Supplemental Fig. S1. Effect of wild-type or a phosphorylation sites mutant CP110 species on responses to CDK2 inhibition. (A) Overexpression of CP110 was detected in ED-1 cells with an anti-HA antibody 24 and 48 hours after transfection. (B) Overexpression of wild-type CP110 significantly reduced apoptosis induced by seliciclib treatment. ED-1 cells overexpressing CP110 were treated with the indicated dosages of seliciclib for 24 hours and analyzed for apoptosis. (C and D). Overexpression of phosphorylation-site mutated CP110 did not have a significant effect on anaphase catastrophe or apoptosis induced by CDK2 inhibition. ED-1 cells overexpressing a mutant-CP110 species were treated with the indicated dosages of seliciclib for 24 hours or transfected individually with two different siRNAs targeting CDK2 for 24 hours and (C) scored for multipolar anaphase and (D) analyzed for apoptosis. (E). Seliciclib treatment did not appreciably affect CP110 basal levels in human and murine lung cancer cells. Hop62 and ED-1 were treated with various dosages of seliclcib and CP110 levels were examined after 24 and 48 hours of treatment.
ARTICLE ABSTRACT
Aneuploidy is frequently detected in human cancers and is implicated in carcinogenesis. Pharmacologic targeting of aneuploidy is an attractive therapeutic strategy, as this would preferentially eliminate malignant over normal cells. We previously discovered that CDK2 inhibition causes lung cancer cells with more than two centrosomes to undergo multipolar cell division leading to apoptosis, defined as anaphase catastrophe. Cells with activating KRAS mutations were especially sensitive to CDK2 inhibition. Mechanisms of CDK2-mediated anaphase catastrophe and how activated KRAS enhances this effect were investigated. Live-cell imaging provided direct evidence that following CDK2 inhibition, lung cancer cells develop multipolar anaphase and undergo multipolar cell division with the resulting progeny apoptotic. The siRNA-mediated repression of the CDK2 target and centrosome protein CP110 induced anaphase catastrophe of lung cancer cells. In contrast, CP110 overexpression antagonized CDK2 inhibitor–mediated anaphase catastrophe. Furthermore, activated KRAS mutations sensitized lung cancer cells to CDK2 inhibition by deregulating CP110 expression. Thus, CP110 is a critical mediator of CDK2 inhibition–driven anaphase catastrophe. Independent examination of murine and human paired normal–malignant lung tissues revealed marked upregulation of CP110 in malignant versus normal lung. Human lung cancers with KRAS mutations had significantly lower CP110 expression as compared with KRAS wild-type cancers. Thus, a direct link was found between CP110 and CDK2 inhibitor antineoplastic response. CP110 plays a mechanistic role in response of lung cancer cells to CDK2 inhibition, especially in the presence of activated KRAS mutations. Cancer Res; 75(10); 2029–38. ©2015 AACR.
