RESUMEN
Prior studies had shown that the Mirk/dyrk1B gene is amplified/upregulated in about 75% of ovarian cancers, that protein levels of this kinase are elevated in quiescent G0 cells and that Mirk maintains tumor cells in quiescence by initiating rapid degradation of cyclin D isoforms and by phosphorylation of a member of the DREAM complex. Depletion of Mirk/dyrk1B led to increased cyclin D levels, an elevated reactive oxygen species (ROS) content and loss of viability. However, many normal cells in vivo are quiescent, and therefore, targeting a kinase found in quiescent cells might be problematic. In our study, Mirk kinase activity was found to be higher in ovarian cancer cells than in normal cells. Pharmacological inhibition of Mirk/dyrk1B kinase increased cyclin D levels both in quiescent normal diploid cells and in quiescent CDKN2A-negative ovarian cancer cells, but led to more active CDK4/cyclin D complexes in quiescent ovarian cancer cells, allowing them to escape G0/G1 quiescence, enter cycle with high ROS levels and undergo apoptosis. The ROS scavenger N-acetyl cysteine reduced both the amount of cleaved poly(ADP-ribose) polymerase (PARP) and the extent of cancer cell loss. In contrast, normal cells were spared because of their expression of cyclin directed kinase (CDK) inhibitors that blocked unregulated cycling. Quiescent early passage normal ovarian epithelial cells and two strains of quiescent normal diploid fibroblasts remained viable after the inhibition of Mirk/dyrk1B kinase, and the few cells that left G0/G1 quiescence were accumulated in G2+M. Thus, inhibition of Mirk kinase targeted quiescent ovarian cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Ováricas/metabolismo , Ovario/citología , Ovario/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Quinasas DyrKRESUMEN
A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they reenter the cell cycle. Earlier studies showed that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G(0) tumor cells. Mirk uses several mechanisms to block cell cycling, and Mirk increases expression of antioxidant genes that decrease reactive oxygen species (ROS) levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G(0) state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1, and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S-phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In previous studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase.