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P27Kip1, regulated by glycogen synthase kinase-3β, results in HMBA-induced differentiation of human gastric cancer cells
© Wei et al; licensee BioMed Central Ltd. 2011
Received: 23 January 2011
Accepted: 27 March 2011
Published: 27 March 2011
Gastric cancer is the second most common cause of global cancer-related mortality. Although dedifferentiation predicts poor prognosis in gastric cancer, the molecular mechanism underlying dedifferentiation, which could provide fundamental insights into tumor development and progression, has yet to be elucidated. Furthermore, the molecular mechanism underlying the effects of hexamethylene bisacetamide (HMBA), a recently discovered differentiation inducer, requires investigation and there are no reported studies concerning the effect of HMBA on gastric cancer.
Based on the results of FACS analysis, the levels of proteins involved in the cell cycle or apoptosis were determined using western blotting after single treatments and sequential combinations of HMBA and LiCl. GSK-3β and proton pump were investigated by western blotting after up-regulating Akt expression by Ad-Akt infection. To investigate the effects of HMBA on protein localization and the activities of GSK-3β, CDK2 and CDK4, kinase assays, immunoprecipitation and western blotting were performed. In addition, northern blotting and RNase protection assays were carried out to determine the functional concentration of HMBA.
HMBA increased p27Kip1 expression and induced cell cycle arrest associated with gastric epithelial cell differentiation. In addition, treating gastric-derived cells with HMBA induced G0/G1 arrest and up-regulation of the proton pump, a marker of gastric cancer differentiation. Moreover, treatment with HMBA increased the expression and activity of GSK-3β in the nucleus but not the cytosol. HMBA decreased CDK2 activity and induced p27Kip1 expression, which could be rescued by inhibition of GSK-3β. Furthermore, HMBA increased p27Kip1 binding to CDK2, and this was abolished by GSK-3β inhibition.
The results presented herein suggest that GSK-3β functions by regulating p27Kip1 assembly with CDK2, thereby playing a critical role in G0/G1 arrest associated with HMBA-induced gastric epithelial cell differentiation.
Gastric cancer is one of the most common cancers in the world and often develops resistance to chemotherapy and radiation treatments. Therefore, combination therapy has been proposed to tackle the disease better and to reduce the probability of developing resistance . Hexamethylene bisacetamide (HMBA), a hybrid polar compound (HPC) originally developed as a differentiation-inducing agent [2–6], causes gastric cell re-differentiation [7–9].
In the stomach, stem cells in the proliferative cell zone of the isthmus region of the gastric glands differentiate and give rise to various cell types [10, 11]. Once the first tumorigenic event takes place, further tumor progression depends on the nature of the initiating event and the developmental stage of the cell that sustained it and additional mutations that could occur. Constant proliferation is a vital feature of stem cells, and in gastrointestinal tissues mutations are likely to result in expansion of altered stem cells, increasing the probability of additional mutations and tumor progression . Therefore, targeting gastric cancer stem cells is likely to be the most effective way of treating gastric cancer. Approximately 50% of the western population develops metaplasia, a key step in cancer development , drawing attention to pathways that control proliferation and thereby cell differentiation. Among these, the TGF-b, Myb, Wnt and Hedgehog pathways are of particular relevance, featuring prominently in cell-fate specification and pattern formation during embryogenesis and adult tissue renewal. The elucidation of complex tumor suppressor and accelerator signaling pathways, which effect differentiation modulation of transitional/progenitor cells, will be pivotal for optimization of therapeutics to treat gastric cancer.
In order for immature gastric cells to differentiate, they require to stay in the G1 phase of the cell cycle for a certain time period. The mammalian cell cycle is regulated by sequential activation and inactivation of a highly conserved family of cyclin dependent kinases (CDKs); progression through early to mid-G1 is dependent on CDK4 and possibly CDK6, while progression through late G1 and the S phase requires activation of CDK2. The activities of CDKs can be inhibited by the binding of CDK inhibitory proteins including the Cip/Kip family (p21Waf1, p27Kip1 and p57Kip2) and INK4 family (p15Ink4b, p16Ink4a, p18Ink4c and p19Ink4d). P27Kip1 is regulated post-transcriptionally by proteolytic degradation. CDK2 binds to p27Kip1 and phosphorylates it on threonine 187 , and HMBA-induced gastric cell differentiation is associated with the up-regulation of p27Kip1 [15, 16] and G0/G1 arrest. However, there are few detailed studies concerning the molecular mechanism of HMBA and there have been no reported studies investigating the effect of HMBA on gastric cancer.
As a downstream target of the phosphatidylinositol-3 kinase/Akt (PI3-kinase/Akt) pathway, GSK-3β regulates cell proliferation and differentiation [17–20]. Accumulating evidence indicates that hypoactive GSK3β signaling, which functions in G1 to receive input from several signaling and developmental pathways, occurs in association with diverse human cancers [21, 22]. GSK-3β has been implicated in multiple biological processes because it phosphorylates a broad range of substrates including several differentiation checkpoints including c-Myc, snail and PI3K . Previously, inhibition of the PI3-kinase pathway was shown to enhance HMBA-mediated gastric cell differentiation . In this study, the role of GSK-3β during gastric cell differentiation was investigated using the human gastric cancer cell line SGC7901, which displays a multipotent phenotype and represents a well-characterized model of gastric differentiation. The results suggest a contributory role for GSK-3β in the p27Kip1 pathway during gastric cell differentiation induced by HMBA.
The gastric cancer cell line SGC7901 was obtained from the cell bank in the Chinese Academy of Sciences and cultured as previously described . SGC7901 cells were infected with an adenovirus encoding the activated form of Akt (Ad-Akt) or the adenoviral control vector encoding β-galactosidase (β-gal) at a multiplicity of infection (MOI) of 10 pfu/cell. After infection with vectors for one hour, followed by replacement of medium and incubation for a further 24 h, cells were treated in the presence or absence of HMBA, and protein and RNA were extracted for western and Northern blotting, respectively.
HMBA, TSA, SB-415286 and LiCl were purchased from Sigma Chemical Company, USA. Adenovirus vectors encoding β-gal and the myristoylated active form of Akt (Ad-Akt) were purchased from Cell BioLabs, USA. The vector encoding the catalytically active mutant of GSK3β (HA-GSK-3βCA) was purchased from Addgene. Non-targeting control siRNA and the SMARTpool for targeting GSK-3β were purchased from Dharmacon, USA. All probes were labeled with a Biotin Random Prime DNA Labeling Kit (Pierce).
Rabbit anti-Akt, anti-phospho-Akt (Ser473) and antiphospho-GSK-3β (Ser9) were purchased from Cell Signaling, USA. Mouse anti-GSK-3β, mouse anti-p27Kip1, mouse anti-Top IIb and mouse anti-p21 Waf1 were purchased from BD Biosciences, USA. Mouse anti-phospho-GSK-3 (Tyr278/Tyr216) was obtained from Upstate, USA. Rabbit anti-cleaved PARP antibody was purchased from Abcam, USA. Anti-proton pump was bought from MBL International (USA). Polyclonal anti-CDK2, anti-CDK4, anti-α-tubulin and anti-caspase-3 were obtained from Santa Cruz Biotechnology (USA).
Sub-cellular protein extraction and western blotting
Nuclear and cytosolic fractions were extracted using a NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce, USA). Cytosolic protein (80 mg) or nuclear protein (20 mg) was resolved on a 10% polyacrylamide gel and transferred to PVDF membranes as previously described . Filters were incubated for one hour at room temperature in blotting solution. Membranes were incubated overnight at 4°C with primary antibodies followed by blotting with a horseradish peroxidase-conjugated secondary antibody for one hour, and visualized using an ECL detection system.
Northern blotting and RNase protection assays (RPA)
Total RNA was prepared using TRIzol reagent (Invitrogen). Samples were run on 1.2% agarose/formaldehyde gels and transferred to supported nitrocellulose. Membranes were hybridized with a biotin labeled gastric proton pump cDNA probe. After hybridization with the GAPDH probe, a loading control, membranes were washed and signals were detected using an ECL detection system. RNase protection experiments were performed using the RPA-III kit from Ambion and the RiboQuant MultiProbe RNase Protection Assay System was utilized to detect multiple specific mRNAs. 32P-labeled antisense RNA probes were prepared using the Human Apoptosis hCC-2 and hCYC-1 Template Sets and hybridizations were performed according to the manufacturer's protocol.
Cell cycle analysis
Gastric cancer cells were harvested using trypsin. Cells were collected, washed twice with ice-cold PBS and fixed in ice-cold 70% ethanol. After being washed twice with ice-cold PBS, resuspended in PBS containing 100 U/ml RNase A and incubated at 37°C for 30 min, cells were stained with PI (20 mg/ml) and analyzed using FACScan (Becton Dickinson, San Jose, CA, USA), as previously described .
In vitrokinase assays
The activities of CDK2, CDK4 and GSK-3β were measured as previously described [26, 27]. Briefly, CDK2, CDK4 or GSK-3β was immunoprecipitated from cytosolic (100 mg of protein) or nuclear (25 mg of protein) extracts. Kinase activity was measured by incubating immunoprecipitated CDK2, CDK4 or GSK-3β in 40 ml of kinase buffer with 4 mg recombinant Snail protein (to measure GSK-3β-associated kinase activity), 5 mg of histone H1 (to measure CDK2-associated kinase activity) or retinoblastoma protein (to measure CDK4-associated kinase activity) at 30°C for 30 min. The samples were processed as described in previous reports .
Inhibition of GSK-3β attenuates HMBA-induced cell cycle arrest and SGC7901 cell differentiation
Akt regulates gastric differentiation induced by HMBA
Treatment with HMBA increased the expression and activity of GSK-3β in the nucleus
Inhibition of GSK-3β overrides HMBA-induced CDK2 inhibition
GSK-3β regulates nuclear p27Kip1protein expression
GSK-3β regulates p27Kip1binding to CDK2
The PI3-kinase/Akt/GSK-3β signaling pathway has been implicated in the regulation of cell growth, apoptosis and differentiation of various cell types . In the present study, inhibition of GSK-3β using complementary approaches (i.e. chemical inhibition and constitutively active Akt over-expression) attenuated proton pump expression, a measure of gastric-like differentiation, in the gastric tumor-derived SGC7901 cell line.
Cell proliferation and differentiation are traditionally perceived as reciprocal processes, with cell-cycle withdrawal being required for terminal differentiation , and P27Kip1 playing an important role [41, 42]. Genetic deletion of p27 but not p21 has been shown to affect gastric cell differentiation, whereas forced p27Kip1 expression leads to differentiation, suggesting that p27Kip1 is more important than p21Waf1 in the regulation of gastric cell differentiation. In agreement with these findings, inhibition of GSK-3β attenuated HMBA-mediated gastric cell differentiation and inhibition of GSK-3β blocked HMBA-mediated nuclear p27Kip1 expression, while over-expression of the active form of GSK-3β increased nuclear p27Kip1 expression, suggesting an important role in the regulation of gastric cell differentiation through the regulation of nuclear p27Kip1 expression. Previous studies have demonstrated that inhibition of the PI3-kinase pathway or over-expression of PTEN increases p27Kip1 levels and enhances cell differentiation [43, 44]. Consistent with the results presented herein, inactivation of GSK-3β using LiCl results in down-regulation of p27Kip1 and cell cycle progression in primary human T cells .
HPC decreases the phosphorylation of Akt [8, 47], and therefore decreases the phosphorylation of GSK-3β at Ser9. Ser9 phosphorylation of GSK-3β decreases GSK-3β activity, whereas Tyr216 phosphorylation increases GSK-3β activity . In the present study, HPC decreased Akt phosphorylation significantly in the cytosol but only a minor effect was noted in the nucleus. These results suggest that the induction of GSK-3β activity in the nucleus may not be due to dephosphorylated Akt, which should result in the dephosphorylation of GSK-3β at Ser9, as treating SGC7901 cells with HPC increased GSK-3β phosphorylation at Ser9. HPC increased total GSK-3β protein expression and GSK-3β phosphorylation at Tyr216, but the mechanisms underlying GSK-3β activation in the nucleus by HPC, which could involve novel pathways or additional regulatory elements for GSK-3β, remain to be elucidated.
Nuclear GSK-3β expression is related to cell cycle progression . GSK-3β is predominantly cytoplasmic during the G1 phase , but a significant fraction enters the nucleus during the S phase [49, 50]. HMBA increased expression and activity of nuclear GSK-3β and induced G1 cell cycle arrest, which was attenuated by GSK-3β inhibition. These results demonstrate a role for GSK-3β in the modulation of G1 cell cycle progression. In addition to attenuation of G1 arrest, treatment with GSK-3β inhibitors increased the cell population in the G2/M phase. GSK-3β may regulate G2/M through the regulation of Chk1 phosphorylation, an important regulator of the G2/M checkpoint [51, 52], as SB-415286 and LiCl enhance Chk1 phosphorylation and G2/M arrest by etoposide. Fluxes in levels of GSK-3β in the nucleus at critical periods could be related to the well-documented capacity of nuclear GSK-3β to activate NF-κB [53, 54]. Inhibition of NF-κB increases the percentage of cancer cells in the G2/M phase . Nuclear GSK-3β phosphorylates cyclin D1, resulting in the export of cyclin D1 from the nucleus [56, 57]. Cyclin D1 plays distinct roles in cell cycle progression through the G1 phase . Collectively, these results reveal an important role for GSK-3β in the regulation of p27Kip1 nuclear localization and CDK2 inhibitory functions on G1/S progression.
The GSK-3β-dependent ubiquitination pathway could serve as a post-transcriptional mechanism regulating the stability of p27Kip1 and its binding to CDK2 in the nucleus. GSK-3β phosphorylates p27Kip1 at S160 and S161, resulting in increased p27Kip1 stability . Therefore, HMBA treatment could result in the phosphorylation of p27Kip1 at S160 and S161 in the nucleus through GSK-3β activation, leading to nuclear p27Kip1 accumulation and increased p27Kip1 binding to CKD2. In conclusion, the present study supports a contributory role of the PI3-kinase/Akt/GSK-3β pathway in the differentiation process of gastric cells. Importantly, the data demonstrated that nuclear GSK-3β increased nuclear p27Kip1 accumulation and p27Kip1 binding to CDK2. Inhibition of CDK2 contributed to HMBA-mediated G1 cell cycle arrest and subsequently to HMBA-mediated gastric cell differentiation.
The present study identified a novel mechanism whereby GSK-3β affects nuclear p27Kip1 proteolysis, and showed that it participates in the regulation of cell cycle progression and cell differentiation in gastric cells induced by HMBA treatment.
This study was supported by the National High Technology Research and Development Program of China (863 Program No. 2006AA02A301 and No. 2007AA02Z179), Science and Technology Commission of Shanghai Municipality (No. 07jc14041, 09DZ1950100), National Natural Science Foundation (No. 30471961, 30772107, 30670939 and 30872476), and Key Projects in the National Science & Technology Pillar Program (No. 2008BA152B03), Shanghai Leading Discipline-Surgery (S30204), Shanghai Key Laboratory of Gastric Neoplasms (09DZ2260200). There are no potential conflicts of interest.
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