Cancer: From genome instability to therapy
2019 ASCB DOORSTEP MEETING
Walter E. Washington Convention Center, East Salon * December 7, 2019
Cancer metastasis, i.e., the spreading of cancer cells from the primary tumor to distant sites, is responsible for the vast majority of cancer deaths. During this process, tumor cells migrate through tight interstitial spaces that require substantial deformation of the cell and its nucleus, which is the largest and stiffest organelle. We have developed microfluidic devices that closely mimic the physical constraints of physiological interstitial environments while providing precise control over the constriction geometry and enabling live-cell imaging at high spatial and temporal resolution. Using these devices, along with fluorescent reporters for nuclear integrity and DNA damage, we demonstrated that the deformability of the nucleus forms a rate-limiting step during migration through confined environments, and that the physical stress associated with confined migration can result in nuclear envelope rupture, DNA damage, and nuclear fragmentation. These findings suggest that tumor cell migration can increase the genomic instability of tumor cells, which could promote cancer progression and resistance to therapy. Here, I will present new findings into the mechanism by which cells are able to squeeze their nucleus through small interstitial spaces, and discuss the functional consequences of the nuclear deformation on nuclear structure and function. Insights gained from this work may improve prognostic approaches and motivate novel therapies to control metastatic disease.
Pericentromeric heterochromatin occupies ~30% of the human genome, and mostly comprises repeated DNA sequences prone to aberrant recombination, with a high potential to induce chromosome rearrangements and tumorigenesis. In Drosophila cells, ‘safe’ homologous recombination (HR) repair of heterochromatic double strand breaks (DSBs) relies on a specialized pathway that relocalizes repair sites to the nuclear periphery before strand invasion. Notably, heterochromatin is highly enriched for ‘silent' chromatin marks (H3K9me2/3 and HP1a), but how this unique epigenetic environment influences repair is mostly unknown and largely understudied. Our studies revealed a critical role for silencing in the spatial and temporal regulation of heterochromatin repair, including by promoting: i) the recruitment of Smc5/6 and SUMOylation activities that block HR progression inside the heterochromatin domain; and ii) loading of actin nucleators and myosins that generate nuclear actin filaments and drive the direct motion of repair sites. Using recently developed site-specific DSB systems we also started exploring chromatin responses to heterochromatic DSBs at high resolution by chromatin immunoprecipitation-sequencing (ChIP-Seq). This analysis revealed that silencing marks are largely retained during heterochromatin repair and are required to maintain low Mu2/Mdc1 levels at heterochromatic DSBs. Together, these studies challenge the common view that silencing and compaction in heterochromatin are an obstacle to repair, and propose a new paradigm where silencing actively contributes to unique repair responses in this domain. Notably, heterochromatin silencing is commonly deregulated at early stages of tumorigenesis, suggesting silencing-related defects as early contributors to genome instability that drives cancer initiation and progression.
The life of any organism depends on the ability of cells to detect and to respond to pathogens. In order to detect the immense variety of pathogenic entities, the innate immune system of mammals has evolved a range of distinct sensing strategies. One major mechanism is based on the recognition of microbial DNA - an invariant and highly immunogenic pathogen-associated molecular pattern. Host cells, however, contain abundant sources of self-DNA. In the context of cellular damage or metabolic derangement, “out-of-the-context” self-DNA can elicit potentially damaging inflammatory responses. Our research focuses on the so-called cGAS-STING system - an evolutionary highly conserved innate DNA sensing system. On DNA binding, cGAS is activated to produce a second messenger cyclic dinucleotide (cyclic GMP-AMP), which stimulates the adaptor protein STING to induce innate immune responses. While this process was originally discovered as a crucial component of immune defense against pathogens, recent work has elucidated a pathogenic role for innate DNA sensing in a variety of sterile inflammatory diseases. In this talk I will discuss recent findings on cellular mechanisms that regulate cGAS activity and present work on the pharmacological manipulation of aberrant cGAS-STING signaling in the context of inflammatory diseases.
Alterations in DNA damage signaling and repair pathways in cancer cells provides a unique therapeutic opportunity for developing tumor-specific treatments. These alterations arise from genetic and epigenetic events in combination with pathway re-wiring that occurs during tumor development and evolution. Identifying these tumor-specific vulnerabilities remains challenging, however, since only a subset of possible targets is revealed by genomic sequencing. An alternative approach is to use systems-based methods to explore altered signaling and drug responses in specific cancer types. Using this approach, we have identified a unique DNA damage-dependent vulnerability in oncogenically-driven tumors by targeting the epigenetic transcriptional co-activator BRD4. Inhibition of BRD4 results in persistent DNA:RNA hybrids (R-loops) that collide with the replication machinery during S-phase, resulting in enhanced replication stress. Cell cycle checkpoint signaling from these stalled replication forks at sites of transcription-replication collision is compromised, however, because BRD4 inhibition also results in downregulation of TopBP1, disrupting the ATR-Chk1 pathway, and leading to cell death in S-phase and mitotic catastrophe in M-phase. These findings highlight a new mechanism by which BRD4 inhibitors work as anti-tumor agents in a Myc-independent manner by leveraging replication stress and the DNA damage response.
Chromosomal instability (CIN) is a hallmark of cancer and it results from ongoing errors in chromosome segregation during mitosis. While CIN is a major driver of tumor evolution, its role in metastasis has not been established. Here we show that CIN promotes metastasis by sustaining a tumor-cell autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING cytosolic DNA-sensing pathway and downstream noncanonical NF-kB signaling. Genetic suppression of CIN significantly delays metastasis even in highly aneuploid tumor models, whereas inducing continuous chromosome segregation errors promotes cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumor cells co-opt chronic activation of innate immune pathways to spread to distant organs.
While driver mutations in cancer genomes were the main focus of cancer research for a long time, passenger mutational signatures - the imprints of DNA damage and DNA repair processes that have been operative during tumorigenesis - are also biologically informative. In this lecture, I provide an update of where we are in untangling the mechanisms underpinning genomic instability in human somatic cells, describing the new insights that we have gained through combinations of computational analysis and experiments in cell-based systems, and showcase how we have developed the concept into applications that we hope to translate into clinical utility in the near future.
DNA entering the cytoplasm of mammalian cells can trigger a potent innate immune response, including the production of type-I interferons and inflammatory cytokines. We have identified the enzyme cGAS as the sensor of cytosolic DNA that triggers the innate immune response. cGAS catalyzes the synthesis of cyclic GMP-AMP, which functions as a second messenger that activates the adaptor protein STING and the downstream pathway. Recent research has revealed an expanding role of the cGAS pathway in immune defense, autoimmune disease, cellular senescence, autophagy and cancer. I will discuss our recent work on understanding the mechanism and functions of the cGAS-STING pathway.