Tumor and Stem Cell Biology D Np 63 Promotes Pediatric Neuroblastoma and Osteosarcoma by Regulating Tumor Angiogenesis

The tumor suppressor gene p53 and its family members p63/p73 are critical determinants of tumorigenesis. DNp63 is a splice variant of p63, which lacks the N-terminal transactivation domain. It is thought to antagonize p53-, p63-, and p73-dependent translation, thus blocking their tumor suppressor activity. In our studies of the pediatric solid tumors neuroblastoma and osteosarcoma, we find overexpression of DNp63; however, there is no correlation ofDNp63 expression with p53mutation status. Our data suggest that DNp63 itself endows cells with a gain-of-function that leads tomalignant transformation, a function independent of any p53 antagonism. Here, we demonstrate that DNp63 overexpression, independent of p53, increases secretion of interleukin (IL)-6 and IL-8, leading to elevated phosphorylation of STAT3 (Tyr-705). We show that elevated phosphorylation of STAT3 leads to stabilization of hypoxia-inducible factor 1a (HIF-1a) protein, resulting in VEGF secretion. We also show human clinical data, which suggest a mechanistic role for DNp63 in osteosarcoma metastasis. In summary, our studies reveal the mechanism by which DNp63, as a master transcription factor, modulates tumor angiogenesis. Cancer Res; 74(1); 320–9. 2013 AACR. Introduction p63 exhibits high sequence and structural homology to the tumor suppressor protein p53 (Trp53), and to anothermember of this family, p73 (Trp73; refs. 1, 2). Studies using genetically engineered mice have elucidated a central role for p63 in skin development and the aging of epithelial tissues (3–7). Through the use of alternative promoters, the p63 gene generates transcripts encoding two major classes of protein isoforms, TAp63 and DNp63. TAp63 contains an N-terminal transactivation domain, whereas DNp63 lacks this domain (2). Additional diversity arises through alternative splicing, which generates proteins with unique C-Termini, designated a, b, and g . Together, these give rise to six unique p63 transcripts. All of thesemaintain a DNA-binding domain with significant homology to p53, but with somewhat altered target specificity (8, 9). Several reports both from in vitro and in vivo experiments have suggested that the TAp63 isoforms act as tumor suppressor genes. For instance, TAp63 isoforms suppress metastasis through induction of senescence (10) and transcriptional activation of Dicer1 andmir-130b (11). TAp63 / mice develop metastatic mammary and lung adenocarcinoma, as well as squamous cell carcinoma (SCC) with metastases to the lung, liver, and brain (11). The roles of the DNp63 isoforms seem to be more complex. Early studies showed that the DNp63 isoforms oppose p53-, TAp63-, and TAp73-mediated transcription (and therefore apoptosis and cell-cycle arrest), suggesting an oncogenic role for DNp63 isoforms (2, 9, 12–14). Other studies have demonstrated effects that are independent of any dominant negative inhibitory activity, such as targeting of the chromatin remodeler Lsh byDNp63, which results in stem cell proliferation and tumorigenesis (15). Some reports show that DNp63 isoforms retain transcriptional activity and can transactivate genes involved in epidermal morphogenesis (16,17) and DNA repair (18). DNp63 is overexpressed in some types of adult human cancer (19), particularly SCC (20), in which it promotes oncogenesis by suppressing TAp73 (21). In other tumor types, such as adenocarcinoma of the breast and prostate, DNp63 expression is lost during the tumorigenic process (22). Althoughmice that exhibit knocked down (17) or total loss of expression (23) of DNp63 have been described, their cancer-associated phenotypes have not yet been reported. Taken together, the role that p63 isoforms play in cancer merits further investigation. Here, we show that two childhood malignancies, neuroblastoma and osteosarcoma, overexpress DNp63. In these tumors we find no correlation between p53 mutation and DNp63 overexpression. We hypothesize that DNp63 is a key modulator of tumorigenesis in these childhood cancers, independent of p53 status. We demonstrate that Authors' Affiliations: Center for Childhood Cancer and Blood Diseases, Nationwide Children's Hospital; and Department of Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). H.K. Bid, R.D. Roberts, and M. Cam contributed equally to this work. Corresponding Author: Hakan Cam, Center for Childhood Cancer and Blood Diseases, The Research Institute, Nationwide Children's Hospital, 700 Children's Drive, Research Building II, Columbus, OH 43205. Phone: 614-355-1998; Fax: 614-355-2927; E-mail: hakan.cam@nationwidechildrens.org doi: 10.1158/0008-5472.CAN-13-0894 2013 American Association for Cancer Research. Cancer Research Cancer Res; 74(1) January 1, 2014 320 on August 30, 2017. © 2014 American Association for Cancer Research. cancerres.aacrjournals. org Downloaded from Published OnlineFirst October 23, 2013; DOI: 10.1158/0008-5472.CAN-13-0894