Platinum-based drugs induce phenotypic alterations in nucleoli and Cajal bodies in prostate cancer cells.

PURPOSE: Platinum-based drugs are cytotoxic drugs commonly used in cancer treatment. They cause DNA damage, effects of which on chromatin and cellular responses are relatively well described. Yet, the nuclear stress responses related to RNA processing are incompletely known and may be relevant for the heterogeneity with which cancer cells respond to these drugs. Here, we determine the type and extent of nuclear stress responses of prostate cancer cells to clinically relevant platinum drugs. METHODS: We study nucleolar and Cajal body (CB) responses to cisplatin, carboplatin, and oxaliplatin with immunofluorescence-based methods in prostate cancer cells. We utilize organelle-specific markers NPM, Fibrillarin, Coilin, and SMN1, and study CB-regulatory proteins FUS and TDP-43 using siRNA-mediated downregulation. RESULTS: Different types of prostate cancer cells have different sensitivities to platinum drugs. With equally cytotoxic doses, cisplatin, and oxaliplatin induce prominent nucleolar and CB stress responses while the nuclear stress phenotypes to carboplatin are milder. We find that Coilin is a stress-specific marker for platinum drug response heterogeneity. We also find that CB-associated, stress-responsive RNA binding proteins FUS and TDP-43 control Coilin and CB biology in prostate cancer cells and, further, that TDP-43 is associated with stress-responsive CBs in prostate cancer cells. CONCLUSION: Our findings provide insight into the heterologous responses of prostate cancer cells to different platinum drug treatments and indicate Coilin and TDP-43 as stress mediators in the varied outcomes. These results help understand cancer drug responses at a cellular level and have implications in tackling heterogeneity in cancer treatment outcomes.

Increased n-6 Polyunsaturated Fatty Acids Indicate Pro- and Anti-Inflammatory Lipid Modifications in Synovial Membranes with Rheumatoid Arthritis.

Emerging evidence suggests that fatty acids (FAs) and their lipid mediator derivatives can induce both beneficial and detrimental effects on inflammatory processes and joint degradation in osteoarthritis (OA) and autoimmune-driven rheumatoid arthritis (RA). The present study characterized the detailed FA signatures of synovial membranes collected during knee replacement surgery of age- and gender-matched OA and RA patients (n = 8/diagnosis). The FA composition of total lipids was determined by gas chromatography and analyzed with univariate and multivariate methods supplemented with hierarchical clustering (HC), random forest (RF)-based classification of FA signatures, and FA metabolism pathway analysis. RA synovium lipids were characterized by reduced proportions of shorter-chain saturated FAs (SFAs) and elevated percentages of longer-chain SFAs and monounsaturated FAs, alkenyl chains, and C20 n-6 polyunsaturated FAs compared to OA synovium lipids. In HC, FAs and FA-derived variables clustered into distinct groups, which preserved the discriminatory power of the individual variables in predicting the RA and OA inflammatory states. In RF classification, SFAs and 20:3n-6 were among the most important FAs distinguishing RA and OA. Pathway analysis suggested that elongation reactions of particular long-chain FAs would have increased relevance in RA. The present study was able to determine the individual FAs, FA groups, and pathways that distinguished the more inflammatory RA from OA. The findings suggest modifications of FA elongation and metabolism of 20:4n-6, glycerophospholipids, sphingolipids, and plasmalogens in the chronically inflamed RA synovium. These FA alterations could have implications in lipid mediator synthesis and potential as novel diagnostic and therapeutic tools.

Reduction in Nuclear Size by DHRS7 in Prostate Cancer Cells and by Estradiol Propionate in DHRS7-Depleted Cells.

Increased nuclear size correlates with lower survival rates and higher grades for prostate cancer. The short-chain dehydrogenase/reductase (SDR) family member DHRS7 was suggested as a biomarker for use in prostate cancer grading because it is largely lost in higher-grade tumors. Here, we found that reduction in DHRS7 from the LNCaP prostate cancer cell line with normally high levels of DHRS7 increases nuclear size, potentially explaining the nuclear size increase observed in higher-grade prostate tumors where it is lost. An exogenous expression of DHRS7 in the PC3 prostate cancer cell line with normally low DHRS7 levels correspondingly decreases nuclear size. We separately tested 80 compounds from the Microsource Spectrum library for their ability to restore normal smaller nuclear size to PC3 cells, finding that estradiol propionate had the same effect as the re-expression of DHRS7 in PC3 cells. However, the drug had no effect on LNCaP cells or PC3 cells re-expressing DHRS7. We speculate that separately reported beneficial effects of estrogens in androgen-independent prostate cancer may only occur with the loss of DHRS7/ increased nuclear size, and thus propose DHRS7 levels and nuclear size as potential biomarkers for the likely effectiveness of estrogen-based treatments.

Nuclear size rectification: A potential new therapeutic approach to reduce metastasis in cancer.

Research on metastasis has recently regained considerable interest with the hope that single cell technologies might reveal the most critical changes that support tumor spread. However, it is possible that part of the answer has been visible through the microscope for close to 200 years. Changes in nuclear size characteristically occur in many cancer types when the cells metastasize. This was initially discarded as contributing to the metastatic spread because, depending on tumor types, both increases and decreases in nuclear size could correlate with increased metastasis. However, recent work on nuclear mechanics and the connectivity between chromatin, the nucleoskeleton, and the cytoskeleton indicate that changes in this connectivity can have profound impacts on cell mobility and invasiveness. Critically, a recent study found that reversing tumor type-dependent nuclear size changes correlated with reduced cell migration and invasion. Accordingly, it seems appropriate to now revisit possible contributory roles of nuclear size changes to metastasis.

MCF10CA Breast Cancer Cells Utilize Hyaluronan-Coated EV-Rich Trails for Coordinated Migration.

Invasion of tumor cells through the stroma is coordinated in response to migratory cues provided by the extracellular environment. One of the most abundant molecules in the tumor microenvironment is hyaluronan, a glycosaminoglycan known to promote many hallmarks of tumor progression, including the migratory potential of tumor cells. Strikingly, hyaluronan is also often found to coat extracellular vesicles (EVs) that originate from plasma membrane tentacles of tumor cells crucial for migration, such as filopodia, and are abundant in tumor niches. Thus, it is possible that hyaluronan and hyaluronan-coated EVs have a cooperative role in promoting migration. In this work, we compared the hyaluronan synthesis, EV secretion and migratory behavior of normal and aggressive breast cell lines from MCF10 series. Single live cell confocal imaging, electron microscopy and correlative light and electron microscopy experiments revealed that migrating tumor cells form EV-rich and hyaluronan -coated trails. These trails promote the pathfinding behavior of follower cells, which is dependent on hyaluronan. Specifically, we demonstrated that plasma membrane protrusions and EVs left behind by tumor cells during migration are strongly positive for CD9. Single cell tracking demonstrated a leader-follower behavior, which was significantly decreased upon removal of pericellular hyaluronan, indicating that hyaluronan promotes the pathfinding behavior of follower cells. Chick chorioallantoic membrane assays in ovo suggest that tumor cells behave similarly in 3D conditions. This study strengthens the important role of extracellular matrix production and architecture in coordinated tumor cell movements and validates the role of EVs as important components and regulators of tumor matrix. The results suggest that tumor cells can modify the extracellular niche by forming trails, which they subsequently follow coordinatively. Future studies will clarify in more detail the orchestrated role of hyaluronan, EVs and other extracellular cues in coordinated migration and pathfinding behavior of follower cells.

Chemical Interrogation of Nuclear Size Identifies Compounds with Cancer Cell Line-Specific Effects on Migration and Invasion.

Background: Lower survival rates for many cancer types correlate with changes in nuclear size/scaling in a tumor-type/tissue-specific manner. Hypothesizing that such changes might confer an advantage to tumor cells, we aimed at the identification of commercially available compounds to guide further mechanistic studies. We therefore screened for Food and Drug Administration (FDA)/European Medicines Agency (EMA)-approved compounds that reverse the direction of characteristic tumor nuclear size changes in PC3, HCT116, and H1299 cell lines reflecting, respectively, prostate adenocarcinoma, colonic adenocarcinoma, and small-cell squamous lung cancer. Results: We found distinct, largely nonoverlapping sets of compounds that rectify nuclear size changes for each tumor cell line. Several classes of compounds including, e.g., serotonin uptake inhibitors, cyclo-oxygenase inhibitors, ß-adrenergic receptor agonists, and Na+/K+ ATPase inhibitors, displayed coherent nuclear size phenotypes focused on a particular cell line or across cell lines and treatment conditions. Several compounds from classes far afield from current chemotherapy regimens were also identified. Seven nuclear size-rectifying compounds selected for further investigation all inhibited cell migration and/or invasion. Conclusions: Our study provides (a) proof of concept that nuclear size might be a valuable target to reduce cell migration/invasion in cancer treatment and (b) the most thorough collection of tool compounds to date reversing nuclear size changes specific to individual cancer-type cell lines. Although these compounds still need to be tested in primary cancer cells, the cell line-specific nuclear size and migration/invasion responses to particular drug classes suggest that cancer type-specific nuclear size rectifiers may help reduce metastatic spread.

Imipridone Anticancer Compounds Ectopically Activate the ClpP Protease and Represent a New Scaffold for Antibiotic Development.

Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as nonmitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide-4 (ADEP4), a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in S. aureus ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, B. subtilis, and Enterococcus faecium) and Gram-negative species (E. coli and Neisseria gonorrhoeae). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.

Cdc48/VCP Promotes Chromosome Morphogenesis by Releasing Condensin from Self-Entrapment in Chromatin.

The morphological transformation of amorphous chromatin into distinct chromosomes is a hallmark of mitosis. To achieve this, chromatin must be compacted and remodeled by a ring-shaped enzyme complex known as condensin. However, the mechanistic basis underpinning condensin's role in chromosome remodeling has remained elusive. Here we show that condensin has a strong tendency to trap itself in its own reaction product during chromatin compaction and yet is capable of interacting with chromatin in a highly dynamic manner in vivo. To resolve this apparent paradox, we identified specific chromatin remodelers and AAA-class ATPases that act in a coordinated manner to release condensin from chromatin entrapment. The Cdc48 segregase is the central linchpin of this regulatory mechanism and promotes ubiquitin-dependent cycling of condensin on mitotic chromatin as well as effective chromosome condensation. Collectively, our results show that condensin inhibition by its own reaction product is relieved by forceful enzyme extraction from chromatin.

Quiescent Saccharomyces cerevisiae forms telomere hyperclusters at the nuclear membrane vicinity through a multifaceted mechanism involving Esc1, the Sir complex, and chromatin condensation.

Like other eukaryotes, Saccharomyces cerevisiae spatially organizes its chromosomes within the nucleus. In G1 phase, the yeast's 32 telomeres are clustered into 6-10 foci that dynamically interact with the nuclear membrane. Here we show that, when cells leave the division cycle and enter quiescence, telomeres gather into two to three hyperclusters at the nuclear membrane vicinity. This localization depends on Esc1 but not on the Ku proteins. Telomere hypercluster formation requires the Sir complex but is independent of the nuclear microtubule bundle that specifically assembles in quiescent cells. Importantly, mutants deleted for the linker histone H1 Hho1 or defective in condensin activity or affected for histone H4 Lys-16 deacetylation are impaired, at least in part, for telomere hypercluster formation in quiescence, suggesting that this process involves chromosome condensation. Finally, we establish that telomere hypercluster formation is not necessary for quiescence establishment, maintenance, and exit, raising the question of the physiological raison d'être of this nuclear reorganization.