Reactivation of p53 mutants by prima-1 [corrected] in thyroid cancer cells.

Most undifferentiated thyroid carcinomas express p53 mutants and thereafter, are very resistant to chemotherapy. p53 reactivation and induction of massive apoptosis (Prima-1) is a compound restoring the tumor-suppressor activity of p53 mutants. We tested the effect of Prima-1 in thyroid cancer cells harboring p53 mutations. Increasing doses of Prima-1 reduced viability of thyroid cancer cells at a variable extent (range 20-80%). Prima-1 up-regulated p53 target genes (p21(WAF1) , BCL2-associated X protein (Bax), and murine double minute 2 (MDM2)), in BC-PAP and Hth-74 cells (expressing D259Y/K286E and K286E p53 mutants) but had no effect in SW1736 (p53 null) and TPC-1 (expressing wild-type p53) thyroid cancer cells. Prima-1 also increased the cytotoxic effects of either doxorubicin or cisplatin in thyroid cancer cells, including the chemo-resistant 8305C, Hth-74 and BC-PAP cells. Moreover, real-time PCR and Western blot indicated that Prima-1 increases the mRNA of thyroid-specific differentiation markers in thyroid cancer cells. Fluorescence-activated cell sorting analysis revealed that Prima-1 effect on thyroid cancer cells occurs via the enhancement of both cell cycle arrest and apoptosis. Small interfering RNA experiments indicated that Prima-1 effect is mediated by p53 mutants but not by the p53 paralog p73. Moreover, in C-643 thyroid cancer cells, forced to ectopically express wild-type p53, Prima-1 prevented the dominant negative effect of double K248Q/K286E p53 mutant. Finally, co-IP experiments indicated that in Hth-74 cells Prima-1 prevents the ability of p53 mutants to sequestrate the p53 paralog TAp73. These in vitro studies imply that p53 mutant reactivation by small compounds may become a novel anticancer therapy in undifferentiated thyroid carcinomas.

DeltaNp73alpha inhibits PTEN expression in thyroid cancer cells.

DeltaNp73 is a N-terminally truncated p53 family member with a dominant negative function, which is upregulated in cancer. PTEN is a lipid phosphatase, which is involved in the attenuation of tyrosine kinase signaling. PTEN expression is increased by p53, and its function is blunted in several malignancies. Because in most of the thyroid carcinomas, DeltaNp73alpha is upregulated, whereas PTEN expression down regulated, we investigated whether DeltaNp73alpha may influence PTEN expression in this cell model. We found that DeltaNp73alpha overexpression in thyroid cancer cells reduces PTEN expression, whereas DeltaNp73alpha down-regulation by siRNA increases PTEN expression. Real-time PCR indicated that overexpression of DeltaNp73alpha is able to reduce PTEN mRNA levels. Moreover, chromatin immunoprecipitation (ChIP) and luciferase assays indicated that DeltaNp73alpha binds to -1031-779 region of the PTEN promoter, which is a different site than that for p53, thereby inhibiting promoter activity. Interestingly, also the transcriptionally active p73 isoforms (TAp73alpha and TAp73beta) bound to this DNA sequence and, at variance with DeltaNp73alpha, stimulated PTEN promoter activity to an extent similar to that of p53. In accordance with its effect on PTEN protein levels, DeltaNp73alpha increased phospho-Akt protein content and, as a consequence, Mdm2-mediated p53 degradation. This effect of DeltaNp73alpha resulted in increased thyroid cancer cell proliferation and reduced apoptosis and was reverted by the PI3-kinase inhibitor LY294002, indicating the role of Akt pathway in this effect. Taken together, these results indicate a novel p73 regulated mechanism for PTEN expression in thyroid cancer cells, and that, also through this mechanism, DeltaNp73alpha exerts its protumorigenic effect.

TAp73 alpha increases p53 tumor suppressor activity in thyroid cancer cells via the inhibition of Mdm2-mediated degradation.

p53 family proteins include p53 tumor suppressor, p63, and p73. Despite the high similarity in structure and function with p53, p63, and p73 function in tumor suppression is still controversial. Here, we show that TAp73alpha, a transcriptionally active p73 isoform, is able to synergize p53 tumor suppressor function in thyroid cancer cells. Indeed, depletion of p73 by small interfering RNA in thyroid cancer cells resulted in a reduced transcriptional activity of p53. Ectopic coexpression of both p53 and TAp73alpha in thyroid cancer cells resulted in increased transcription and tumor suppressor function compared with p53 or TAp73alpha alone, as well as in increased p53 protein levels. The enhancing effect of TAp73alpha on p53 activity is Mdm2 dependent because it is prevented by Mdm2 depletion by small interfering RNA. At least two mechanisms may explain the interference of TAp73alpha with p53 function. First, in thyroid cancer cells, TAp73alpha inhibits the effect of p53 on Mdm2 induction by antagonizing p53 at the Mdm2 promoter level. Second, a TAp73alpha mutant (G264W), which is devoid of DNA binding capability, is still able to increase p53 protein levels by competing with p53 for Mdm2 protein binding. Taken together, these results indicate that in thyroid cancer cells, TAp73alpha is able to increase p53 protein level and function by interfering with Mdm2-mediated p53 degradation. These results may be useful for designing gene therapies aimed at restoring a normal p53 function in thyroid cancer cells.