Molecular and Cellular Pathobiology High-Mobility Group A1 Proteins Regulate p53-Mediated Transcription of Bcl-2 Gene Francesco Esposito 1 , Mara Tornincasa 2 , Paolo Chieffi 3 , Ivana De Martino 2 , Giovanna Maria Pierantoni 2 , and Alfredo Fusco 1,4 Abstract We have previously described a mechanism through which the high-mobility group A1 (HMGA1) proteins inhibit p53-mediated apoptosis by delocalizing the p53 proapoptotic activator homeodomain-interacting pro- tein kinase 2 from the nucleus to the cytoplasm. By this mechanism, HMGA1 modulates the transcription of p53 target genes such as Mdm2, p21 waf1 , and Bax, inhibiting apoptosis. Here, we report that HMGA1 antag- onizes the p53-mediated transcriptional repression of another apoptosis-related gene, Bcl-2, suggesting a nov- el mechanism by which HMGA1 counteracts apoptosis. Moreover, HMGA1 overexpression promotes the reduction of Brn-3a binding to the Bcl-2 promoter, thereby blocking the Brn-3a corepressor function on Bcl-2 expression following p53 activation. Consistently, a significant direct correlation between HMGA1 and Bcl-2 overexpression has been observed in human breast carcinomas harboring wild-type p53. Therefore, this study suggests a novel mechanism, based on Bcl-2 induction, by which HMGA1 overexpression contri- butes to the escape from apoptosis leading to neoplastic transformation. Cancer Res; 70(13); 5379–88. ©2010 AACR. Introduction The high-mobility group A (HMGA) family is comprised of three proteins: HMGA1a, HMGA1b, and HMGA2. They are encoded by two distinct genes, HMGA1a and HMGA1b proteins being products of the same gene generated through alternative splicing (1). These proteins bind the mi- nor groove of AT-rich DNA sequences through their DNA- binding domain, the so-called “AT-hooks.” HMGA proteins do not have transcriptional activity per se; however, by in- teracting with the transcription machinery, they alter the chromatin structure and thereby regulate the transcription- al activity of several genes (2, 3). In normal cells and adult tissues, the levels of HMGA proteins are low or absent (4). In contrast, in neoplastically transformed cells as well as in embryonic cells, the constitutive expression of HMGA pro- teins is exceptionally high (5). Their overexpression is main- ly associated with a highly malignant phenotype, also representing a poor prognostic index because HMGA over- expression often correlates with the presence of metastasis as well as reduced survival (6). Moreover, both in vitro and in vivo studies have established the oncogenic role of HMGA genes (7–11). Recently, we have shown a novel mechanism, based on HMGA1-p53 interaction, by which HMGA1 proteins have a role in the process of carcinogenesis. In fact, HMGA1 binds p53 and interferes with the p53-mediated transcrip- tion of Bax, p21 waf1 , and Mdm2, leading to a reduction of p53-dependent apoptosis (12). HMGA1 is also able to inter- fere with the apoptotic function of p53 by another mecha- nism that involves the p53 proapoptotic activator homeodomain-interacting protein kinase 2 (HIPK2), which binds to and phosphorylates p53 on Ser 46 leading to apo- ptosis (13, 14). Indeed, HMGA1 overexpression promotes HIPK2 relocalization from the nucleus to the cytoplasm, in- hibiting p53 apoptotic function, whereas HIPK2 overexpres- sion re-established HIPK2 nuclear localization and sensitivity to apoptosis (15). Consistently, a strong correla- tion among HMGA1 overexpression, HIPK2 cytoplasmic lo- calization, and a low spontaneous apoptosis index was observed in wild-type p53-expressing human breast carcino- mas (15). On the basis of these data, we looked for other genes reg- ulated by HMGA1 proteins among p53 target genes involved in cell apoptosis. In particular, we have focused our attention on B-cell lymphoma gene 2 (Bcl-2). Bcl-2 represents a good candidate as the target for HMGA1 proteins because its pro- moter region contains some AT-rich DNA sequences and its transcription is regulated by p53 (16, 17). The Bcl-2 protein exerts an antiapoptotic function: it inhibits the release of cy- tochrome c into the cytosol which, in turn, activates caspase- 9 and caspase-3, leading to apoptosis (18). The Bcl-2 gene has been found to be overexpressed in different cancers, includ- ing B-cell lymphoma (19), melanoma (20), neuroblastoma Authors' Affiliations: 1 Istituto di Endocrinologia ed Oncologia Sperimentale del CNR c/o 2 Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia di Napoli, Università degli Studi di Napoli “Federico II”, 3 Dipartimento di Medicina Sperimentale, Seconda Università di Napoli, and 4 Naples Oncogenomic Center-Centro di Ingegneria Genetica, Biotecnologie Avanzate-Napoli, and the European School of Molecular Medicine, Naples, Italy Corresponding Author: Alfredo Fusco, Istituto di Endocrinologia ed On- cologia Sperimentale “Gaetano Salvatore” Del Consiglio Nazionale delle Ricerche, via Pansini 5, 80131 Naples, Italy. Phone: 39-081-746-3602; Fax: 39-081-229-6674; E-mail: afusco@napoli.com. doi: 10.1158/0008-5472.CAN-09-4199 ©2010 American Association for Cancer Research. Cancer Research www.aacrjournals.org 5379 on May 24, 2016. © 2010 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst June 8, 2010; DOI: 10.1158/0008-5472.CAN-09-4199