Telomere, Replication Stress and Cancer stem cell

SMARCAL1 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A-Like 1) is an ATP-dependent DNA-annealing helicase that reverses stalled replication forks. Its loss of function genetic alterations occurs in a subset of glioblastomas (GBMs) and has been found to be associated with alterative lengthening of telomeres (ALT+) in tumor cells. ALT tumors exploit homologous recombination to maintain telomere length and share common characteristics, including compromised telomere shelterin protein and increased replication stress in the telomere region.We established a SMARCAL1-null, ALT+ primary GBM culture. We show that the primary GBM culture displays stable ALT features and maintains mostly consistent karyotypes after their growth in mice. Transcriptomic profiling of the ALT+ primary GBM cells that have been propagated in vitro and those that have undergone propagation in vivo revealed the effects of microenvironments on the gene expression of these tumor cells. By using a doxycycline-inducible expression system, we show that the ALT+ features in the GBM primary culture can be turned off and on by the restoration or withdraw of exogenous wild-type SMARCAL1, but not its enzymatic dead mutant counterpart. Telomere pull down assays demonstrated the expression of SMARCAL1 effectively attenuates the process of ALT in tumor cells. In supporting the critical roles of ALT for tumor progression, induced restoration of wild-type SMARCAL1, but not its enzymatic dead mutant, effectively suppresses tumor progression in vivo. By taking advantage of our well characterized ALT model system, we are investigating the role of intrinsic DNA damage in tumorigenesis. Intrinsic DNA replicative stress occurs constantly in tumor cells. However, the pathogenic ramifications of replicative stress and the strategies cancer cells undertake to adapt remain to be fully defined. Here, we attempt to address these questions, using isogenic sarcoma and glioma cell line models differing in their intrinsic telomeric replicative stress levels, we show that intrinsic replicative stress promotes cancer stemness in human sarcoma and glioma cells. Further, molecular profiling analysis of human gliomas supports that human gliomas with higher levels of intrinsic replicative stress levels have increased stemness. We show this intrinsic replicative stress-stimulated stemness is accompanied by nonrandom segregation of chromosomes in mitotic cells. More notably, this nonrandom chromosome segregation is associated with asymmetric partition of CD133, a canonical marker for cancer cell stemness, in that the newly synthesized set of chromosomes are placed in one progeny cell while the set serving as templates for DNA replication turns to co-segregate with CD133 in another. We further reveal that this asymmetric co-segregation of chromosomes and CD133 depends on the Wnt/β-catenin signaling pathway. Collectively, these findings identify intrinsic DNA replicative stress as a driver of cancer cell stemness, and suggest a coordinated, Wnt/β-catenin signal-driven process of asymmetrically partitioning DNA and proteins in these cells, potentially as a way of maintaining cellular heterogeneity and population fitness in response to DNA damage. They also highlight and provide new insights into the roles of the Wnt/β-catenin pathway in maintaining tumor cells stemness, and suggest strategies for therapeutically targeting DNA damage-driven stemness in gliomas.