Browsing by Subject "Replication stress"

Understanding the differences between normal and malignant tissue is required to find vulnerabilities in cancer that can be exploited. One of the hallmarks of cancer is its ability to sustain proliferative signaling, leading to unbridled cellular replication. This puts an increased pressure on the cell’s ability to maintain genome integrity and creates a potential vulnerability to be targeted by cancer therapies. Targeting how a cancer cell prevents or responds to DNA damage is one way to take advantage of this vulnerability.

My dissertation work aims to better understand this DNA damage response in cancer and tests two hypotheses: The first is whether inhibition of the transcriptional regulator BRD4 leads to an increase in transcription-replication conflicts, DNA damage, and cell death. The second is whether the tumor microenvironment alters the way cancer cells respond to DNA damage induced by radiation therapy in glioblastoma.

Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintain genomic integrity and cell survival. My work shows that BRD4, a BET bromodomain protein and known transcriptional regulator, is important for preventing dysregulation of these systems leading to conflicts between the transcription and replication machinery in S-phase. We demonstrate that inhibition or degradation of BET bromodomain proteins leads to an accumulation of RNA:DNA hybrids, a known cause of transcription-replication conflicts, and causes increased DNA damage and cell death in cancer cells actively undergoing replication. Furthermore,

over-expression of full-length BRD4, which contains a P-TEFb interacting domain known to activate efficient transcription, is necessary and sufficient to rescuing this effect. These results give mechanistic insight into chemotherapeutics that target BRD4 currently in clinical trials.

In complementary work, we explored the effect that the extracellular environment of cancer plays in its response to DNA damage caused by radiation therapy. Standard methods of culturing cancer cells, which do not replicate the extracellular environment of a native tumor, have led to an incomplete understanding of response to therapies such as ionizing radiation in vivo. To understand the role that the tumor environment plays on the radiation response, we used both human and murine glioblastoma cells to show that organotypic brain slice culture was better able to recapitulate the expression profiles of in vivo tumors. Specifically, we saw that pathways involved in multicellular processes, cell morphogenesis, and the extracellular matrix were not only significantly upregulated in glioblastoma cells cultured on brain slices compared to in vitro culture but were also critically important to radiation survival.

Collectively, this dissertation provides novel understandings of how cancer cells prevent and respond to DNA damage as well as a framework for future work in cancer biology.

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.