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First published online 12 February 2008
doi: 10.1242/jcs.013029

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Mechanisms underlying p53 regulation of PIK3CA transcription in ovarian surface epithelium and in ovarian cancer

¹ Department of Obstetrics and Gynecology
² Centre for Molecular Medicine and Therapeutics
³ Laboratory for Oncogenomic Research, Department of Pediatrics, CFRI, University of British Columbia, Vancouver BC V6H3V5, Canada
⁴ AstraZeneca, Macclesfield, Cheshire, UK
⁵ Department of Molecular Therapeutics, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA

View larger version (62K): [in this window] [in a new window]   Fig. 1. p53 induction and overexpression cause a decrease in p110 protein levels. (A) Western blot analyses of a panel of cells subjected to 10 Gy -irradiation show that p21 and PTEN levels increased, and p110 levels decreased as p53 became activated in three primary OSE cultures (OSE 457, OSE 384 and OSE 392), in the ovarian cancer cell line (A2780) and in the breast cancer cell line (MCF7), all of which express endogenous wild-type p53. By contrast, in the OVCAR3 ovarian cancer cell line, which contains a loss-of-function p53 mutation, no decrease in p110 was observed. PTEN levels were again increased over time in OVCAR3 cells. (B) Western blot analyses of OVCAR4, A2780 and MCF7 cells with endogenous wild-type p53, and SKOV3, CaOV3, OVCAR3, OVCAR5 and OVCAR8 cells with mutated p53 (loss of function) show that overexpression of wild-type p53 via infection with an adenoviral vector resulted in a decrease in p110 levels 48 hours post-infection in all lines. Adenoviral GFP was used as control. Details of the cell lines, including mutations in the cancer lines, are listed in Tables 1 and 2.

View larger version (53K): [in this window] [in a new window]   Fig. 2. Conditional activation of p53 causes a decrease in p110 protein and PIK3CA transcript levels. (A) Western blot analyses of ts OSEC2, IOSE 166h and IOSE 166a cells show that, after a switch from 34 to 39°C, p53 became functional, p21 and PTEN levels increased, whereas p110 levels decreased. IOSE lines with non-functional p53 (IOSE 80pc and IOSE 397) and WI38 cells with wild-type p53 were used as controls to rule out direct effects of temperature on p110 levels. There was no decrease in p110 levels with the increase in temperature in these non-ts control cells. WI38 cells expressed low levels of p110 protein; these levels did not change in response to increased temperature. Furthermore, in the control cells, PTEN levels remained unchanged (IOSE 80pc) or decreased (IOSE 397 and WI38) at 39°C. +/–indicate functional/non-functional SV40 TAg or p53. (B) Real-time quantitative RT-PCR of the cells shown in A demonstrates that, upon temperature shift to 39°C, which results in p53 activation, PIK3CA transcript levels significantly decreased in the ts lines (OSEC2, IOSE 166h, IOSE 166a). PIK3CA transcript levels increased at 39°C in non-ts control cells (IOSE 80pc, IOSE 397 and WI38). At 34°C, at which p53 remained inactive, all the ts cell lines had higher PIK3CA transcript levels on day 5 at 34°C compared to day 1 at 34°C. The expression level of the ribosomal RNA gene was used as control.

View larger version (67K): [in this window] [in a new window]   Fig. 3. Conditional activation of p53 causes a decline in PI3K activity. (A) Western blot analyses demonstrate maximum AKT phosphorylation in OSEC2 cells after 60 minutes, and in IOSE 166h and IOSE 166a cells after 30 minutes, of stimulation with 10% FBS following serum starvation (0.5% FBS). Phosphorylated AKT (P-AKT) levels were lower at 39°C compared with at 34°C. (B) Western blots of OSEC2, IOSE 166h and IOSE 166a cells show that AKT-P levels decreased after a shift to 39°C, whereas total AKT (T-AKT) levels remained unchanged.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Conditional activation of p53 causes a reduction in AKT membrane localization. (A) OSEC2 cells at 34°C in the absence of serum stimulation did not show membrane localization of AKT-PH-GFP protein. Upon serum stimulation (10% FBS) at 34°C, the AKT-PH-GFP protein was targeted to the membrane, indicative of increased PI3K activity in the absence of functional p53. When serum-stimulated OSEC2 cells at 34°C were treated with 20 µM of the PI3K inhibitor LY294002 for 1 hour, membrane localization was minimal. OSEC2 cells with functional p53 at 39°C did not demonstrate membrane localization in either the absence or presence of serum stimulation. Similar to the OSEC2 cells, IOSE 166h (B) and IOSE 166a (C) cells showed a significant reduction in AKT-PH-GFP membrane localization at 39°C compared with at 34°C after serum stimulation. (D) Between 300 and 400 cells per group in each cell line (OSEC2, IOSE 166h and IOSE 166a) in the preparations shown in A, B and C were grouped by percentage into three categories: strong, weak and no membrane localization (ML). Only with serum stimulation at 34°C was there a highly significant increase in the percentage of cells with strong membrane localization (P<0.001). Statistical analysis was by one-way ANOVA test (Tukey).

View larger version (48K): [in this window] [in a new window]   Fig. 5. Conditional activation of p53 causes decreased proliferation and increased apoptosis. (A) Ki-67 immunofluorescence of OSEC2 cells demonstrates that, on day 1, OSEC2 cells at 34°C had many positively stained nuclei (red), whereas OSEC2 cells at 39°C had decreased numbers of Ki-67-positive nuclei and, thus, had decreased proliferation. Ki-67 staining of non-ts IOSE 397 and WI38 cells did not show the same pattern: with the switch in temperature from 34 to 39°C, the number of Ki-67-positive nuclei in the IOSE 397 cultures increased, whereas the number of positive nuclei in WI38 cells remained unchanged. (B) Cell death detection ELISA to quantify levels of apoptosis of the cells demonstrated that the percentage of apoptotic OSEC2 cells at 39°C was significantly higher than at 34°C. Treatment of cells at 34°C with LY294002 also resulted in increased apoptosis.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Identification of two alternate upstream PIK3CA exons. 5' RACE analysis was used to determine the presence of upstream exons. (A) A 1:100 dilution of the primary PCR product of the tailed cDNA was amplified with GSP3 and AUAP primers, demonstrating the presence of a single band of approximately 500 bp in size. The diluted PCR product of no tail cDNA amplified with GSP3 and AUAP was the negative control. (B) The presence of putative exon1b was confirmed using a 1:100 dilution of the primary PCR product of the tailed cDNA amplified with GSP3 and GSP5, resulting in a 442 bp band. GSP3 binds to exon2(1), whereas GSP6 and GSP5 bind to exons1a and 1b, respectively. GSP3 and GSP6 were used as positive control (429 bp) to amplify the newly identified exon1a. (C) A diagram of the first three exons of PIK3CA showing the nucleotide positions of the newly identified exons 1a and 1b, thereby demonstrating their nucleotide distance from exon2(1). The first nucleotide of exon1a is marked as 1 and the positions of the first and last nucleotides of the first three exons [1a, 1b and 2(1)] are accordingly labeled. The exons are indicated by the black boxes labeled 1a, 1b and 2(1); the introns are indicated by the horizontal lines connecting them. Exon2(1) [coordinates, chr3:180,399,240-180,399,668 (human genome assembly 17)] contains the translational start site (arrow). Exon1a [coordinates, chr3:180,348,661-180,348,709 (human genome assembly 17)] is in the 5' UTR and is 50,579 bp upstream of exon2(1). Exon1b [coordinates, chr3:180,349,013-180,349,093 (human genome assembly 17)] is also in the 5' UTR and is 50,227 bp upstream of exon2(1). Exon1a and exon1b are highly conserved, and their first nucleotides are 352 bp apart. (D) A diagram showing that there are two alternate PIK3CA transcripts. Exons 1a and 1b splice alternatively to exon2(1). (E) Phylogenetic footprinting and p53-binding-site analysis of promoter-proximal sequences. p53 interacts with DNA through binding to two tandem copies of a well-defined half-site. The PIK3CA promoter region was analyzed with two half-site filters to predict candidate binding sites. The specific profile (lines) requires the presence of two nucleotides at critical positions within the recognition sequence (CnnG). Because empirical observation suggests that some bonafide p53 target sequences diverge from the constrain in one of the two half-sites, the sensitive profile (circles) requires only one perfect match to the core sequences. The locations of the oligonucleotides used in mobility shift assays and the amplified PCR products from the ChIP experiments are indicated.

View larger version (49K): [in this window] [in a new window]   Fig. 7. p53 binds to the PIK3CA promoter and suppresses its activity. (A) ChIP results for OSEC2 cells. Antibodies used for immunoprecipitation (IP) are indicated below the panels. OSEC2 cells at 34°C have reduced p53 activity, whereas the cells at 39°C have full activity. PCR amplification with GAPDH primers of samples immunoprecipitated with RNA polymerase II antibody is presented as a positive control. Input DNA acts as control for levels of DNA present in each sample. Amplification with primers around the known p53-binding sites on the p21 promoter were used as positive controls. (Ba) An electrophoretically retarded complex (shift) was formed with recombinant p53 and biotin-labeled oligo4. Formation of this complex was inhibited with an excess of unlabeled wild-type oligo4. However, the unlabeled mutant oligo4, with point mutations in the core (CnnG) binding region, did not compete with the biotin-labeled oligo4 to the same extent. In addition, the biotin-labeled mutant oligo4 showed reduced interaction with p53 compared to wild-type oligo4. (Bb) Similarly to the recombinant p53, the use of nuclear lysates from OSEC2 cells at 39°C with p53 resulted in the formation of an electrophoretically retarded complex, which was competed with excess unlabeled wild-type oligo4. Similarly, the unlabeled mutant oligo4 did not compete with the biotin-labeled oligo4 to the same extent; and the biotin-labeled mutant oligo4 showed reduced interaction with p53 compared with the wild-type oligo4. The use of nuclear lysates from OSEC2 cells at 34°C without p53 did not lead to formation of an electrophoretically retarded complex (shift). Addition of an anti-p53 antibody (PAb421) to the reaction mixture induced a supershift of the protein-DNA complex, indicating the specificity of oligonucleotide for p53. (C) OSEC2 cells transfected transiently with promoter1a construct (pGL3-P1A) showed 83% less luminescence (PIK3CA-promoter activity) at 39°C compared with at 34°C. pGL3-P1A-mut4 construct showed significantly less decrease in promoter activity after the switch in temperature to 39°C (less than 50% decrease). pGL3-control was used as positive control and pGL3-basic was used as negative control. The values presented were normalized with the internal control (phRL-TK).

View larger version (13K): [in this window] [in a new window]   Fig. 8. The p53 and PI3K pathways regulate one another. p53 positively regulates PTEN levels, which reverse PI3K action by dephosphorylating PtdIns(3,4,5)P3 (Stambolic et al., 2001). Our contribution to this figure is in bold with an asterisk: this study demonstrates that p53 directly regulates PIK3CA transcription, resulting in decreased p110 and thus PtdIns(3,4,5)P3 levels. PtdIns(3,4,5)P3 recruits AKT to the membrane, where AKT becomes phosphorylated and activated. In turn, activated AKT phosphorylates MDM2, which leads to the nuclear import of MDM2. In the nucleus, MDM2 binds to and degrades p53 (Zhou et al., 2001).

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