. Parental, vector control, MCF10A/RBP-Jκ, and MCF10A/NICD cells were transiently transfected with either an empty vector or an expression plasmid encoding a dominant negative form of Akt. Transfected cells were identified by the co-transfection of these plasmids with pEGFP-C1 (Promega). The percentage of GFP positive cells that were apoptotic was determined. Average of three independent experiments.

Dr. Gianluca Civenni (Friedrich Miescher Institute), Prof. Boudewijn Bur- p199-N1IC-i2E-SVzeo and p201-doubleTET plasmids. MCF10DCIS.com gering (University Medical Centre Utrecht), Dr. Ulrike Rennefahrt (Univer- cells were grown in a 1:1 mixture of Ham's F-12 medium and DMEM sität Würzburg), Dr. Steven Anderson (University of Colorado), and Dr. supplemented with 100 μg/mL penicillin, 100 units/mL streptomycin, Hidenori Ichijo (University of Tokyo), respectively. Lentiviral vectors for 5% horse serum, and 2 mmol/L L-glutamine (Asterand; ref. 30). MCF7 the inducible expression of NICD (p201-doubleTET and p199-N1IC-i2E- cell lines were maintained as described previously (10). MCF7/Numb SVzeo) were based on vectors from Wolfgang Hillen and Stephen Elledge and MCF7/dnMAML cell lines were generated by selecting cells that sta- and were a generous gift from Manfred Gessler (Universität of Würzburg). bly carry the pcDNA3.1+Numb and pcDNA3.1(hygro)+dnMAML vectors, The NICD, RPB-Jκ/VP16, and Numb expression plasmids have been de- respectively. scribed previously (10). For conditioned medium experiments, culture medium was recovered Cell culture conditions. MCF10A cell lines, including those stably ex- from cells that had been detached, washed twice in PBS, plated in serum-free pressing NICD or the RBP-Jκ/VP16 fusion protein (MCF10A/NICD and medium, and cultured for 48 h. The conditioned medium was passed through MCF10A/RBP-Jκ), were maintained as described previously (10). A new a45μm filter to remove cellular debris before being added to MCF10A cells MCF10A/NICD cell line was derived by lentiviral infection using the that had been starved by culturing for 48 h in serum-free medium.

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For coculture experiments, parental MCF10A were stained for 30 min then analyzed for cell death as described below counting only CellTracker- with 5 μmol/L CellTracker Orange (Molecular Probes), and 2 × 104 of these stained cells. stained cells were plated with an equal number of nonstained parental Transfection conditions. Transient transfection of MCF7 and DCIS. MCF10A or nonstained MCF10A/NICD cells. Twenty-four hours after seed- com cells was carried out using Lipofectamine 2000 (Invitrogen). DNA ing, cells were treated with melphalan to induce DNA damage. Cells were (2 μg) was mixed in serum-free medium with 6 μL Lipofectamine 2000

Figure 2. Notch inhibition decreased Akt phosphorylation and increased apoptosis sensitivity in breast cancer cells. A, Akt phosphorylation on ser473 was decreased in breast cancer cells when Notch signaling was inhibited. Whole cell lysates from MCF7 or DCIS.com cells treated with 1 μmol/L DAPT or 0.1% DMSO for 48 h and from DCIS.com cells transiently transfected with an empty pcDNA3.1 vector, or expression plasmids encoding Numb or dnMAML, were analyzed by Western blotting for Akt phosphorylation. B, inhibiting Notch signaling in breast cancer cell lines increased their sensitivity to melphalan-induced apoptosis. MCF7 and DCIS. com cells were treated with 1 μmol/L DAPT or 0.1% DMSO for 48 h before treatment with or without 25 μmol/L melphalan for 16 h. The percentage of apoptotic cells was determined. A two-way ANOVA showed that the DAPT-induced increase in cell death following melphalan treatment was significant (*, P < 0.05; **, P < 0.01). C, expressing Numb or dnMAML increased the sensitivity of breast cancer cell lines to melphalan-induced apoptosis. DCIS.com and MCF7 cells stably expressing Numb or dnMAML were treated with or without 25 μmol/L melphalan for 16 h and the percentage of apoptotic cells was determined. A two-way ANOVA showed that expression of NUMB or dnMAML induced a significant increase in apoptosis (*, P < 0.05; ***, P < 0.001). D, expression of a constitutively active form of Akt (Myr-Akt) blocked the increased sensitivity to melphalan following Notch inhibition. DCIS.com cells transiently transfected with an empty pcDNA3.1 vector or expression vector encoding MYR-AKT were treated with 1 μmol/L of DAPT or 0.1% DMSO for 48 h. Cells were then treated with or without 25 μmol/L melphalan for 8 h and the percentage of apoptotic cells was determined.

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Figure 3. Inhibition of DNA damage-induced apoptosis by Akt signaling was not mediated by HDM2. Inhibiting the HDM2/p53 interaction through Nutlin-3 treatment did not resensitize MCF10A/NICD and MCF10A/RBP-Jκ cells to melphalan-induced apoptosis. MCF10A, MCF10A/NICD, and MCF10A/RBP-Jκ cells were treated with 10 μmol/L Nutlin-3 or 0.1% DMSO for 1 h before treatment with or without 100 μmol/L melphalan for a further 16 h. The percentage of apoptotic cells was determined and whole cell lysates were analyzed by Western blotting for p53 accumulation, Noxa expression, and caspase-3 cleavage.

for 20 min at room temperature. Lipofectamine 2000/DNA complexes were apoptosis to a comparable extent in parental, vector control, added to cells dropwise in serum free-medium. This medium was removed MCF10A/NICD, and MCF10A/RPB-Jκ cells (Fig. 1C); the apparently and complete growth medium was added after 6 h. more potent effects of a dominant-negative form of Akt than SH6 Apoptosis analysis. Microscopic detection of apoptosis was carried out are most likely due to it actively antagonizing Akt signaling, caus- on both detached and adherent cells recovered after treatment. These cells ing a more profound drop in Akt function than SH6. Altogether, were spun down onto slides using a Cytospin 2 (Shandon), fixed for 10 min these data show that Notch-induced apoptosis resistance in with methanol/acetone (50:50, v/v) solution at -20°C, and stained with 1 μg/mL Hoechst 33342 for 15 min at 37°C. Stained cells were observed using MCF10A cells requires Akt function. a Hamamatsu ORCA-ER digital camera on a Zeiss Axioplan2 microscope. Notch inhibition decreased Akt signaling and sensitized Apoptotic cells were recognized due to their nuclear condensation and frag- breast cancer cells to apoptosis. The Notch pathway is constitu- mentation and could be distinguished easily and reliably from necrotic cells. tively active in most breast epithelial cancer cell lines (10, 15). Con- In addition, this method allowed the detection of all cells that had under- sequently, we looked to see whether Notch inhibition would reduce gone apoptosis during the experiment. The percentage of apoptotic cells was Akt function in breast cancer cell lines. We focused our analysis on calculated and presented with SE. Differences between samples were ana- the MCF7 and DCIS.com cell lines, which are both wild-type for lyzed using two-way ANOVA followed by a Bonferroni post-test. p53 and thus are expected to respond to DNA damage in a similar manner as MCF10A cells. Pretreatment with DAPT, a γ-secretase Results inhibitor that prevents Notch cleavage and thus NICD production, Akt activation was necessary for Notch-conferred resistance induced a decrease of Akt phosphorylation in MCF7 and DCIS.com to apoptosis in normal breast epithelial cells. We have shown cell lines (Figs. 2A and 5A). Moreover, DAPT treatment induced an previously that increased RPB-Jκ–dependent Notch signaling in- increase in sensitivity to melphalan treatment (Fig. 2B). We also duces resistance to various apoptotic stimuli (10). We thus inves- inhibited Notch signaling by expressing Numb or dnMAML (25) tigated the activation of general survival pathways such as Akt in DCIS.com and MCF7 cells. Expression of either Numb or and extracellular signal-regulated kinase (ERK) signaling (22, dnMAML induced a decrease in Akt phosphorylation (Fig. 2A) 31). RBP-Jκ–dependent Notch signaling was activated in MCF10A and a sensitization to melphalan treatment (Fig. 2C). To show cells as described previously (10). Although we found no change that the increased sensitivity to melphalan-induced apoptosis in ERK phosphorylation in MCF10A/NICD and MCF10A/RPB-Jκ was linked to reduced Akt activity, we expressed a constitutively cells (data not shown), Akt signaling was clearly activated as active form of Akt (myr-Akt) in the presence or absence of mel- shown by Ser473 phosphorylation (Fig. 1A). The Akt targets phalan and DAPT treatment. Expression of myr-Akt completely GSK-3β, ASK1, and HDM2 were also phosphorylated on Ser9, abrogated the increase in melphalan-induced cell death ob- Ser83, and Ser166, respectively (Fig. 1A). served following DAPT treatment (Fig. 2D,comparecolumns 4 We next showed that inhibiting Akt by pretreatment with SH6 and 8). efficiently inhibited Akt phosphorylation on Ser473 in MCF10A/ Inhibition of ASK1/JNK signaling was responsible for Notch- NICD and MCF10A/RBP-Jκ cells (Fig. 1B, compare lanes 5 and 6 conferred resistance to apoptosis in MCF10A cells. As Akt sig- and lanes 9 and 10). This reduction in Akt signaling also restored naling can regulate p53 function through the phosphorylation of the sensitivity of MCF10A/NICD and MCF10A/RBP-Jκ cells to mel- HDM2 and ASK1 (22), we next investigated the role of these down- phalan-induced apoptosis leading to JNK phosphorylation, accu- stream proteins in Notch-induced resistance to DNA damage. We mulation of the p53 target Noxa, and caspase-3 cleavage (Fig. 1B, used Nutlin-3 to disrupt the p53-HDM2 interaction and thus pre- compare lanes 3, 8, and 12). Similarly, we found that inhibiting Akt vent p53 degradation. Although pretreatment with Nutlin-3 did signaling by expressing a dominant-negative form of Akt induced induce a stabilization of p53, it failed to either restore Noxa

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst June 2, 2009; DOI: 10.1158/0008-5472.CAN-08-3478 Akt Activation by Notch in Breast Epithelial Cells induction and cleavage of caspase-3 following melphalan treat- Notch signaling was not sufficient to confer resistance to DNA ment or to resensitize MCF10A/NICD and MCF10A/RBP-Jκ cells damage. to DNA damage-induced apoptosis (Fig. 3). We thus conclude that We next asked whether changes in ASK1 function downstream phosphorylation of HDM2 by Akt in MCF10A cells in response to of Akt were required for the resistance to DNA damage-induced

Figure 4. Suppression of the ASK1/JNK pathway by Akt mediates the Notch induced resistance to DNA damage-induced apoptosis in breast epithelial cells. A, inhibiting JNK signaling prevented melphalan-induced apoptosis in MCF10A cells. MCF10A cells were treated overnight with 10 μmol/L SP600125 or 0.1% DMSO prior to treatment or not with 100 μmol/L melphalan for 8 h. Cells were retreated with SP600125 every 2 h to maintain a strong inhibition of JNK or DMSO as a control. The percentage of apoptotic cells was determined and whole cell lysates were analyzed by Western blotting for c-jun, JNK and p53 phosphorylation, p53 accumulation, Noxa expression, and caspase-3 cleavage. A two-way ANOVA showed that the SP600125-induced decrease in apoptosis was significant (***, P < 0.001). B, expressing a constitutively active form of JNK induced apoptosis in MCF10A/NICD and MCF10A/RBP-Jκ cells. Parental MCF10A, vector control MCF10A cells, MCF10A/NICD, and MCF10A/RBP-Jκ cells were transiently transfected with expression vectors encoding a JNK/MKK7 fusion protein which constitutively activates JNK signaling (CA-JNK) or a JNK/MKK7 fusion protein that carries point mutations within JNK and MKK7 rendering the protein inactive as a control. Transfected cells were identified by the co-transfection of these plasmids with pEGFP-C1 (Promega). The percentage of GFP positive cells that were apoptotic was determined in three independent experiments. C, inhibiting Notch signaling increased the response of breast cancer cell lines to melphalan. DCIS.com cells transiently transfected with an empty pcDNA3.1 vector, or expression plasmids encoding Numb or dnMAML, were treated with or without 25 μmol/L melphalan. Similarly, DCIS.com cells pretreated for 48 h with 1 μmol/L DAPT were treated with or without 25 μmol/L melphalan. Finally, MCF7 cells carrying an empty pcDNA3.1 vector or stably expressing dnMAML were treated with melphalan. Whole cell lysates from these cell lines were analyzed by Western blotting for JNK and ASK1 phosphorylation. D, expressing a form of ASK1, which cannot be regulated by Akt phosphorylation (ΔN-ASK1), increased the sensitivity DCIS.com and MCF10A/NICD cells to melphalan-induced apoptosis. DCIS.com cells and MCF10A/NICD cells were transiently transfected with either an empty pcDNA3.1 vector or an expression plasmid encoding ΔN-ASK1. Cells were treated with or without 25 μmol/L melphalan 48 h later and the percentage of apoptotic cells was determined. www.aacrjournals.org 5019 Cancer Res 2009; 69: (12). June 15, 2009

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst June 2, 2009; DOI: 10.1158/0008-5472.CAN-08-3478 Cancer Research apoptosis. ASK1 is activated following DNA damage and stimulates JNK activity (32), which in turn phosphorylates and activates p53 (33). Akt can phosphorylate ASK1 on Ser83, preventing its activation (34). As ASK1 was phosphorylated on Ser83 following Notch activation (Fig. 1A), we examined whether inhibition of ASK1/ JNK signaling in MCF10A cells was sufficient to mimic Notch-induced apoptosis resistance. As expected, treatment with the JNK inhibitor SP600125 reduced phosphorylation of the JNK target c-Jun on Ser73 (Fig. 4A). Furthermore, inhibition of JNK prevented phosphorylation of p53 on Thr81, p53 accumulation, and expression of Noxa in parental MCF10A cells (Fig. 4A). Moreover, JNK inhibition completely abrogated cell death induction (Fig. 4A). On the other hand, expression of a constitutively active form of JNK induced apoptosis in MCF10A/NICD and MCF10A/RPB-Jκ cells as readily as in parental MCF10A cells (Fig. 4B). This suggests that Notch signaling may also inhibit the ASK1/JNK pathway in breast cancer cell lines. Indeed, inhibiting Notch signaling in the breast cancer cell lines DCIS.com and MCF7, by expressing Numb or dnMAML, led to increased JNK phosphorylation following melphalan treatment (Fig. 4C). JNK phosphorylation was similarly increased in response to melphalan in DCIS.com cells treatedwithDAPTfor48h(Fig.4C). In keeping with this, ASK1 phosphorylation on Ser83 following melphalan treatment was reduced when Numb or dnMAML was expressed (Fig. 4C). Finally, restoring ASK1 signaling in DCIS.com or MCF10A/NICD by expressing a form of ASK1 that can no longer be regulated by Akt phosphorylation (ΔN-ASK1) sensitized these cells to melphalan treatment (Fig. 4D). Altogether, these data show that inhibition of ASK1/JNK signaling was sufficient to mimic the resistance to DNA damage-induced apoptosis that occurs in response to Notch activation. Akt activation in response to Notch signaling was due to the release of an autocrine factor. Akt activation by Notch has been described in several different systems (19, 20, 35, 36). In T-cell acute lymphoblastic leukemia, Notch has been shown to inhibit PTEN expression (19). However, we saw no changes in PTEN expression in MCF10A cells when Notch signaling was activated (Fig. 5A). Simi- larly, we saw no modification of PTEN expression when Notch signaling was inhibited following DAPT treatment of MCF7 cells (Fig. 5A). We thus looked at other phosphatases that regulate PIP3 levels, SHIP2 and INPP4B (37, 38). Again, activation of Notch signaling in MCF10A cells or inhibition of Notch signaling in MCF7 cells did not alter SHIP2 or INPP4B expression (Fig. 5A). This suggests that the activation of Akt following Notch signaling was not due to changes in the expression of these phosphatases. Figure 5. Notch-induced Akt activation was dependent on the release of an A, We have, however, noticed that MCF10A/NICD and MCF10A/ autocrine factor into the medium. Notch signaling did not alter the expression of PTEN, SHIP2, or INPP4B in breast epithelial cells. Whole cell lysates from parental RBP-Jκ cells can tolerate growth factor withdrawal (data not MCF10A and MCF10A/NICD cells, and from MCF7 cells treated with 1 μmol/L DAPT shown). We thus hypothesized that Akt activation could be or 0.1% DMSO for 48 h were analyzed by Western blotting for the activation of Akt, phosphorylation of GSK-3β, and level of PTEN, SHIP2, and INPP4B. B, conditioned due to the secretion of a growth factor that signals through medium from MCF10A/NICD cells increased Akt signaling. Conditioned medium an autocrine loop. To test this hypothesis, we starved MCF10A from MCF10A or MCF10A/NICD cells was added to parental MCF10A cells that cells in serum-free and growth factor-free medium for 48 h and had been starved for 48 h in serum- and growth factor-free medium. As a positive control starved MCF10A cells were treated with complete growth medium. Whole cell stimulated these cells with complete growth medium or serum- lysates were then prepared after the indicated times and analyzed by Western free and growth factor-free medium that had been conditioned blotting for Akt and GSK-3β phosphorylation. C, DCIS.com cells secrete a factor in a by parental MCF10A cells or MCF10A/NICD cells. As expected, Notch-dependent fashion that induced Akt signaling. DCIS.com cells were cultured in serum- and growth factor-free medium for 48 h in the presence of 1 μmol/L DAPT complete growth medium induced phosphorylation of Akt on or 0.1% DMSO. Conditioned medium was harvested and added to starved parental Ser473 and phosphorylation of the Akt target GSK-3β, whereas MCF10A cells for the indicated times. Whole cell lysates were prepared and analyzed by Western blotting for Akt phosphorylation. D, co-culturing MCF10A cells with conditioned medium from parental MCF10A cells did not (Fig. MCF10A/NICD cells protects them from melphalan-induced apoptosis. MCF10A 5B). In contrast, conditioned medium from MCF10A/NICD cells cells were stained with CellTracker Orange and then co-cultured with unstained induced robust Akt signaling (Fig. 5B). Furthermore, conditioned MCF10A or MCF10A/NICD cells for 24 h. Cells were then treated with 100 μmol/L melphalan for 16 h and the percentage of apoptotic stained cells was determined. medium from DCIS.com cells stimulated phosphorylation of Akt A two-way ANOVA showed that the presence of MCF10A/NICD cells reduced the on Ser473 in starved parental MCF10A cells (Fig. 5C), whereas sensitivity of MCF10A cells to melphalan (**, P <0.01).

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst June 2, 2009; DOI: 10.1158/0008-5472.CAN-08-3478 Akt Activation by Notch in Breast Epithelial Cells conditioned medium from DCIS.com cells treated with DAPT for ed on Ser166 in response to Notch signaling (Fig. 1A). However, 48 h was ineffective (Fig. 5C), suggesting that these cells also inhibiting the interaction between HDM2 and p53 by treating with secrete a Notch-dependent factor that activates Akt. Finally, co- Nutlin-3 was not sufficient to restore the sensitivity of MCF10A/ culturing parental MCF10A cells with MCF10A/NICD cells pro- NICD and MCF10A/RBP-Jκ cells to DNA damage-induced apopto- tected parental cells from DNA damage-induced apoptosis (Fig. sis (Fig. 3). 5D). Together, these data show that activating Notch signaling In contrast, the inhibition of JNK signaling does appear to be caused breast epithelial cells to secrete a factor into the culture significant (Fig. 4B; ref. 10). Inhibiting JNK signaling in parental medium and protected MCF10A cells from apoptosis. MCF10A cells was sufficient to mimic the Notch-induced apoptosis resistance (Fig. 4A), whereas expressing a constitutively active form of JNK induced apoptosis in MCF10A/NICD and MCF10A/RPB-Jκ cells (Fig. 4B). Furthermore, JNK signaling was activated more Discussion strongly in DCIS.com and MCF7 cells in response to melphalan Here, we have shown that Akt signaling was activated in MCF10A treatment when Notch signaling was inhibited (Fig. 4C). cells, which were transformed by RPB-Jκ–dependent Notch sig- Notch signaling has been shown to inhibit JNK function in both naling. This activation of Akt led to the inhibition of ASK1 by Drosophila and mammalian cells (42, 43). Within mammalian cells, phosphorylation on Ser83, which in turn inhibited JNK activation, NICD inhibits JNK by interfering with the scaffold function of JNK- phosphorylation of p53 on Thr81, and subsequent induction of ap- interacting protein 1 (17). However, we suspect that this is not the optosis in response to DNA damage. Finally, the activation of Akt only way Notch can limit JNK signaling, as we found that expres- was due to an autocrine loop involving the release of a secreted sing the RBP-Jκ/VP16 fusion protein inhibited JNK as potently as factor, which may be a growth factor. NICD (Fig. 1B). We describe here an alternative mechanism where- Akt activation by Notch signaling has been described in several by Notch signaling inhibits JNK function through the induction of different cellular models (19, 20, 39). However, the mechanism by Akt signaling, which, in turn, phosphorylates and inhibits upstream which Akt is activated is not well understood. It has recently been kinases of the JNK pathway such as ASK1 (Figs. 1A and 4C). In shown that Notch signaling induces transcription of the epidermal keeping with this, we found that expression of a NH2-terminally growth factor (EGF) receptor in glioma cells (40). However, in truncated ASK1 molecule, which lacks the Akt phosphorylation site MCF10A cells, expressing either NICD or the RPB-Jκ/VP16 fusion at Ser83, restores the sensitivity of cells in which Notch signaling is protein did not alter EGF receptor or HER-2 expression levels (data activated, DCIS.com and MCF10A/NICD, to DNA damage (Fig. 4D). not shown). On the other hand, Notch signaling induces a c-Myc- Notch signaling has also been shown to regulate p53 signaling by and HES1-dependent down-regulation of PTEN expression in T- several other mechanisms (18, 35, 44, 45). Mungamuri and collea- cell acute lymphoblastic leukemia, leading to an increase in Akt gues showed that Notch inhibited p53 function through the activa- phosphorylation and activity (19). We did not see any changes in tion of mTORC1 and eIF4E (35). However, rapamycin did not PTEN expression either when Notch signaling was activated in sensitize MCF10A/NICD to apoptosis, suggesting that this mecha- MCF10A cells or following DAPT treatment to inhibit Notch in nism does not play a role in these cells (data not shown). NICD has breast cancer cells (Fig. 5A). Similarly, we saw no change in the also been observed to directly interact with p53, inhibiting its expression of INPP4B and SHIP2, which can also regulate PIP3 le- phosphorylation and subsequent activation (18). As expression of vels and thus Akt activation (Fig. 5A; ref. 37, 38). It has also been RPB-Jκ/VP16 in MCF10A cells was as effective as expression of shown that NICD can directly interact with p56lck and phosphati- NICD, we again suspect that this mechanism does not contribute dylinositol 3-kinase in T cells to activate phosphatidylinositol 3-ki- significantly to the regulation of p53 function in these cells. nase and hence Akt (20). A similar mechanism is unlikely to play a We therefore conclude that, in breast epithelial cells, Notch ac- role in breast epithelial cells, as we have shown that expression of tivation causes a general apoptosis resistance through the activa- the RPB-Jκ/VP16 fusion protein can activate Akt to the same ex- tion of Akt and specifically inhibits p53-mediated apoptosis in tent as NICD (Fig. 1A). response to DNA damage through an Akt-dependent inhibition Together, these observations suggest that Akt signaling is acti- of JNK. Finally, as Notch is now considered as an attractive target vated in response to Notch signaling by a novel mechanism in for cancer therapy (5), the results presented here may help with the breast epithelial cells. Furthermore, it is likely to be through the design of treatment strategies that can be combined with Notch transcription of a Notch target gene, as the effects of expressing pathway inhibitors to improve therapy and reduce unwanted side NICD and an active form of RPB-Jκ were indistinguishable effects. (Fig. 1A). Although the identity of the target gene is currently not clear, our results suggest that it is either a secreted factor or a protein required for the secretion of the appropriate factor, as Disclosure of Potential Conflicts of Interest serum-free and growth factor-free conditioned medium from No potential conflicts of interest were disclosed. MCF10A/NICD cells was sufficient to activate Akt signaling in parental MCF10A cells (Fig. 5B). Furthermore, parental MCF10A cells cocultured with NICD-expressing MCF10A cells were protected Acknowledgments from apoptosis (Fig. 5D). Received 9/10/08; revised 2/12/09; accepted 4/6/09; published OnlineFirst 6/2/09. To elucidate the molecular mechanism underlying the resistance Grant support: Breast Cancer Campaign. The costs of publication of this article were defrayed in part by the payment to DNA damage-induced apoptosis and the failure to activate p53 of page charges. This article must therefore be hereby marked advertisement in (10), we determined which downstream targets of Akt were re- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. quired. In particular, changes in ASK1/JNK signaling and HDM2 We thank Gianluca Civenni, Ulrike Rennefahrt, Boudewijn Burgering, Hidenori Ichijo, Manfred Gessler, and Asterand for providing plasmids and cell lines used in activity were examined, which have both been shown to regulate this research and Charles Streuli for suggestions that helped the development of p53 function downstream of Akt (33, 41). HDM2 was phosphorylat- the project. www.aacrjournals.org 5021 Cancer Res 2009; 69: (12). June 15, 2009

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Cancer Res 2009; 69: (12). June 15, 2009 5022 www.aacrjournals.org

Notch Activation Induces Akt Signaling via an Autocrine Loop to Prevent Apoptosis in Breast Epithelial Cells

Olivier Meurette, Spyros Stylianou, Rebecca Rock, et al.

Cancer Res 2009;69:5015-5022. Published OnlineFirst June 2, 2009.

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