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Chromosome compaction is a key feature of mitosis and critical for accurate chromosome segregation. However, a precise quantitative analysis of chromosome geometry during mitotic progression is lacking. Here, we use volume electron microscopy to map, with nanometer precision, chromosomes from prometaphase through telophase in human RPE1 cells. During prometaphase, chromosomes acquire a smoother surface, their arms shorten, and the primary centromeric constriction is formed. The chromatin is progressively compacted, ultimately reaching a remarkable nucleosome concentration of over 750 µM in late prometaphase that remains relatively constant during metaphase and early anaphase. Surprisingly, chromosomes then increase their volume in late anaphase prior to deposition of the nuclear envelope. The plateau of total chromosome volume from late prometaphase through early anaphase described here is consistent with proposals that the final stages of chromatin condensation in mitosis involve a limit density, such as might be expected for a process involving phase separation., (© 2024 Cisneros-Soberanis et al.)

Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain unclear, KRAS is far more frequently mutated than the other Ras isoforms in cancer and RASopathies. We have quantified HRAS, NRAS, KRAS4A and KRAS4B protein abundance across a large panel of cell lines and healthy tissues. We observe consistent patterns of KRAS > NRAS»HRAS protein expression in cells that correlate with the rank order of Ras mutation frequencies in cancer. Our data provide support for the model of a sweet-spot of Ras dosage mediating isoform-specific contributions to cancer and development. We suggest that in most cases, being the most abundant Ras isoform correlates with occupying the sweet-spot and that HRAS and NRAS expression is usually insufficient to promote oncogenesis when mutated. However, our results challenge the notion that rare codons mechanistically underpin the predominance of KRAS mutant cancers. Finally, direct measurement of mutant versus wildtype KRAS protein abundance revealed a frequent imbalance that may suggest additional non-gene duplication mechanisms for optimizing oncogenic Ras dosage., (© 2023. The Author(s).)

Approximately 15% of all cancer patients harbor mutated KRAS. Direct inhibitors of KRAS have now been generated and are beginning to make progress through clinical trials. These include a suite of inhibitors targeting the KRASG12C mutation commonly found in lung cancer. We investigated emergent resistance to representative examples of different classes of Ras targeted therapies. They all exhibited rapid reactivation of Ras signaling within days of exposure and adaptive responses continued to change over long-term treatment schedules. Whilst the gene signatures were distinct for each inhibitor, they commonly involved up-regulation of upstream nodes promoting mutant and wild-type Ras activation. Experiments to reverse resistance unfortunately revealed frequent desensitization to members of a panel of anti-cancer therapeutics, suggesting that salvage approaches are unlikely to be feasible. Instead, we identified triple inhibitor combinations that resulted in more durable responses to KRAS inhibitors and that may benefit from further pre-clinical evaluation., (© 2022 The Author(s).)

In this review, we analyse the impact of oncogenic Ras mutations in mediating cancer drug resistance and the progress made in the abrogation of this resistance, through pharmacological targeting. At a physiological level, Ras is implicated in many cellular proliferation and survival pathways. However, mutations within this small GTPase can be responsible for the initiation of cancer, therapeutic resistance and failure, and ultimately disease relapse. Often termed "undruggable," Ras is notoriously difficult to target directly, due to its structure and intrinsic activity. Thus, Ras-mediated drug resistance remains a considerable pharmacological problem. However, with advances in both analytical techniques and novel drug classes, the therapeutic landscape against Ras is changing. Allele-specific, direct Ras-targeting agents have reached clinical trials for the first time, indicating there may, at last, be hope of targeting such an elusive but significant protein for better more effective cancer therapy. LINKED ARTICLES: This article is part of a themed issue on New avenues in cancer prevention and treatment (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.12/issuetoc., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The recruitment of DRP1 to mitochondrial membranes prior to fission is facilitated by the wrapping of endoplasmic reticulum (ER) membranes around the mitochondria. To investigate the complex interplay between the ER membranes and DRP1 in the context of mitochondrial structure and function, we downregulate two key ER shaping proteins, RTN4 and CLIMP-63, and demonstrate pronounced mitochondrial hyperfusion and reduced ER-mitochondria contacts, despite their differential regulation of ER architecture. Although mitochondrial recruitment of DRP1 is unaltered in cells lacking RTN4 or CLIMP-63, several aspects of mitochondrial function, such as mtDNA-encoded translation, respiratory capacity and apoptosis are significantly hampered. Further mechanistic studies reveal that CLIMP-63 is required for cristae remodeling (OPA1 proteolysis) and DRP1-mediated mitochondrial fission, whereas both RTN4 and CLIMP-63 regulate the recruitment of BAX to ER and mitochondrial membranes to enable cytochrome c release and apoptosis, thereby performing novel and distinct roles in the regulation of mitochondrial structure and function., (© 2022. The Author(s).)

Surface engineering is a promising strategy to limit or prevent the formation of biofilms. The use of topographic cues to influence early stages of biofilm formationn has been explored, yet many fundamental questions remain unanswered. In this work, we develop a topological model supported by direct experimental evidence, which is able to explain the effect of local topography on the fate of bacterial micro-colonies of Staphylococcus spp. We demonstrate how topological memory at the single-cell level, characteristic of this genus of Gram-positive bacteria, can be exploited to influence the architecture of micro-colonies and the average number of surface anchoring points over nano-patterned surfaces, formed by vertically aligned silicon nanowire arrays that can be reliably produced on a commercial scale, providing an excellent platform to investigate the effect of topography on the early stages of Staphylococcus spp. colonisation. The surfaces are not intrinsically antimicrobial, yet they delivered a topography-based bacteriostatic effect and a significant disruption of the local morphology of micro-colonies at the surface. The insights from this work could open new avenues towards designed technologies for biofilm engineering and prevention, based on surface topography.

Purpose: B-cell receptor (BCR) signaling is critical for the pathogenesis of chronic lymphocytic leukemia (CLL), promoting both malignant cell survival and disease progression. Although vital, understanding of the wider signaling network associated with malignant BCR stimulation is poor. This is relevant with respect to potential changes in response to therapy, particularly involving kinase inhibitors. In the current study, we describe a novel high-resolution approach to investigate BCR signaling in primary CLL cells and track the influence of therapy on signaling response., Experimental Design: A kinobead/mass spectrometry-based protocol was used to study BCR signaling in primary CLL cells. Longitudinal analysis of samples donated by clinical trial patients was used to investigate the impact of chemoimmunotherapy and ibrutinib on signaling following surface IgM engagement. Complementary Nanostring and immunoblotting analysis was used to verify our findings., Results: Our protocol isolated a unique, patient-specific signature of over 30 kinases from BCR-stimulated CLL cells. This signature was associated with 13 distinct Kyoto Encyclopedia of Genes and Genomes pathways and showed significant change in cells from treatment-naïve patients compared with those from patients who had previously undergone therapy. This change was validated by longitudinal analysis of clinical trials samples where BCR-induced kinome responses in CLL cells altered between baseline and disease progression in patients failing chemoimmunotherapy and between baseline and treatment in patients taking ibrutinib., Conclusions: These data comprise the first comprehensive proteomic investigation of the BCR signaling response within CLL cells and reveal unique evidence that these cells undergo adaptive reprogramming of this signaling in response to therapy., (©2021 The Authors; Published by the American Association for Cancer Research.)

Background: Local sequence context is known to have an impact on the mutational pattern seen in cancer. The RAS genes and a smoking carcinogen, Benzo[a]pyrene diol epoxide (BPDE), have been utilised to explore these context effects. BPDE is known to form an adduct at the guanines in a number of RAS gene sites, KRAS codons 12, 13 and 14, NRAS codon 12, and HRAS codons 12 and 14., Results: Molecular modelling techniques, along with multivariate analysis, have been utilised to determine the sequence influenced differences between BPDE-adducted RAS gene sequences as well as the local distortion caused by the adducts., Conclusions: We conclude that G:C > T:A mutations at KRAS codon 12 in the tumours of lung cancer patients (who smoke), proposed to be predominantly caused by BPDE, are due to the effect of the interaction methyl group at the C5 position of the thymine base in the KRAS sequence with the BPDE carcinogen investigated causing increased distortion. We further suggest methylated cytosine would have a similar effect, showing the importance of methylation in cancer development., (© 2021. The Author(s).)

In this work, we introduce a one-step strategy that is suitable for continuous flow manufacturing of antimicrobial PDMS materials. The process is based on the intrinsic capacity of PDMS to react to certain organic solvents, which enables the incorporation of antimicrobial actives such as salicylic acid (SA), which has been approved for use in humans within pharmaceutical products. By combining different spectroscopic and imaging techniques, we show that the surface properties of PDMS remain unaffected while high doses of the SA are loaded inside the PDMS matrix. The SA can be subsequently released under physiological conditions, delivering a strong antibacterial activity. Furthermore, encapsulation of SA inside the PDMS matrix ensured a diffusion-controlled release that was tracked by spatially resolved Raman spectroscopy, Attenuated Total Reflectance IR (ATR-IR), and UV-Vis spectroscopy. The biological activity of the new material was evaluated directly at the surface and in the planktonic state against model pathogenic bacteria, combining confocal laser scanning microscopy, electron microscopy, and cell viability assays. The results showed complete planktonic inhibition for clinically relevant strains of Staphylococcus aureus and Escherichia coli, and a reduction of up to 4 orders of magnitude for viable sessile cells, demonstrating the efficacy of these surfaces in preventing the initial stages of biofilm formation. Our approach adds a new option to existing strategies for the antimicrobial functionalisation of a wide range of products such as catheters, wound dressings and in-dwelling medical devices based on PDMS.

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane β-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche., Competing Interests: The authors have declared that no competing interests exist.

The generation of effective and safe nanoagents for biological applications requires their physicochemical characteristics to be tunable, and their cellular interactions to be well characterized. Here, the controlled synthesis is developed for preparing high-aspect ratio gold nanotubes (AuNTs) with tailorable wall thickness, microstructure, composition, and optical characteristics. The modulation of optical properties generates AuNTs with strong near infrared absorption. Surface modification enhances dispersibility of AuNTs in aqueous media and results in low cytotoxicity. The uptake and trafficking of these AuNTs by primary mesothelioma cells demonstrate their accumulation in a perinuclear distribution where they are confined initially in membrane-bound vesicles from which they ultimately escape to the cytosol. This represents the first study of the cellular interactions of high-aspect ratio 1D metal nanomaterials and will facilitate the rational design of plasmonic nanoconstructs as cytosolic nanoagents for potential diagnosis and therapeutic applications., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Ras is frequently mutated in cancer, however, there is a lack of consensus in the literature regarding the cancer mutation frequency of Ras, with quoted values varying from 10%-30%. This variability is at least in part due to the selective aggregation of data from different databases and the dominant influence of particular cancer types and particular Ras isoforms within these datasets. To provide a more definitive figure for Ras mutation frequency in cancer, we cross-referenced the data in all major publicly accessible cancer mutation databases to determine reliable mutation frequency values for each Ras isoform in all major cancer types. These percentages were then applied to current U.S. cancer incidence statistics to estimate the number of new patients each year that have Ras-mutant cancers. We find that approximately 19% of patients with cancer harbor Ras mutations, equivalent to approximately 3.4 million new cases per year worldwide. We discuss the Ras isoform and mutation-specific trends evident within the datasets that are relevant to current Ras-targeted therapies., (©2020 American Association for Cancer Research.)

Activation of trypsinogen (formation of trypsin) inside the pancreas is an early pathological event in the development of acute pancreatitis. In our previous studies we identified the activation of trypsinogen within endocytic vacuoles (EVs), cellular organelles that appear in pancreatic acinar cells treated with the inducers of acute pancreatitis. EVs are formed as a result of aberrant compound exocytosis and subsequent internalization of post-exocytic structures. These organelles can be up to 12 μm in diameter and can be actinated (i.e. coated with F-actin). Notably, EVs can undergo intracellular rupture and fusion with the plasma membrane, providing trypsin with access to cytoplasmic and extracellular targets. Unraveling the mechanisms involved in cellular processing of EVs is an interesting cell biological challenge with potential benefits for understanding acute pancreatitis. In this study we have investigated autophagy of EVs and discovered that it involves a non-canonical LC3-conjugation mechanism, reminiscent in its properties to LC3-associated phagocytosis (LAP); in both processes LC3 was recruited to single, outer organellar membranes. Trypsinogen activation peptide was observed in approximately 55% of LC3-coated EVs indicating the relevance of the described process to the early cellular events of acute pancreatitis. We also investigated relationships between actination and non-canonical autophagy of EVs and concluded that these processes represent sequential steps in the evolution of EVs. Our study expands the known roles of LAP and indicates that, in addition to its well-established functions in phagocytosis and macropinocytosis, LAP is also involved in the processing of post-exocytic organelles in exocrine secretory cells., Abbreviations: AP: acute pancreatitis; CCK: cholecystokinin; CLEM: correlative light and electron microscopy; DPI: diphenyleneiodonium; EV: endocytic vacuole; LAP: LC3-associate phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PACs: pancreatic acinar cells; PFA: paraformaldehyde; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; Res: resveratrol; TAP: trypsinogen activation peptide; TEM: transmission electron microscopy; TLC-S: taurolithocholic acid 3-sulfate; TRD: Dextran Texas Red 3000 MW Neutral; ZGs: zymogen granules.

The peritrophic matrix of blood-feeding insects is a chitinous structure that forms a protective barrier against oral pathogens and abrasive particles 1 . Tsetse flies transmit Trypanosoma brucei, which is the parasite that causes human sleeping sickness and is also partially responsible for animal trypanosomiasis in Sub-Saharan Africa. For this parasite to establish an infection in flies, it must first colonize the area between the peritrophic matrix and gut epithelium called the ectoperitrophic space. Although unproven, it is generally accepted that trypanosomes reach the ectoperitrophic space by penetrating the peritrophic matrix in the anterior midgut 2-4 . Here, we revisited this event using fluorescence- and electron-microscopy methodologies. We show that trypanosomes penetrate the ectoperitrophic space in which the newly made peritrophic matrix is synthesized by the proventriculus. Our model describes how these proventriculus-colonizing parasites can either migrate to the ectoperitrophic space or become trapped within peritrophic matrix layers to form cyst-like bodies that are passively pushed along the gut as the matrix gets remodelled. Furthermore, early proventricular colonization seems to be promoted by factors in trypanosome-infected blood that cause higher salivary gland infections and potentially increase parasite transmission.

Smart antimicrobial surfaces are a powerful tool to prevent bacterial colonization at surfaces. In this work, we report a successful strategy for the functionalization of polydimethylsiloxane (PDMS) surfaces, widely used in medical devices, with salicylic acid (SA), a biocide approved for use in humans. Antimicrobial PDMS surfaces were fabricated via a rational design in which bifunctional silane linker molecules were covalently grafted onto the PDMS via one end, while soft intermolecular interactions with SA were generated at the other end to enable reversible load and release of the biocide. A molecular level understanding of the interface was obtained using attenuated total reflectance Fourier transform infrared, Raman, and X-ray photoelectron spectroscopies, alongside density functional theory calculations. These reveal that the linker molecules dock the SA molecules at the surface via a 1:1 complexation interaction. Furthermore, each 1:1 complex acts as a nucleation point onto which multiple stacks of the biocide are subsequently stabilized via a combination of H-bonding and π-π stacking interactions, thus significantly enhancing SA uptake at the interface. The antimicrobial activity of these surfaces against model Gram-negative and Gram-positive bacteria represented by Escherichia coli , Staphylococcus aureus , and Staphylococcus epidermidis is demonstrated by a log 6 reduction of planktonic bacterial populations and an efficient anti-biofilm activity at the surface.

Due to cell-cycle dysregulation, many cancer cells contain more than the normal compliment of centrosomes, a state referred to as centrosome amplification (CA). CA can drive oncogenic phenotypes and indeed can cause cancer in flies and mammals. However, cells have to actively manage CA, often by centrosome clustering, in order to divide. Thus, CA is also an Achilles' Heel of cancer cells. In recent years, there have been many important studies identifying proteins required for the management of CA and it has been demonstrated that disruption of some of these proteins can cause cancer-specific inhibition of cell growth. For certain targets therapeutically relevant interventions are being investigated, for example, small molecule inhibitors, although none are yet in clinical trials. As the field is now poised to move towards clinically relevant interventions, it is opportune to summarise the key work in targeting CA thus far, with particular emphasis on recent developments where small molecule or other strategies have been proposed. We also highlight the relatively unexplored paradigm of reversing CA, and thus its oncogenic effects, for therapeutic gain., (© 2019 The Author(s).)

Specific molecular interactions that underpin the switch between ER stress-triggered autophagy-mediated cellular repair and cellular death by apoptosis are not characterized. This study reports the unexpected interaction elicited by ER stress between the plasma membrane (PM)-localized apoptosis effector PERP and the ER Ca 2+ pump SERCA2b. We show that the p53 effector PERP, which specifically induces apoptosis when expressed above a threshold level, has a heterogeneous distribution across the PM of un-stressed cells and is actively turned over by the lysosome. PERP is upregulated following sustained starvation-induced autophagy, which precedes the onset of apoptosis indicating that PERP protein levels are controlled by a lysosomal pathway that is sensitive to cellular physiological state. Furthermore, ER stress stabilizes PERP at the PM and induces its increasing co-localization with SERCA2b at ER-PM junctions. The findings highlight a novel crosstalk between pro-survival autophagy and pro-death apoptosis pathways and identify, for the first time, accumulation of an apoptosis effector to ER-PM junctions in response to ER stress., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest.

The structure, ultrastructure and function of hyaline articular cartilage (HAC) and subchondral bone (SCB), and their involvement in the pathogenesis of osteoarthritis (OA) have been extensively researched. However, much less attention has been focused on the intervening tissue, articular calcified cartilage (ACC) and its role in the initiation and progression of OA. Using both light microscopy (LM) and transmission electron microscopy (TEM), a study of ACC in wild type (WT) mice, and mice with genetic osteoarthropathies (AKU) was undertaken to further understand the role played by ACC in the early stages of OA.Tibio-femoral joints were obtained from BALB/c WT and BALB/c AKU mice aged between 7 and 69 weeks. One joint was processed for routine histological analysis. The tip of the medial femoral condyle (MFC), which contained HAC, ACC, and SCB, was dissected from the contra-lateral joint and processed for TEM.In WT and AKU mice novel microanatomical structures, designated concentric lamellae, were identified surrounding chondrocytes in the ACC. The lamellae appeared to be laid down in association with advancement of the tidemark indicating they may be formed during calcification of cartilage matrix. The lamellae were associated with hypertrophic chondrocytes throughout the ACC.Novel microanatomical structures, termed concentric lamellae, which were present around hypertrophic chondrocytes in the ACC are described for the first time. Their apparent association with mineralisation, advancement of the tidemark, and greater abundance in a model of osteoarthropathy indicate their formation could be important in the pathogenesis of OA and AKU.

Maintenance of mitochondrial integrity is critical for normal cellular homoeostasis. Most cells respond to stress stimuli and undergo apoptosis by perturbing mitochondrial structure and function to release proteins, such as cytochrome c , which are essential for the execution of the intrinsic apoptotic cascade. Cancer cells evade these events by overexpressing the anti-apoptotic BCL-2 family of proteins on mitochondrial membranes. Inhibitors of the anti-apoptotic BCL-2 family proteins, also known as BH3 mimetics, antagonise the pro-survival functions of these proteins and result in rapid apoptosis. Although the precise mechanism by which BH3 mimetics induce apoptosis has been well characterised, not much is known in terms of the structural changes that occur in mitochondria during apoptosis. Using a panel of highly selective BH3 mimetics and a wide range of cell lines, we demonstrate that BH3 mimetics induce extensive mitochondrial fission, accompanied by swelling of the mitochondrial matrix and rupture of the outer mitochondrial membrane. These changes occur in a BAX/ BAK-dependent manner. Although a major mitochondrial fission GTPase, DRP-1, has been implicated in mitochondrial apoptosis, our data demonstrate that DRP-1 might function independently/downstream of BH3 mimetic-mediated mitochondrial fission to facilitate the release of cytochrome c and apoptosis. Moreover, downregulation of DRP-1 prevented cytochrome c release and apoptosis even when OPA1, a protein mediating mitochondrial fusion, was silenced. Although BH3 mimetic-mediated displacement of BAK and other BH3-only proteins from BCL-X L and MCL-1 was unaffected by DRP-1 downregulation, it prevented BAK activation significantly, thus placing DRP-1 as one of the most critical players, along with BAX and BAK, that governs BH3 mimetic-mediated cytochrome c release and apoptosis., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest.

The rapid evolution of super-resolution light microscopy has narrowed the gap between light and electron microscopy, allowing the imaging of molecules and cellular structures at high resolution within their normal cellular and tissue context. Multimodal imaging approaches such as correlative light electron microscopy (CLEM) combine these techniques to create a tool with unique imaging capacity. However, these approaches are typically reserved for specialists, and their application to the analysis of neural tissue is challenging. Here we present SuperCLEM , a relatively simple approach that combines super-resolution fluorescence light microscopy (FLM), 3D electron microscopy (3D-EM) and rendering into 3D models. We demonstrate our workflow using neuron-glia cultures from which we first acquire high-resolution fluorescent light images of myelinated axons. After resin embedding and re-identification of the region of interest, serially aligned EM sections are acquired and imaged using a serial block face scanning electron microscope (SBF-SEM). The FLM and 3D-EM datasets are then combined to render 3D models of the myelinated axons. Thus, the SuperCLEM imaging pipeline is a useful new tool for researchers pursuing similar questions in neuronal and other complex tissue culture systems., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

HRAS, NRAS, and KRAS isoforms are almost identical proteins that are ubiquitously expressed and activate a common set of effectors. In vivo studies have revealed that they are not biologically redundant; however, the isoform specificity of Ras signaling remains poorly understood. Using a novel panel of isogenic SW48 cell lines endogenously expressing wild-type or G12V-mutated activated Ras isoforms, we have performed a detailed characterization of endogenous isoform-specific mutant Ras signaling. We find that despite displaying significant Ras activation, the downstream outputs of oncogenic Ras mutants are minimal in the absence of growth factor inputs. The lack of mutant KRAS-induced effector activation observed in SW48 cells appears to be representative of a broad panel of colon cancer cell lines harboring mutant KRAS. For MAP kinase pathway activation in KRAS-mutant cells, the requirement for coincident growth factor stimulation occurs at an early point in the Raf activation cycle. Finally, we find that Ras isoform-specific signaling was highly context dependent and did not conform to the dogma derived from ectopic expression studies.

In malignant transformation, cellular stress-response pathways are dynamically mobilized to counterbalance oncogenic activity, keeping cancer cells viable. Therapeutic disruption of this vulnerable homeostasis might change the outcome of many human cancers, particularly those for which no effective therapy is available. Here, we report the use of fibroblast growth factor 2 (FGF2) to demonstrate that further mitogenic activation disrupts cellular homeostasis and strongly sensitizes cancer cells to stress-targeted therapeutic inhibitors. We show that FGF2 enhanced replication and proteotoxic stresses in a K-Ras-driven murine cancer cell model, and combinations of FGF2 and proteasome or DNA damage response-checkpoint inhibitors triggered cell death. CRISPR/Cas9-mediated K-Ras depletion suppressed the malignant phenotype and prevented these synergic toxicities in these murine cells. Moreover, in a panel of human Ewing's sarcoma family tumor cells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE-821 (ATR inhibitor) induced cell death when combined with FGF2. Sustained MAPK-ERK1/2 overactivation induced by FGF2 appears to underlie these synthetic lethalities, as late pharmacological inhibition of this pathway restored cell homeostasis and prevented these described synergies. Our results highlight how mitotic signaling pathways which are frequently overridden in malignant transformation might be exploited to disrupt the robustness of cancer cells, ultimately sensitizing them to stress-targeted therapies. This approach provides a new therapeutic rationale for human cancers, with important implications for tumors still lacking effective treatment, and for those that frequently relapse after treatment with available therapies., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

RAS proteins are small GTPases that regulate signalling networks that control cellular proliferation and survival. They are frequently mutated in cancer and a commonly occurring group of developmental disorders called RASopathies. We discuss recent findings describing how RAS isoforms and different activating mutations differentially contribute to normal and disease-associated biology and the mechanisms that have been proposed to underpin this., (© 2018 The Author(s).)

Ras signaling originates from transient nanoscale compartmentalized regions of the plasma membrane composed of specific proteins and lipids. The highly specific lipid composition of these nanodomains, termed nanoclusters, facilitates effector recruitment and therefore influences signal transduction. This suggests that Ras nanocluster proteolipid composition could represent a novel target for future chemoprevention interventions. There is evidence that consumption of fish oil containing long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) such as eicosapentaenoic acid (EPA, 20:5 Δ5,8,11,14,17 ) and docosahexaenoic acid (DHA, 22:6 Δ4,7,10,13,16,19 ) may reduce colon cancer risk in humans, yet the mechanism underlying this effect is unknown. Here, we demonstrate that dietary n-3 PUFA reduce the lateral segregation of cholesterol-dependent and -independent nanoclusters, suppressing phosphatidic acid-dependent oncogenic KRas effector interactions, via their physical incorporation into plasma membrane phospholipids. This results in attenuation of oncogenic Ras-driven colonic hyperproliferation in both Drosophila and murine models. These findings demonstrate the unique properties of dietary n-3 PUFA in the shaping of Ras nanoscale proteolipid complexes and support the emerging role of plasma membrane-targeted therapies. Significance: The influence of dietary long chain n-3 polyunsaturated fatty acids on plasma membrane protein nanoscale organization and KRas signaling supports development of plasma membrane-targeted therapies in colon cancer. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3899/F1.large.jpg Cancer Res; 78(14); 3899-912. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Ubiquitin-specific protease 15 (USP15) is a widely expressed deubiquitylase that has been implicated in diverse cellular processes in cancer. Here we identify topoisomerase II (TOP2A) as a novel protein that is regulated by USP15. TOP2A accumulates during G2 and functions to decatenate intertwined sister chromatids at prophase, ensuring the replicated genome can be accurately divided into daughter cells at anaphase. We show that USP15 is required for TOP2A accumulation, and that USP15 depletion leads to the formation of anaphase chromosome bridges. These bridges fail to decatenate, and at mitotic exit form micronuclei that are indicative of genome instability. We also describe the cell cycle-dependent behaviour for two major isoforms of USP15, which differ by a short serine-rich insertion that is retained in isoform-1 but not in isoform-2. Although USP15 is predominantly cytoplasmic in interphase, we show that both isoforms move into the nucleus at prophase, but that isoform-1 is phosphorylated on its unique S229 residue at mitotic entry. The micronuclei phenotype we observe on USP15 depletion can be rescued by either USP15 isoform and requires USP15 catalytic activity. Importantly, however, an S229D phospho-mimetic mutant of USP15 isoform-1 cannot rescue either the micronuclei phenotype, or accumulation of TOP2A. Thus, S229 phosphorylation selectively abrogates this role of USP15 in maintaining genome integrity in an isoform-specific manner. Finally, we show that USP15 isoform-1 is preferentially upregulated in a panel of non-small cell lung cancer cell lines, and propose that isoform imbalance may contribute to genome instability in cancer. Our data provide the first example of isoform-specific deubiquitylase phospho-regulation and reveal a novel role for USP15 in guarding genome integrity.

The human protein kinome comprises 535 proteins that, with the exception of approximately 50 pseudokinases, control intracellular signaling networks by catalyzing the phosphorylation of multiple protein substrates. While a major research focus of the last 30 years has been cancer-associated Tyr and Ser/Thr kinases, over 85% of the kinome has been identified to be dysregulated in at least one disease or developmental disorder. Despite this remarkable statistic, for the majority of protein kinases and pseudokinases, there are currently no inhibitors progressing toward the clinic, and in most cases, details of their physiologic and pathologic mechanisms remain at least partially obscure. By curating and annotating data from the literature and major public databases of phosphorylation sites, kinases, and disease associations, we generate an unbiased resource that highlights areas of unmet need within the kinome. We discuss strategies and challenges associated with characterizing catalytic and noncatalytic outputs in cells, and describe successes and new frontiers that will support more comprehensive cancer-targeting and therapeutic evaluation in the future. Cancer Res; 78(1); 15-29. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Post-translational modification of proteins by ubiquitylation is increasingly recognised as a highly complex code that contributes to the regulation of diverse cellular processes. In humans, a family of almost 100 deubiquitylase enzymes (DUBs) are assigned to six subfamilies and many of these DUBs can remove ubiquitin from proteins to reverse signals. Roles for individual DUBs have been delineated within specific cellular processes, including many that are dysregulated in diseases, particularly cancer. As potentially druggable enzymes, disease-associated DUBs are of increasing interest as pharmaceutical targets. The biology, structure and regulation of DUBs have been extensively reviewed elsewhere, so here we focus specifically on roles of DUBs in regulating cell cycle processes in mammalian cells. Over a quarter of all DUBs, representing four different families, have been shown to play roles either in the unidirectional progression of the cell cycle through specific checkpoints, or in the DNA damage response and repair pathways. We catalogue these roles and discuss specific examples. Centrosomes are the major microtubule nucleating centres within a cell and play a key role in forming the bipolar mitotic spindle required to accurately divide genetic material between daughter cells during cell division. To enable this mitotic role, centrosomes undergo a complex replication cycle that is intimately linked to the cell division cycle. Here, we also catalogue and discuss DUBs that have been linked to centrosome replication or function, including centrosome clustering, a mitotic survival strategy unique to cancer cells with supernumerary centrosomes., (© 2017 The Author(s).)