Reactivation of p53 mutants by p53 reactivation and induction of massive apoptosis in thyroid cancer cells Rosa Linda Messina 1 , Mariangela Sanfilippo 1 , Veronica Vella 1 , Giuseppe Pandini 1 , Paolo Vigneri 2 , Maria Luisa Nicolosi 1 , Fiorenza Gianı ` 1 , Riccardo Vigneri 1 and Francesco Frasca 1 1 Department of Clinical and Molecular Biomedicine, Endocrinology Unit, University of Catania, Catania, Italy 2 Section of General Pathology, University of Catania, Catania, Italy 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. Thyroid cancer is the most frequent endocrine malignancy, and its incidence is increasing faster than any other malig- nancy (3.8% per year in the period 1992–2001). 1 Among men, also age-adjusted mortality for thyroid cancer has increased faster than any other cancer (2.3% per year in the period 1992–2001). 2 Although well-differentiated thyroid car- cinomas are efficiently cured by surgery and radioiodine, un- differentiated thyroid carcinomas (10–20% of total) are very resistant to chemotherapy. 3 Therefore, novel and more effica- cious therapies are urgently needed for these tumors. In differentiated thyroid cancer, the most frequent genetic alteration is the overactivation of the RAt sarcoma (RAS)/V- raf 1 murine leukemia viral oncogene homolog 1 (RAF)/ extracellular signal-regulated kinase (ERK) pathway, 4 which includes B-RAF and RAS mutations and Ret and Trk tyrosine kinase rearrangements. 4 In these tumors, the genetic alteration of tumor suppressors is relatively rare. 5 In contrast, p53 muta- tions are frequent in undifferentiated thyroid carcinomas. 6,7 Because of p53 dual role as a guardian of genome integrity and as a key mediator of apoptosis, defective p53 activity promotes resistance to chemo- and radio-therapies and a more malignant phenotype of cancer cells. 8 Therefore, several efforts have been made to restore p53 function in cancer 9 including thyroid can- cer cells. 10–12 These strategies include introduction of wild-type p53 or rescue of mutant p53 function by the induction of a wild-type conformation. 13,14 Although wild-type p53 reconstitu- tion has been shown to inhibit tumor growth in clinical trials, 15 the absence of efficient delivery systems 16 and the immune response against viral vectors have limited the clinical utility of this type of gene therapy. 17 Alternative approaches were addressed to reactivate endogenous wild-type p53, overcoming the p53 blockade by MDM2 using small molecules such as Nut- lins 18 and MI-219. 19 The screening of chemical libraries in cell lines carrying p53 mutations led to the discovery of potent death-inducing small molecules such as CP-31398 20 and p53 reactivation and induction of massive apoptosis (Prima-1). 13 Prima-1 is a chemical compound that was originally identified, because it is able to induce apoptosis in cancer cells harboring p53 mutants 21,22 by reactivating the transcriptional transactivation Key words: Prima-1, thyroid cancer, p53 mutant, p73, cell proliferation, apoptosis Grant sponsor: Associazione Italiana Ricerca Sul Cancro (AIRC; to R.V.) DOI: 10.1002/ijc.26228 History: Received 26 Aug 2010; Accepted 20 May 2011; Online 6 Jun 2011 Correspondence to: Francesco Frasca, Endocrinologia – Dipartimento di Medicina Interna e Medicina Specialistica, University of Catania, PO Garibaldi Nesima, Via Palermo 636, Catania 95122, Italy, Tel.: þ39 095 759 8702; Fax: þ39 095 472988, E-mail: f.frasca@unict.it Cancer Cell Biology Int. J. Cancer: 130, 2259–2270 (2012) V C 2011 UICC International Journal of Cancer IJC