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

Abstract

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 mechanism 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. Re-expression 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 shRNAs 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.

KAP1 Promotes Proliferation and Metastatic Progression of Breast Cancer Cells

“…Injection of cells expressing control shRNA into the mammary fat pad of NSG mice resulted in rapid tumor growth as reported previously (33). Inhibition of KAP1 expression using shRNA reduced tumor growth significantly (Fig.…”

“…Mice were injected with MDA-MB-231LN cells and imaged as described (33). Primary tumor and organs with metastases were collected at the end point of study and analyzed as described (33). …”

Issues in interpreting thein vivoactivity of Aurora-A

“…Overexpression of NEDD9 induces Aurora-A hyperactivation and T288 phosphorylation, and produces cells with both multipolar spindles and supernumerary centrosomes and failure of cytokinesis [54, 64]. NEDD9 is commonly overexpressed in cancer, and promotes invasion and metastasis [63, 65]. …”

“…Two degradation-targeting sequences on Aurora-A mediate destruction by the APC/C complex: the carboxy-terminal D-box (destruction box) and an amino-terminal A-box [53, 65, 79]. Phosphorylation of Aurora-A on S51 in the A-box inhibits CDH1-APC/C–mediated ubiquitination and consequent degradation [78].…”

“…Several proteins are involved in the regulation of AURKA stability either by direct deubiquitination of Aurora-A (USP2a) [80] or through interference with Aurora-A ubiquitination by APC/C (NEDD9, PUM2, TPX2, LIMK2, PHDL1) [40, 65, 81–83]. As an example, NEDD9 associates with the Aurora-A through mitosis, supports its T288 phosphorylation, and hampers the ability of the APC/C complex to ubiquitinate Aurora-A by preventing CDH1 binding [65].…”