Carcinogenesis

Carcinogenesis Advance Access originally published online on July 16, 2007
Carcinogenesis 2007 28(11):2313-2320; doi:10.1093/carcin/bgm152

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Id-1 activation of PI3K/Akt/NFB signaling pathway and its significance in promoting survival of esophageal cancer cells

Bin Li, Pak Yan Cheung, Xianghong Wang, Sai Wah Tsao, Ming Tat Ling, Yong Chuan Wong and Annie L.M. Cheung^*

Cancer Biology Group, Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong, SAR, China

^* To whom correspondence should be addressed. Tel: +852 28199293; Fax: +852 28170857; Email: lmcheung{at}hkucc.hku.hk

	Abstract

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Inhibitor of differentiation or DNA binding (Id-1) is a helix-loop-helix protein that is over-expressed in many types of cancer including esophageal cancer. This study aims to investigate its effects on the phosphatidylinositol-3-kinase (PI3K)/Akt/ nuclear factor kappa B (NFB) signaling pathway and the significance in protecting esophageal cancer cells against apoptosis. We found elevated expression of phosphorylated forms of Akt, glycogen synthase kinase 3ß and inhibitor of kappa B, as well as increased nuclear translocation of NFB subunit p65 and NFB DNA-binding activity, in esophageal cancer cells with stable ectopic Id-1 expression. Transient transfection of Id-1 into HEK293 cells confirmed activation of PI3K/Akt/NFB signaling and the effects were counteracted by the PI3K inhibitor LY294002. Treatment with tumor necrosis factor- (TNF-) elicited a significantly weaker apoptotic response, following a marked and sustained activation of Akt and NFB in the Id-1-over-expressing cells, compared with the vector control. The effects of Id-1 on the PI3K/Akt/NFB signaling pathway and apoptosis were reversed in esophageal cancer cells transfected with siRNA against Id-1. In addition, inhibition of PI3K or NFB signaling using the PI3K inhibitor LY294002 or the NFB inhibitor Bay11-7082 increased the sensitivity of Id-1-over-expressing esophageal cancer cells to TNF--induced apoptosis. Our results provide the first evidence that Id-1 induces the activation of PI3K/Akt/NFB signaling pathway, and protects esophageal cancer cells from TNF--induced apoptosis in vitro. Inactivation of Id-1 may provide us with a novel strategy to improve the treatment and survival of patients with esophageal cancer.

Abbreviations: Id-1, inhibitor of differentiation or DNA binding; PI3K, phosphatidylinositol-3-kinase; NFB, nuclear factor kappa B; GSK3ß, glycogen synthase kinase 3ß; IB, inhibitor of kappa B; ESCC, esophageal squamous cell carcinoma; MDM2, mouse double minute 2; mTOR, mammalian target of rapamycin; PARP, poly (ADP-ribose) polymerase; TUNEL, TdT-mediated dUTP nick-end labeling; TNF-, tumor necrosis factor-

	Introduction

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Inhibitor of differentiation or DNA binding (Id-1) is a member of the helix-loop-helix proteins. It lacks the basic domain for DNA binding and functions as a dominant inhibitor of the basic helix-loop-helix transcription factors by forming heterodimers, thus inhibiting gene expression (1). Increased expression of Id-1 has been found in many types of human cancer including esophageal squamous cell carcinoma (ESCC) (2). Recently, Id-1 has been shown to play an important role in the regulation of cell proliferation and survival in human cancers (3,4). In addition, Id-1 promotes invasion and metastasis of human cancers (5), and the level of Id-1 protein expression is associated with poor prognosis in several types of cancers (6,7). Id-1, therefore, may play a critical role in tumorigenesis and cancer progression. The oncogenic function of Id-1 may involve multiple signaling pathways. In prostate, nasopharyngeal and hepatocellular cancer cells, for example, Id-1 induces serum-independent cell proliferation that is associated with inactivation of the p16/Rb pathway (3,8,9). The growth promoting function of Id-1 also involves activation of the mitogen-activated protein kinase (MAPK) signaling pathway in prostate cancer cells (3,10). In our recent paper, we reported that Id-1-induced cell proliferation of esophageal cancer cells was associated with up-regulation of mouse double minute 2 (MDM2), but not the key members of the p16/Rb pathway (11). Our findings suggest that the oncogenic function of Id-1 in esophageal cancer may preferentially involve signaling pathways different from that of other cancers.

Phosphatidylinositol-3-kinase (PI3K) is a lipid kinase that generates second messengers involved in regulation of a wide spectrum of cellular functions including proliferation, survival and invasion (12). One of its major effectors is Akt (protein kinase B). The PI3K/Akt pathway is frequently activated in many types of human cancers including ESCC (13–15), and has been linked to cancer development for some time. The pathway controls several growth-regulatory transcription factors. One of the prominent examples is nuclear factor kappa B (NFB) (16,17), a heterodimeric transcription factor that is sequestered in the cytoplasm as an inactive form by inhibitor of kappa B (IB) (18). Phosphorylation of IB frees NFB, and allows its nuclear translocation, binding and subsequent activation of target genes (19). In addition to being involved in immune and inflammatory responses, NFB also regulates cell proliferation, apoptosis and migration, and is constitutively activated in a number of human cancers including ESCC (20–22). Therefore, these evidences suggest that the PI3K/Akt/NFB signaling pathway may be associated with tumorigenesis in ESCC. However, the mechanism responsible for PI3K/Akt/NFB activation in ESCC is largely unknown.

Esophageal cancer ranks as the eighth most common cancer in the world (23), and ESCC is the most common form throughout the Asia–Pacific region. Since both Id-1 over-expression and PI3K/Akt/NFB activation are frequent events in ESCC, and are associated with tumor progression of ESCC (14,15,21,24), we investigated whether there is any association between Id-1 and PI3K/Akt/NFB signaling. We examined the effects of ectopic and down-regulated Id-1 expression on the PI3K/Akt/NFB pathway and the anti-apoptotic role of Id-1 in ESCC cells. In addition, we studied the effects of pharmaceutical inhibitors of PI3K/Akt and NFB on the anti-apoptotic function of Id-1 in ESCC cells. Our results suggest that Id-1 induces the activation of the PI3K/Akt/NFB signaling pathway, leading to increased resistance to tumor necrosis factor- (TNF-)-induced apoptosis.

	Materials and methods

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Cell culture and drugs
Human ESCC cell line, HKESC-3, was established from a well-differentiated ESCC (25). The cell line has serum-dependent Id-1 expression. The establishment of stable Id-1-expressing clones and empty vector control clones from this cell line was described previously (11). The pooled stable transfectants, designated HKESC-3-Id-1 and HKESC-3-pBabe, respectively, and another ESCC cell line KYSE510 with high endogenous Id-1 expression even under serum-starved condition (26) were maintained in RPMI 1640 (Sigma, St Louis, MO) supplemented with 10% fetal bovine serum (Invitrogen, Gaithersburg, MD) at 37°C in 5% CO₂. The HEK293 cell line, obtained from American Type Culture Collection (Rockville, MD), was maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum. TNF- (PeproTech EC, London, UK) was diluted in culture medium to obtain the desired concentration. Unless specified otherwise, the HKESC-3-Id-1 and HKESC-3-pBabe cells were cultured in serum-free medium for 24 h before treatment or collection.

Expression vector and transient transfection
The vector containing full-length Id-1 cDNA (pcDNA3-Id-1) or empty vector control (pcDNA3) (27) was transiently transfected into HEK293 cells using Fugene 6 transfection reagent (Roche Diagnostics, GmbH, Mannheim, Germany) according to the manufacturer's protocol. Cells were collected 48 h after transfection for Western blot.

Knock down of Id-1 expression using siRNA
A siRNA duplex targeting Id-1 (si-Id-1) was purchased from Dharmacon (Chicago, IL) and dissolved in RNase-free distilled water. The siRNA target site is 5'-UAAACGUGCUGCUCUACGA-3', and the si-genome duplex RNA sequence used was validated previously to successfully inhibit Id-1 in a nasopharyngeal carcinoma cell line (28). The Dharmacon siCONTROL non-targeting siRNA (catalog number D-001210-02) was used as irrelevant siRNA control (si-CON). Cells were seeded into six-well plates and left for 24 h until 50% confluent. A 5 µl aliquot of siRNA solution (20 µM) and 4 µl of Lipofectamine 2000 (Invitrogen) were each mixed with 250 µl of RPMI 1640 culture medium. The two mixtures were combined and incubated for 20 min at room temperature, then added to the cells to give a final concentration of 100 nM siRNA. The siRNA-transfected cells were used 48 h post-transfection.

Preparation of nuclear extracts
Cells (5 x 10⁶) collected after trypsinization were washed with ice-cold phosphate-buffered saline and the pellet resuspended in 200 µl of ice-cold Buffer A (10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid pH 7.9, 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid, 2.5 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM dithiothreitol, 1 µg/ml NaF and 0.5 mM phenylmethylsulfonyl fluoride). After incubation on ice for 15 min, 10 µl of 1% Nonidet P-40 was added. The mixture was left at room temperature for 4 min, and then vortexed vigorously for 20 s. The cell nuclear pellet was collected by centrifugation at 3000 rpm for 3 min, washed with 100 µl of Buffer A, and collected again by centrifugation. The pellet was then resuspended in 50 µl of Buffer C (20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.9, 0.4 M NaCl, 1 mM ethylenediaminetetraacetic acid, 2.5 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM dithiothreitol, 1 µg/ml NaF and 1 mM phenylmethylsulfonyl fluoride), shaken vigorously at 4°C for 15 min and then centrifuged at 14 000 r.p.m. for 15 min at room temperature. The supernatant was recovered as nuclear extract and then transferred to a fresh Eppendorf tube containing 10% v/v of glycerol. The protein concentration was assayed using Bio-Rad Dc protein assay kit (Bio-Rad, Hercules, CA) before use for Western blotting or electrophoretic mobility shift assay.

Western blot analyses
Preparation of whole-cell lysates and immunoblotting were described previously (11,29). The primary antibodies used include p-Akt (Ser-473), Akt, p-glycogen synthase kinase 3ß (GSK3ß) (Ser-9), GSK3ß, p-IB (Ser-32/36), caspase-3, cleaved caspase-3, poly (ADP-ribose) polymerase (PARP) (Cell signaling Technology, Beverly, MA), and Id-1, Id-2, Id-3, Id-4, NFB/p65, histone H1 and actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Electrophoretic mobility shift assay
NFB DNA-binding activity was detected using an EMSA kit (Panomics, Redwood, CA) according to manufacturer's instruction. Briefly, the nuclear extract was incubated in 1x binding reaction mixture including biotin-labeled double-stranded NFB consensus oligonucleotide for 30 min at 20°C. The mixture was separated on a non-denaturing polyacrylamide gel and then transferred to a nylon membrane (Amersham). The membrane was baked for 1 h at 80°C in a dry oven, then transferred to a UV crosslinker oven for 3 min. The shifted bands corresponding to the protein–DNA complexes were visualized relative to the unbound dsDNA. The bands were visualized after exposure to BioMax Light Film (Kodak, Rochester, NY).

TdT-mediated dUTP nick-end labeling assay
End labeling of exposed 3'-OH ends of DNA fragments was undertaken with the TdT-mediated dUTP nick-end labeling (TUNEL) in situ cell death detection kit Fluorescein (Roche Diagnostics) as described by the manufacturer. Briefly, the cells were fixed with 4% paraformaldehyde for 1 h, and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline for 10 min before incubating in TUNEL reaction mixture. The cells were subsequently stained with 4',6-diamidino-2-phenylindole and visualized under a fluorescence microscopy with a x40 objective. Eight representative areas were randomly selected. At least 500 4',6-diamidino-2-phenylindole-positive cells were scored. The percentage of apoptotic cells was determined by dividing the number of TUNEL-positive cells by the total number of cells (4',6-diamidino-2-phenylindole-positive cells) in the corresponding area.

	Results

Top Abstract Introduction Materials and methods Results Discussion Funding References

 
Effect of Id-1 over-expression and silencing on Akt signaling pathway
Esophageal cancer cells with stable ectopic Id-1 expression (HKESC-3-Id-1) and the vector control cells (HKESC-3-pBabe) were used to investigate whether ectopic Id-1 expression had any effect on the Akt signaling pathway. These pooled populations of stably transfected clones were derived from parental cells that showed nearly undetectable Id-1 expression under serum-free condition (Figure 1A). Since Akt is activated through phosphorylation (30), we detected the expression of the Ser 473 phosphorylated form of Akt and its downstream target, GSK3ß, in the cell lysates. As shown in Figure 1B, there was increased phosphorylation of Akt and GSK3ß in HKESC-3-Id-1, although the total levels of each protein remained similar to those of the vector control (HKESC-3-pBabe). To eliminate the possibility that the activation of Akt pathway was due to the drug selection during the establishment of the stable cell lines, we repeated the experiments by transiently transfecting HEK293 cells with different amounts of Id-1 expression vector. As shown in Figure 1C, phosphorylated Akt was up-regulated in these cells, and the increase corresponded to the level of Id-1 expression. We next determined the effect of Id-1 gene silencing on Akt signaling pathway. Inhibition of Id-1 expression was achieved through transient transfection with siRNA against Id-1 (si-Id-1) in KYSE510, an ESCC cell line with high inherent Id-1 expression (Figure 1A). The siRNA used was specific for Id-1, and did not affect the expression of other Id proteins. Compared with control cells transfected with an irrelevant siRNA (si-CON), the pooled si-Id-1 transiently transfected cells showed decreased expression of the phosphorylated form of Akt and GSK3ß (Figure 1D). Taken together, these results indicate that Id-1 activates Akt signaling pathway, and that this function is not confined to ESCC cells.

View larger version (49K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1. Effects of Id-1 modulation on Akt signaling pathway as detected by Western blot. (A) Parental esophageal cancer cell lines HKESC-3 and KYSE510, which were used to generate stable Id-1-expressing clones and transient transfectants for Id-1 knockdown experiments, respectively, were compared for the expression of Id-1, Akt, phospho-Akt (p-Akt) and phospho-IB (p-IB) after 24 h culture in serum-free medium. Actin was included as internal loading control. The HKESC-3 cell line had markedly lower expression of Id-1, p-Akt and p-IB than the KYSE510 cell line. (B) Id-1-over-expressing ESCC cells HKESC-3-Id-1 and vector control cells HKESC-3-pBabe were examined for the expression of Akt, GSK3ß and their phosphorylated forms (p-Akt and p-GSK3ß). Higher expression levels of p-Akt and p-GSK3ß, but not total Akt or GSK3ß, were detected in HKESC-3-Id-1. (C) Different doses of pcDNA3-Id-1 expression vector, with the addition of empty vector (pcDNA3) to normalize the total amount of DNA across all transfections, were transiently transfected into HEK293 cells. A dose-dependent upregulation of p-Akt and p-GSK3ß, but not total Akt or GSK3ß, was detected. (D) KYSE510 cells were transiently transfected with siRNA against Id-1 (si-Id-1) or irrelevant RNA (si-CON). Down-regulation of Id-1 led to reduced phosphorylation of Akt and GSK3ß, whereas the total levels of these proteins remained unchanged. Western blots of Id-2, Id-3 and Id-4 were included to verify that the siRNA used was specific to Id-1 but not the other Id proteins.

 
PI3K is required for Id-1 activation of Akt signaling pathway
Since activated Akt is the predominant and essential mediator of PI3K function (31), we hypothesized that Id-1 activates Akt through the PI3K signaling pathway. To examine whether PI3K was involved in the induction of Akt in the Id-1-over-expressing cells, esophageal cancer cells with stable ectopic Id-1 expression (i.e. HKESC-3-Id-1) were treated with a PI3K-specific inhibitor LY294002. As shown in Figure 2A, treatment with LY294002 attenuated Id-1-induced phosphorylation of Akt and GSK3ß in HKESC-3-Id-1, indicating the significance of PI3K in the Id-1 activation of Akt pathway. To confirm this observation, we transiently transfected HEK293 cells with different amounts of Id-1 expression vector and studied the effects on the expression levels of Akt, GSK3ß and their phosphorylated forms in the presence of LY294002. Figure 2B shows that inhibition of PI3K with LY294002 abolished the dose-dependent up-regulation of p-Akt and p-GSK3ß by Id-1 seen in a parallel experiment without the addition of LY294002. These data show that PI3K is involved in the Id-1 induction of Akt activity.

View larger version (50K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2. Effects of PI3K inhibition on Akt signaling pathway in cells over-expressing Id-1. (A) Id-1-over-expressing ESCC cells (HKESC-3-Id-1) were treated with 50 µM PI3K inhibitor LY294002 for 12 h. Western blot analysis showed reduced expression of phosphorylated forms of Akt and GSK3ß in the LY294002-treated cells, compared with untreated cells and vehicle (dimethyl sulfoxide)-treated cells. (B) HEK293 cells were transiently transfected with different doses of pcDNA3-Id-1 expression vector in the absence (lanes 1–3) or presence of LY294002 (lanes 4–6). Comparison of p-Akt and p-GSK3ß expression levels in the Western blots showed that treatment with LY294002 attenuated the effect of Id-1 on Akt signaling pathway.

 
Effect of Id-1 expression on NFB signaling pathway
We next investigated whether Id-1 expression had any effect on NFB activity. Since NFB translocates to the nucleus upon activation, we studied the expression of the most abundant subunit of NFB, p65, in the nuclear extracts and whole lysates of Id-1-over-expressing cells and the vector control cells by Western blot. As shown in Figure 3A, there was increased nuclear translocation of p65 in HKESC-3-Id-1, as indicated by increased p65 expression in the nuclear extract, compared with that of the HKESC-3-pBabe vector control cells. Since phosphorylation of IB is required for NFB activation (19), we also compared the expression level of the phosphorylated form of IB in the whole-cell lysates of the two cell lines, and found increased phosphorylated IB in HKESC-3-Id-1 (Figure 3A). Moreover, transient transfection of HEK293 cells with increasing amounts of Id-1 expression plasmids resulted in a dose-dependent increase in nuclear p65 (Figure 3B), thus demonstrating that Id-1 was directly involved in the activation of NFB signaling pathway. Furthermore, electrophoretic mobility shift assay showed that the HKESC-3-Id-1 cells had higher NFB DNA-binding activity than the empty vector control cells (Figure 3C, lane 3 compared with lane 2). We also tested the effect of Id-1 gene silencing on NFB in the KYSE510 ESCC cells transiently transfected with si-Id-1 and found decreased phosphorylation of IB and reduced nuclear translocation of p65 (Figure 3D). Taken together, these observations support that Id-1 functions as an upstream regulator of the NFB signaling pathway.

View larger version (52K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3. Effects of Id-1 modulation on NFB signaling pathway. (A) Id-1-over-expressing ESCC cells (HKESC-3-Id-1) and vector control cells (HKESC-3-pBabe) were compared for the expression of phosphorylated IB (p-IB) and nuclear p65 and by Western blot. Nuclear histone and actin were included as loading controls. The Id-1-over-expressing cells showed up-regulated p-IB and increased nuclear translocation of p65, indicating increased NFB activity, compared with vector control cells. (B) HEK293 cells transiently transfected with increasing doses of Id-1 expression vector were also compared for nuclear p65 expression, and the increase was found to be proportional to the level of Id-1 expressed. (C) Electrophoretic mobility shift assay was carried out to determine the effect of Id-1 on NFB DNA-binding activity. Binding activity was indicated by a labeled shifted band at the top of the gel. Higher binding activity was evident in the nuclear extract/DNA mixture of HKESC-1-Id-1 (lane 3), compared with that of HKESC-3-pBabe (lane 2). In lane 4, addition of excess unlabeled dsDNA probe eliminated the shifted band, thus confirming that the intensity of the shifted bands was a measure of NFB DNA binding. (D) The effect of Id-1 gene silencing on NFB activity was assessed in the KYSE510 ESCC cells transiently transfected with siRNA against Id-1 by Western blot. Knock down of Id-1 expression through transfection with siRNA resulted in reduced p-IB and nuclear p65, indicating suppression of NFB activity. (E) HKESC-3-Id-1 cells were treated with 50 µM PI3K inhibitor LY294002, 20 µM NFB inhibitor Bay11-7082 or vehicle (dimethyl sulfoxide) for 12 h. Western blot showed decreased expression of p-IB and reduced nuclear translocation of p65 in LY294002-treated cells (lane 3), compared with untreated (lane 1) and vehicle-treated cells (lane 2). Treatment with NFB inhibitor Bay11-7082 did not affect Akt activity, bust decreased phosphorylation of IB and nuclear translocation of p65.

 
Id-1-induced NFB activation is mediated through PI3K/Akt
Since NFB is a downstream target of PI3K/Akt signaling pathway (16,17), and our results above indicated that Id-1 activated both PI3K/Akt and NFB, we hypothesized that PI3K/Akt signaling pathway is required for the Id-1 activation of NFB. As shown in Figure 3E, inhibition of PI3K activity in HKESC-3-Id-1 cells using the PI3K inhibitor LY294002 resulted in reduced phosphorylation of Akt and IB, as well as decreased nuclear translocation of p65 (lane 3). On the other hand, treatment with the NFB inhibitor Bay11-7082 attenuated phosphorylation of IB and nuclear translocation of NFB subunit p65, but did not affect Akt activity (lane 4). This suggests that Id-1-induced activation of NFB is mediated through the PI3K/Akt signaling pathway.

Id-1 protects ESCC cells from TNF--induced apoptosis through activation of PI3K/Akt/NFB signaling pathway
It has been documented that the PI3K/Akt/NFB signaling pathway plays a role in preventing cells from undergoing apoptosis (32). Since our results indicated that Id-1 induced up-regulation of the PI3K/Akt/NFB signaling pathway in ESCC cells, we reasoned that activation of PI3K/Akt/NFB by Id-1 may protect ESCC cells against apoptosis. The parental cell line HKESC-3 was tested for sensitivity to TNF--induced apoptosis using the TUNEL assay. The cells showed a dose-dependent response to increasing doses of TNF- (up to 200 ng/ml) in serum-free medium (Figure 4A). We then treated HKESC-3-Id-1 (consisting of pooled stable clones of Id-1 over-expressing ESCC cells) and HKESC-3-pBabe (vector control cells) with TNF- at a dose of 50 ng/ml for up to 24 h. As shown in Figure 4B and C, the treatment elicited a significantly weaker apoptotic response in HKESC-3-Id-1, compared with the vector control (4.8 versus 24.5% apoptotic cells at 24 h). Western blot analysis of apoptosis-related proteins, caspase 3, PARP and their cleaved fragments in the cell lysates at different time points showed increasing levels of cleaved fragments of caspase 3 and PARP in the vector control cells, which was not apparent in the Id-1-over-expressing cells (Figure 4D). These results demonstrated that the Id-1-over-expressing cells were more resistant to TNF--induced apoptosis than the control cells. The time course experiment also showed a rapid and marked elevation of phosphorylated Akt and nuclear p65 in the HKESC-3-Id-1 cells, and the increase was sustained throughout the remaining duration of the treatment. In contrast, the vector control cells showed a relatively delayed and weaker stimulation of Akt phosphorylation, and only a transient increase in p65 nuclear translocation. Notably, the p65 expression level in the nuclear extract of the control cells returned to basal level at 4 h, prior to increased apoptosis indicated by elevated expression levels of cleaved fragments of caspase 3 and PARP.

View larger version (67K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4. Up-regulation of Id-1 induced PI3K/Akt/NFB signaling pathway and protected ESCC cells from TNF--induced apoptosis. (A) HKESC-3 parental cells showed dose-dependent increase in percentage of apoptotic cells after treatment with 50–200 ng/ml TNF- for 24 and 48 h. (B–D) Pooled Id-1 stable transfectant (HKESC-3-Id-1) and vector control cells (HKESC-3-pBabe) were treated with TNF- (50 ng/ml) for up to 24 h. (B) The Id-1-over-expressing cells had significantly lower percentage of apoptotic cells than the vector control at 12 and 24 h after TNF- treatment (*P < 0.001, Student's t-test). Data represent mean ± SD of three independent experiments. (C) Apoptotic cells were detected by TUNEL staining (green) and nuclei of all cells counterstained with 4',6-diamidino-2-phenylindole (blue). (D) Phosphorylation of Akt, nuclear p65 as well as apoptotic markers were detected using Western blot, with the inclusion of actin and nuclear histone as loading controls. Note rapid and pronounced induction of Akt and NFB activities in HKESC-3-Id-1, compared with the vector control. The control cells showed elevated levels of cleaved caspase-3 and cleaved PARP at later time points, indicating increased apoptosis.

 
If the anti-apoptotic function of Id-1 is indeed mediated through Akt/NFB, we would expect suppression of Id-1 to reverse the effects seen in Figure 4. Compared with HKESC-3 (Figure 4A), the KYSE510 cell line was more resistant to TNF- treatment (Figure 5A), which made it a good model for studying the effect of Id-1 gene silencing on apoptotic response. As shown in Figure 5B and C, knock down of Id-1 expression in KYSE510 cells transiently transfected with si-Id-1 significantly increased the sensitivity to TNF--induced apoptosis. Moreover, whereas induction of Akt and NFB activities was sustained after 12 h of TNF- treatment in the control cells, the effect was more transient in the si-Id-1 transfected KYSE510 cells and was followed by increased expressions of cleaved caspase 3 and PARP (Figure 5D).

View larger version (50K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5. Knock down of Id-1 expression led to suppression of PI3K/Akt/NFB signaling pathway and increased sensitivity of ESCC cells to TNF--induced apoptosis. (A) The parental cell line KYSE510, used to generate transient si-Id-1 transfectants, was relatively resistant to apoptosis induced by increasing doses of TNF- under serum-free condition. (B–D) KYSE510 cells transiently transfected with si-Id-1 or si-CON were treated with TNF- (50 ng/ml) for up to 36 h, and the effects on apoptosis and PI3K/Akt/NFB signaling pathway were analyzed using TUNEL staining and Western blot as described in Figure 4. (B and C) Significantly higher percentages of apoptotic cells were found in the si-Id-1-transfected cells after exposure to TNF- for 24 and 36 h (*P < 0.005 and **P < 0.001, respectively, Student's t-test). (D) Increased expressions of cleaved caspase 3 and PARP fragment were detected in the KYSE510 cells transfected with si-Id-1 after a transient elevation in p-Akt, p-GSK3ß, p-IB and nuclear p65.

 
To provide further proof that activation of PI3K/Akt/NFB mediates the anti-apoptotic effect of Id-1, we investigated whether suppression of PI3K/Akt/NFB could increase the sensitivity of Id-1-over-expressing cells to TNF--induced apoptosis in HKESC-3-Id-1 cells (Figure 6). In the presence of TNF-, LY294002 treatment decreased the protein expression levels of phosphorylated Akt and its downstream target GSK3ß, as well as that of phosphorylated IB and nuclear p65, indicating suppression of the PI3K/Akt/NFB signaling pathway. Treatment with Bay11-7082 did not affect Akt but suppressed NFB activity, indicated by down-regulation of phosphorylated IB and nuclear p65. The results also demonstrated that, in the presence of TNF-, regardless of whether the PI3/Akt or the NFB part of the PI3/Akt/NFKB pathway was inhibited, there was increased apoptosis, accompanied by increase in cleaved caspase 3 and PARP. Taken together, these results illustrate that PI3K/Akt acts upstream of NFB, and that this pathway plays a role in mediating the anti-apoptotic effect of Id-1.

View larger version (35K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6. PI3K/Akt/NFB mediated the anti-apoptotic effect of Id-1. In the presence of TNF- (50 ng/ml), Id-1-over-expressing cells were treated with 50 µM LY294002 (lane 4), 20 µM Bay11-7082 (lane 5) or vehicle (dimethyl sulfoxide) (lane 3) for 12 h. (A) Both inhibitors increased the sensitivity of HKESC-3-Id-1 cells to TNF--induced apoptosis (*P < 0.001, compared with vehicle control). (B) Western blot analyses showed that inhibition of PI3K/Akt/NFB signaling pathway using specific inhibitors (indicated by decreased p-Akt, p-GSK3ß, p-IB and nuclear p65) led to increased expressions of apoptotic markers, cleaved caspase3 and PARP.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion Funding References

 
In this study, our results indicate for the first time that Id-1 induces activation of the PI3K/Akt/NFB signaling pathway, which may be one of the mechanisms responsible for protecting ESCC cells from TNF--induced apoptosis.

Several novel points arise from this study. First, we provide the first evidence that Id-1 is an upstream regulator of the PI3K/Akt pathway (Figures 1 and 2). Our finding that Id-1 activates PI3K/Akt is significant since activation of PI3K/Akt is a frequent event in many types of human cancers (13,20), but the mechanisms underlying its activation are not fully understood. Recently, it was reported that the Evi1 oncogene increases the sensitivity of colon cancer cells to taxol-mediated apoptosis through activation of PI3K/Akt (33). Whether a similar association exists between the PI3K/Akt pathway and other known oncogenes warrants further investigation.

Second, in discovering an association between Id-1 and PI3K/Akt pathway, we might have identified an important missing link in the downstream signaling of Id-1 that is responsible for some of its oncogenic functions. One of downstream target implicated in the anti-apoptotic function of Id-1 in cancer cells is NFB, but the association has only been demonstrated in prostate cancer cells (29). Our results showing that Id-1 over-expression induced phosphorylation of IB, nuclear translocation of p65 and NFB DNA-binding activity in ESCC and HEK293 cells confirmed this (Figure 3), but a critical question remains as to how Id-1 activates NFB. Since our data showed that ectopic Id-1 expression also activated the Akt pathway and protected ESCC cells from TNF--induced apoptosis (Figures 4 and 5), and treatment with specific PI3K and NFB inhibitors indicated that PI3K/Akt acts upstream of NFB (Figure 6), we believe that the PI3K/Akt pathway serves as the link between Id-1 and NFB in promoting cancer cell survival. Previously, we reported that Id-1 up-regulates MDM2 and promotes cell proliferation in ESCC (11). Since MDM2 is a direct target of Akt (12) and is known to induce cell proliferation by directly stimulating E2F-1 (34), it is possible that Id-1 acts through PI3K/Akt and MDM2 to drive G₁/S progression. In addition to growth stimulation and anti-apoptotic function, Id-1 is known to promote invasion and metastasis of breast cancer cells (35,36). Since PI3K/Akt/NFB is documented to be involved in the regulation of cellular invasion and metastasis (12,37), it may be one of the mechanisms by which Id-1 exerts its effects on cancer progression.

Third, our findings that endogenous Id-1 expression could be inhibited by siRNA to suppress the PI3K/Akt/NFB signaling pathway and hence increase the sensitivity of ESCC cells to TNF-induced apoptosis suggest a potentially novel therapeutic strategy for esophageal cancer. Although PI3K/Akt/NFB is unlikely to be the sole mediator of Id-1-dependent cell survival, its functions in regulating cell cycle control, driving tumorigenesis and imparting chemoresistance to anticancer treatment makes it an attractive target for cancer therapy (12). A number of candidate drugs targeting this pathway, such as inhibitors of PI3K, epidermal growth factor receptor, platelet-derived growth factor receptor and mammalian target of rapamycin (mTOR), as well as monoclonal HER2 antibody, have been studied. Although the PI3K inhibitors wortmannin and LY294002 have been extensively evaluated in cultured cells as research tools, the non-selectivity of these compounds within the PI3K family and the short half-life of wortmannin or poor water solubility of LY294002 have limited their clinical use. Rapamycin, an inhibitor of the Akt downstream mTOR, also has poor aqueous solubility and chemical stability, although it has significant anti-proliferative activity in several murine tumor systems (38). A rapamycin analog, CCI-779, with improved pharmaceutical properties and comparable efficacy was approved in phase I and II of clinical studies; phase III trials are in progress (39). However, inhibitors of mTOR may not block all the functions of PI3K/Akt pathway because they only affect one of the many downstream pathways of PI3K/Akt signaling. While new reagents targeting this pathway are being developed and tested, perhaps, consideration should be given to targeting Id-1 as an alternative strategy in cancer therapy since evidence to date indicates that Id-1 has multiple effects on tumor progression including tumor growth, invasion, angiogenesis, metastasis and drug resistance. This notion is supported by a study showing that systemically targeting Id-1 expression using antisense Id-1 cDNA reduced the metastatic spread of 4T1 breast cancer cells in syngeneic BALB/c mice (5). From a therapeutic standpoint, since Id-1 is over-expressed in many types of human cancer but present at very low levels in normal adult tissues (2), inhibition of Id-1 should have very little side effects on normal tissues. Furthermore, with the development of improved delivery systems in RNA interference technology and the recent success in application of therapeutic siRNA in non-human primates (40), RNAi-based therapeutic reagents targeting Id-1 may be a promising alternative or adjunct to cytotoxic chemotherapy for esophageal cancer.

	Funding

Top Abstract Introduction Materials and methods Results Discussion Funding References

 
The University of Hong Kong University Research Committee Seed Funding Programme for Basic Research (No. 200511159063); the University of Hong Kong Small Project Funding (No. 200607176143); and partly by the Research Grants Council of the Hong Kong Special Administrative Region, China (Central Allocation Project No. HKUST 2/06C).

	Acknowledgments

 
We thank Prof. G. Srivastava (Department of Pathology, The University of Hong Kong) for providing the HKESC-3 cell line, and Ms Alla Li for excellent technical assistance.

Conflict of Interest Statement: None declared.

	References

Top Abstract Introduction Materials and methods Results Discussion Funding References

 

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Received March 9, 2007; revised May 4, 2007; accepted June 22, 2007.

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