Increased Protein Expression of the PTEN Tumor Suppressor in the ...

[Cell Cycle 4:10, 1389-1395, October 2005]; ©2005 Landes Bioscience

Increased Protein Expression of the PTEN Tumor Suppressor in the Presence of Constitutively Active Notch-1 Report

ABSTRACT Mammalian Notch-1 is part of an evolutionarily conserved family of transmembrane receptors best known for involvement in cell fate decisions. Mutations that result in Notch-1 activation result in T-lineage oncogenesis. In other cell lineages, however, studies have indicated that cooperation with cellular signaling pathways, such as Ras, is necessary for Notch-mediated oncogenesis and in some settings, Notch-1 has been reported to function as a tumor suppressor. In order to test the hypothesis that the Notch-1 pathway exhibits cross-talk with Ras/Raf/MEK/ERK, the constitutively active cytoplasmic portion of Notch-1 was introduced into 293 HEK fibroblasts via retroviral transduction. ERK-1,-2 activation was markedly increased in cells expressing constitutively active Notch-1. These cells exhibited a more rounded morphology as compared to 293 cells transduced with an empty vector or parental 293 cells. These observations correlated with decreased total and phosphorylated focal adhesion kinase protein (FAK). Subsequent examination of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) revealed that total and phosphorylated PTEN protein was elevated in cells expressing constitutively active Notch-1. Loss of Akt phosphorylation was also observed in cells bearing activated Notch-1. Two potential binding sites for the Notch effector CBF-1 were identified in the human PTEN promoter sequence. A PTEN promoter luciferase reporter exhibited increased activity in the presence of Notch-1 signaling. These data indicate that Notch-1 can participate in cross-talk with other signaling pathways such as Ras/Raf/MEK/ERK through the regulation of the PTEN tumor suppressor.

Received 07/05/05; Accepted 07/13/05

Notch, CBF-1, RBP-Jκ∫, PTEN, FAK, experimental therapeutics

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2028

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*Correspondence to: Fred E. Bertrand; Department of Microbiology & Immunology; The Brody School of Medicine at East Carolina University; Greenville, North Carolina 27834 USA; Tel.: 252.744.2703; Fax: 252.744.3104; Email: [email protected]

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Department of Microbiology & Immunology; The Brody School of Medicine at East Carolina University; Greenville, North Carolina USA

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William H. Chappell Thomas D. Green Justin D. Spengeman James A. McCubrey Shaw M. Akula Fred E. Bertrand*

The human Notch family consists of four family members termed Notch-1, -2, -3 and -4.1,2 There are at least three human ligands, jagged-1, jagged-2 and delta, although additional ligands likely exist. Notch-1 is the most widely studied of the Notch receptors and functional differences between Notch receptors and different ligand combinations are not well understood. The Notch receptor is assembled on the cell surface as a 180 kDa extracellular fragment and a 120 kDa fragment consisting of 12 extracellular amino acids, a transmembrane region and a cytoplasmic region.3 The cytoplasmic region of Notch is also referred to as intracytoplasmic notch (ICN) and when expressed alone is constitutively active.1-3 Upon ligand binding, Notch undergoes a series of proteolytic cleavages that result in the release of the cytoplasmic portion of Notch-1 and the subsequent conversion of the CBF-1 transcription factor from a repressor to an activator.4-9 CBF-1 in turn activates members of the HES gene family, of which HES-1 is the most-well studied.1 HES-1 goes on to activate a variety of other transcription factors; many in a cell type specific fashion.1 Notch-1 was originally identified in humans as the result of a chromosomal translocation, t(7;9), that fuses the constitutively active cytoplasmic region of Notch-1 with the T-cell receptor β locus in a subset of T-cell acute leukemia (T-ALL).10 Constitutively active Notch-1 has subsequently been shown to be oncogenic and mutations in Notch-1 have been identified in 80% of T-ALL.11 Oncogenic Notch has also been implicated in pancreatic and breast cancer.12,13 Interestingly, in mammalian skin, Notch-1 has been shown to function as a powerful tumor suppressor.14 There is evidence that Notch may interact with other cellular signaling pathways. Notch has been implicated in activating PI3K and Akt, and has also been reported to both activate and repress NF-κB.15-18 There is genetic evidence in fruit flies and nematodes linking Notch with Ras signaling.19-21 In mammalian systems evidence is beginning to emerge linking Notch signaling with the Ras/Raf/MEK/ERK pathway. Some studies have

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F.E.B. and S.M.A. were supported in part by New Investigator Awards from an American Cancer Society Institutional Grant awarded to East Carolina University. J.A.M. was supported in part by NIH grant RO1 CA98195.

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Increased Protein Expression of the PTEN Tumor Suppressor in the Presence of Constitutively Active Notch-1

reported that oncogenic Notch requires activated Ras, while others have suggested that activation of growth factor receptors that act through Ras, such as the epidermal growth factor receptor (EGFR), require downstream Notch activation.22,23 Cross-talk between Ras/Raf/MEK/ERK and PI3K/AKT and JAK/STAT has been reported.24-26 Such cross-talk between signaling pathways can have a profound influence on growth and apoptotic sensitivity of cells. For instance, in B-lineage acute leukemia signals from both MEK and PI3K are required for stromal cell induced survival and proliferation.25 Simultaneous activation of PI3K/AKT and Ras/Raf/MEK/ERK is profoundly effective at promoting cytokine independence in acute myeloid leukemia cell lines.24,26 PTEN was first identified through common deletions on chromosome 10 among several different tumor types.27-29 Deletions of PTEN, inactivating point mutations in the PTEN codon or the PTEN promoter have subsequently been identified in a variety of cancers.30-32 Loss of PTEN expression or activity correlates well with oncogenesis. This 54 kDa protein has been reported to have at least three distinct roles: (1) a lipid phosphatase activity that mediates inactivation of PI3K; (2) a protein phosphatase activity (e.g.: dephosphorylation of FAK); and (3) a substrate for phosphorylation (reviewed in Steelman et al.).30 Upon phosphorylation, PTEN is degraded through the ubiquitin/proteasome pathway. Other mechanism(s) regulating PTEN are poorly characterized. The human PTEN promoter has been identified and contains several p53-binding sites.33 TGF-β family growth factors have been reported to regulate PTEN protein levels,34 but the precise pathways involved regulation of PTEN have remained elusive. Lipid phosphatase activity is the most well characterized function of PTEN.28,30 This protein removes phosphates from PI3K, rendering PI3K inactive and inactivating the pathway. This results in increased apoptosis, reduced proliferation and exit from the cell cycle, and is one major pathway by which PTEN exerts its function as a tumor suppressor.30 Studies have reported that PTEN restoration in PTEN-defective cells will result in loss of proliferation and increased apoptosis. PTEN has been reported to regulate other pathways as well. PTEN can remove phosphates from FAK, rendering it inactive and incapable of activating the MEK/ERK MAPK pathway.35 FAK is typically activated through integrin signaling which plays a major role in cell adhesion and motility.36 Thus, PTEN can function as a tumor suppressor by inactivating signals that may promote metastasis. PTEN can also directly inhibit MAPK signaling through interactions with GAB1, regulating activation of Ras/Raf/MEK/ERK.30 Herein, we have tested the hypothesis that cross-talk exists between Notch-1 and Ras/Raf/MEK/ERK signaling, using a 293 HEK fibroblast model. Stable expression of constitutively active Notch-1 resulted in increased ERK-1, -2 activation. Inhibition of MEK blocked ERK-1, -2 activation, but did not block activation of a CBF-1 reporter indicating that MEK/ERK activation was not required for Notch signaling. 293 cells expressing activated Notch exhibited an altered morphology, which prompted us to examine FAK and PTEN protein expression. Levels of FAK protein were decreased, but both total and phosphorylated PTEN protein was increased in the presence of a constitutive Notch signal. Phosphorylation of Akt was not observed in cells expressing activated Notch-1. The identification of two potential CBF-1 binding sites in the PTEN promoter sequence and increased PTEN promoter activity in the presence of constitutively active Notch-1 provides evidence that the PTEN tumor suppressor may be regulated through the Notch-1 signaling pathway. 1390

METHODS Cell lines. 293 cells were purchased from the ATCC and maintained in T-75 flasks coated with 0.1% gelatin in DMEM (Invitrogen, Carlsbad CA) supplemented with 10% heat inactivated fetal calf serum. Retroviral constructs and transduction. MIGR1 and human Notch-1/ GFP retroviral vectors have been described previously and were a kind gift from Dr. Warren Pear, University of Pennsylvania.37 Packaged retroviral viral supernatant was prepared by cotransfection of MIGR1 or Notch-1/GFP with a vector expressing the amphotropic coat into 293 cells via calcium phosphate precipitation.38-39 Supernatants bearing packaged particles were collected 48 hours later.38-39 Subconfluent 293 cells were overlaid with a 1:1 mixture of packaged viral particles and growth medium plus 1 µg/ml polybrene. After a six to 14 hour incubation, medium was removed and replaced with DMEM + 10% serum. Successfully transduced cells were purified on the basis of GFP expression by FACS sorting using a FACS Vantage instrument (Becton-Dickenson, Moutainview, CA). Luciferase assays. Parental 293 cells, 293/MIGR1 or 293/Notch-1 cells were transfected via calcium phosphate precipitation with 20 pmol of either HES AB, HES ∆AB,40 kind gift from Dr. Jon Aster, Harvard University) or pGL2-Basic (Promega, Madison, WI) along with 10 µg of CMV-β-gal plasmids. pGL2-Basic contains only the luciferase gene with no promoter or enhancer and was used as a background control. CMV-β-gal has the β-galactosidase gene driven by the CMV promoter and was used as a control for transfection efficiency. HES AB bears two CBF-1 bidning sites fused to the luciferase gene.40 HES ∆AB contains CBF-1 binding sites is used as a background control.40 Luciferase and β-galactosidase assays were performed using the Luciferase Assay System and β-Galactosidase Assay System from Promega as per manufacturer’s instruction. Luciferase was read in a Turner Systems Luminometer (Sunnyvale, CA), as per manufacturer’s instruction. Luciferase values were normalized to β-galactosidase for each experiment, and adjusted for fold expression over that of the pGL2-Basic reporter (background) for each cell line tested. In experiments examining Notch signal transduction, normalized luciferase expression was recorded as fold HES AB over HES ∆AB. To examine PTEN promoter activity, cells were transfected as described above using a PTEN/pGL2-Basic reporter along with the CMV-β gal plasmid. These cells were then cultured for 24 hours in medium in the absence of serum prior to luciferase and β-galactosidase analysis. The PTEN/pGL2-Basic reporter (PTEN-LR) was constructed by PCR cloning the PTEN promoter (GenBank AF067844) into the Xho I and Hind III sites of PGL2-basic (Promega). Statistical significance was tested with the unpaired t test using GraphPad Software (San Diego, CA). Western blot analysis. Whole cell lysates were prepared in lysis buffer and separated on 8% SDS-PAGE gels followed by transfer to nitrocellulose membranes.24 Membranes were blocked overnight at 4˚C in 1% BSA. Blots were probed sequentially with a phospho-specific antibody, the corresponding total antibody, and anti β-tubulin or actin as a loading control. Antibodies against phosphorylated and total ERK-1,-2, PTEN, and Akt were purchased from Cell Signaling Technologies (Beverly, MA). FAK and phospo-FAK were purchased from Signal Transduction Laboratories (BD Biosciences, San Diego, CA). Notch-1 was detected using rat anti-human Notch-1 IC, clone btan-20,41 purchased from the University of Iowa Developmental Studies Hybridoma Bank. Rabbit anti β-actin was purchased from Sigma (St. Louis MO). Immunofluorescence. Three hundred thousand 293 cells per well were added to each well of a six well plate containing a sterile glass cover slip. Following overnight culture, the cover slips were rinsed in PBS and fixed in 3.7% formaldehyde at R.T. for 10 minutes. Cover slips were washed twice with PBS, and were permeabilized with 0.1% triton X-100 + 0.1% BSA at R.T. for 3 minutes. Following two PBS washes, the cells were blocked in 0.1% BSA for 20 minutes at R.T. The cover slips were stained for 20 minutes with Rhodamine Phalloidin (Molecular Probes, Eugene, OR) at R.T., then washed twice with PBS and mounted onto slides using mount + DAPI (Molecular Probes). Slides were viewed on a Zeiss fluorescent microscope. Digital images were merged using Image J Software (NIH, Bethesda, MD).

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Figure 1. Establishment of stable constitutively active Notch-1 retrovirally transduced 293 cells. (A) Retroviral constructs. The constructs used are IRES/GFP constructs in which the gene of interest and GFP are transcribed as a single mRNA, but are translated as separate proteins due to the presence of the IRES sequence. MIGR1 is the empty vector control and Notch-1 contains the constitutively active cytoplasmic tail of human Notch-1.37 (B) Notch-1 transduced 293 cells. Transduced cells were FACS purified based on GFP expression 48 hour post-transduction and then placed into culture. Two weeks post-sort, both the vector control (MIGR1) and the Notch-1 transduced cells were greater than 95% GFP positive. (C) Exogenous HES-1 promoter activity is increased in Notch-1 transduced 293 cells. The function of the transduced Notch-1 retrovirus was confirmed using a CBF-1 reporter assay consisting of the murine HES-1 promoter fused to the luciferase gene (HES AB).40 An inactive version of the HES promoter (HES ∆AB) in which the two CBF-1 binding sites are mutated to prevent binding was used as a background control.40 Data are shown as fold luciferase activity of HES AB/HES ∆AB. N = 4. **denotes statistical significance between 293 versus 293/Notch-1 and 293/MigR1 versus 293/Notch-1 (p = 0.023). (D) Western blot analysis of transfected 293 cells. Whole cell lysates were prepared and subjected to western blot analysis for Notch-1 expression. Transfected Notch-1 is readily detectable in 293/Notch-1 cells, but Notch-1 is not detected in the parental 293 and 293/MigR1 control cells.

RESULTS

Figure 2. Notch-1 transduced 293 cells exhibit altered morphology and actin localization. Cells stably transduced with constitutively active Notch-1 exhibited a more rounded morphology as compared with vector control (MIGR1) transduced cells or parental 293 cells. This was marked by a lack of fine filopodia (indicated by dark arrows). Fluorescent staining revealed that actin appeared evenly distributed over the cell surface of 293/Notch-1 cells, but that actin was concentrated in the filopodia of parental 293 or MIGR1 transduced 293 cells.


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Generation of 293 cells expressing constitutively active Notch-1. In order to study the effects of constitutively active Notch-1 on MAPK signaling pathways, we generated 293 HEK fibroblasts that expressed the constitutively active cytoplasmic region of human Notch-1 (293/Notch-1) and 293 HEK fibroblasts that expressed the empty vector alone (293/MIGR1). The constructs are illustrated in Figure 1A.37 Stably transduced cells were identified and isolated by FACS sorting based on GFP expression (Fig. 1B). Notch-1 signal transduction was verified using a HES-1 promoter luciferase reporter assay (Fig. 1C). As expected, 293/Notch-1 cells exhibited a large increase in luciferase expression, confirming that the Notch-1 signaling pathway is activated in these cells. The presence of transfected, consitutively active Notch-1 protein was confirmed by western blot analysis (Fig. 1D). 293/Notch-1 cells exhibit altered morphology. 293 fibroblasts expressing constitutively active Notch-1 exhibited an altered morphology as compared with 293/MIGR1 or parental 293 cells. The 293/Notch-1 cells typically exhibited a more rounded morphology and tended to aggregate into syncytia (Fig. 2). In contrast the parental 293 cells and the control 293/MIGR1 cells exhibited extended hair-like projections called filopodia that were more apparent following actin staining with rhodamine-phalloidin (Fig. 2). Thus, constitutively active Notch-1 resulted in an altered morphology in fibroblasts.

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Figure 4. Cells expressing constitutively active Notch-1 exhibit decreased expression of focal adhesion kinase protein. Parental 293 cells, 293/ MIGR1 and 293/Notch-1 were examined by western blotting under standard culture conditions and serum deprivation for expression and activation of focal adhesion kinase (FAK). Serum deprived conditions were preformed in duplicate and are shown as two individual lanes for each cell type on the blot. For cells cultured in 10% serum, phosphorylated and total FAK protein levels were similar. However, under conditions of serum deprivation, cells stably transduced with constitutively active Notch-1 (Notch-1) expressed lower level of phosphorylated and total FAK as compared with parental 293 cells (293) or empty vector transduced cells (MIGR1). Data are representative of four independent experiments.

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Figure 3. (A) Notch-1 signaling promotes ERK-1,-2 phosphorylation. Whole cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with antibodies against phosphorylated and total ERK-1,-2, human Notch-1 and β-tubulin. Cells expressing Notch-1 exhibited an increase in the amount of phosphorylated ERK-1,-2. In contrast, ERK-1,-2 activation appeared similar among parental 293 cells and control MIGR1 293 cells. Endogenous Notch-1 is below the threshold of detection via western blotting and does not show up on these blots. Data is representative of more than three independent experiments. B-C) ERK-1,-2 activation is not required for CBF-1 dependent Notch signaling. 293/Notch-1 cells were transiently transfected with the CBF-1 HES-AB reporter or the HES-∆AB control and treated for 24 hours with the MEK inhibitor U0126. (B) Inhibition of MEK was confirmed via western blot analysis to examine phosphorylated ERK-1,-2. Cells treated with U0126 (5 µg/ml) exhibited no detectable phosphorylated ERK-1,-2. (C) CBF-1 reporter assay. Cells treated with the MEK inhibitor U0126 did not loose the ability to transactivate a CBF-1 reporter in the presence of constitutively active Notch-1. Data is presented as fold luciferase expression of HES AB/HES ∆AB. N = 3.

ERK-1,-2 phosphorylation is increased in 293 fibroblasts bearing constitutively active Notch-1. Several lines of evidence have linked Notch signaling with activated Ras and vice versa.19-23 Western blot analysis (Fig. 3A) revealed that 293/Notch-1 cells exhibited higher levels of phosphorylated ERK-1,-2 as compared with 293/MIGR1 cells or the parental 293 cell line. ERK-1,-2 activation is not necessary for CBF-1 mediated Notch signaling. To determine if activated ERK-1,-2 was required for Notch-1 signal transduction, 293/Notch-1 cells were transfected via calcium phosphate precipitation with the HES promoter luciferase reporters. These were then treated with U0126 (5 µg/ml) to inhibit phosphorylation of ERK-1,-2. U0126 was titrated in order to determine the lowest dose that inhibited MEK activity in 293 cells, as determined by western blot analysis of ERK-1,-2 phosphorylation (data not shown). Western blot analysis demonstrated that treatment with the MEK inhibitor resulted in undetectable levels of phosphorylated ERK-1,-2 (Fig. 3B). However, luciferase assays demonstrated CBF-1 activation even in the presence of U0126 (Fig. 3C). These data indicate that MEK/ERK activation is not required for Notch-1 signal transduction. FAK and phospho-FAK protein is decreased the presence of Notch-1 signaling. The altered morphology and activation of ERK-1,-2 observed in 293/Notch-1 cells prompted us to examine expression and activation of FAK in 293 cells expressing constitutively active Notch-1. FAK is a key mediator of integrin signaling and acts to channel integrin signals into other

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Figure 5. PTEN protein expression is increased in 293 cells expressing constitutively active Notch-1. Parental 293 cells, 293/MIGR1 and 293/ Notch-1 were examined by western blotting under standard culture conditions and serum deprivation for expression and activation of PTEN. Cells cultured in 10% serum exhibited similar levels of PTEN protein expression (right panel). Upon serum deprivation, 293 cells expressing constitutively active Notch-1 (Notch-1) exhibited an increase in phosphorylated and total PTEN protein (left panel).

signaling pathways, including Ras/Raf/MEK/ERK.36 Because serum components are known to activate FAK, cells were plated at 70% confluency, then serum starved for 24 hours prior to harvesting the cells to make whole cell protein lysates. Serum starved cells expressing constitutively active Notch-1 exhibited lower levels of total and phosphorylated (Tyr 397) FAK protein expression as compared with parental 293 cells or 293 cells transduced with an empty vector (293/MIGR1), as shown in Figure 4. In contrast, under conditions of 10% serum expression of total and phosphorylated FAK was similar between parental 293 cells, 293/MIGR1 cells or 293/Notch-1 cells. PTEN protein is increased in 293 cells expressing constitutively active Notch-1. It has been reported previously that PTEN can modulate FAK activity.35 Parental 293 cells, 293/MIGR1 and 293/Notch-1 were plated as subconfluent levels then serum starved for 24 hours followed by western blot analysis for total and phosphorylated PTEN expression. In the presence of serum, no significant changes in PTEN expression were observed between parental 293 cells, 293/MIGR1 and 293/Notch-1 cells (Fig. 5,

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Figure 6. Akt phosphorylation is lost in 293 cells expressing constitutively active Notch-1. Whole cell lysate from 293, 293/MigR1 and 293/Notch-1 cells were examined for phosphorylated and total Akt protein expression. Loss of Akt phosphorylation at serine 473 and threonine 308 was observed in cells expressing constitutively active Notch-1 (293/Notch-1). Total Akt protein levels were similar among the three 293 cell variants.

right panel). However, both total and phosphorylated PTEN protein species were increased in cells expressing activated Notch-1 under conditions of serum deprivation (Fig. 5, left panel). 293 cells expressing constitutively active Notch-1 exhibit loss of Akt phosphorylation. PTEN is best characterized for reducing the activity of PI3K, which in turn results in loss of phosphorylation and inactivation of Akt. Parental 293 cells, 293/MigR1 control cells and 293/Notch-1 cells bearing constitutively active Notch-1 were cultured as above, then analyzed by western blotting Akt phosphorylation. As seen in Figure 6, 293 cells transfected with constitutively active Notch-1 exhibited a loss of Akt phosphorylation at serine 473 and threonine 308. Akt was phosphorylated at both sites in parental 293 cells and control 293/MigR1 cells. Levels of total Akt protein were similar among the three 293 variants. Loss of Akt phosphorylation in 293/Notch-1 cells is consistent with the observed increase in PTEN protein. The PTEN promoter region contains two CBF-1 binding sites. The minimal PTEN promoter has been characterized and reported to be contained within a 600 bp fragment located about 745 bp 5' of the first ATG start site.33 Visual inspection of this region identified two potential CBF-1 binding sites (Fig. 7A) signifying that PTEN may be a direct target for regulation through the Notch-1 signaling pathway. Indeed, the juxtaposition of two CBF-1 sites in the human PTEN promoter is reminiscent of the two CBF-1 sites found in the murine HES-1 promoter, an extremely well characterized target of Notch regulation.40 In contrast, no CBF-1 binding sites were identified in the reported human FAK promoter.42 PTEN promoter activity is increased in the presence of constitutively active Notch-1. The PTEN promoter encompassing the two CBF-1 binding sites was cloned 5' of the luciferase gene into the PGL2-Basic (Promega) reporter vector (PTEN-LR). Parental 293 cells, 293/MIGR1 and 293/ Notch-1 cells were transfected with PTEN-LR, cultured for 24 hours without serum and subsequently analyzed for luciferase expression. Cells were cotransfected with a β-galactosidase expressing plasmid as a normalization control for transfection efficiency. Increased luciferase expression was observed in 293/Notch-1 cells as compared with parental 239 cells or 293/ MIGR1 cells under conditions of serum deprivation (Fig. 7B). The difference between the 293/MigR control cells and the 293/Notch-1 cells was found to be statistically significant with a p value of 0.009. The luciferase values between parental 293 cells and 293/Notch-1 cells fell just outside of statistical significance with a p value of 0.061. These results are consistent with our findings in Figure 5, that PTEN protein levels are increased in 293 cells expressing constitutively active Notch under conditions of cellular stress


Figure 7. (A) The human PTEN promoter contains two CBF-1 binding sites. Illustration indicating the relative location of two CBF-1 binding site within the minimal human PTEN promoter. Figure is based on Genbank accession number AF067844 and the reported PTEN promoter.33 Figure is not drawn to scale. (B) The PTEN promoter exhibited increased activation in the presence of Notch-1 signaling. Parental 293 cells, 293/MIGR1 and 293/Notch-1 were cotransfected with a PTEN promoter luciferase reporter. Cells were cultured 24 hours post-transfection in the absence of serum, and then analyzed for luciferase expression. Luciferase expression is reported as fold expression over that seen with the pGL2-Basic reporter that has no promoter and thus is a measure of background luciferase expression. 293/Notch-1 cells (Notch-1) that bear constitutively active Notch-1 exhibited more PTEN promoter activity as compared with the parental 293 cell line (293) or the 293/MIGR1 (MIGR1) vector control cell line. Data is shown for at least three independent experiments. ** denotes statistical significance (p = 0.009).

(e.g.: serum deprivation). Moreover, these results indicate that the PTEN promoter may likely be a direct target of Notch-1 signaling through the CBF-1 transcription factor.

DISCUSSION Notch signaling has been linked to oncogenesis.12 A constitutive Notch signal is sufficient to cause T-lineage oncogenesis and more recent studies have demonstrated that constitutive expression of any component of the direct Notch signaling cascade (e.g.: CBF-1, HES-1, MAML-1) will result in T-lineage leukemogenesis.43-44 However, the oncogenic properties of Notch appear to be extremely cell type specific.12 In pancreatic cells Notch is thought to be oncogenic only in the presence of activated Ras,23,45 but in epithelial cells Notch is thought to act as a tumor suppressor.14 PTEN plays a central role as a tumor suppressor and is best characterized for its role as a lipid phosphatase that acts to oppose activation of the PI3K/Akt pathway.30-32 However, there is emerging evidence that PTEN also functions as a protein phosphatase and may regulate other pathways such as FAK and Ras/Raf/ MEK/ERK.30 Thus, PTEN is increasingly being viewed as a master tumor suppressor gene that can regulate the activation/deactivation of multiple signaling pathways associated with oncogenic progression. Regulation of normal PTEN expression and function is not well characterized. In the present work we provide evidence that PTEN protein expression is regulated through the Notch-1 signaling pathway. Overexpression of human Notch-1 IC in 293 cells resulted in an

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increase of total and phosphorylated PTEN protein (Fig. 5) and a corresponding decrease in phosphorylated Akt (Fig. 6). Two CBF-1 binding sites were identified within the sequence of the human PTEN minimal promoter and an increase in PTEN promoter reporter activity was observed in 293 cells bearing constitutively active Notch-1 (Fig. 7). These observations support the hypothesis the PTEN promoter may be a direct target for CBF-1 mediated Notch-1 signaling. However, other mechanisms such as Notch mediated regulation of PTEN phosphorylation and ubiquitination governing PTEN protein stability may also be involved. The Notch induced effects on PTEN expression were associated with an altered morphology of 293 cells, decreased FAK expression and activation of ERK-1,-2. These present data do not indicate if these effects are due to a common cascade initiated by Notch-1 signaling effects on PTEN protein expression or if they are independent events that result from activated Notch-1. The effects of constitutive Notch-1 signaling on PTEN protein expression were observed under conditions of cell stress (e.g.: serum deprivation). This suggests that other factors activated by cell stress may cooperate with Notch-1 signals to regulate PTEN expression. One potential candidate may be p53, which has several potential binding sites in the PTEN promoter33 and which was activated in the serum deprived cultures (Bertrand, unpublished observations). Notch-1 expression and that of Notch ligands is often increased in various oncogenic settings.12 Its expression is increased in B-cell acute leukemia as compared with normal human bone marrow B-lineage precursors.46 Increased Notch expression is also observed in cervical, breast and pancreatic cancers.12 P53 is mutated in many cancers and wild type p53 is activated by the action of various chemotherapeutic drugs.47 Understanding how Notch signaling may promote PTEN expression in the context of chemotherapeutic induced cell stress may be particularly relevant in defining pathways and molecular interactions that promote increased drug susceptibility in tumors. It has been proposed that endogenous Notch-1 and Ras interact as part of a feed-back mechanism.45 Because PTEN functions upstream of Ras in the regulation of MAPK signaling, our novel finding that Notch-1 can regulate PTEN is consistent with these studies. Pharmacologic agents that inhibit Notch signaling have been developed48 and our data presented herein further valid the Notch signaling pathway as a potential therapeutic target in cancer. References 1. Mumm J, Kopan R. Notch signaling: From the outside in. Dev Biol 2000; 228:151-65. 2. Allman D, Aster J, Pear W. Notch signaling in hematopoiesis and early lymphocyte development. Immunol Rev 2002; 187:75-86. 3. Logeat F, Bessia C, Brou C, LeBail O, Jarriault S, Seidah NG, Israel Alain. The Notch1 receptor is cleaved constitutively by a furin-like convertase. Proc Natl Acad Sci USA 1998; 95:8108-12. 4. Mumm JS, Schroeter EH, Saxena MT, Griesemer A, Tain X, Pan DJ, Ray WJ, Kopan R. A ligand induced-extracellular cleavage regulates γ-secretase-like proteolytic activation of Notch1. Mol Cell 2000; 5:207-16. 5. Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR, Cumano A, Roux P, Black RA, Israel A. A novel proteolytic cleavage involved in Notch signaling: The role of the disintegrin-metalloprotease TACE. Mol Cell 2000; 5:207-16. 6. Struhl G, Adachi A. Nuclear access and activation of Notch in vivo. Cell 1998; 93:649-60. 7. Furukawa T, Maruyama S, Kawiachi M, Honjo T. The drosophila homolog of the immunoglobulin recombination signal-binding protein regulates peripheral nervous system develoment. Cell 1992; 69:1191-7. 8. Kao HY, Ordentlich P, Koyano-Nakagawa N, Tang Z, Downes M, Kintner C, Evans R, Kadesch T. A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev 1998; 12:2269-77. 9. Wu L, Griffin JD. Modulation of Notch signaling by mastermind-like (MAML) transcriptional coactivators and their involvement in tumorigensis. Semin Cancer Biol 2004; 14:248-56.

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10. Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD, Sklar J. TAN-1, the homolog of the Drosophila notch gene, is broken by chromosomal translocation in T lymphoblastic neoplasms. Cell 1991; 66:649-61. 11. Weng AP, Ferrando AA, Lee W, Morris IVth JP, Silverman LB, Sanchez-Irizarry C, Blacklow SC, Look AT, Aster JC. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 2004; 30:269-71. 12. Weng AP, Aster JC. Multiple niches for Notch in cancer: Context is everything. Curr Opin Genet Dev 2004; 14:48-54. 13. Politi K, Feirt N, Kitajewski J. Notch in mammary gland development and breast cancer. Semin Cancer Biol 2004; 14:341-47. 14. Nicolas M, Wolfer A, Raj K, Kummer JA, Mill P, Noort M, Hui C, Clevers H, Dotto GP, Radtke F. Notch1 functions as a tumor suppressor in mouse skin. Nature Gen 2003; 33:416-21. 15. Cheng P, Zlobin A, Volgina V, Gottipati S, Osborne B, Simel EJ, Miele L, Gabrilovich DI. Notch-1 regulates NF-κB Activity in hemopoietic progenitor cells. J Immunol 2001; 167:4458-67. 16. Wang J, Shelly L, Miele L, Boykins R, Norcross MA, Guan E. Human Notch-1 inhibits NF-κB activity in the nucleus through a direct interaction involving a novel domain. J Immunol 2001; 167:289-95. 17. Hadassah S, Krishna S, Sarin A. The anti-apoptotic effect of Notch-1 requires p56lck-dependent Akt/PKB-mediated signaling in T cells. J Biol Chem 2004; 279:2937-44. 18. Nair P, Somasundaram K, Krishna S. Activated Notch1 inhibits p53-induced apoptosis and sustains transformation by human papillomavirus type 16 E6 and E7 oncogenes through a PI3K-PKB/Akt-dependent pathway. J Virol 2003; 77:7106-12. 19. Berset T, Hoier EF, Battu G, Canevascini S, Hajnal A. Notch inhibition of RAS signaling through MAP kinase phosphatase LIP-1 during C. elegans vulval development. Science 2001; 291:1055-8. 20. Tomlinson A, Struhl G. 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