Molecular and Cellular Pathobiology Candidate Tumor Suppressor and pVHL Partner Jade-1 Binds and Inhibits AKT in Renal Cell Carcinoma

The vonHippel–Lindau (VHL) tumor suppressor pVHL is lost in themajority of clear-cell renal cell carcinomas (RCC). Activation of the PI3K/AKT/mTOR pathway is also common in RCC, with PTEN loss occurring in approximately 30% of the cases, but other mechanisms responsible for activating AKT at a wider level in this setting are undefined. Plant homeodomain protein Jade-1 (PHF17) is a candidate renal tumor suppressor stabilized by pVHL. Here, using kinase arrays, we identified phospho-AKT1 as an important target of Jade-1. Overexpressing or silencing Jade-1 in RCC cells increased or decreased levels of endogenous phospho-AKT/AKT1. Furthermore, reintroducing pVHL into RCC cells increased endogenous Jade-1 and suppressed endogenous levels of phospho-AKT, which colocalized with and bound to Jade-1. The N-terminus of Jade-1 bound both the catalytic domain and the C-terminal regulatory tail of AKT, suggesting a mechanism through which Jade-1 inhibited AKT kinase activity. Intriguingly, RCC precursor cells where Jade-1 was silenced exhibited an increased capacity for AKT-dependent anchorage-independent growth, in support of a tumor suppressor function for Jade-1 in RCC. In support of this concept, an in silico expression analysis suggested that reduced Jade-1 expression is a poor prognostic factor in clear-cell RCC that is associated with activation of an AKT1 target gene signature. Taken together, our results identify 2 mechanisms for Jade-1 fine control of AKT/AKT1 in RCC, through loss of pVHL, which decreases Jade-1 protein, or through attenuation in Jade-1 expression. These findings help explain the pathologic cooperativity in clear-cell RCC between PTEN inactivation and pVHL loss, which leads to decreased Jade-1 levels that superactivate AKT. In addition, they prompt further investigation of Jade-1 as a candidate biomarker and tumor suppressor in clear-cell RCC. Cancer Res; 73(17); 5371–80. 2013 AACR. Introduction Renal cancer is a major clinical problem (1). In the United States, 65,150 new cases and 13,680 deathswere predicted from cancer of the kidney and renal pelvis for 2013 (2). More than 90% of kidney cancers are believed to originate from renal epithelial cells and are therefore referred to as renal cell carcinomas (RCC; ref. 3). The majority of cases of clear-cell RCC, the most common subtype of RCC, are characterized genetically by the loss, mutation, or silencing of the VHL gene (4, 5), making the von Hippel–Lindau tumor suppressor (pVHL) the major renal tumor suppressor in adults. However, the pathogenesis of renal cancer remains unresolved. Serine/threonine kinase AKT is a key factor of perhaps the most frequently activated proliferation and survival pathway in cancer (6). Elevated AKT activity is also found in RCC and kidney cysts. Cystic lesions of patients with VHL show hyperactivated PI3K/AKT signaling (7). Increased phospho-AKT levels were found in about 50% of RCC tumor samples, and most commonly in the clear-cell subtype (8). Combined mutations of VHL and PTEN, a phosphatidylinositol (3–5)-trisphosphate phosphatase that negatively regulates the AKT/PKB signaling pathway, leads to kidney cysts in mice (7). PTENinactivating mutations (9, 10) or decreased PTEN expression have been identified in about 30% of clear-cell RCCs (11–13). Hyperactivation of AKT due to conditional knockout of neurofibromatosis type II (Nf2) in mouse renal proximal tubules leads to invasive RCC (14). Human renal cancer cell lines also show constitutive activation of AKT, and PI3K/AKT inhibitor treatment induces apoptosis and inhibits cell growth in vitro and in xenografts (15). Thus, AKT is activated in clear-cell RCC, but the mechanism has not always been apparent. Jade-1, a short-lived protein most highly expressed in renal proximal tubules, was identified as a novel strong binding partner of pVHL (16). Wild-type pVHL stabilizes Jade-1, whereas renal cancer-causing forms cannot (17). Jade-1 is a candidate renal tumor suppressor and promotes apoptosis (18). Jade-1 functions as a ubiquitin ligase to inhibit canonical Wnt signaling (19) and as a transcription factor associated with Authors' Affiliations: Renal and Hematology/Oncology Sections, Departments of Medicine and Pathology, Boston Medical Center and Boston University School of Medicine; Division of Rheumatology, Immunology and Allergy and Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts; and Division of Nephrology & Hypertension, Department of Medicine, Oregon Health Science University, Portland, Oregon Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Herbert T. Cohen, 650 Albany Street, Rm X-535, Boston, MA 02118. Phone: 617-638-7322; Fax: 617-638-7326; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-4707 2013 American Association for Cancer Research. Cancer Research www.aacrjournals.org 5371 on April 20, 2017. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst July 1, 2013; DOI: 10.1158/0008-5472.CAN-12-4707