Inhibition of Chk1 kills tetraploid tumor cells through a p53-dependent pathway.

Ilio Vitale,Ken A Olaussen,Lisa Nanty,Sonia Vivet,Maria Castedo,Laura Senovilla,Michelle Prudhomme,Vladimir Lazar,Lorenzo Galluzzi,Roy M Golsteyn,Guido Kroemer,Philippe Dessen,Nils Cordes

doi:10.1371/journal.pone.0001337

Abstract

Tetraploidy constitutes an adaptation to stress and an intermediate step between euploidy and aneuploidy in oncogenesis. Tetraploid cells are particularly resistant against genotoxic stress including radiotherapy and chemotherapy. Here, we designed a strategy to preferentially kill tetraploid tumor cells. Depletion of checkpoint kinase-1 (Chk1) by siRNAs, transfection with dominant-negative Chk1 mutants or pharmacological Chk1 inhibition killed tetraploid colon cancer cells yet had minor effects on their diploid counterparts. Chk1 inhibition abolished the spindle assembly checkpoint and caused premature and abnormal mitoses that led to p53 activation and cell death at a higher frequency in tetraploid than in diploid cells. Similarly, abolition of the spindle checkpoint by knockdown of Bub1, BubR1 or Mad2 induced p53-dependent apoptosis of tetraploid cells. Chk1 inhibition reversed the cisplatin resistance of tetraploid cells in vitro and in vivo, in xenografted human cancers. Chk1 inhibition activated p53-regulated transcripts including Puma/BBC3 in tetraploid but not in diploid tumor cells. Altogether, our results demonstrate that, in tetraploid tumor cells, the inhibition of Chk1 sequentially triggers aberrant mitosis, p53 activation and Puma/BBC3-dependent mitochondrial apoptosis.

Highlights

Cancer results from the accumulation of genetic and epigenetic alterations in which genomic instability conditions the progressive deterioration towards an ever more aggressive phenotype
We show that inhibition of checkpoint kinase-1 (Chk1) by small interfering RNAs, dominant-negative mutant Chk1, or pharmacological compounds is toxic on tetraploid tumor cells that otherwise are relatively resistant against genotoxic agents
The mechanism accounting for the preferential killing of tetraploid cells appears to involve the abolition of the spindle assembly checkpoint (SAC), premature entry in mitosis, failed and catastrophic mitotic events, as well as p53 activation, transcription of pro-apoptotic p53 target genes including the BH3 only protein Puma and induction of the mitochondrial pathway of apoptosis

Summary

Introduction

Cancer results from the accumulation of genetic and epigenetic alterations in which genomic instability conditions the progressive deterioration towards an ever more aggressive phenotype. One of the mechanisms of genomic instability involves a transient phase of polyploidization (in most cases tetraploidization), which may result from endoreplication (DNA replication without mitosis), endomitosis (karyokinesis without cytokinesis) [1] or aberrant cell fusion [2,3]. A variety of endogenous tumor suppressor gene products prevent the generation of tetraploid cells. The loss of the tumor suppressor proteins p21 [14], Bax [8], APC [15], or Lats2 [12] facilitates the generation of tetraploid cells. One possible interpretation of these findings is that a hypothetical ‘‘tetraploidy checkpoint’’ [9] would prevent the generation or propagation of tetraploid cells, by mean of a stable cell cycle arrest or their elimination by apoptosis. Chk is involved in tumor suppression, and its inhibition can sensitize tumor cells to DNA damage [21,22,23,24], presumably because the failure to arrest the cell cycle upon DNA damage is a lethal event

Methods

Results

Conclusion