Increased Circulating Osteopontin Levels Promote Primary Tumour Growth, but Do Not Induce Metastasis in Melanoma.

Osteopontin (OPN) is a phosphoprotein with diverse functions in various physiological and pathological processes. OPN expression is increased in multiple cancers, and OPN within tumour tissue has been shown to promote key stages of cancer development. OPN levels are also elevated in the circulation of cancer patients, which in some cases has been correlated with enhanced metastatic propensity and poor prognosis. However, the precise impact of circulating OPN (cOPN) on tumour growth and progression remains insufficiently understood. To examine the role of cOPN, we used a melanoma model, in which we stably increased the levels of cOPN through adeno-associated virus-mediated transduction. We found that increased cOPN promoted the growth of primary tumours, but did not significantly alter the spontaneous metastasis of melanoma cells to the lymph nodes or lungs, despite an increase in the expression of multiple factors linked to tumour progression. To assess whether cOPN has a role at later stages of metastasis formation, we employed an experimental metastasis model, but again could not detect any increase in pulmonary metastasis in animals with elevated levels of cOPN. These results demonstrate that increased levels of OPN in the circulation play distinct roles during different stages of melanoma progression.

Actin cytoskeleton in angiogenesis.

Actin, one of the most abundant intracellular proteins in mammalian cells, is a critical regulator of cell shape and polarity, migration, cell division, and transcriptional response. Angiogenesis, or the formation of new blood vessels in the body is a well-coordinated multi-step process. Endothelial cells lining the blood vessels acquire several new properties such as front-rear polarity, invasiveness, rapid proliferation and motility during angiogenesis. This is achieved by changes in the regulation of the actin cytoskeleton. Actin remodelling underlies the switch between the quiescent and angiogenic state of the endothelium. Actin forms endothelium-specific structures that support uniquely endothelial functions. Actin regulators at endothelial cell-cell junctions maintain the integrity of the blood-tissue barrier while permitting trans-endothelial leukocyte migration. This review focuses on endothelial actin structures and less-recognised actin-mediated endothelial functions. Readers are referred to other recent reviews for the well-recognised roles of actin in endothelial motility, barrier functions and leukocyte transmigration. Actin generates forces that are transmitted to the extracellular matrix resulting in vascular matrix remodelling. In this review, we attempt to synthesize our current understanding of the roles of actin in vascular morphogenesis. We speculate on the vascular bed specific differences in endothelial actin regulation and its role in the vast heterogeneity in endothelial morphology and function across the various tissues of our body.

Histone H4 lysine 20 mono-methylation directly facilitates chromatin openness and promotes transcription of housekeeping genes.

Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction.

Chromatin Landscaping At Mitotic Exit Orchestrates Genome Function.

Chromatin architecture is highly dynamic during different phases of cell cycle to accommodate DNA-based processes. This is particularly obvious during mitotic exit, where highly condensed rod-like chromatids need to be rapidly decondensed. Such chromatin structural transitions are tightly controlled and organized as any perturbance in this dynamic process can lead to genome dysfunction which may culminate in loss of cellular fitness. However, the mechanisms underlying cell cycle-dependent chromatin structural changes are not fully understood. In this mini review, we highlight our current knowledge of chromatin structural organization, focusing on mitotic exit. In this regard, we examine how nuclear processes are orchestrated during chromatin unfolding and compartmentalization and discuss the critical importance of cell cycle-controlled chromatin landscaping in maintaining genome integrity.

Chromatin Dynamics in Genome Stability: Roles in Suppressing Endogenous DNA Damage and Facilitating DNA Repair.

Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage.

Telomere Replication Stress Induced by POT1 Inactivation Accelerates Tumorigenesis.

Genome sequencing studies have revealed a number of cancer-associated mutations in the telomere-binding factor POT1. Here, we show that when combined with p53 deficiency, depletion of murine POT1a in common lymphoid progenitor cells fosters genetic instability, accelerates the onset, and increases the severity of T cell lymphomas. In parallel, we examined human and mouse cells carrying POT1 mutations found in cutaneous T cell lymphoma (CTCL) patients. Inhibition of POT1 activates ATR-dependent DNA damage signaling and induces telomere fragility, replication fork stalling, and telomere elongation. Our data suggest that these phenotypes are linked to impaired CST (CTC1-STN1-TEN1) function at telomeres. Lastly, we show that proliferation of cancer cells lacking POT1 is enabled by the attenuation of the ATR kinase pathway. These results uncover a role for defective telomere replication during tumorigenesis.

Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination.

The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions. Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Polθ; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes.

Identification and characterization of MUS81 point mutations that abolish interaction with the SLX4 scaffold protein.

MUS81-EME1 is a conserved structure-selective endonuclease with a preference for branched DNA substrates in vitro that correspond to intermediates of DNA repair. Cells lacking MUS81 or EME1 show defects in the repair of DNA interstrand crosslinks (ICL) resulting in hypersensitivity to agents such as mitomycin C. In metazoans, a proportion of cellular MUS81-EME1 binds the SLX4 scaffold protein, which is itself instrumental for ICL repair. It was previously reported that mutations in SLX4 that abolished interaction with MUS81 affected ICL repair in human cells but not in murine cells. In this study we looked the other way around by pinpointing amino acid residues in MUS81 that when mutated abolish the interaction with SLX4. These mutations fully rescued the mitomycin C hypersensitivity of MUS81 knockout murine cells, but they were unable to rescue the sensitivity of two different human cell lines defective in MUS81. These data support an SLX4-dependent role for MUS81 in the repair, but not the induction of ICL-induced double-strand breaks. This study sheds light on the extent to which MUS81 function in ICL repair requires interaction with SLX4.

Cooperative control of holliday junction resolution and DNA repair by the SLX1 and MUS81-EME1 nucleases.

Holliday junctions (HJs) are X-shaped DNA structures that arise during homologous recombination, which must be removed to enable chromosome segregation. The SLX1 and MUS81-EME1 nucleases can both process HJs in vitro, and they bind in close proximity on the SLX4 scaffold, hinting at possible cooperation. However, the cellular roles of mammalian SLX1 are not yet known. Here, we use mouse genetics and structure function analysis to investigate SLX1 function. Disrupting the murine Slx1 and Slx4 genes revealed that they are essential for HJ resolution in mitotic cells. Moreover, SLX1 and MUS81-EME1 act together to resolve HJs in a manner that requires tethering to SLX4. We also show that SLX1, like MUS81-EME1, is required for repair of DNA interstrand crosslinks, but this role appears to be independent of HJ cleavage, at least in mouse cells. These findings shed light on HJ resolution in mammals and on maintenance of genome stability.