HDAC1/HDAC2 and PR130 modulate checkpoint kinase-dependent cell fate decisions during replicative stress
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Abstract
Replicative stress (RS) activates a complex signalling network to maintain the integrity of replicating DNA and to protect cells from premature mitosis. The checkpoint kinases ATM, ATR, CHK1 and CHK2 orchestrate the replicative stress response (RSR). They promote cell cycle arrest, the repair of stalled and collapsed replication forks and eventually processes that lead to the elimination of cells with irreparable DNA damage. The high proliferation rate of cancer cells makes them especially susceptible to RS and replication-associated DNA damage. Accordingly, the induction of RS, for example with the ribonucleotide reductase inhibitor hydroxyurea (HU), is a widely used strategy in cancer therapy.
Prior reports have revealed that histone deacetylase inhibitors (HDACis) like MS-275 modulate checkpoint kinase activity in cancer cells. In the case of ATM, this was attributed to an interaction with PR130, a regulatory subunit of protein phosphatase PP2A.
The present study demonstrates that MS-275 counteracts the phosphorylation of a subset of checkpoint kinases (ATM, CHK1, CHK2) upon RS while the phosphorylation of ATR remains mostly unaffected. Western blot analyses of additional factors that are involved in the RSR reveal that MS-275 deregulates multiple pathways including WEE1 and p53 signalling, which are involved in the inhibition of cell cycle-promoting CDK activity during RS. The combined loss of these cell cycle regulatory mechanisms by MS-275 triggers an aberrant progression of HCT116 colon cancer cells from HU-induced replicative arrest into mitotic catastrophe. The inhibition of individual checkpoint kinases with chemical inhibitors partially mimics the effect of MS-275.
Moreover, the results in this study demonstrate for the first time the dephosphorylation of pS1981-ATM by the PR130-PP2A complex. The discovery that PR130 is acetylated and that this posttranslational modification is augmented when the HDACs1-3 are inhibited suggests a previously unrecognised acetylation-dependent mechanism for the interaction between pS1981-ATM and PR130.
The generation of a PR130-knockout cell line (HCT116ΔPR130) by CRISPR-based genome editing reveals a to-date unknown, regulatory function of PR130 in the RSR. The loss of PR130 sensitises cells to HU-induced replicative arrest that was accompanied by an excessive accumulation of RPA foci and an increased phosphorylation of H2AX; these factors indicate single stranded DNA (ssDNA) and stalled to broken replication forks, respectively. The enhanced RS levels in PR130-knockout cells are linked to the hyperphosphorylation of CHK1 and p53. Thus, the elimination of PR130 sensitised HCT116 cells to the cell cycle deregulation and RS-induced apoptosis, which was associated with a loss of the DNA repair protein RAD51 by MS-275.
In sum, this work demonstrates that class I histone deacetylase activity maintains the integrity of cell cycle checkpoints upon RS through transcriptional and post-transcriptional mechanisms that control PR130. Hence, PR130 is a novel regulator of cell fate decisions in cancer cells during RS.