In breast cancer, the HER2 (human epidermal growth factor receptor 2) receptor tyrosine kinase is associated with extremely poor prognosis and survival. Notch signalling has a key role in cell-fate decisions, especially in cancer-initiating cells. The Notch intracellular domain produced by Notch cleavage is translocated to the nucleus where it activates transcription of target genes. To determine the combinatory effect of HER2 and Notch signalling in breast cancer, we investigated the effect of HER2 on Notch-induced cellular phenomena. We found the down-regulation of Notch-dependent transcriptional activity by HER2 overexpression. Also, the HER2/ERK (extracellular-signal-regulated kinase) signal pathway down-regulated the activity of γ-secretase. When we examined the protein level of Notch target genes in HER2-overexpressing cells, we observed that the level of survivin, downstream of Notch, increased in HER2 cells. We found that activation of ERK resulted in a decrease in XAF1 [XIAP (X-linked inhibitor of apoptosis)-associated factor 1] which reduced the formation of the XIAP–XAF1 E3 ligase complex to ubiquitinate survivin. In addition, Thr34 of survivin was shown to be the most important residue in determining survivin stability upon phosphorylation after HER2/Akt/CDK1 (cyclin-dependent kinase 1)–cyclin B1 signalling. The results of the present study show the combinatorial effects of HER2 and Notch during breast oncogenesis.

INTRODUCTION

Proteins in the IAP (inhibitor of apoptosis protein) family are vital in the blockade of apoptosis [1]. Survivin is the smallest IAP (16.5 kDa) containing a single baculovirus IAP-repeat domain [2]. Survivin down-regulates the activity of caspases 3/7/9 and survivin overexpression induces resistance to apoptosis caused by various cell death stimuli [3]. Significantly, survivin expression is up-regulated in most human cancers, and associated with aggressive tumours and poor clinical prognosis [4,5].

HER2 {human EGFR [EGF (epidermal growth factor) receptor] 2}, which belongs to the EGFR family, is associated with aggressive tumours and poor clinical prognosis, especially in breast cancer patients [6]. HER2 activates several intracellular signalling pathways including the PI3K (phosphoinositide 3-kinase)/Akt pathway and the ERK (extracellular-signal-regulated kinase) pathway [7]. Both pathways are significantly associated with cell proliferation, migration, invasion and angiogenesis [8]. The HER2 receptor is reported to up-regulate survivin expression through the PI3K/Akt or ERK pathway [9,10].

Notch is a single-transmembrane domain protein that regulates cell-fate decisions, proliferation and survival [11]. Upon ligand (Jagged1/2 and Delta-like1/3/4) binding, proteolytic cleavage of Notch occurs by the γ-secretase complex, resulting in release of the NICD (Notch-intracellular domain) [12]. NICD translocates to the nucleus and activates the transcription of target genes, including genes in the Hes and Hey familes and survivin [11,13,14]. Inhibition of HER2 kinase activity by herceptin up-regulates Notch-dependent transcriptional activity in breast cancer cells [15].

In the present study, to investigate the paradox of HER2 up-regulation of the Notch target gene survivin while inhibiting Notch transcriptional activity, we examined mRNA and protein levels of survivin during co-expression of HER2 and Notch1. We observed that, in HER2-overexpressing cells, the level of survivin protein downstream of Notch increased, but survivin (HGNC approved symbol BIRC5) mRNA decreased. We demonstrated that HER2 downstream signalling enhanced the stability of survivin, whereas transcription of the survivin gene decreased by inhibition of Notch-dependent transcription by HER2.

MATERIALS AND METHODS

Cell lines

MCF7 vec, MCF7 HER2, BT474, SKBR3, MDA-MB-231 vec and MDA-MB-231 Akt1 human breast cancer cells and HEK (human embryonic kideny)-293T, Cos7 and HeLa cells were grown and routinely maintained in DMEM (Dulbecco's modified Eagle's medium; Thermo Scientific HyClone) supplemented with 10% FBS (fetal bovine serum), 100 units/ml penicillin and 100 mg/ml streptomycin (all Thermo Scientific HyClone). MCF10A vec and MCF10A HER2 normal mammary epithelial cells were cultured in DMEM/ Ham's F12 (1:1) medium (Invitrogen) containing 5% horse serum (Invitrogen), 20 ng/ml EGF (Peprotech), 0.5 mg/ml hydrocortisone (Sigma), 100 ng/ml cholera toxin (Sigma), 10 μg/ml insulin (Sigma), 100 units/ml penicillin and 100 mg/ml streptomycin. Cells were incubated at 37°C with 5% CO2 and 95% humidified air. HER2- and Akt1-overexpressing cells were generated as described previously [1618]. Survivin-silenced cells were generated by transfection with a survivin shRNA (small hairpin RNA) by Lipofectamine™ 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions.

Antibodies and reagents

Growth factors were EGF and heregulin (Peprotech). Kinase inhibitors, antibodies and reagents were U0126, LY294002, wortmannin, Akt inhibitor VIII, rapamycin, rottlerin and JAK3 (Janus kinase 3) inhibitor II (Calbiochem); lapatinib (LC Laboratories); stattic, SB203580 and SP600125 (Tocris Bioscience); herceptin (Roche); gefitinib (Biaffin); antibodies against Notch1, Hes1, Hes5, p-survivin (p- indicates phosphorylated protein), survivin, p-ERK, ERK, ubiquitin, XAF1 [XIAP (X-inhibitor of apoptosis)-associated factor 1], p-CDK1 (cyclin-dependent kinase 1), CDK1, p-p21, p21, actin and tubulin, and survivin [a pool of three target-specific siRNAs (small interfering RNAs)] and control siRNA (Santa Cruz Biotechnology); antibodies against p-HER2, p-Akt, Akt, p-p38 MAPK (mitogen-activated protein kinase), p38 MAPK, p-mTOR (mammalian target of rapamycin), mTOR, nicastrin, PEN2 (presenilin enhancer 2), presenilin1, presenilin2, c-Jun, XIAP, Bcl-Xl and caspase 3 (Cell Signaling Technology); antibodies against p-EGFR, EGFR, p-HER3, HER3, p-STAT3, STAT3, p-JNK (c-Jun N-terminal kinase) and JNK (Epitomics,); anti-NICD and anti-Hey1 antibodies (Abcam); Cy3 (indocarbocyanine)-conjugated anti-mouse IgG antibody (Zymed); anti-PARP [poly(ADP-ribose) polymerase] antibody (BD Pharmingen); and anti-HER2 antibody (Lab Vision).

Plasmids

The 4× CSL (C promoter-binding factor/suppressor of hairless/Lag-1)-Luc (luciferase) and 6× NRE (Notch-response element)-Luc vectors have been described previously [19]. HER2-WT (wild type), HER2-CA (constitutively active) and HER2-KD (kinase dead) were from Addgene. Akt1-CA, Akt1-KD, MEK1 (MAPK/ERK kinase 1)-CA and MEK2-CA have been described previously [20,21]. ERK1-WT, ERK2-WT, p38 MAPK-WT and p38 MAPK-KD were provided by Dr Su-Jae Lee (Hanyang University, Seoul, Korea). NotchΔE-GVP and UAS-Luc were provided by Dr Inhee Mook-Jung (Seoul National University, Seoul, Korea). Survivin-WT, XAF1, XIAP-WT, XIAP-C450A and CDK1 were provided by Dr Yong-Keun Jung (Seoul National University, Seoul, Korea). pSuper-survivin-shRNA was provided by Dr Hyunggee Kim (Korea University, Seoul, Korea). A plasmid containing human cyclin B1 was made by cloning the gene by PCR from HEK-293T cells with the primers 5′-ATAAAGCTTATGGCGCTCCGAGTCAC-3′ (forward) and 5′-CACTCGAGACCTTTGCCACAGC-3′ (reverse) and cloning into the pcDNA3.1 ver.B-Myc-His vector (Invitrogen) using HindIII and XhoI enzymes. Using the survivin-WT construct as a backbone, survivin mutants were produced using a QuikChange® kit (Stratagene) with the following mutagenic primers: T34A, 5′-GGGCTGCGCCTGCGCCCCGGAGCGGATG-3′ (forward) and 5′-CATCCGCTCCGGGGCGCAGGCGCAGCCC-3′ (reverse); D53A, 5′-GAGAACGAGCCAGCCTTGGCCCAGTGTTTC-3′ (forward) and 5′-GAAACACTGGGCCAAGGCTGGCTCGTTCTC-3′ (reverse); H77A, 5′-GACCCCATAGAGGAAGCTAAAAAGCATTCG-3′ (forward) and 5′-CGAATGCTTTTTAGCTTCCTCTATGGGGTC-3′ (reverse); and C84A, 5′-ATTCGTCCGGTGCCGCTTTCCTTTCTG-3′ (forward) and 5′-CAGAAAGGAAAGCGGCACCGGACGAAT-3′ (reverse).

Tumour tissue collection

A total of 18 primary invasive breast cancer tissues from different individuals were used to evaluate the expression of survivin and HER2 in human breast cancer. Tissue specimens were randomly selected from the tissue archives of the Cancer Research Institute, Seoul National University. All tumours were excised between 2004 and 2006 and were histopathologically confirmed. Informed consent was obtained for all participants before operation and the study protocol was approved by the Institutional Review Board of Seoul National University Hospital (H-0512-502-163).

Proliferation assay

For MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assays, cells were seeded in 96-well culture plates at 5×103 cells/well, exposed to the antibodies indicated and incubated for 72 h. Cell viability was assessed by adding 20 μl of 10 mg/ml MTT (Sigma) to 100 μl of culture medium, and incubating for 3 h at 37°C in a CO2 incubator. Medium was removed, formazan was dissolved in DMSO (Sigma), and absorbance was measured at 590 nm using a Multiskan EX (Thermo Scientific).

Cell-cycle analyses by flow cytometry

Cells were harvested with 0.25% trypsin and washed once with PBS. After centrifugation (600 g for 5 min at 4°C), cells were fixed in 100% ice-cold methanol overnight at −20°C. Fixed cells were incubated with 50 μg/ml propidium iodide in PBS and 1 mg/ml RNase in PBS for 30 min. Cell-cycle analyses were performed on a BD FACSCalibur flow cytometer (BD Biosciences).

Western blot analysis

Cells were lysed in lysis buffer [50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM sodium orthovanadate and protease inhibitor cocktail (Roche)]. Samples were separated by SDS/PAGE (8, 10 or 12% gels) and transferred on to nitrocellulose membranes (Whatman). After blocking with 5% skimmed milk in TBS-T [Tris buffered saline (50 mM Tris/HCl and 150 mM NaCl, pH 7.6) containing 0.05% Tween 20], membranes were incubated with appropriate primary antibodies overnight, followed by 2 h incubation with HRP (horseradish peroxidase)-conjugated secondary antibodies. Protein bands were visualized with a WEST ZOL plus System (iNtRON).

Immunoprecipitation

Cell lysates (1 mg) were precleared by adding 30 μl of Protein A– or Protein G–Sepharose (Invitrogen) for 2 h at 4°C. After centrifugation at 1000 g, the supernatant was incubated with either primary antibodies or normal rabbit/mouse IgG overnight at 4°C. Protein A– or Protein G–Sepharose (30 μl) was added and incubated for 3 h at 4°C. After centrifugation, the pellet was washed three times with cell lysis buffer. Immunoprecipitated proteins were resolved by SDS/PAGE (10% gels) and subsequently analysed by Western blotting.

Immunocytochemistry

Cells were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 15 min. Fixed samples were blocked with 3% skimmed milk in PBS for 1 h, followed by incubation with primary antibody diluted in 1% skimmed milk in PBS for 1 h. After washing with PBS, samples were treated with the Cy3-conjugated anti-rabbit IgG. For DNA staining, samples were incubated with Hoechst 33342 (1 μg/ml) for an additional 10 min. Immunofluorescence was monitored with an Olympus upright fluorescence microscope (BX50F).

Dual-luciferase assays

Cells in 12-well plates were transfected with 0.2 μg of reporter constructs and 0.002 μg of pCMV-Rl as an internal control by using Lipofectamine™ 2000. At 48 h post-transfection, dual-luciferase assays were performed according to the manufacturer's protocol (Promega).

Cell fractionation

Cells were washed with ice-cold PBS, harvested in cytoplasmic extraction buffer [10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT (dithiothreitol) and 0.5 mM PMSF] and agitated for 10 min at 4°C. After the addition of Nonidet P40 (final concentration 0.5%), the samples were agitated for 10 min at 4°C. Samples were centrifuged at 16000 g in a microcentrifuge (Eppendorf) for 5 min. The supernatant was collected as the cytosolic fraction. Nuclear pellets were washed twice with ice-cold PBS and resuspended in a nuclear extraction buffer [20 mM Hepes (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF]. Nuclear extracts were agitated for 10 min at 4°C and centrifuged at 16000 g at 4°C and supernatants were collected as the nuclear fraction.

RT (reverse transcription)–PCR

Expression of transcripts was assessed using the following primers: Notch1, 5′-GCCGCCTTTGTGCTTCTGTTC-3′ (forward) and 5′-CCGGTGGTCTGTCTGGTCGTC-3′ (reverse); Hes1, 5′-CGGCATTCCAAGCTGGAGAAGG-3′ (forward) and 5′-GGAAGCGGGTCACCTCGTTCATG-3′ (reverse); Hes5, 5′-CGCATCAACAGCAGCATC-3′ (forward) and 5′-TGGAAGTGGTACAGCAGC-3′ (reverse); Hey1, 5′-AGAGTGCGGACGAGAATGGA-3′ (forward) and 5′-GGGAAGCGTAGTTGTTGAGA-3′ (reverse); and survivin, 5′-CCGACGTTGCCCCCTGC-3′ (forward) and 5′-TCGATGGCACGGCGCAC-3′ (reverse). Quantification of actin expression was performed as an internal control using an RT–PCR primer and control set (Invitrogen).

Statistic analyses

All experiments were repeated at least three times independently. Data were presented as mean±S.D. Comparison of results from treated compared with control cells was performed using Student's t tests. P<0.05 was considered statistically significant.

RESULTS

Survivin up-regulates cell survival in HER2-overexpressing cells

We compared the proliferation of cells in which survivin was silenced by shRNA transfection and HER2 was overexpressed (MCF7 HER2 survivin-shRNA, BT474 survivin-shRNA and SKBR3 survivin-shRNA cells) with control cells. Western blot analysis confirmed the silencing of survivin in HER2-overexpressing cells (Figure 1A). Silencing of survivin did not significantly affect cell proliferation in HER2-overexpressing human breast cancer cells (Figure 1B). To determine the effect of silencing survivin on drug sensitivity of human breast cancer cells, cells were treated with doxorubicin, which induces apoptosis by damaging DNA. Treatment with doxorubicin resulted in massive cell death of survivin-silenced cells, whereas induction of cell death in control cells was significantly attenuated in a dose- and time-dependent manner compared with survivin-silenced cells (Figures 1C and 1D). Using flow cytometry for cell-cycle analyses, we confirmed that the sub-G1 apoptotic fraction was significantly increased in survivin-silenced cells after doxorubicin treatment, whereas control cells showed a less prominent increase in the apoptotic fraction (Figure 1E). These results suggested that silencing of survivin enhanced drug-induced cell death by increased apoptosis.

Survivin increases cell survival in HER2-overexpressing cells

Figure 1
Survivin increases cell survival in HER2-overexpressing cells

(A) Silencing of survivin was comfirmed by Western blot analysis using an anti-survivin antibody. (B) Cells were seeded at 5×103 cells per well in 96-well dishes and analysed using MTT assays for 5 days. (C) Cells were seeded at 2×104 cells per well in 96-well dishes and analysed using MTT assays for 5 days. At 24 h after cell seeding, doxorubicin was added to 0.5 μM. (D) Cells were seeded at 2×104 cells per well in 96-well dishes and treated with various concentration of doxorubicin (0, 0.1, 0.2, 0.5 and 1 μM) for 48 h and subjected to MTT assays. (E) Doxorubicin was added at 0.5 μM for 48 h. Cells were fixed in methanol and incubated in 50 μg/ml propodium iodide and 1 mg/ml RNase. Propodium-iodide-labelled nuclei were analysed by flow cytometry. Histograms represent DNA from three independent experiments. (F) MCF10A HER2, MCF7 HER2, BT474 or SKBR3 cells were transfected with survivin siRNA for 48 h and analysed by MTT assay and Western blot analysis with the antibodies indicated. Doxorubicin was added at 1 μM for 24 h. Cont, control; Doxo, doxorubicin. *P<0.05, **P<0.005 and ***P<0.0005.

Figure 1
Survivin increases cell survival in HER2-overexpressing cells

(A) Silencing of survivin was comfirmed by Western blot analysis using an anti-survivin antibody. (B) Cells were seeded at 5×103 cells per well in 96-well dishes and analysed using MTT assays for 5 days. (C) Cells were seeded at 2×104 cells per well in 96-well dishes and analysed using MTT assays for 5 days. At 24 h after cell seeding, doxorubicin was added to 0.5 μM. (D) Cells were seeded at 2×104 cells per well in 96-well dishes and treated with various concentration of doxorubicin (0, 0.1, 0.2, 0.5 and 1 μM) for 48 h and subjected to MTT assays. (E) Doxorubicin was added at 0.5 μM for 48 h. Cells were fixed in methanol and incubated in 50 μg/ml propodium iodide and 1 mg/ml RNase. Propodium-iodide-labelled nuclei were analysed by flow cytometry. Histograms represent DNA from three independent experiments. (F) MCF10A HER2, MCF7 HER2, BT474 or SKBR3 cells were transfected with survivin siRNA for 48 h and analysed by MTT assay and Western blot analysis with the antibodies indicated. Doxorubicin was added at 1 μM for 24 h. Cont, control; Doxo, doxorubicin. *P<0.05, **P<0.005 and ***P<0.0005.

To investigate further the effect of silencing survivin on drug sensitivity, cells were transfected with survivin siRNA to transiently suppress survivin expression. Similar to the results obtained with cell lines silenced with the shRNA retroviral vector, cells in which survivin was silenced by siRNA transfection were more sensitive to doxorubicin than control cells (Figure 1F). We observed effective down-regulation of survivin and Bcl-xl (an anti-apoptotic protein), and enhanced proteolytic cleavage of caspase 3 and PARP (apoptosis markers) after doxorubicin treatment of survivin-silenced cells compared with control cells. These results indicated that silencing of survivin might confer sensitivity to DNA-damage-induced apoptosis by regulating apoptosis-related intracellular effector molecules in HER2-overexpressing cells.

HER2 is coupled with survivin expression

To determine the effect of HER2 expression on survivin expression, we measured the level of survivin in MCF10A vec, MCF10A HER2, MCF7 vec and MCF7 HER2 cells by Western blot analysis. We found that survivin levels were up-regulated in HER2-overexpressing cells compared with control cells (Figures 2A and 2B). To determine the effect of HER2 activity on survivin expression, EGFR/HER2/HER3-positive cells (MCF10A HER2 and SKBR3 cells) were treated with EGF, an EGFR agonist [22], or heregulin, an HER3 agonist [23]. HER2/HER3-positive cells (MCF7 HER2 and BT474 cells) were treated with heregulin. Expression levels of HER family members are shown in Supplementary Figure S1 (at http://www.biochemj.org/bj/451/bj4510123add.htm). We found that survivin expression was also up-regulated by the increase in HER2 kinase activity upon agonist stimulation (Figures 2A and 2B). To confirm the effect of HER2 activity on survivin expression, cells were treated with herceptin, lapatinib or gefitinib to inhibit HER2 kinase activity. All inhibitor-treated cells exhibited decreased survivin expression compared with control cells (Figure 2C and Supplementary Figure S2A at http://www.biochemj.org/bj/451/bj4510123add.htm). We confimed these results in vivo using a mouse model that revealed HER2/neu in mammary glands. Western blot analysis revealed that survivin was up-regulated in the mammary gland of MMTV (mouse mammary tumour virus)-HER2/neu mice compared with their wild-type littermates (Figure 2D). HER2 levels in human breast tumour tissue samples were also strongly correlated with survivin levels (Figure 2E). These results indicated that HER2 increased survivin expression.

HER2 activity is coupled to survivin expression

Figure 2
HER2 activity is coupled to survivin expression

(A) MCF10A vec, MCF10A HER2 and SKBR3 cells were treated with EGF (10 ng/ml) for 24 h and subjected to Western blot analysis with the antibodies indicated. (B) MCF10A vec, MCF10A HER2, MCF7 vec, MCF7 HER2, BT474 or SKBR3 cells were treated with heregulin (50 ng/ml) for 24 h and subjected to Western blot analysis with the antibodies indicated. (C) MCF10A HER2, MCF7 HER2, BT474 and SKBR3 cells were treated with herceptin (20 μg/ml), lapatinib (1 μM) or gefitinib (10 μM) for 24 h and subjected to Western blot analysis with the antibodies indicated. (D) Total tissue lysates were prepared from MMTV-HER2/neu transgenic mice (n=5) and their WT littermates (n=4, 10 weeks) mammary gland tissues. Samples were resolved by SDS/PAGE and subjected to Western blot analysis with the antibodies indicated. (E) Western blot analysis showing expression levels of HER2 and survivin protein in primary tumour lysates from breast cancer patients (n=18). Actin was used to verify equal loading. Cont, control; Gefi, gefitinib; Herc, herceptin; HRG, heregulin; Lapa, lapatinib. *P<0.05.

Figure 2
HER2 activity is coupled to survivin expression

(A) MCF10A vec, MCF10A HER2 and SKBR3 cells were treated with EGF (10 ng/ml) for 24 h and subjected to Western blot analysis with the antibodies indicated. (B) MCF10A vec, MCF10A HER2, MCF7 vec, MCF7 HER2, BT474 or SKBR3 cells were treated with heregulin (50 ng/ml) for 24 h and subjected to Western blot analysis with the antibodies indicated. (C) MCF10A HER2, MCF7 HER2, BT474 and SKBR3 cells were treated with herceptin (20 μg/ml), lapatinib (1 μM) or gefitinib (10 μM) for 24 h and subjected to Western blot analysis with the antibodies indicated. (D) Total tissue lysates were prepared from MMTV-HER2/neu transgenic mice (n=5) and their WT littermates (n=4, 10 weeks) mammary gland tissues. Samples were resolved by SDS/PAGE and subjected to Western blot analysis with the antibodies indicated. (E) Western blot analysis showing expression levels of HER2 and survivin protein in primary tumour lysates from breast cancer patients (n=18). Actin was used to verify equal loading. Cont, control; Gefi, gefitinib; Herc, herceptin; HRG, heregulin; Lapa, lapatinib. *P<0.05.

HER2 down-regulates NICD-dependent transcriptional activity

To confirm the effect of HER2 on Notch1-dependent transcriptional activity, reporter assays were performed in MCF10A vec, MCF10A HER2, MCF7 vec, MCF7 HER2, HEK-293T, HeLa, Cos7, BT474 and SKBR3 cells using luciferase reporter constructs: 4× CSL-Luc with four copies of CBF1, suppressor of hairless and Lag-1-binding sites [24] and 6× NRE-Luc with six NRE sites [25]. In MCF10A and MCF7 cells, Notch1-dependent transcriptional activity was down-regulated by HER2 overexpression using two different reporter constructs (Figure 3A). Transient transfection experiments using HER2-KD and HER2-CA revealed that the kinase activity of HER2 was essential for down-regulation of Notch1-dependent transcriptional activity in HEK-293T, HeLa and Cos7 cells (Figure 3B). The decrease in Notch1-dependent transcriptional activity by EGF or heregulin, and the increase by herceptin, lapatinib or gefitinib revealed an inverse correlation between the activity of the HER2 tyrosine kinase and Notch1-dependent transcription activity in MCF10A HER2, MCF7 HER2, BT474 and SKBR3 cells (Figures 3C–3E).

HER2 down-regulates NICD-dependent transcription

Figure 3
HER2 down-regulates NICD-dependent transcription

(A) MCF10A vec, MCF10A HER2, MCF7 vec or MCF7 HER2 cells were transfected with 4× CSL or 6× NRE luciferase reporter constructs, harvested after 48 h and analysed by dual-luciferase assay. (B) HEK-293T, HeLa and Cos7 cells were transfected with pcDNA, HER2-WT, HER2-CA or HER2-KD and 4× CSL or 6× NRE luciferase reporter constructs, harvested after 48 h and analysed by dual-luciferase assay. (CE) MCF10A HER2, MCF7 HER2, BT474 and SKBR3 cells were transfected with 4× CSL or 6× NRE luciferase reporter constructs for 48 h, treated with EGF (10 ng/ml), heregulin (50 ng/ml), herceptin (20 μg/ml), lapatinib (1 μM) or gefitinib (10 μM) at 24 h post-transfection for 24 h and subjected to dual-luciferase assays. (F) MCF10A vec, MCF10A HER2, MCF7 vec, MCF7 HER2, BT474 and SKBR3 cells were treated with heregulin (50 ng/ml) for 24 h and analysed by RT–PCR using specific primers. MCF10A HER2, MCF7 HER2, BT474 and SKBR3 cells were treated with herceptin (20 μg/ml), lapatinib (1 μM) or gefitinib (10 μM) for 24 h and analysed by RT–PCR using specific primers. Cont, control; Gefi, gefitinib; Herc, herceptin; HRG, heregulin; Lapa, lapatinib. *P<0.05, **P<0.005 and ***P<0.0005.

Figure 3
HER2 down-regulates NICD-dependent transcription

(A) MCF10A vec, MCF10A HER2, MCF7 vec or MCF7 HER2 cells were transfected with 4× CSL or 6× NRE luciferase reporter constructs, harvested after 48 h and analysed by dual-luciferase assay. (B) HEK-293T, HeLa and Cos7 cells were transfected with pcDNA, HER2-WT, HER2-CA or HER2-KD and 4× CSL or 6× NRE luciferase reporter constructs, harvested after 48 h and analysed by dual-luciferase assay. (CE) MCF10A HER2, MCF7 HER2, BT474 and SKBR3 cells were transfected with 4× CSL or 6× NRE luciferase reporter constructs for 48 h, treated with EGF (10 ng/ml), heregulin (50 ng/ml), herceptin (20 μg/ml), lapatinib (1 μM) or gefitinib (10 μM) at 24 h post-transfection for 24 h and subjected to dual-luciferase assays. (F) MCF10A vec, MCF10A HER2, MCF7 vec, MCF7 HER2, BT474 and SKBR3 cells were treated with heregulin (50 ng/ml) for 24 h and analysed by RT–PCR using specific primers. MCF10A HER2, MCF7 HER2, BT474 and SKBR3 cells were treated with herceptin (20 μg/ml), lapatinib (1 μM) or gefitinib (10 μM) for 24 h and analysed by RT–PCR using specific primers. Cont, control; Gefi, gefitinib; Herc, herceptin; HRG, heregulin; Lapa, lapatinib. *P<0.05, **P<0.005 and ***P<0.0005.

To determine the effect of HER2 on mRNA and protein levels of Notch1-target genes, we treated MCF10A vec, MCF10A HER2, MCF7 vec, MCF7 HER2, BT474 and SKBR3 cells with EGF, heregulin, herceptin, lapatinib or gefitinib and examined the levels of Notch1-target gene product by RT–PCR and Western blotting. EGF and heregulin decreased mRNA and protein levels of three Notch1-target genes, Hes1, Hes5 and Hey1 (Figures 2A, 2B and 3F). In the same manner, herceptin, lapatinib and gefitinib increased the mRNA and protein levels of Hes1, Hes5 and Hey1 (Figures 2C and 3F). Although survivin is a Notch1-target gene, the protein level of survivin was inversely proportional to its mRNA level (Figures 2 and 3F, and Supplementary Figure S2B). Survivin protein levels were increased by HER2 kinase activity, whereas the mRNA levels of survivin were down-regulated by HER2. The expression of NICD decreased with EGF and heregulin, and increased with herceptin, lapatinib and gefitinib, similar to the protein levels of Notch1-target genes, except for survivin (Figure 2). However, the total mRNA and protein levels of Notch1 were not changed by HER2 kinase activity (Figures 2 and 3F). These results indicated that HER2 activity specifically down-regulated Notch1-dependent transcriptional activity, but not Notch1 gene expression.

ERK and Akt signalling up-regulate the survivin level with concomitant reduction in transcriptional activity

Using protein kinase inhibitors [U0126 (MEK inhibitor), SP600125 (JNK inhibitor), SB203580 (p38 MAPK inhibitor), LY294002 and wortmannin (PI3K inhibitors), Akt inhibitor VIII (Akt inhibitor), rapamycin (mTOR inhibitor), rottlerin (protein kinase C -δ inhibitor), JAK3 I #II (JAK3 inhibitor) and stattic (STAT3 inhibitor)], we investigated whether HER2 downstream kinase activity was responsible for the down-regulation of Notch1-dependent transcription. Supplementary Figure S3 (at http://www.biochemj.org/bj/451/bj4510123add.htm) confirms the effects of the kinase inhibitors. RT–PCR revealed that blockade of ERK or Akt signals by U0126, LY294002, wortmannin or Akt inhibitor VIII increased mRNA and protein from the Notch1-target genes Hes1, Hes5 and Hey1 (Figure 4A), whereas the other inhibitors had no effect. However, only the protein level of survivin was decreased by blockade of ERK or Akt signalling (Figure 4A, bottom lanes 2, 5, 6 and 7 from the left).

ERK and Akt signalling increase survivin levels with concomitant reduction in survivin gene transcription

Figure 4
ERK and Akt signalling increase survivin levels with concomitant reduction in survivin gene transcription

(A) SKBR3 cells were treated with U0126 (10 μM), SP600125 (10 μM), SB203580 (10 μM), LY294002 (10 μM), wortmannin (1 μM), Akt inhibitor VIII (10 μM), rottlerin (10 μM), rapamycin (1 μM), JAK3 inhibitor II (10 μM) or stattic (10 μM) for 24 h and analysed by RT–PCR using specific primers or Western blotting with the antibodies indicated. (B) MCF10A vec, MCF10A HER2, MCF7 vec and MCF7 HER2 cells were transfected with NotchΔE-GVP and UAS-Luc luciferase reporter constructs, harvested after 48 h and analysed by dual-luciferase assay. (CE) MCF10A HER2, MCF7 HER2, BT474 or SKBR3 cells were transfected with NotchΔE-GVP and UAS-Luc luciferase reporter constructs for 48 h, treated with EGF (10 ng/ml), heregulin (50 ng/ml), herceptin (20 μg/ml), lapatinib (1 μM) or gefitinib (10 μM) at 24 h post-transfection for 24 h and subjected to dual-luciferase assays. (F) HEK-293T cells were transfected with the kinase constructs indicated, or NotchΔE-GVP and UAS-Luc luciferase reporter constructs, harvested after 48 h, and analysed by dual-luciferase assay. (G) MDA-MB-231 vec and Akt1 cells were analysed by Western blotting with the antibodies indicated. (H) MDA-MB-231 vec and Akt1 cells were immunostained using anti-NICD antibodies. (I) MDA-MB-231 vec and Akt1 cells were fractionated into cytoplasmic (C) and nuclear (N) fractions. Fractions were subjected to Western blot analysis with antibodies against NICD, c-Jun (as a nuclear marker) and actin (as a cytosol marker). (J) MDA-MB-231 vec or Akt1 cells were transfected with NotchΔE-GVP and UAS-Luc luciferase reporter constructs, harvested after 48 h and analysed by dual-luciferase assay. Cont, control; Gefi, gefitinib; Herc, herceptin; HRG, heregulin; Lapa, lapatinib. *P<0.05, **P<0.005 and ***P<0.0005.

Figure 4
ERK and Akt signalling increase survivin levels with concomitant reduction in survivin gene transcription

(A) SKBR3 cells were treated with U0126 (10 μM), SP600125 (10 μM), SB203580 (10 μM), LY294002 (10 μM), wortmannin (1 μM), Akt inhibitor VIII (10 μM), rottlerin (10 μM), rapamycin (1 μM), JAK3 inhibitor II (10 μM) or stattic (10 μM) for 24 h and analysed by RT–PCR using specific primers or Western blotting with the antibodies indicated. (B) MCF10A vec, MCF10A HER2, MCF7 vec and MCF7 HER2 cells were transfected with NotchΔE-GVP and UAS-Luc luciferase reporter constructs, harvested after 48 h and analysed by dual-luciferase assay. (CE) MCF10A HER2, MCF7 HER2, BT474 or SKBR3 cells were transfected with NotchΔE-GVP and UAS-Luc luciferase reporter constructs for 48 h, treated with EGF (10 ng/ml), heregulin (50 ng/ml), herceptin (20 μg/ml), lapatinib (1 μM) or gefitinib (10 μM) at 24 h post-transfection for 24 h and subjected to dual-luciferase assays. (F) HEK-293T cells were transfected with the kinase constructs indicated, or NotchΔE-GVP and UAS-Luc luciferase reporter constructs, harvested after 48 h, and analysed by dual-luciferase assay. (G) MDA-MB-231 vec and Akt1 cells were analysed by Western blotting with the antibodies indicated. (H) MDA-MB-231 vec and Akt1 cells were immunostained using anti-NICD antibodies. (I) MDA-MB-231 vec and Akt1 cells were fractionated into cytoplasmic (C) and nuclear (N) fractions. Fractions were subjected to Western blot analysis with antibodies against NICD, c-Jun (as a nuclear marker) and actin (as a cytosol marker). (J) MDA-MB-231 vec or Akt1 cells were transfected with NotchΔE-GVP and UAS-Luc luciferase reporter constructs, harvested after 48 h and analysed by dual-luciferase assay. Cont, control; Gefi, gefitinib; Herc, herceptin; HRG, heregulin; Lapa, lapatinib. *P<0.05, **P<0.005 and ***P<0.0005.

The activity of γ-secretase complex induced proteolytic cleavage of Notch, resulting in release of NICD [26], which translocates to the nucleus and activates transcription of target genes [27]. To determine whether HER2 and its downstream signalling decrease γ-secretase activity, reporter assays were performed in MCF10A vec, MCF10A HER2, MCF7 vec, MCF7 HER2, BT474, SKBR3 and HEK-293T cells using NotchΔE-GVP and UAS-Luc constructs [28]. In MCF10A and MCF7 HER2-overexpressing cells, γ-secretase activity was reduced (Figure 4B). The decrease in γ-secretase activity by EGF or heregulin, and the increase by herceptin, lapatinib or gefitinib correlated with the activity of HER2 tyrosine kinase and its downstream signals in MCF10A HER2, MCF7 HER2, BT474 and SKBR3 cells. These results indicated that HER2 activity downregulated γ-secretase activity (Figures 4C–4E). Experiments using HER2, Akt, MEK, ERK or p38 MAPK constructs and a transient transfection system revealed that the ERK pathway was essential for down-regulation of γ-secretase activity in HEK-293T cells (Figure 4F). Only the blockade of the ERK pathway by U0126 increased NICD expression in SKBR3 cells (Figure 4A, bottom lane 2 from the left). Inhibitors of protein kinases that do not affect expression of γ-secretase component complex proteins include nicastrin, PEN2, presenilin1 and presenilin2 [29] (Supplementary Figure S4 at http://www.biochemj.org/bj/451/bj4510123add.htm). These results suggested that the HER2/ERK pathway decreased the following intracellular cascade events: γ-secretase activity, proteolytic cleavage of Notch1, expression of NICD and activity of Notch1-dependent transcription.

As shown in Figures 4(A) and 4(F), Akt signalling did not seem to affect γ-secretase activity directly. We previously reported, using transient transfection systems, that Akt kinase activity down-regulated Notch1-dependent transcriptional activity by Akt-mediated phosphorylation of NICD and subsequent inhibition of proper nuclear translocation of NICD [19]. When levels of Notch1 downstream target proteins were assessed using MDA-MB-231 cells, which constitutively overexpress active Akt1 [18], survivin was up-regulated, but other Notch1-target proteins were down-regulated in Akt-overexpressing cells (Figure 4G). NICD subcellular localization appeared to be significantly different between Akt1 and control cells, as determined by both immunocytochemistry and cell fractionation. These results clearly showed that Akt1 inhibited proper nuclear translocation of NICD in MDA-MB-231 cells (Figures 4H and 4I). NICD expression and γ-secretase activity were not different between Akt1 and control cells (Figures 4G and 4J). We confirmed that Akt1 inhibited proper NICD nuclear localization using cell lines stably expressing Akt1, in agreement with our previous report using transiently transfected cells [19]. These results suggested that the HER2/Akt pathway inhibited nuclear translocation of NICD and subsequent down-regulation of Notch1-dependent transcriptional activity. The two HER2-downstream signalling pathways, ERK and Akt, operated independently of each other for down-regulation of Notch1-dependent transcriptional activity.

ERK and Akt signalling up-regulates survivin stability

We found that the HER2/ERK and HER2/Akt pathways down-regulated Notch1-dependent transcription, inhibiting expression of Notch1-target genes. However, although survivin mRNA decreased, survivin protein was up-regulated by the activity of HER2, ERK and Akt (Figures 24). We next evaluated the possible modulation of survivin protein stability by ERK and/or Akt. Survivin degradation is regulated by the ubiquitin–proteosome pathway in a cell-cycle-dependent manner [30]. Treatment with MG132, a 26S proteasome inhibitor, resulted in increased survivin protein levels as well as increased survivin ubiquitination (Figure 5A). Decreased survivin ubiquitination was evident in HER2- or Akt1-overexpressing cells, suggesting that ubiquitinated survivin was less actively processed in HER2- or Akt1-overexpressing cells.

HER2 downstream signalling increases survivin stability

Figure 5
HER2 downstream signalling increases survivin stability

(A) MCF10A vec, MCF10A HER2, MCF7 vec, MCF7 HER2, MDA-MB-231 vec and MDA-MB-231 Akt1 cells were treated with MG132 at 10 μM for 12 h and lysed. The supernatant was incubated with anti-survivin antibody and Protein A–Sepharose. Immunoprecipitates were analysed by Western blotting with the antibodies indicated. (B) SKBR3 cells were treated with EGF (10 ng/ml), heregulin (50 ng/ml), herceptin (20 μg/ml), lapatinib (1 μM), gefitinib (10 μM), Akt inbihitor VIII (10 μM) or U0126 (10 μM) for 24 h and lysed. The supernatant was incubated with antibodies against survivin, XIAP or XAF1 and Protein A–Sepharose. Immunoprecipitates and lysates were analysed by Western blotting with the antibodies indicated. (C) HEK-293T cells were transfected with plasmids encoding survivin-WT or T34A. At 48 h post-transfection, 20 μg/ml cycloheximide was added to block further protein synthesis. Cells were harvested at the time points indicated and subjected to Western blot analysis with antibodies against p-survivin or survivin. (D) HEK-293T cells were co-transfected with the constructs indicated. Lysates were analysed by Western blotting or co-immunoprecipitation with the antibodies indicated. Cont, control; Gefi, gefitinib; Herc, herceptin; HRG, heregulin; IB, immunoblot; IP, immunoprecipitation; Lapa, lapatinib; Ub, ubiquitin.

Figure 5
HER2 downstream signalling increases survivin stability

(A) MCF10A vec, MCF10A HER2, MCF7 vec, MCF7 HER2, MDA-MB-231 vec and MDA-MB-231 Akt1 cells were treated with MG132 at 10 μM for 12 h and lysed. The supernatant was incubated with anti-survivin antibody and Protein A–Sepharose. Immunoprecipitates were analysed by Western blotting with the antibodies indicated. (B) SKBR3 cells were treated with EGF (10 ng/ml), heregulin (50 ng/ml), herceptin (20 μg/ml), lapatinib (1 μM), gefitinib (10 μM), Akt inbihitor VIII (10 μM) or U0126 (10 μM) for 24 h and lysed. The supernatant was incubated with antibodies against survivin, XIAP or XAF1 and Protein A–Sepharose. Immunoprecipitates and lysates were analysed by Western blotting with the antibodies indicated. (C) HEK-293T cells were transfected with plasmids encoding survivin-WT or T34A. At 48 h post-transfection, 20 μg/ml cycloheximide was added to block further protein synthesis. Cells were harvested at the time points indicated and subjected to Western blot analysis with antibodies against p-survivin or survivin. (D) HEK-293T cells were co-transfected with the constructs indicated. Lysates were analysed by Western blotting or co-immunoprecipitation with the antibodies indicated. Cont, control; Gefi, gefitinib; Herc, herceptin; HRG, heregulin; IB, immunoblot; IP, immunoprecipitation; Lapa, lapatinib; Ub, ubiquitin.

XIAP is an IAP family member and a dominant antagonist of apoptotic activity including caspase activity [31]. XAF1 functions as a tumour suppressor via XIAP–XAF1 association [32]. The XIAP–XAF1 complex induces the E3 ligase activity of XIAP and targets survivin for ubiquitination [33]. To determine whether HER2 activity regulates the expression of XIAP, XAF1 and the XIAP–XAF1 complex through ERK and/or Akt signalling, we treated SKBR3 cells with EGF, heregulin, herceptin, lapatinib, gefitinib, Akt inhibitor VIII or U0126. Both Akt and ERK signalling increase XIAP levels [34,35]. Interestingly, XAF1 expression is up-regulated by inhibition of the ERK pathway [36]. Herceptin, lapatinib, gefitinib and U0126 appeared to up-regulate the expression of XAF1 and the XIAP–XAF1 complex, although the total amount of XIAP was down-regulated by the inhibitors (Figure 5B). As a result of increased XIAP–XAF1 association, ubiquitinated survivin was increased by MG132 treatment. These results indicated that ERK inhibition resulted in XAF1 expression that induced the XIAP–XAF1 E3 ligase complex to ubiquitinate survivin with subsequent proteosomal degradation.

Previous studies have reported that phosphorylation of survivin on Thr34 by CDK1 [37] is required to maintain survivin protein stability [38]. The CDK inhibitor p21 decreases activation and phosphorylation of CDK1 on Thr161 [39]. The phosphorylation of p21 on Thr145 by Akt induces cytoplasmic localization of p21 and its subsequent inactivation and degradation [40]. To confirm these cascade reactions in human breast cancer cells, the cells were treated with EGF or heregulin to activate HER2/Akt signalling. The activation of HER2/Akt induced phosphorylation of p21 on Thr145, CDK1 on Thr161 and survivin on Thr34 (Figure 5B). Upon HER2/Akt activation, ubiquitinated survivin decreased. These results indicated that survivin stability was regulated by survivin phosphorylation on Thr34 mediated by the HER2/Akt/p21/CDK1 cascade.

We next asked whether survivin ubiquitination requires dephosphorylation of Thr34. We confirmed that survivin-T34A exhibited decreased protein stability compared with survivin-WT in transient transfection experiments of HEK-293T cells using cycloheximide, an inhibitor of protein biosynthesis (Figure 5C). When XIAP constructs (WT or C450A as an E3 ligase or ubiquitin protein ligase mutant [41]) were co-transfected with genes for XAF1 and survivin-WT or survivin-T34A, we observed association between XIAP-C450A and XAF1 as well as between XIAP-WT and XAF1 (Figure 5D, lanes 5, 6, 10 and 11 from the left). This suggested that ligase mutants of XIAP could still bind XAF1. However, XIAP-C450A and XAF1 co-expressing cells showed decreased levels of survivin (both WT and T34A) and increased levels of ubiquitinated survivin (both WT and T34A) in the presence of MG132 (Figure 5D, lane 5 compared with 6, and lane 10 compared with 11; numbering from the left). Overall, survivin-WT-expressing cells exhibited higher levels of survivin and decreased ubiquitinated survivin in the presence of MG132 regardless of XIAP and XAF1 co-expression (Figure 5D, lanes 2–6 compared with lanes 7–11; numbering from the left). These results suggested that the dephosphorylation of survivin on Thr34 is a prerequisite for survivin ubiquitination and subsequent degradation. Previous reports found that mutation of survivin to D53A, H77A or C84A, as well as T34A, induced apoptosis by reducing survivin through a proteosome-dependent degradation pathway [30,38,42] in a cell-cycle-dependent manner [43,44]. We further confirmed the effects of these point mutants on survivin stability using transient transfection experiments in HEK-293T cells. As indicated in Figure 6(A) and Supplementary Figure S2(C), all survivin point mutants exhibited increased ubiquitination compared with survivin-WT. However, when CDK1 and cyclin B1 were co-transfected with survivin-WT or mutants, all mutants and WT showed reduced ubiquitination upon MG132 treatment. With the exception of survivin-T34A, phosphorylation on Thr34 and survivin levels increased. These results suggested that co-expression of CDK1–cyclin B1 increased survivin stability by phosphorylation on survivin Thr34. HER2-CA or Akt1-CA exhibited similar effects as CDK1–cyclin B1 on survivin phosphorylation, ubiquitination upon MG132 treatment and protein levels, but HER2-KD or Akt1-KD did not. These results indicated that Thr34 of survivin is the most important residue for determining survivin stability upon phosphorylation after HER2/Akt/CDK1–cyclin B1 signalling. The overall scheme of the HER2-induced increase in survivin protein levels is depicted in Figure 6(B).

Survivin stability is regulated by phosphorylation on Thr34 through HER2/Akt/CDK1–cyclin B1 signalling

Figure 6
Survivin stability is regulated by phosphorylation on Thr34 through HER2/Akt/CDK1–cyclin B1 signalling

(A) HEK-293T cells were co-transfected with the constructs indicated. Lysates were analysed by Western blotting (IB) or co-immunoprecipitation (IP) with the antibodies indicated. (B) A schematic model showing the mechanism by which HER2 induced survivin stabilization.

Figure 6
Survivin stability is regulated by phosphorylation on Thr34 through HER2/Akt/CDK1–cyclin B1 signalling

(A) HEK-293T cells were co-transfected with the constructs indicated. Lysates were analysed by Western blotting (IB) or co-immunoprecipitation (IP) with the antibodies indicated. (B) A schematic model showing the mechanism by which HER2 induced survivin stabilization.

DISCUSSION

The present study demonstrates that HER2 downstream signalling, including in the Akt and ERK pathways, enhanced survivin stability. At the same time, the transcription of the survivin gene was decreased by inhibition of Notch1-dependent transcriptional activity by HER2 downstream signalling in the Akt and ERK pathways. HER2/ERK signalling decreased γ-secretase activity and HER2/Akt signalling inhibited nuclear translocation of NICD. These two signalling pathways collectively down-regulated expression of Notch1-target genes, including survivin. ERK-mediated XAF1 inhibition reduced formation of the E3 ligase XIAP–XAF1 complex, which catalyses the ubiquitination of survivin. This led to inhibition of survivin degradation. Activation of the HER2/Akt/CDK1–cyclin B1 signalling cascade induced phosphorylation of survivin on Thr34. The suppression of survivin Thr34 phosphorylation was shown to reduce survivin levels in cancer cells, using the CDK inhibitor flavopiridol [38]. We also showed that a T34A survivin mutant had the least stability of several survivin point mutants (D53A, H77A and C84A) that are reported to decrease survivin stability. Therefore we conclude that survivin Thr34 is the most important residue for determining survivin stability and cell function.

Survivin, a protein of the IAP family [2], is generally considered to be a cell-cycle regulatory protein that functions through interaction with the mitotic spindle microtubules during mitosis [45]. A study using knockout mice found that survivin is required for mitosis during development [46]. In agreement with the present study, expression of survivin was shown to be up-regulated by HER2-downstream ERK/PI3K signalling in breast cancer and by the EGF-induced ERK pathway in pancreatic β-cells [10,47]. In the present study we found that treatment with doxorubicin resulted in massive apoptosis of survivin-silenced cells, whereas induction of apoptosis in control cells was significantly attenuated compared with survivin-silenced cells. In contrast, silencing of survivin did not significantly affect the cell cycle or proliferation in HER2-overexpressing human breast cancer cells in the present study. These results suggested the possibility that the components of HER2 or its downstream signalling might be responsible for the lack of a survivin-mediated effect on the cell cycle.

A previous study in keratinocytes and skin squamous cell carcinomas indicated that EGFR down-regulates transcription of the Notch1 gene and Notch1-target genes by down-regulating p53 via the EGFR/MEK/ERK/c-Jun pathway [48]. Notch1 is generally known as a p53 target gene [49]. Like EGFR, HER2 activates the downstream ERK pathway [7]. However, in contrast with EGFR, which down-regulates p53 levels through the EGFR/ERK pathway, HER2 increases p53 levels by an unknown mechanism(s) [50]. In addition, whereas EGFR decreases total Notch1 levels [48], HER2 did not affect the total Notch1 level in the present study. Therefore we conclude that HER2 might reduce Notch1-dependent transcription in HER2-overexpressing cells in a p53-independent manner. Other members of the transcription complex (except for p53) might also be able to regulate Notch1 gene transcription in HER2-overexpressing cells.

Abbreviations

     
  • CA

    constitutively active

  •  
  • CDK1

    cyclin-dependent kinase 1

  •  
  • CSL

    C promoter-binding factor/suppressor of hairless/Lag-1

  •  
  • Cy3

    indocarbocyanine

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • DTT

    dithiothreitol

  •  
  • EGF

    epidermal growth factor

  •  
  • EGFR

    EGF receptor

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • HEK

    human embryonic kideny

  •  
  • HER2

    human EGFR 2

  •  
  • IAP

    inhibitor of apoptosis protein

  •  
  • JAK3

    Janus kinase 3

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • KD

    kinase dead

  •  
  • Luc

    luciferase

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MEK

    MAPK/ERK kinase

  •  
  • MMTV

    mouse mammary tumour virus

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • MTT

    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide

  •  
  • NICD

    Notch-intracellular domain

  •  
  • NRE

    Notch-response element

  •  
  • p-

    phosphorylated

  •  
  • PARP

    poly(ADP-ribose) polymerase

  •  
  • PEN2

    presenilin enhancer 2

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • RT

    reverse transcriptase

  •  
  • shRNA

    small hairpin RNA

  •  
  • siRNA

    small interfering RNA

  •  
  • WT

    wild-type

  •  
  • XAF1

    XIAP (X-linked inhibitor of apoptosis)-associated factor 1

  •  
  • XIAP

    X-linked inhibitor of apoptosis

AUTHOR CONTRIBUTION

Ji-hyun Ju and Incheol Shin were involved in the conception and design of the study, development of the methodology, analysis and interpretation of data, and writing and revising the paper. Ji-hyun Ju, Wonseok Yang, Sunhwa Oh, KeeSoo Nam and Kyung-min Lee acquired the data. Ji-hyun Ju, Dong-young Noh and Incheol Shin providided administrative, technical and material support. Incheol Shin supervised the study.

FUNDING

This work was supported by the Converging Research Center Program [grant number 2011-K001445], a National Research Foundation grant [grant number 2011-0015515] and the Basic Research Program of the Korea Science and Engineering Foundation [grant number 2008-05943].

References

References
1
Crook
N. E.
Clem
R. J.
Miller
L. K.
An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif
J. Virol.
1993
, vol. 
67
 (pg. 
2168
-
2174
)
2
Ambrosini
G.
Adida
C.
Altieri
D. C.
A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma
Nat. Med.
1997
, vol. 
3
 (pg. 
917
-
921
)
3
Li
F.
Ambrosini
G.
Chu
E. Y.
Plescia
J.
Tognin
S.
Marchisio
P. C.
Altieri
D. C.
Control of apoptosis and mitotic spindle checkpoint by survivin
Nature
1998
, vol. 
396
 (pg. 
580
-
584
)
4
Kawasaki
H.
Altieri
D. C.
Lu
C. D.
Toyoda
M.
Tenjo
T.
Tanigawa
N.
Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer
Cancer Res.
1998
, vol. 
58
 (pg. 
5071
-
5074
)
5
Kennedy
S. M.
O'Driscoll
L.
Purcell
R.
Fitz-Simons
N.
McDermott
E. W.
Hill
A. D.
O'Higgins
N. J.
Parkinson
M.
Linehan
R.
Clynes
M.
Prognostic importance of survivin in breast cancer
Br. J. Cancer
2003
, vol. 
88
 (pg. 
1077
-
1083
)
6
Menard
S.
Pupa
S. M.
Campiglio
M.
Tagliabue
E.
Biologic and therapeutic role of HER2 in cancer
Oncogene
2003
, vol. 
22
 (pg. 
6570
-
6578
)
7
Hudis
C. A.
Trastuzumab: mechanism of action and use in clinical practice
N. Engl. J. Med.
2007
, vol. 
357
 (pg. 
39
-
51
)
8
Roberts
E. C.
Shapiro
P. S.
Nahreini
T. S.
Pages
G.
Pouyssegur
J.
Ahn
N. G.
Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis
Mol. Cell. Biol.
2002
, vol. 
22
 (pg. 
7226
-
7241
)
9
Asanuma
H.
Torigoe
T.
Kamiguchi
K.
Hirohashi
Y.
Ohmura
T.
Hirata
K.
Sato
M.
Sato
N.
Survivin expression is regulated by coexpression of human epidermal growth factor receptor 2 and epidermal growth factor receptor via phosphatidylinositol 3-kinase/AKT signaling pathway in breast cancer cells
Cancer Res.
2005
, vol. 
65
 (pg. 
11018
-
11025
)
10
Siddiqa
A.
Long
L. M.
Li
L.
Marciniak
R. A.
Kazhdan
I.
Expression of HER-2 in MCF-7 breast cancer cells modulates anti-apoptotic proteins Survivin and Bcl-2 via the extracellular signal-related kinase (ERK) and phosphoinositide-3 kinase (PI3K) signalling pathways
BMC Cancer
2008
, vol. 
8
 pg. 
129
 
11
Artavanis-Tsakonas
S.
Rand
M. D.
Lake
R. J.
Notch signaling: cell fate control and signal integration in development
Science
1999
, vol. 
284
 (pg. 
770
-
776
)
12
Blaumueller
C. M.
Qi
H.
Zagouras
P.
Artavanis-Tsakonas
S.
Intracellular cleavage of Notch leads to a heterodimeric receptor on the plasma membrane
Cell
1997
, vol. 
90
 (pg. 
281
-
291
)
13
Ohtsuka
T.
Ishibashi
M.
Gradwohl
G.
Nakanishi
S.
Guillemot
F.
Kageyama
R.
Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation
EMBO J.
1999
, vol. 
18
 (pg. 
2196
-
2207
)
14
Chen
Y.
Li
D.
Liu
H.
Xu
H.
Zheng
H.
Qian
F.
Li
W.
Zhao
C.
Wang
Z.
Wang
X.
Notch-1 signaling facilitates survivin expression in human non-small cell lung cancer cells
Cancer Biol. Ther.
2011
, vol. 
11
 (pg. 
14
-
21
)
15
Osipo
C.
Patel
P.
Rizzo
P.
Clementz
A. G.
Hao
L.
Golde
T. E.
Miele
L.
ErbB-2 inhibition activates Notch-1 and sensitizes breast cancer cells to a γ-secretase inhibitor
Oncogene
2008
, vol. 
27
 (pg. 
5019
-
5032
)
16
Shin
I.
Arteaga
C. L.
Expression of active Akt protects against tamoxifen-induced apoptosis in MCF-7 Cells
IUBMB Life
2006
, vol. 
58
 (pg. 
664
-
669
)
17
Ueda
Y.
Wang
S.
Dumont
N.
Yi
J. Y.
Koh
Y.
Arteaga
C. L.
Overexpression of HER2 (erbB2) in human breast epithelial cells unmasks transforming growth factor β-induced cell motility
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
24505
-
24513
)
18
Yang
W.
Ju
J. H.
Lee
K. M.
Shin
I.
Akt isoform-specific inhibition of MDA-MB-231 cell proliferation
Cell. Signalling
2011
, vol. 
23
 (pg. 
19
-
26
)
19
Song
J.
Park
S.
Kim
M.
Shin
I.
Down-regulation of Notch-dependent transcription by Akt in vitro
FEBS Lett.
2008
, vol. 
582
 (pg. 
1693
-
1699
)
20
Shin
I.
Yakes
F. M.
Rojo
F.
Shin
N. Y.
Bakin
A. V.
Baselga
J.
Arteaga
C. L.
PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization
Nat. Med.
2002
, vol. 
8
 (pg. 
1145
-
1152
)
21
Abbott
D. W.
Holt
J. T.
Mitogen-activated protein kinase kinase 2 activation is essential for progression through the G2/M checkpoint arrest in cells exposed to ionizing radiation
J. Biol. Chem.
1999
, vol. 
274
 (pg. 
2732
-
2742
)
22
Davies
R. L.
Grosse
V. A.
Kucherlapati
R.
Bothwell
M.
Genetic analysis of epidermal growth factor action: assignment of human epidermal growth factor receptor gene to chromosome 7
Proc. Natl. Acad. Sci. U.S.A.
1980
, vol. 
77
 (pg. 
4188
-
4192
)
23
Kita
Y. A.
Barff
J.
Luo
Y.
Wen
D.
Brankow
D.
Hu
S.
Liu
N.
Prigent
S. A.
Gullick
W. J.
Nicolson
M.
NDF/heregulin stimulates the phosphorylation of Her3/erbB3
FEBS Lett.
1994
, vol. 
349
 (pg. 
139
-
143
)
24
Tang
Z.
Kadesch
T.
Identification of a novel activation domain in the Notch-responsive transcription factor CSL
Nucleic Acids Res.
2001
, vol. 
29
 (pg. 
2284
-
2291
)
25
Lecourtois
M.
Schweisguth
F.
The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by Notch signaling
Genes Dev.
1995
, vol. 
9
 (pg. 
2598
-
2608
)
26
Tamm
I.
Wang
Y.
Sausville
E.
Scudiero
D. A.
Vigna
N.
Oltersdorf
T.
Reed
J. C.
IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs
Cancer Res.
1998
, vol. 
58
 (pg. 
5315
-
5320
)
27
Lieber
T.
Kidd
S.
Alcamo
E.
Corbin
V.
Young
M. W.
Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei
Genes Dev.
1993
, vol. 
7
 (pg. 
1949
-
1965
)
28
Kim
S. K.
Park
H. J.
Hong
H. S.
Baik
E. J.
Jung
M. W.
Mook-Jung
I.
ERK1/2 is an endogenous negative regulator of the γ-secretase activity
FASEB J.
2006
, vol. 
20
 (pg. 
157
-
159
)
29
St George-Hyslop
P.
Fraser
P. E.
Assembly of the presenilin γ-/∊-secretase complex
J. Neurochem.
2012
, vol. 
120
 
Suppl. 1
(pg. 
84
-
88
)
30
Zhao
J.
Tenev
T.
Martins
L. M.
Downward
J.
Lemoine
N. R.
The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner
J. Cell Sci.
2000
, vol. 
113
 (pg. 
4363
-
4371
)
31
Deveraux
Q. L.
Takahashi
R.
Salvesen
G. S.
Reed
J. C.
X-linked IAP is a direct inhibitor of cell-death proteases
Nature
1997
, vol. 
388
 (pg. 
300
-
304
)
32
Liston
P.
Fong
W. G.
Kelly
N. L.
Toji
S.
Miyazaki
T.
Conte
D.
Tamai
K.
Craig
C. G.
McBurney
M. W.
Korneluk
R. G.
Identification of XAF1 as an antagonist of XIAP anti-caspase activity
Nat. Cell Biol.
2001
, vol. 
3
 (pg. 
128
-
133
)
33
Arora
V.
Cheung
H. H.
Plenchette
S.
Micali
O. C.
Liston
P.
Korneluk
R. G.
Degradation of survivin by the X-linked inhibitor of apoptosis (XIAP)–XAF1 complex
J. Biol. Chem.
2007
, vol. 
282
 (pg. 
26202
-
26209
)
34
Carter
B. Z.
Milella
M.
Tsao
T.
McQueen
T.
Schober
W. D.
Hu
W.
Dean
N. M.
Steelman
L.
McCubrey
J. A.
Andreeff
M.
Regulation and targeting of antiapoptotic XIAP in acute myeloid leukemia
Leukemia
2003
, vol. 
17
 (pg. 
2081
-
2089
)
35
Dan
H. C.
Sun
M.
Kaneko
S.
Feldman
R. I.
Nicosia
S. V.
Wang
H. G.
Tsang
B. K.
Cheng
J. Q.
Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP)
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
5405
-
5412
)
36
Yu
L. F.
Wang
J.
Zou
B.
Lin
M. C.
Wu
Y. L.
Xia
H. H.
Sun
Y. W.
Gu
Q.
He
H.
Lam
S. K.
, et al. 
XAF1 mediates apoptosis through an extracellular signal-regulated kinase pathway in colon cancer
Cancer
2007
, vol. 
109
 (pg. 
1996
-
2003
)
37
O'Connor
D. S.
Grossman
D.
Plescia
J.
Li
F.
Zhang
H.
Villa
A.
Tognin
S.
Marchisio
P. C.
Altieri
D. C.
Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin
Proc. Natl. Acad. Sci. U.S.A.
2000
, vol. 
97
 (pg. 
13103
-
13107
)
38
Wall
N. R.
O'Connor
D. S.
Plescia
J.
Pommier
Y.
Altieri
D. C.
Suppression of survivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis
Cancer Res.
2003
, vol. 
63
 (pg. 
230
-
235
)
39
Smits
V. A.
Klompmaker
R.
Vallenius
T.
Rijksen
G.
Makela
T. P.
Medema
R. H.
p21 inhibits Thr161 phosphorylation of Cdc2 to enforce the G2 DNA damage checkpoint
J. Biol. Chem.
2000
, vol. 
275
 (pg. 
30638
-
30643
)
40
Zhou
B. P.
Liao
Y.
Xia
W.
Spohn
B.
Lee
M. H.
Hung
M. C.
Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells
Nat. Cell Biol.
2001
, vol. 
3
 (pg. 
245
-
252
)
41
Owens
T. W.
Foster
F. M.
Valentijn
A.
Gilmore
A. P.
Streuli
C. H.
Role for X-linked Inhibitor of apoptosis protein upstream of mitochondrial permeabilization
J. Biol. Chem.
2010
, vol. 
285
 (pg. 
1081
-
1088
)
42
Tu
S. P.
Jiang
X. H.
Lin
M. C.
Cui
J. T.
Yang
Y.
Lum
C. T.
Zou
B.
Zhu
Y. B.
Jiang
S. H.
Wong
W. M.
, et al. 
Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer
Cancer Res.
2003
, vol. 
63
 (pg. 
7724
-
7732
)
43
Song
Z.
Liu
S.
He
H.
Hoti
N.
Wang
Y.
Feng
S.
Wu
M.
A single amino acid change (Asp53→ Ala53) converts Survivin from anti-apoptotic to pro-apoptotic
Mol. Biol. Cell
2004
, vol. 
15
 (pg. 
1287
-
1296
)
44
Zhang
R.
Wang
T.
Li
K. N.
Qin
W. W.
Chen
R.
Wang
K.
Jia
L. T.
Zhao
J.
Wen
W. H.
Meng
Y. L.
, et al. 
A survivin double point mutant has potent inhibitory effect on the growth of hepatocellular cancer cells
Cancer Biol. Ther.
2008
, vol. 
7
 (pg. 
547
-
554
)
45
Li
F.
Ackermann
E. J.
Bennett
C. F.
Rothermel
A. L.
Plescia
J.
Tognin
S.
Villa
A.
Marchisio
P. C.
Altieri
D. C.
Pleiotropic cell-division defects and apoptosis induced by interference with survivin function
Nat. Cell Biol.
1999
, vol. 
1
 (pg. 
461
-
466
)
46
Uren
A. G.
Wong
L.
Pakusch
M.
Fowler
K. J.
Burrows
F. J.
Vaux
D. L.
Choo
K. H.
Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype
Curr. Biol.
2000
, vol. 
10
 (pg. 
1319
-
1328
)
47
Wang
H.
Gambosova
K.
Cooper
Z. A.
Holloway
M. P.
Kassai
A.
Izquierdo
D.
Cleveland
K.
Boney
C. M.
Altura
R. A.
EGF regulates survivin stability through the Raf-1/ERK pathway in insulin-secreting pancreatic β-cells
BMC Mol. Biol.
2010
, vol. 
11
 pg. 
66
 
48
Kolev
V.
Mandinova
A.
Guinea-Viniegra
J.
Hu
B.
Lefort
K.
Lambertini
C.
Neel
V.
Dummer
R.
Wagner
E. F.
Dotto
G. P.
EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer
Nat. Cell Biol.
2008
, vol. 
10
 (pg. 
902
-
911
)
49
Yugawa
T.
Handa
K.
Narisawa-Saito
M.
Ohno
S.
Fujita
M.
Kiyono
T.
Regulation of Notch1 gene expression by p53 in epithelial cells
Mol. Cell. Biol.
2007
, vol. 
27
 (pg. 
3732
-
3742
)
50
Casalini
P.
Botta
L.
Menard
S.
Role of p53 in HER2-induced proliferation or apoptosis
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
12449
-
12453
)

Supplementary data