Replication stress triggers microsatellite destabilization and hypermutation leading to clonal expansion in vitro

Abstract

Mismatch repair (MMR)-deficient cancers are characterized by microsatellite instability (MSI) and hypermutation. However, it remains unclear how MSI and hypermutation arise and contribute to cancer development. Here, we show that MSI and hypermutation are triggered by replication stress in an MMR-deficient background, enabling clonal expansion of cells harboring ARF/p53-module mutations and cells that are resistant to the anti-cancer drug camptothecin. While replication stress-associated DNA double-strand breaks (DSBs) caused chromosomal instability (CIN) in an MMR-proficient background, they induced MSI with concomitant suppression of CIN via a PARP-mediated repair pathway in an MMR-deficient background. This was associated with the induction of mutations, including cancer-driver mutations in the ARF/p53 module, via chromosomal deletions and base substitutions. Immortalization of MMR-deficient mouse embryonic fibroblasts (MEFs) in association with ARF/p53-module mutations was ~60-fold more efficient than that of wild-type MEFs. Thus, replication stress-triggered MSI and hypermutation efficiently lead to clonal expansion of cells with abrogated defense systems.

Mismatch repair dependence of replication stress-associated DSB recognition and repair

“…By contrast, MSI is defined as changes in the lengths of microsatellite fragments that contain short repetitive sequences (1-6 bases) [6]. Importantly, such genomic destabilization is associated with mutation induction and clonal evolution of cells with abrogated defense systems, such as the ARF/p53 module [7]. Given that most cancers develop with genomic instability, mutagenesis associated with genomic destabilization is probably the major risk factor for step-wise cancer development.…”

“…Such cellular senescence is further associated with aging and aging-associated disorders [10,11]. Both CIN and MSI inductions are triggered by replication stress-associated DNA double-strand breaks (DSBs) [7,12,13,14,15], which are generally targeted by homologous recombination (HR) factors [16,17].…”

“…By contrast, identical DSBs in MMR-deficient cells are erroneously repaired by microhomology-mediated end joining (MMEJ) and this induces MSI, i.e., specific insertions/deletions at microsatellite loci that are potential micro-homologous sites [22]. Since MSI is induced with eliminating DSBs, CIN is suppressed during the MSI induction [7]. Thus, CIN and MSI are induced when those DSBs are persisted or erroneously repaired by MMEJ.…”

“…Even in MMR-deficient cells, MSI is blocked upon suppression of MMEJ by PolQ knockdown (KD) and PARP inhibition [7]. Under these conditions, cells are similar to MMR-proficient cells.…”

“…Under these conditions, cells are similar to MMR-proficient cells. Specifically, replication stress-associated DSBs persist, leading to CIN induction (tetraploidization) [7]. This implies that MMEJ specifically occurs in an MMR-deficient background to induce MSI and suppress CIN, posing the question of how MMR deficiency is associated with MMEJ induction.…”