RESUMEN
Insulin receptor substrate 2 (IRS2) is an essential adaptor that mediates signaling downstream of the insulin receptor and other receptor tyrosine kinases. Transduction through IRS2-dependent pathways is important for coordinating metabolic homeostasis, and dysregulation of IRS2 causes systemic insulin signaling defects. Despite the importance of maintaining proper IRS2 abundance, little is known about what factors mediate its protein stability. We conducted an unbiased proteomic screen to uncover novel substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that controls the abundance of key cell cycle regulators. We found that IRS2 levels are regulated by APC/C activity and that IRS2 is a direct APC/C target in G1 Consistent with the APC/C's role in degrading cell cycle regulators, quantitative proteomic analysis of IRS2-null cells revealed a deficiency in proteins involved in cell cycle progression. We further show that cells lacking IRS2 display a weakened spindle assembly checkpoint in cells treated with microtubule inhibitors. Together, these findings reveal a new pathway for IRS2 turnover and indicate that IRS2 is a component of the cell cycle control system in addition to acting as an essential metabolic regulator.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Secuencias de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase/efectos de los fármacos , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Línea Celular , Cromatografía Liquida , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Humanos , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Fosforilación , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad Proteica , Proteómica , Piridinas/farmacología , Espectrometría de Masas en Tándem , Imagen de Lapso de Tiempo , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genéticaRESUMEN
Purpose: Acquired resistance to cisplatin is a major barrier to success in treatment of various cancers, and understanding mitotic mechanisms unique to cisplatin-resistant cancer cells can provide the basis for developing novel mitotic targeted therapies aimed at eradicating these cells.Experimental Design: Using cisplatin-resistant models derived from primary patient epithelial ovarian cancer (EOC) cells, we have explored the status of mitotic exit mechanisms in cisplatin-resistant cells.Results: We have uncovered an unexpected role of long-term cisplatin treatment in inducing mitotic exit vulnerability characterized by increased spindle checkpoint activity and functional dependency on Polo-like kinase 1 (PLK1) for mitotic exit in the presence of anaphase promoting complex/cyclosome (APC/C) dysfunction in a cisplatin-resistant state. Accordingly, PLK1 inhibition decreased the survival of cisplatin-resistant cells in vitro and in vivo and exacerbated spindle checkpoint response in these cells. APC/CCDC20 inhibition increased sensitivity to pharmacologic PLK1 inhibition, further confirming the existence of APC/C dysfunction in cisplatin-resistant cells. In addition, we uncovered that resistance to volasertib, PLK1 inhibitor, is due to maintenance of cells with low PLK1 expression. Accordingly, stable PLK1 downregulation in cisplatin-resistant cells induced tolerance to volasertib.Conclusions: We provide the first evidence of APC/C dysfunction in cisplatin-resistant state, suggesting that understanding APC/C functions in cisplatin-resistant state could provide a basis for developing novel mitotic exit-based therapies to eradicate cisplatin-resistant cancer cells. Our results also show that PLK1 downregulation could underlie emergence of resistance to PLK1-targeted therapies in cancers. Clin Cancer Res; 24(18); 4588-601. ©2018 AACR.