Tumor and Stem Cell Biology NEDD9 Depletion Destabilizes Aurora A Kinase and Heightens the Efficacy of Aurora A Inhibitors: Implications for Treatment of Metastatic Solid Tumors

Aurora A kinase (AURKA) is overexpressed in 96% of human cancers and is considered an independent marker of poor prognosis. While the majority of tumors have elevated levels of AURKA protein, few have AURKA gene amplification, implying that posttranscriptional mechanisms regulating AURKA protein levels are significant. Here, we show that NEDD9, a known activator of AURKA, is directly involved in AURKA stability. Analysis of a comprehensive breast cancer tissue microarray revealed a tight correlation between the expression of both proteins, significantly corresponding with increased prognostic value. A decrease in AURKA, concomitant with increased ubiquitination and proteasome-dependent degradation, occurs due to depletion or knockout of NEDD9. Reexpression of wild-type NEDD9 was sufficient to rescue the observed phenomenon. Binding of NEDD9 to AURKA is critical for AURKA stabilization, as mutation of S296E was sufficient to disrupt binding and led to reduced AURKA protein levels. NEDD9 confers AURKA stability by limiting the binding of the cdh1–substrate recognition subunit of APC/C ubiquitin ligase to AURKA. Depletion of NEDD9 in tumor cells increases sensitivity to AURKA inhibitors. Combination therapy with NEDD9 short hairpin RNAs and AURKA inhibitors impairs tumor growth and distant metastasis in mice harboring xenografts of breast tumors. Collectively, our findings provide rationale for the use of AURKA inhibitors in treatment of metastatic tumors and predict the sensitivity of the patients to AURKA inhibitors based on NEDD9 expression. Cancer Res; 73(10); 3168–80. 2013 AACR. Introduction The serine/threonine kinase, AURKA, is a proto-oncoprotein that is overexpressed in most cancers (1–3). High AURKA expression is strongly associated with decreased survival and is an independent prognostic marker (4). AURKA overexpression disrupts the spindle checkpoint activated by paclitaxel or nocodazole, inducing resistance to these compounds (5). Inhibition or depletion of AURKA protein may therefore improve the survival of patients resistant to paclitaxel (5). While 94% of the primary invasive mammary carcinomas have elevated AURKA protein levels (6), only 13.6% show AURKA gene amplification (1, 3). Thus, posttranscriptional mechanisms of AURKA stabilization are important in breast cancer. AURKA is polyubiquitinated by the anaphase-promoting complex/cyclosome (APC/C) complex and targeted for degradation by the proteasome (7). APC/C-dependent degradation of AURKA requires cdh1, which acts as a substrate recognition subunit for a number ofmitotic proteins, including Plk1 and cyclin B. Overexpression of cdh1 reduces AURKA levels (8), whereas cdh1 knockdown ormutation of the AURKA cdh1–binding site results in elevated AURKA expression (7–9). AURKA is ubiquitinated through the recognition of a carboxylterminal D-box (destruction box) and an amino-terminal Abox, specific for the destruction of AURKA (10, 11). Phosphorylation of AURKA on Ser51 in the A-box inhibits cdh1-APC/C– mediated ubiquitination and consequent AURKA degradation (9). Cancer cells express high levels of AURKA independently of a cell cycle, which suggests that there are additionalmechanisms of AURKA stabilization. Recently, a number of proteins were documented to be involved in the regulation of AURKA stability either by direct deubiquitination of AURKA (12) or through interference with AURKA ubiquitination by APC/C (PUM2, TPX2, LIMK2; refs. 13–15.) NEDD9 is a member of metastatic gene signature identified in breast adenocarcinomas and melanomas (16–18). NEDD9 is a cytoplasmic docking protein of the CAS family. NEDD9 regulates proliferation directly by binding to and activating AURKA (19). In nontransformed cells, activation Authors' Affiliations: Departments of Biochemistry, Statistics, Pathology, and Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, Morgantown, West Virginia Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Elena N. Pugacheva, Department of Biochemistry, POBox9142, 1MedicalCenterDrive,West VirginiaUniversitySchool of Medicine, Morgantown, WV 26506. Phone: 304-293-5295; Fax: 304-2934667; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-12-4008 2013 American Association for Cancer Research. Cancer Research Cancer Res; 73(10) May 15, 2013 3168 on April 15, 2017. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst March 28, 2013; DOI: 10.1158/0008-5472.CAN-12-4008