Phosphatidylinositol 3'-kinase, mTOR, and Glycogen synthase kinase-3β mediated regulation of p21 in human urothelial carcinoma cells
© Yohn et al; licensee BioMed Central Ltd. 2011
Received: 11 March 2011
Accepted: 24 August 2011
Published: 24 August 2011
The PTEN/Phosphatidylinositol 3'-kinase (PI3-kinase) growth factor signaling pathway plays a critical role in epithelial tumor development in a multitude of tissue types. Deletion of the Pten tumor suppressor gene in murine urothelial cells in vivo results in upregulation of cyclin-dependent kinase inhibitor p21. We have previously shown in mice that p21 expression blocks an increase in urothelial cell proliferation due to Pten deletion. In this study, we utilized human urothelial carcinoma cells UMUC-3 and UMUC-14 to identify the signaling pathways downstream of PI3-kinase that regulate p21.
Cells were treated with a combination of PI3-kinase stimulating growth factors and kinase inhibitors, or transfected with exogenous genes in order to identify the signaling events that are necessary for p21 induction. Mice with conditional deletion of Pten in bladder urothelium were also examined for evidence of PI3-kinase pathway signaling events that affect p21 expression.
When cells were treated with PI3-kinase activating growth factors EGF or PDGF, we found that p21 levels increased, in a manner similar to that observed in mice. We used the inhibitors LY294002, Akti-1/2, and rapamycin, to show that p21 induction is dependent upon PI3-kinase and AKT activity, and partially dependent on mTOR. We treated the cells with proteasome inhibitor MG-132 and found that p21 may be degraded in the proteasome to regulate protein levels. Importantly, our findings show that GSK-3β plays a role in diminishing p21 levels in cells. Treatment of cells with the GSK-3β inhibitor SB-216763 increased p21 levels, while exogenous expression of GSK-3β caused a decrease in p21, indicating that GSK-3β actively reduces p21 levels. We found that a combined treatment of LY294002 and SB-216763 improved the cytotoxic effect against UMUC-3 and UMUC-14 carcinoma cells over LY294002 alone, suggesting potential therapeutic uses for GSK-3β inhibitors. Immunohistochemical staining in bladders from wild-type and Pten-deleted mice indicated that GSK-3β inhibitory phosphorylation increases when Pten is deleted.
PI3-kinase and AKT cause an upregulation of p21 by suppressing GSK-3β activity and activating mTOR in both cultured human urothelial carcinoma cells and mouse urothelial cells in vivo.
It has been well established that the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) gene plays an important role in suppressing tumor development in multiple human cell types and organs such as the endometrium, brain, skin, and prostate . Studies in the last few years have shown that PTEN mutation is also associated with bladder cancer [2, 3]. Multiple studies utilizing tissue microarray analysis and immunohistochemistry have shown that PTEN expression is diminished in late bladder cancers of higher tumor stage and grade [4–7]. Screens of human bladder cancer cell lines have also revealed that PTEN expression is often lost [8–10]. Exogenous expression of PTEN in bladder cancer cells results in decreased invasiveness , providing an explanation for why PTEN loss in advanced cancers is common. The finding that PTEN expression is reduced in bladder cancer is consistent with PTEN's known functions not only in inhibiting cell migration but also in suppressing cell proliferation and apoptosis, as well as maintaining genomic integrity .
The principal manner in which PTEN appears to suppress cell growth is through its lipid phosphatase activity . PTEN removes the phosphate from the D3 position of phosphatidylinositol-3,4,5-trisphosphate (PIP3) to generate phosphatidylinositol-4,5-bisphosphate (PIP2) . The reverse reaction is catalyzed by Class I phosphatidylinositol 3-kinases (PI3-kinases) in response to activation by receptor tyrosine kinases and G-protein coupled receptors . PIP3 in the plasma membrane generated by PI3-kinase leads to the recruitment and activation of the AKT serine/threonine kinase . AKT in turn phosphorylates numerous substrates that lead to cell proliferation, growth, and survival. One known substrate of AKT is glycogen synthase kinase-3 beta (GSK-3β) , a serine-threonine kinase that plays an important role in insulin signaling. Phosphorylation of GSK-3β by AKT inactivates its kinase activity . There is a second isoform of glycogen synthase kinase called GSK-3α that is also inhibited by phosphorylation by AKT, but its function is less clear . Another important kinase that is activated downstream of AKT is mTOR; it mediates an increase in protein synthesis and cell growth, among other functions .
In a previous study, we generated mice in which Pten was conditionally deleted in bladder urothelium in order to study the effects on tumorigenesis and PI3-kinase signaling . Notably, we found that the cyclin-dependent kinase p21 was consistently upregulated in the PTEN-deficient cells. As in normal urothelial cells, the p21 remained in the nucleus, indicating that activation of the PI3-kinase pathway was not leading to relocalization of p21 to the cytoplasm . Other subsequent studies in the liver  and in kidney cells  have also shown that activation of the PI3-kinase/AKT pathway and PTEN knockdown lead to an increase in p21 levels.
This increase in p21 levels is important because it suppresses bladder urothelial proliferation elicited by the Pten deletion , and may contribute to reduced tumorigenesis in the bladder. The p21 protein inhibits cell proliferation by functioning as a cyclin-dependent kinase inhibitor , and p21 exhibits tumor suppressor functions as shown by the finding that p21-/- 129Sv/C57Bl6 mice develop spontaneous tumors at 16 months of age . Immunohistochemical studies of human tumors suggest that the p21 induction we observed in mice may occur in human bladder cells as well, since p21 levels are increased in bladder tumors [25, 26] and in transitional cell carcinoma cell lines  compared to normal urothelium. Importantly, bladder tumors with increased grade [25, 28] and/or stage [25, 29] have reduced p21 levels compared to lower grade or stage noninvasive tumors, suggesting that p21 expression is selectively lost in advanced tumors. Furthermore, tumors that have lost p21 expression are associated with decreased probability of survival .
In many cell types, PI3-kinase/AKT signaling leads to increased cell proliferation, so the fact that it induces p21 and inhibits cell proliferation in bladder urothelial cells is surprising. The commonly accepted model that PI3-kinase/AKT signaling induces cell cycle progression does not apply to urothelial cells. Understanding how urothelial cells will respond to stimulation or inhibition of this signaling pathway is important for tailoring therapy for tumors originating from the urothelium. We therefore aimed to elucidate the mechanism by which PI3-kinase/AKT signaling leads to p21 increase in human urothelial cells.
Human UMUC-3 urinary bladder transitional cell carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were passaged in DMEM (Hyclone) supplemented with 10% newborn calf serum (Hyclone), penicillin (100 U/ml) and streptomycin (100 mg/L) (Sigma) in a humidified incubator containing 5% CO2 and maintained at 37°C. Human UMUC-14 urothelial carcinoma cells were generously provided by Herbert Grossman (MD Anderson Cancer Center). These cells were maintained in DMEM with 10% fetal calf serum (Hyclone) and penicillin (100 U/mL)/streptomycin (100 mg/L).
PDGF-BB and EGF were obtained from Peprotech (Rocky Hill, NJ). LY294002, MG-132, and SB-216763 were purchased from Enzo Life Sciences (Plymouth Meeting, PA). Akti-1/2 was purchased from EMD Biosciences (San Diego, CA). Rapamycin was obtained from LKT Labs (St. Paul, MN). The monoclonal p21 antibody was obtained from BD Biosciences (San Diego, CA). The phospho-AKT ser 473, GAPDH, β-catenin, GSK-3α, GSK-3β, and phospho-GSK-3 α/β ser 9/21 antibodies were purchased from Cell Signaling Technology (Danvers, MA). The 12G10 α- tubulin and α-actin antibodies were obtained from the Developmental Studies Hybridoma Bank (University of Iowa). The anti-phospho-Histone H3 antibody was obtained from Upstate (Lake Placid, NY). The mammalian expression vectors GSK3 alpha pMT2 (15896) and HA GSK3 beta wt pcDNA3 (14753) were purchased from Addgene (Cambridge, MA). siRNA (siGENOME smartpool) was purchased from Dharmacon (Lafayette, CO).
Cells were treated as described and then washed in phosphate-buffered saline. Cells were lysed in ice-cold RIPA buffer (50 mM Tris pH 7.5, 1% NP-40, 150 mM NaCl, 1 mM EDTA pH8, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium fluoride). After centrifugation at 13,000 rpm for 5 min, supernatants were isolated, and an aliquot was measured for protein concentration using a BCA Assay (Pierce). The lysates were mixed in a 1:1 ratio with 2 × electrophoresis sample buffer (125 mM Tris-Cl pH 6.8, 20% glycerol, 4% SDS, 10% beta mercaptoethanol, 0.0025% bromophenol blue). Approximately 25 μg of protein from each sample was loaded into each well for SDS-PAGE, and the proteins were transferred to nitrocellulose membranes (Protran). Membranes were rocked for 1 h at room temperature in blocking buffer (3% nonfat milk dissolved in TBS-T: 50 mM Tris pH 8, 137.5 mM NaCl, 2.7 mM KCl, 0.08% Tween 20). Membranes were incubated with primary antibody diluted in either 3% milk/TBS-T (tubulin antibody) or 5% BSA/TBS-T (all other antibodies) for 1 h at room temperature. Then membranes were incubated with goat secondary antibody conjugated to HRP (Jackson ImmunoResearch Labs) diluted in 3% milk/TBS-T for 1 h at room temperature. Finally blots were washed in TBS-T, exposed to chemiluminescent substrate (Immobilon) and visualized on an imaging system (AlphaInnotech Fluorchem). Spot densitometry quantitation was performed, by subtracting background signal from each band of interest and dividing that value by the background-subtracted tubulin signal for the same lane, in order to normalize for protein loading.
4000 cells were plated in each well in a 96 well cell culture plate. Cells were transfected with siRNA (Dharmacon) using RNAiMAX (Invitrogen) in antibiotic-free medium.
4000 cells were plated in each well in 96 well cell culture plates. The following day, cells were treated with drugs as described in the Results. Every type of treatment was performed in quadruplicate wells. After 48 or 72 h, cell viability was measured using the Promega CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTS). 1 to 3 h after the MTS solution was added, the absorbance of the wells was measured in a plate reader at 450 nm.
All mice were maintained in a specific pathogen free animal facility at Harvard Medical School. Research was performed in accordance with guidelines and policies as set by the Harvard Medical Area Standing Committee on Animals (approval reference #02460). Mice homozygous for floxed Pten exon 5 (Pten loxp/loxp )  were crossed to mice transgenic for Fabpl-Cre . F1 Pten loxp/+ ;Fabpl-Cre+ mice were crossed with Pten loxp/loxp mice to obtain Pten loxp/loxp ;Fabpl-Cre+ mice.
Murine bladders were fixed in 4% paraformaldehyde/PBS and stored in 70% ethanol until they were embedded in paraffin and sectioned (Harvard Rodent Histopathology Core). Sections were deparaffinized and subject to 0.5% hydrogen peroxide/PBS treatment for 10 min. Antigen retrieval was accomplished by boiling sections in 0.01 M Citrate pH 6 buffer for 10 min in a microwave. Sections were blocked in 3% donkey serum/PBS/0.2% triton-X. Primary antibody was added in blocking solution for 2 h at room temperature or overnight at 4C, followed by washes and incubation with biotinylated secondary antibody for 1 h at room temperature. The Vectastain ABC system (Vector Labs) was used in conjunction with either Vector Red substrate for alkaline phosphatase or 3,3'-diaminobenzidine (DAB) substrate for peroxidase. All sections were further stained with hematoxylin, dehydrated, and mounted with Permount™.
Cells were counted, and 2 × 105 cells were plated in 60 mm dishes. The following day 2 μg of DNA was mixed with 6 μl of Fugene in Opti-MEM solution (Invitrogen). After 15 min, the mixture was dropped onto cells and left to incubate for 48 h. Cells were then washed with PBS and depending on treatment, some cells were incubated in serum-free DMEM for 24 additional hours. Cells were lysed and subject to western blotting as described above.
All statistical tests (Oneway ANOVA, Tukey-Kramer HSD, and Student's t-test) were done using JMP9 software.
It was important to see if the increased p21 in the cells affects cell proliferation, so we transfected the UMUC-3 cells with either control siRNA or p21 siRNA to knock down p21 expression. Two days after transfection, cells were washed in PBS and serum starved for 24 hours. Then EGF (10 ng/ml) was added to some of the wells for 24 hours, and an MTS assay was run to measure cell proliferation. As seen in Figure 1C, EGF treatment actually significantly decreased the number of viable cells, and knock down of p21 prevented this effect of EGF (ANOVA; p = .002), showing that the induction of p21 in response to EGF results in reduced cell proliferation.
In order to more directly test the effects of GSK-3α and β individually on p21, we transiently transfected GSK-3 expression vectors into UMUC-14 cells for 48 hours. The exogenous GSK-3β has a slightly higher molecular weight than does endogenous GSK-3β due to the presence of a C-terminal HA tag (Figure 5B). When p21 levels were tested in the presence of 10% fetal calf serum, no effects of GSK-3 expression on p21 were evident. However, when transfected cells were serum starved for 24 hours in order to minimize GSK-3 inhibition by AKT, exogenous expression of GSK-3β resulted in a massive decrease in p21 levels (Figure 5B). GSK-3α had a lesser effect but also appeared to diminish p21 levels in experimental replicates. This indicates that GSK-3β downregulates p21, but that activity is inhibited in the presence of serum and an active PI3-kinase pathway. Interestingly, β catenin levels were unaffected by exogenous GSK-3 expression (Figure 5B), which suggests that β catenin did not play a role in the induction of p21.
The ability of the GSK-3 inhibitor SB216763 to induce p21 led us to speculate that SB216763 should inhibit urothelial carcinoma cell proliferation. We further hypothesized that the combination of SB216763 with a PI3-kinase inhibitor such as LY294002 should more effectively inhibit cell proliferation and/or induce cytotoxicity than LY294002 alone. In order to test these hypotheses, we treated UMUC-3 and UMUC-14 cells with various concentrations of SB216763 in the presence or absence of LY294002. After 48 hours (for UMUC-14 cells) or 72 hours (for UMUC-3 cells), we performed an MTS assay to measure cell viability (Figure 5C). The UMUC-3 cells had a longer drug treatment because they showed relatively little cytotoxicity at 48 hours. After 72 hours, the UMUC-3 cells did not show significant decreases in viability due to treatment with LY294002 alone or SB216763 alone, even at the highest concentrations used in this assay, although there was a trend toward decreased viability for both drugs. A combination of the two drugs was most effective for cell cytotoxicity; 2 μM LY294002 plus SB216763 at any tested concentration caused significant decreases in UMUC-3 cell viability compared to 2 μM LY294002 alone (Oneway ANOVA; p < .0001). While treatment of the UMUC-14 cells with 2 μM LY294002 alone had no significant effect on cell viability compared to untreated cells, there was a dose-dependent cytotoxicity response to SB216763 alone (Oneway ANOVA; p < .0001), and there was a significant decrease in cell viability at 10 μM SB216763 (Tukey-Kramer HSD; p = .0004) and at 40 μM SB216763 (Tukey-Kramer HSD; p < .0001) compared to the untreated control cells. A combination of the two drugs was once again more effective for cytotoxicity; when 2 μM LY294002 was added to the UMUC-14 cells together with 10 μM SB216763, there was a significant further decrease in cell viability compared to LY294002 alone (Oneway ANOVA; p < .0001; Tukey-Kramer HSD; p = .0007). In addition, there was significantly more cytotoxicity at 40 μM SB216763 when comparing cells in the presence of LY294002 compared to the cells that received no LY294004 (Student's t-test; p = .02). This suggests that increased inhibition of cell viability can be attained with a combination of the LY294002 PI3-kinase inhibitor and the SB216763 GSK-3 inhibitor.
Deletion of Pten causes increased tumorigenesis in a mouse model of bladder cancer , and there is a great deal of evidence of decreased PTEN expression in human bladder carcinomas [4–7]. However, those same studies on human tumors have suggested that testing PTEN expression levels in urothelial tumors is not useful for clinical management, since it does not predict disease progression or survival. While one reason for this discrepancy may be that multiple mechanisms besides diminished PTEN lead to overstimulation of the PI3-kinase/AKT pathway , we propose that an additional consideration may be the increased expression of p21 in bladder epithelium when the PI3-kinase pathway is activated. We have now shown in both the mouse in vivo and in human urothelial cell lines that stimulation of the PI3-kinase/AKT pathway leads to an increase in p21 levels. Our studies in the mouse  and multiple studies by others in human and rat bladder cancer cell lines [37–39] have shown that induction of p21 inhibits cell proliferation. Therefore, when bladder cells acquire mutations that cause overactive PI3-kinase/AKT signaling, the ensuing p21 expression provides a protective response by suppressing proliferation, and it may delay tumorigenesis. This p21 induction from PI3-kinase signaling could partially explain why early stage and low grade bladder tumors demonstrate elevated levels of p21 [25, 26], whereas advanced tumors have reduced p21 expression [25, 28, 29]; the p21 provides a protective function and suppresses tumor growth. Future studies should examine both the PTEN/PI3-kinase pathway and p21 expression levels in human tumors simultaneously in order to more accurately predict the course of disease.
In this paper, we examined both a PTEN negative cell line, UMUC-3, and a PTEN positive cell line, UMUC-14, and found similar results in terms of mechanism of p21 induction in both cell types. This does not contradict our initial finding in mice that p21 induction occurs in the Pten deficient bladders, because in this paper, the growth factors such as EGF and PDGF may have overwhelmed PTEN's ability to counteract the PI3-kinase stimulation in the UMUC-14 cells. It is possible that the p21 levels may eventually fall again in the PTEN positive UMUC-14 cells as PTEN downregulates the PI3-kinase signaling pathway. It was our observation that in the Pten deficient animals, the p21 levels remained elevated for over a year. It is worth noting here that the UMUC-3 cells usually exhibited superior p21 induction and GSK-3 phosphorylation compared to the UMUC-14 cells, which is consistent with the PTEN negative status of UMUC-3 cells. Nevertheless, these pathways were functional in the UMUC-14 cells. Our finding that the UMUC-14 cells were initially more susceptible to LY294002 cytotoxicity than the UMUC-3 cells was surprising, since UMUC-14 cells show low levels of AKT activation (8). A recent study indicated that UMUC-14 cells were also more susceptible than UMUC-3 cells to growth inhibition by mTOR inhibitor RAD001 , hinting at a possible "addiction" to downstream signaling in the PI3-kinase/AKT pathway. In the future, these studies should be performed in primary human urothelial cells instead of cell lines, to rule out the possibility that the mechanisms observed here do not accurately reflect what occurs in normal urothelial cells. In addition, cell culture does not take into effect the possible role of differentiation in affecting p21 levels.
A fuller understanding of the mechanisms by which p21 is upregulated by PI3-kinase/AKT signaling will allow potential therapeutic intervention to increase p21 expression in tumors that have lost it. Although GSK-3β is generally considered to negatively regulate cell growth, GSK-3β inhibitors have been found to reduce colon and ovarian tumor cell growth ; these drugs may be appropriate in bladder cancer because of their effects on p21. It will be helpful to examine GSK-3β activation levels in urothelial tumors in order to assess the possible utility of GSK-3β inhibitors. This pathway should also be considered where urothelial cancer patients are treated with PI3-kinase inhibitors, since the decrease in p21 as a result of the drugs may limit the effectiveness of the therapy. Our studies suggest that combination therapy with PI3-kinase and GSK-3 inhibitors could be more effective than PI3-kinase inhibitors alone.
This is the first description of GSK-3β mediated regulation of p21 in bladder cells. We have found evidence that inhibition of GSK-3β either through activation of the PI3-kinase/AKT signaling pathway or through the use of pharmacological inhibitors of GSK-3β leads to an increase in p21 levels, while an overexpression of GSK-3β in cells with reduced PI3-kinase/AKT signaling leads to a decrease in p21. Signaling through mTOR also contributes to the induction of p21. This information will be useful in predicting outcomes and tailoring chemotherapy for bladder cancer patients.
Acknowledgements and funding
We thank Hong Wu and Junying Yuan for the mice, H. Barton Grossman for the UMUC-14 cells, and Jim Woodgett for the GSK-3 expression plasmids. We are grateful to Guim Kwon and Christine Weingart for critical reading of the manuscript and to SueJeanne Koh and Ho Jung Yoo for editorial assistance. This work was supported by the Denison University Research Foundation to LY and the Anderson/Bowen Endowment to NY, CB, and AD.
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