Two novel genes, named p63 and p73, have been discovered as members of the p53 tumor suppressor family because of their remarkable similarity with p53 structure and functional domains.1 Although the structural homology suggests that they may share similar tumor suppressor functions, the functions of these proteins are not entirely redundant because each of them can behave as a p53 agonist or antagonist, and can also perform its own unique biological tasks.2, 3 Like p53, p63 and p73 also contain 3 major domains: the TAD (trans activation domain), the DBD (DNA binding domain) and the OD (oligomerization domain).4 p63 and p73 are expressed as multiple isoforms, the products of alternative splicing, and the use of 2 different promoters. P1 promoter, upstream of exon 1, yields full-length proteins (TAp63 and TAp73, containing the TA domain), whereas P2, spanning intron 3, produces NH2-terminally truncated forms ΔNp63 and ΔNp73 (missing TAD). The latter isoforms may play a dominant negative function3 by both competition for DNA binding and oligomerization with isoforms containing the TA domain. Additional complexity to this network of protein isoforms arises from multiple splicing of the COOH terminus, skipping one or several exons.3 For instance, several COOH-terminus transcripts have been identified for p73 family members α, β, γ, δ, 3, ζ, η and ϕ. With respect to p73 activity, it depends on many factors, including expression pattern and functional crosstalk between full-length protein and other isoforms. Several reports have indicated that although p53 protein is ubiquitously expressed, p73 expression is more restricted and dependent on cell differentiation and development stage.5 Indeed, p73 has its own distinct development functions.6p73 knockout mice show site-specific development defects in hippocampus, immune system and behavior.6, 7 Moreover, ΔNp73 has been shown to inhibit neuronal apoptosis by blocking p53 proapoptotic function.8, 9 These observations suggest that p73 and ΔNp73 play an important role in development and differentiation, by protecting neuronal precursors from apoptotic death.9
Different isoforms of p53 family members can play opposite roles, depending on various conditions. Ectopic TAp73 are able to bind to p53-responsive promoters and cause p53-like functions (tumor suppressor) in human cells, whereas the N-terminally truncated ΔNp73 isoform may have an oncogenic role by antagonizing full-length TAp73 and TAp53.7 Thus, the tumor suppressor function of these proteins depends on the balance between the different isoforms expressed in different cells and tissues. Recent findings suggest that p73 may be involved in the acquisition and maintenance of the transformed phenotype. Indeed, p73 is very rarely mutated in cancer cells and often overexpressed. These observations are in accordance with a possible role of these protein in tumorigenesis.6, 10–12 Interestingly, in some tumors, this overexpression is concomitant with that of the dominant negative isoform ΔNp73, which may override the effects of the transcriptionally active isoforms explaining why, as a final effect, p73 may function as oncogenes.11
Thyroid cancer is one of the malignancies expressing ΔNp7313 and the most common endocrine malignancy.14–17 Thyroid carcinomas originating from the follicular epithelium are divided into 3 main histotypes: papillary (80–90%), follicular (5–10%) and anaplastic (1–3%) thyroid carcinomas. Rarely, a tumor can arise from the parafollicular calcitonin (CT)-secreting cells of the thyroid gland: medullary cancer (5–8%). As thyroid cancer may display different histotypes, variable differentiation degree and many genetic abnormalities, it is a suitable model for studies on human carcinogenesis and tumor progression. Genetic alterations leading to loss of function of tumor suppressors are rare in thyroid carcinomas and mainly involve p53 mutations in anaplastic and poorly differentiated thyroid carcinomas.18–20 However, inhibition of p53 tumor suppressor function by mutation-independent mechanisms is a frequent event in well-differentiated thyroid carcinomas.21 Indeed, p53 protein in thyroid cancer is inhibited by the presence of p53 family isoforms with dominant negative function, including TAp63α and ΔNp73α.13, 18, 22
Inhibition of the tumor suppressor PTEN may also contribute to thyroid tumorigenesis. PTEN is a lipid phosphatase inhibiting PI3-K/Akt pathway and PTEN activity inhibition results in Akt overactivation.23, 24 Indeed, germline inactivating mutation of PTEN causes Cowden Disease, an inherited disease characterized by the onset of several tumors including thyroid carcinomas.25, 26 In accordance with this observation, mice with targeted suppression of PTEN expression in thyroid gland develop thyroid follicular tumors.27, 28 Moreover, a reduced PTEN expression was found in ∼50% human thyroid cancer specimens.29–32 Although PTEN down regulation in thyroid cancer has been partially attributed to promoter methylation,32, 33 the mechanisms underlying this phenomenon remain to be elucidated. Conversely, upregulation of PTEN by the use of PPARγ agonists results in the reversion of epithelial-mesenchymal phenotype of poorly differentiated thyroid cancer cells.34
Interestingly, a cooperation between PTEN and p53 tumor suppressors has been described: p53 stimulates PTEN promoter activity,35–37 whereas PTEN increases p53 effect by inhibiting Mdm2-mediated degradation.38–40 In the light of these results, it is reasonable to hypothesize that the reduced PTEN expression in thyroid cancer may be due to the inhibition of p53 activity. Because one of the major mechanisms of p53 inactivation in thyroid cancer is the expression of ΔNp73α, we investigated whether the expression of this p73 isoform is able to affect PTEN expression. We found that expression of ΔNp73α is able to directly down regulate PTEN levels in thyroid cancer cells in a p53 independent manner. As a consequence, down regulation of PTEN by ΔNp73α results in decreased p53 protein levels and increased cell proliferation and resistance to apoptosis. These data unravel a novel mechanism for the role of ΔNp73α in (thyroid) cancer progression.
Material and methods
Plasmids and cell lines
pcDNA3.1-HA-TAp73α, pcDNA3.0-HA-TAp73β, pcDNA3.0-HA-ΔNp73α and pcDNA3.1-p53 were kindly provided by Dr. J. Wang (UCSD, La Jolla, CA). Full length PTENLuc, minimal PTENLuc and HEXLuc promoters were provided by Dr. G Tell (University of Udine, Udine, Italy). In these plasmids, the indicated promoters drive the transcription of the luciferase reporter gene (Luc). The pIND-TAp73α, pIND-TAp73β, pIND-ΔNp73α and pIND-p53 expression vectors were constructed using standard techniques by cloning the TAp73α, TAp73β, ΔNp73α and p53 cDNAs into the Kpn I/Not I cloning site of the ecdysone-inducible expression vector pIND (Invitrogen).
Papillary (TPC-1) and anaplastic (ARO) thyroid cancer cells were provided by Drs. A. Fusco and M. Santoro, University of Naples, Italy. ARO cells have been recently reclassified as colon cancer cells.41 Anaplastic thyroid cancer cells SW-1736 and C-643 were provided by Dr. N.E. Heldin, (University of Uppsala, Sweden). These cell lines were grown in 10% FBS RPMI 1640. Human osteosarcoma Saos-2 cells were cultured in DMEM/F12 (1/1 ratio), and NIH3T3 mouse fibroblasts were cultured in complete 10% FBS DMEM (Sigma). The inducible C643 and TPC-1 cell lines were constructed by cotransfecting the pIND-TAp73α, pIND-TAp73β, pIND-ΔNp73α and pIND-p53 expression vectors along with the pVgRXR vector (Invitrogen) and selecting cell clones in G418-zeocin-containing media. Cells were subcloned and screened for TAp73α, TAp73β, ΔNp73α and p53 protein expression by Western blot after ponasterone A induction (2.5 μM, for 24 hr). Inducible cell clones were maintained in G418 (300 μg/ml) and Zeocin (200 μg/ml).
Transfections were performed by the Fugene6 (Roche Biochemical, Basel, Switzerland) or Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. In promoter studies, the full-length PTENLuc, minimal-PTENLuc and HEXLuc constructs were cotransfected with either pcDNA3.1, pcDNA3.1-p53, pcDNA3.0-HA-TAp73α, pcDNA3.0-HA-TAp73β or pcDNA3.0-HA-ΔNp73α (DNA ratio 1:1). The transfection efficiency was normalized by cotransfecting the CMV-β-GAL plasmid, which contains the cytomegalovirus (CMV) promoter, linked to the β-galactosidase (β-GAL) gene. Cells were harvested 48 hr after transfection, and cell extracts were prepared by a standard freeze and thaw procedure. β-GAL protein levels were measured by ELISA (Amersham-Pharmacia). Luc activity was measured by a standard chemiluminescence procedure.
Cell lysates were prepared in complete RIPA and subjected to SDS-PAGE. Membranes were incubated with primary antibodies in 5% milk-TBST (1 μg/ml). HRP-conjugated secondary antibodies were used for protein detection by enhanced chemiluminescence (Pierce, Rockford, IL).
The following antibodies were used: monoclonal antibody DO-1 against the N-terminus of p53 and polyclonal anti-Mdm2 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-HA monoclonal antibody (CRP), anti-β actin monoclonal antibody (Sigma), a mixture of anti-p73 monoclonal antibodies (Ab-4) (Neomarkers), anti-phospho-Akt (Ser473) and anti-Akt polyclonal antibody (3G2) (Cell Signaling), anti-PTEN polyclonal antibody (Santa Cruz). Densitometry was performed by ImageJ Software (available at http://rsb.info.nih.gov/ij/). Numbers were expressed as arbitrary densitometric units. When indicated, numbers were also normalized for β-actin and expressed as either % of basal or % of total.
Real-time polymerase chain reaction
Total RNA (5 μg) was reverse transcribed by ThermoScript RT (Invitrogen) and oligo(dT) primers. Synthesized cDNA (0.15 μl) was then combined in a PCR reaction using primers 5′-CCC AGT CAG AGG CGC TAT GTG TAT -3′ (forward), 5′-GTT CCG CCA CTG AAC ATT GG-3′ (reverse) specific for PTEN and 5′-TGA TCC ACA TCT GCT GGA AGG T-3′ (forward) and 5′-GAC AGG ATG CAG AGG AGA TCA CT-3′ (reverse) specific for actin. Quantitative real-time PCR was done on an ABI Prism 7700 (PE Applied Biosystems, Foster City, CA) using SYBR Green PCR Master Mix (PE Applied Biosystems) following the manufacturer's instructions. Amplification reactions were checked for the presence of nonspecific products by agarose gel electrophoresis. Relative quantitative determination of target gene levels was done by comparing ΔCt.
Absolute real-time polymerase chain reaction
Calibration curves were made on known concentrations of DNA plasmids (pcDNA3.1-HA-ΔNp73α and pRV-PTEN-EGF, kindly donated by Dr. Santos Mañes, University of Madrid). The range of the calibration curves was from 101 to 1,010 molecules. Total RNA (5 μg) from snap frozen thyroid tissues was reverse transcribed by ThermoScript RT (Invitrogen) and oligo(dT) primers. Synthesized cDNA (3 μl) was then combined in a PCR reaction using the same primers specific for PTEN and actin used for relative Real-time Polymerase Chain Reaction and 5′-CAA ACG GCC CGC ATG TTC CC-3′ (forward) and 5′-TTG AAC TGG GCC GTG GCG AG-3′ (reverse) specific for ΔNp73α.
Gene silencing by siRNA
Cells were plated onto 6-well plates (105 cells/well), maintained in antibiotic free medium for 24 hr and transfected with a mixture containing serum-free OptiMEM (Invitrogen), 8 μl/well Lipofectamine (Lipofectamine 2000, Invitrogen) and either 0.5 μg/well Scrambled-siRNA or either p53-siRNA or p73-siRNA (Smart Pool, Dharmacon Research, USA) for 5 hr. The sequence of these siRNAs is available from the manufacturer. Cells were processed 48 hr after transfection.
Chromatin immunoprecipitation assay (ChIP)
TPC-1 cells (1 × 106 cells/150 mm PD) were transfected with the indicated constructs by Fugene6 reagent according to the manufacturer's instructions (Roche). 24 hr after transfection, DNA and proteins were cross-linked by the addition of formaldehyde to a final concentration of 1% for 10 min before harvesting. Crosslinking reaction was stopped by adding Glycine at 0.125 M final concentration for 5 min at room temperature. Plates were rinsed twice with ice cold 1× PBS and cells were scraped off the plates, resuspended in cell lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40 and protease inhibitors). Cells were dounced on ice with a B dounce several times to aid nuclei release. Then nuclei were spun down and resuspended in nuclei lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCL pH 8.1 and protease inhibitor mixture) and sonicated to generate chromatin to an average length of about 200–600 bp. After centrifugation at 14,000 rpm for 10 min at 4°C, samples (2–4 mg of protein extracts) were precleared with 10 μl protein-G-Sepharose beads (preblocked with 1 μg/μl BSA and 1 μg/μl Salmon Sperm DNA) for 2 hr at 4°C and immunoprecipitated overnight at 4°C with either 2 μg anti-p53 monoclonal antibody (DO-1 from Santa Cruz) or 2 μg anti-HA polyclonal antibody (CRP). Two micrograms Non immune serum (NIS) was used as a negative control for nonspecific DNA immunoprecipitation. Immunoprecipitates were washed with RIPA buffer and immunoprecipitation washing buffer (100 mM Tris-HCl pH 8.0, 500 mM LiCl, 1% NP-40, 1% deoxycolic acid). 20% supernatant from the rabbit IgG immunoprecipitation was saved as total input of chromatin and was processed with the eluted immunoprecipitates beginning at the crosslink reversal step. Immunocomplexes were eluted with elution buffer (1% SDS, 50 mM NaHCO3). 0.1 μg/μl of Salmon Sperm DNA and 10 μg of RNAse A were added to the pooled eluates and crosslinks were reverted by incubation at 65°C for 6 hr. Samples were diluted with 125 μl water containing 0.16 μg/μl proteinase K and incubated for 1 hr at 50°C. DNA was purified with phenol/chloroform and a fraction (5 μl) was used as PCR template to detect the presence of promoter sequences of PTEN and Thymidine Kinase genes using the primers listed below:
The PCR products were resolved in 2% agarose gels and visualized by ethidium bromide staining.
Cell viability was measured by the methyl thiazolyl tetrazolium test (MTT, Amersham Biosciences). 103 cells were seeded in 96-well plates. After 24 hr, complete medium was replaced with medium containing the various compounds. Cells were then incubated with complete medium containing 0.5 mg/ml MTT at 37°C plus 5%CO2. Four hours later, cells were dissolved in 100 μl of a solution containing dimethylsulfoxide plus 2.5% complete medium, and formazan absorbance was then read at 405 nm. The following inhibitors were used: LY 294002 (PI3-Kinase inhibitor); LY 303511 (cell-permeable negative control for the inhibitor LY 294002); PD098059 (MEK inhibitor); PP2 (Src inhibitor); all of them were purchased from Calbiochem, (Nottingham, UK).
Results were compared by two-way analysis of variance. Significance was obtained by T-test (*p < 0.05; **p < 0.01; ***p < 0.001). Statistical analysis was carried out with Microsoft Excel Software.
ΔNp73 inhibits PTEN promoter activity
To confirm previous data13, 22, 27, 29–31, 33 we measured ΔNp73α and PTEN RNA in thyroid cancer tissues by real time PCR (see Methods). In 15 papillary thyroid carcinomas (PTCs), the average of ΔNp73α transcript molecules was 6.8 × 103 (range 1.1 × 103–3.0 × 104), while in 10 normal counterpart thyroid tissues, it was 3.3 × 103 (range 0.7 × 103–1.5 × 104). In contrast, in PTCs, the average of PTEN transcript molecules was 4.6 × 105 (range 1.3 × 105–1.2 × 106) and in normal counterpart thyroid tissues 8.0 × 105 (range 2.0 × 105–1.5 × 106). Taken together, these results indicate that in thyroid cancer, ΔNp73α upregulation is concomitant with PTEN down-regulation.
To test whether ΔNp73α may affect PTEN promoter activity, we performed luciferase assays (Fig. 1b) with full-length PTEN promoter (-1978-1) and devoid of the canonical p53 binding sequences (-1031-779), but containing the Egr-1 binding sequence alone (minimal PTEN promoter, see Fig. 1a).42 The minimal PTEN promoter was used to verify whether the effect of ΔNp73α on PTEN promoter depends on p53 DNA binding. p53 family members were transfected in NIH3T3 cells along with the indicated promoters. In accordance with previous reports,36 ectopic p53 activated full-length PTEN promoter (Fig. 1b). Also TAp73α and TAp73β activated full-length PTEN promoter (Fig. 1b, white bars). In contrast, ΔNp73α significantly reduced full-length PTEN promoter activity (Fig. 1b, white bars). Interestingly, experiments performed with the minimal PTEN promoter showed a reduced effect of p53, whereas TAp73α and TAp73β effect was similar to that displayed with full length promoter (Fig. 1b). These results suggest that, at variance with p53, p73 family members regulate PTEN promoter mainly by binding to −1031-779 region. In accordance with the results obtained with TAp73α and TAp73β, the inhibitory activity of ΔNp73α was also observed with minimal PTEN promoter (Fig. 1b, black bars), suggesting that this phenomenon is also independent from p53 promoter site.
Further, to confirm that the effect of ΔNp73α on PTEN promoter is independent from an antagonism with p53, similar experiments were performed in Saos-2 human osteosarcoma cells, which are p53 null (Fig. 1c, white bars). Indeed, in this cell model, ΔNp73α was still able to inhibit PTEN promoter, indicating that the effect of ΔNp73α is p53 independent (Fig. 1c, white bars). The lack of p53 family member effect on the unrelated HEX promoter confirms the specificity of effect on PTEN promoter activity (Fig. 1c, gray bars). Similar results were also obtained in TPC-1 and ARO thyroid cancer cells (not shown).
To test the hypothesis that p73 family members may directly bind to PTEN promoter, ChIP experiments were performed in TPC-1 thyroid cancer cells, transfected with either p53, TAp73α, or ΔNp73α (Fig. 1d). In these experiments, p53 bound to full length PTEN promoter (-1978-1, Fig. 1d) but only weakly to the minimal promoter (-1031-799, Fig. 1d). In contrast, TAp73α and ΔNp73α bound to the minimal promoter in a manner similar to that of full-length promoter (Fig. 1d, densitometry results are shown on the right), in accordance with luciferase assays (Fig. 1b).
Taken together, these results suggest that ΔNp73α may directly inhibit PTEN promoter activity in a manner independent from binding competition with p53. Moreover, ΔNp73α may directly interact with the −1031-799 PTEN promoter region, which may be considered specific for p73 family proteins.
ΔNp73 down regulates PTEN expression in thyroid cancer cells
To test the effect of ΔNp73α on PTEN transcript, an inducible system was used.43 C-643 thyroid cancer cell line was used because they are p53 deficient (as they express R248Q and K286E p53 mutants) and are p73 negative.13 These cells were stably transfected with ecdysone-inducible TAp73α, TAp73β, ΔNp73α, p53 and incubated in the presence or the absence of 2.5 μM ecdysone (see methods). Western Blot analysis with anti-HA and anti-p53 antibodies confirmed the inducible expression of the transfected genes (Fig. 2b, top panels). Real-time PCR (Fig. 2a) and Western Blot experiments (Fig. 2b) showed that, in accordance with results obtained by luciferase assay, TAp73α, TAp73β and p53 induction caused a significant increase in both PTEN RNA (Fig. 2a) and protein level (Fig. 2a, densitometry results on the right). In contrast, induction of ΔNp73α caused a decreased expression of both PTEN RNA (Fig. 2a) and protein (Fig. 2b, densitometry results are shown on the top right), as expected. These results confirm that ΔNp73α, in contrast with TAp73 isoforms and p53 decreases PTEN expression both at RNA and protein level. These data suggest a novel mechanism for the pro-tumorigenic effect of ΔNp73 in thyroid cancer cells, by inhibiting PTEN expression.
PTEN, in fact, is a lipid phosphatase that inhibits the PI-3K pathway: when PTEN is down-regulated, the downstream target of PI3-K, namely the Akt/PKB kinase should be overactivated.23, 24, 28 In accordance with this hypothesis, induction of TAp73α, TAp73β and p53 resulted in reduced Akt phosphorylation, whereas induction of ΔNp73α resulted in increased Akt phosphorylation (Fig. 2b, densitometry results are shown on the bottom right). Reblot with anti-Akt antibody did not reveal changes in the total amount of Akt protein expression (Fig. 2b). Taken together, these results suggested that ΔNp73α, by down regulating PTEN expression, increases Akt phosphorylation in thyroid cancer cells.
To confirm the possibility that p53 family members may regulate PTEN expression in thyroid cancer cells, transient transfection experiment were performed in ARO (carrying R273H p53 mutant) and TPC-1 (carrying a wild type p53) thyroid cancer cells. Anti-PTEN western blot (Fig. 3a) indicated that ectopic expression of p53 in these cells caused a significant increase in PTEN protein expression (Fig. 3a), in accordance with previous reports obtained in other cell types.35–37 The effect of ΔNp73α was different, as it significantly decreased PTEN protein (Fig. 3a, densitometry results are shown on the right). Anti-HA and anti-p53 blots confirmed the expression of ectopic proteins (Fig. 3a). These results confirmed that ΔNp73α, at variance with p53, may decrease PTEN expression in thyroid cancer cells.
To confirm the results obtained by protein overexpression, the opposite approach of siRNA technique was attempted (Fig. 3b). Thyroid cancer cells with a suitable genetic background were first selected: TPC-1 cells, which express wild type p53, but not p73; SW-1736 cells, which express both TAp73α and ΔNp73α, but not p53.13, 44 In TPC-1 cells, down regulation of p53 by p53-siRNA resulted in decreased PTEN protein levels (Fig. 3b), whereas no change was observed with ΔNp73-down regulation, as expected (Fig. 3b). In contrast, p73-siRNA transfection in SW-1736 cells (which down regulated both TAp73α and ΔNp73α) caused increased PTEN protein level, whereas p53-siRNA caused no change, as expected (Fig. 3b, densitometry results are shown on the right). As TAp73α effect on PTEN promoter is marginal and opposite to that of ΔNp73α, the effect of p73 siRNA in SW-1736 cells is attributable mainly to the down-regulation of ΔNp73α. Western blot with anti-p53 and anti-p73 antibodies confirmed that these siRNAs effectively reduced endogenous p53 and p73 proteins (Fig. 3b). Taken together these results confirm that ΔNp73α is able to reduce PTEN protein expression in thyroid cancer cells. As a consequence, PTEN down regulation by ΔNp73α results in increased levels of phosphorylated Akt.
ΔNp73α decreases p53 protein level in thyroid cancer cells
It has been reported that activation of Akt enhances Mdm2-mediated ubiquitination and degradation of p53 protein leading to a decrease in p53 protein levels.38, 39, 45 Because ΔNp73α expression results in increased Akt activation, we evaluated the effect of ΔNp73α on p53 protein level. To this end, inducible TPC-1 cell clones were used as they express a wild type p53, which is subjected to Mdm2-mediated regulation. Western blot analysis indicated that induction of ΔNp73α in TPC-1 cells caused a significant decrease of PTEN expression, increased Akt phosphorylation (Fig. 4a, compare lanes 5 and 6 to 7 and 8) and decreased p53 protein expression (Fig. 4a, see lane 7 and densitometry results on the right). Down regulation of p53 was not observed in empty-transfected cells (Fig. 4a, see lanes 1–4). Interestingly, p53 down regulation by ΔNp73α was prevented by the incubation with the PI-3 Kinase inhibitor LY294002 (Fig. 4a, compare lane 7 to lane 8), in accordance with the effect of ΔNp73α on Akt activation. Similar results were also obtained in response to doxorubicin (Fig. 4b). In particular, induction of TAp73β resulted in increased PTEN expression (Fig. 4b, compare lanes 13–16 to lanes 9–12 and see densitometry results on the top). As a consequence, induction of TAp73β resulted in increased p53 protein levels in cells exposed to doxorubicin (Fig. 4b), which is able to cause an increased p53 protein level. Because of the short exposition time, the basal level of p53 is not visible. In accordance with the effect on Akt phosphorylation, the increase in p53 protein level caused by TAp73β was prevented by the incubation with LY294002 (Fig. 4b, compare lane 14 to lane 16). In contrast, induction of ΔNp73α resulted in a reduced p53 protein levels in response to doxorubicin (Fig. 4b, lane 22), which was prevented by the incubation with LY294002 (Fig. 4b, compare lane 22 to lane 24). To test the hypothesis that the effect of ΔNp73α on p53 protein levels was dependent on Mdm2-mediated p53 degradation, Mdm2-p53 co-immunoprecipitation experiments were also performed (Fig. 4c). Indeed, induction of ΔNp73α resulted in increased presence of p53 in anti-Mdm2 immunoprecipitates (Fig. 4c, Lane 6 and densitometry results on the left), which was prevented by the incubation with LY294002, suggesting that this phenomenon is Akt dependent. Taken together these results indicate that, in thyroid cancer cells, ΔNp73α is able to decrease PTEN expression, increase Akt phosphorylation and, as a consequence decrease p53 protein levels by enhanced Mdm2-mediated degradation.46
ΔNp73α increases viability of thyroid cancer cells
Increased Akt phosphorylation in thyroid cancer cells is a mechanism responsible for increased cell proliferation and chemo resistance.47–49 We therefore evaluated the Akt-mediated effect of ΔNp73α on both thyroid cancer cell proliferation and survival in a context independent from the effect on p53. To this end, inducible C-643 cell clones, expressing R248Q and K286E p53 mutants, were subjected to MTT assay both under basal conditions and after exposure to chemotherapy drugs (Figs. 5a and 5b). As expected, induction of p53 and TAp73β resulted in decreased number of viable cells (Fig. 5a). In contrast, induction of ΔNp73α resulted in increased number of viable cells at different time points (Fig. 5a). A similar effect was observed on the cytotoxic effects of chemotherapy drugs, including doxorubicin, cisplatin, cyclophosphamide and taxol (Fig. 5b). Induction of p53 and TAp73β reduced while induction of ΔNp73α increased cell survival after exposure to chemotherapy compounds (open circles, Fig. 5b). These results indicated that in thyroid cancer cells ΔNp73 expression is related to increased cell proliferation and resistance to chemotherapy.
To test whether the effect of ΔNp73α on cell viability and survival was dependent on PTEN/Akt pathway, the PI3-Kinase inhibitor LY294002 was used in MTT experiments (Fig. 5c). As a control, unrelated inhibitors were used, including PD98059 (MEK inhibitor) and PP2 (Src inhibitor). We also used a negative control for the PI3-Kinase inhibitor LY294002: the LY303511, as it does not inhibit PI3K even at high concentrations. Incubation of C-643 thyroid cancer cells with PI3-kinase inhibitor LY294002 completely abolished the protective effect of ΔNp73α against the cytotoxic effect of staurosporin. In contrast, the unrelated inhibitors PD98059 and PP2 were without effect (Fig. 5c). Taken together, these results indicate that the effect of ΔNp73α on thyroid cancer cell phenotype depends (at least partially) on the PTEN/Akt pathway.
The role of p73 in tumor progression is still under debate.50 Several reports have shown that p73 is overexpressed in human malignancies, including thyroid carcinomas.13, 21, 50 Further analysis indicated that tumors preferentially express ΔNp73α, a p73 isoform devoid of the transactivation domain (TA) and acting as a dominant negative toward full-length p73 isoforms and p53.8, 50–52 Therefore, the presence of ΔNp73α in tumors may be regarded as a mechanism to abrogate p53 tumor suppressor function.8, 50–52 However, the role of ΔNp73α in tumor progression is not fully elucidated and it is not clear whether ΔNp73α may directly affect gene transcription independently from its effect on p53 activities.
Here, we report that, in thyroid cancer cells, p73 isoforms are able to regulate PTEN expression. In particular, the N-terminally truncated p73 isoform ΔNp73α is able to repress PTEN promoter by binding to a DNA region different of the canonical one for p53. These results are in line with data reporting differences in DNA sequence selectivity between p53 and p73,53, 54 as it is accepted that p73 has a number of unique target genes that are distinct from p53.55, 56 Our data support the possibility that, in vivo, DNA sequences for p73 may be distinct from those of p53 and may play a role in tumor progression. These findings unravel different novel mechanisms for (thyroid) cancer progression and may explain the previous observation indicating that, in ∼40–50% thyroid carcinomas, PTEN is down-regulated29–31 and, as a consequence, Akt hyperphosphorylated.23 These data are also in line with previous studies indicating that ΔNp73α is expressed in the majority of thyroid carcinomas.13, 21 In addition, data obtained with ecdysone-inducible cells unravel an additional and novel mechanism for the dominant negative effect of ΔNp73α. Indeed, PTEN down regulation by ΔNp73α results in hyper-phosphorylation of Akt, and, as a consequence, Mdm2 phosphorylation and its nuclear accumulation. Nuclear Mdm2 is protected by degradation and may effectively target p53 to proteasome and degradation39, 45 (Fig. 6). In this respect, ΔNp73α is able to reduce p53 protein levels in both basal conditions and after exposure to the DNA damaging agent doxorubicin. However, the per se protumorigenic effect of ΔNp73α, which occurs independently from the abrogation of p53 tumor suppressor function, may be important in poorly differentiated thyroid carcinomas, which often harbor p53 mutation. In these poorly differentiated thyroid carcinomas, ΔNp73α may be an additional mechanism to obtain a further decrease in PTEN expression. As a consequence, Akt hyper-phosphorylation in poorly differentiated thyroid cancer may be responsible for cell proliferation and resistance to chemotherapy58–61
The mechanisms underlying the effect of ΔNp73α on PTEN promoter remain to be elucidated. It is interesting to note that TAp73α activates PTEN minimal promoter with a potency higher than that of p53 and that this promoter is down-regulated by ΔNp73α. This suggests that the −1031-779 DNA sequence is specific for p73 isoforms and may be activated by TA and repressed by ΔN isoforms. Several mechanisms may account for the specific effects of p73 isoforms on this regulatory DNA sequence. A simple possibility could be that p73 proteins bind to DNA sequences that are not bound by p53. This is supported by the notion that the DNA-binding domains of the various member of the p53 protein family may have distinct functional properties.62 Accordingly, it has been recently shown that p73 isoforms, but not p53 or p63, regulates the activity of caspase-2s promoter by direct binding to a GC-rich sequence.63 A second possibility, not mutually-exclusive to the first one, could be the interaction between p73 isoforms and other transcriptional regulators that bind to the PTEN minimal promoter. Interestingly, this regulatory DNA sequence contains the binding site for Egr-1 transcription factor, which accounts for PTEN inducible transcription on genotoxic stress.57 It is tempting to speculate that an inhibitory effect of ΔNp73α over Egr-1 transcriptional activity on this site may explain, at least in part, our present data. The existence of a regulatory network between p73, p53 and Egr-164 in regulating cancer apoptosis in tumor cells, reinforces this hypothesis. Thus, the minimal PTEN promoter could represent the functional target for a regulatory mechanism based on functional interactions between Egr-1 and the different p73 isoforms. The regulation of PTEN promoter activity by p73 proteins may be important in cancer, which often displays ΔNp73 preferential expression. These results explain one mechanism for PTEN down regulation in thyroid cancer and, possibly, in other tumors. These mechanisms should be kept into account in designing novel anti-cancer therapies aimed at reactivating p53 tumor suppressor function and inhibiting the Akt pathway.
This work was supported by a grant from Associazione Italiana Ricerca Sul Cancro (AIRC) to Riccardo Vigneri and by a MIUR grant to Giuseppe Damante. This work was also supported by a fellowship from Associazione Italiana Ricerca Sul Cancro to Cinzia Puppin and a post-doctoral research fellowship from the American-Italian Cancer Foundation (AICF) to Veronica Vella. The authors thank Dr. Santos Mañes (Madrid, Spain) for PTEN plasmid used to perform the curve of Real Time PCR experiments.