Bortezomib exerts its anti-cancer activity through the regulation of Skp2/p53 axis in non-melanoma skin cancer cells and C. elegans.

Non-melanoma skin cancer (NMSC), encompassing basal and squamous cell carcinoma, is the most prevalent cancer in the United States. While surgical removal remains the conventional therapy with a 95% 5-year cure rate, there is a growing interest in exploring alternative treatment strategies. In this study, we investigated the role of Bortezomib (BTZ), a proteasome inhibitor, in NMSC. Using two NMSC cell lines (A431 and A388), we examined the effects of BTZ treatment. Our results demonstrated that 48 h of BTZ treatment led to downregulating Skp2 expression in both A431 and A388 cells while upregulating p53 expression, specifically in A388 cells. These alterations resulted in impaired cellular growth and caspase-dependent cell death. Silencing Skp2 in A388 cells with siRNA confirmed the upregulation of p53 as a direct target. Furthermore, BTZ treatment increased the Bax to Bcl-2 ratio, promoting mitochondrial permeability and the subsequent release of cytochrome C, thereby activating caspases. We also found that BTZ exerted its antitumor effects by generating reactive oxygen species (ROS), as blocking ROS production significantly reduced BTZ-induced apoptotic cell death. Interestingly, BTZ treatment induced autophagy, which is evident from the increased expression of microtubule-associated proteins nucleoporin p62 and LC-3A/B. In addition to cell lines, we assessed the impact of BTZ in an in vivo setting using Caenorhabditis elegans (C. elegans). Our findings demonstrated that BTZ induced germline apoptosis in worms even at low concentrations. Notably, this increased apoptosis was mediated through the activity of CEP-1, the worm's counterpart to mammalian p53. In summary, our study elucidated the molecular mechanism underlying BTZ-induced apoptosis in NMSC cell lines and C. elegans. By targeting the skp2/p53 axis, inducing mitochondrial permeability, generating ROS, and promoting autophagy, BTZ demonstrates promising anti-cancer activity in NMSC. These findings provide novel insights into potential therapeutic strategies for controlling the unregulated growth of NMSC.

Tryptophanyl-tRNA synthetase-1 (WARS-1) depletion and high tryptophan concentration lead to genomic instability in Caenorhabditis elegans.

The fidelity of translation is ensured by a family of proteins named aminoacyl-tRNA synthetases (ARSs), making them crucial for development and survival. More recently, mutations in the tryptophanyl-tRNA synthetase 1 (WARS1) have been linked to various human diseases, from intellectual disability to various types of cancer. To understand the function of WARS1, we investigated the effect of WARS-1 depletion during the mitotic and meiotic cell cycle in the developing germline of Caenorhabditis elegans (C. elegans) and demonstrated the role of WARS-1 in genome integrity. wars-1 knockdown results in cell cycle arrest of the mitotically active germ cells. Such mitotic arrest is also associated with canonical DNA damage-induced checkpoint signaling in mitotic and meiotic germ cells. Significantly, such DNA checkpoint activation is associated with the morphological anomalies in chromatin structures that are the hallmarks of genome instability, such as the formation of chromatin bridges, micronuclei, and chromatin buds. We demonstrated that knocking down wars-1 results in an elevation of the intracellular concentration of tryptophan and its catabolites, a surprising finding emphasizing the impact of cellular amino acid availability and organismal/individual dietary uptake on genome integrity. Our result demonstrates that exposing C. elegans to a high tryptophan dosage leads to DNA damage checkpoint activation and a significant increase in the tryptophan metabolites. Targeting tryptophan catabolism, the least utilized amino acid in nature, can be important in developing new cancer therapeutic approaches. All in all, we have strong evidence that knocking down wars-1 results in defects in genomic integrity.

Exploring the in vivo anti-cancer potential of Neosetophomone B in leukemic cells using a zebrafish xenograft model.

Neosetophomone B (NSP-B) is a unique meroterpenoid fungal secondary metabolite that has previously demonstrated promising anti-cancer properties against various cancer cell lines in vitro. However, its in vivo anti-cancer potential remaines unexplored. To fill this gap in our knowledge, we tested NSP-B's in vivo anti-cancer activity using a zebrafish model, an organism that has gained significant traction in biomedical research due to its genetic similarities with humans and its transparent nature, allowing real-time tumor growth observation. For our experiments, we employed the K562-injected zebrafish xenograft model. Upon treating these zebrafish with NSP-B, we observed a marked reduction in the size and number of tumor xenografts. Delving deeper, our analyses indicated that NSP-B curtailed tumor growth and proliferation of leukemic grafted xenograft within the zebrafish. These results show that NSP-B possesses potent in vivo anti-cancer properties, making it a potential novel therapeutic agent for addressing hematological malignancies.

Accelerated epigenetic aging and DNA methylation alterations in Berardinelli-Seip congenital lipodystrophy.

Berardinelli-Seip congenital lipodystrophy type 2 (CGL2) is a very rare human genetic disorder with potential significance to the understanding of the pathobiology of aging. CGL2 patients display characteristic progeroid features and suffer from type 2 diabetes, insulin resistance and fatty liver. In this study, we profiled genome-wide DNA methylation levels in CGL2 patients with BSCL2 mutations to study epigenetic age acceleration and DNA methylation alterations. This analysis revealed significant age acceleration in blood DNA of CGL2 patients using both first- and second-generation epigenetic clocks. We also observed a shortened lifespan of Caenorhabditis elegans following knockdown of the BSCL2 homolog seip-1 on a daf-16/forkhead box, class O mutant background. DNA methylation analysis revealed significant differentially methylated sites enriched for lyase activity, kinase regulator activity, protein kinase regulator activity and kinase activator activity. We could also observe significant hypomethylation in the promoter of the dual specificity phosphatase 22 gene when comparing CGL2 patients versus controls. We conclude that in line with the observed progeroid features, CGL2 patients exhibit significant epigenetic age acceleration and DNA methylation alterations that might affect pathways/genes of potential relevance to the disease.

Biallelic variants in WARS1 cause a highly variable neurodevelopmental syndrome and implicate a critical exon for normal auditory function.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for faithful assignment of amino acids to their cognate tRNA. Variants in ARS genes are frequently associated with clinically heterogeneous phenotypes in humans and follow both autosomal dominant or recessive inheritance patterns in many instances. Variants in tryptophanyl-tRNA synthetase 1 (WARS1) cause autosomal dominantly inherited distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. Presently, only one family with biallelic WARS1 variants has been described. We present three affected individuals from two families with biallelic variants (p.Met1? and p.(Asp419Asn)) in WARS1, showing varying severities of developmental delay and intellectual disability. Hearing impairment and microcephaly, as well as abnormalities of the brain, skeletal system, movement/gait, and behavior were variable features. Phenotyping of knocked down wars-1 in a Caenorhabditis elegans model showed depletion is associated with defects in germ cell development. A wars1 knockout vertebrate model recapitulates the human clinical phenotypes, confirms variant pathogenicity, and uncovers evidence implicating the p.Met1? variant as potentially impacting an exon critical for normal hearing. Together, our findings provide consolidating evidence for biallelic disruption of WARS1 as causal for an autosomal recessive neurodevelopmental syndrome and present a vertebrate model that recapitulates key phenotypes observed in patients.

Over Fifty Years of Life, Death, and Cannibalism: A Historical Recollection of Apoptosis and Autophagy.

Research in biomedical sciences has changed dramatically over the past fifty years. There is no doubt that the discovery of apoptosis and autophagy as two highly synchronized and regulated mechanisms in cellular homeostasis are among the most important discoveries in these decades. Along with the advancement in molecular biology, identifying the genetic players in apoptosis and autophagy has shed light on our understanding of their function in physiological and pathological conditions. In this review, we first describe the history of key discoveries in apoptosis with a molecular insight and continue with apoptosis pathways and their regulation. We touch upon the role of apoptosis in human health and its malfunction in several diseases. We discuss the path to the morphological and molecular discovery of autophagy. Moreover, we dive deep into the precise regulation of autophagy and recent findings from basic research to clinical applications of autophagy modulation in human health and illnesses and the available therapies for many diseases caused by impaired autophagy. We conclude with the exciting crosstalk between apoptosis and autophagy, from the early discoveries to recent findings.

Spatiotemporal coupling and decoupling of gene transcription with DNA replication origins during embryogenesis in C. elegans.

The primary task of developing embryos is genome replication, yet how DNA replication is integrated with the profound cellular changes that occur through development is largely unknown. Using an approach to map DNA replication at high resolution in C. elegans, we show that replication origins are marked with specific histone modifications that define gene enhancers. We demonstrate that the level of enhancer associated modifications scale with the efficiency at which the origin is utilized. By mapping replication origins at different developmental stages, we show that the positions and activity of origins is largely invariant through embryogenesis. Contrary to expectation, we find that replication origins are specified prior to the broad onset of zygotic transcription, yet when transcription initiates it does so in close proximity to the pre-defined replication origins. Transcription and DNA replication origins are correlated, but the association breaks down when embryonic cell division ceases. Collectively, our data indicate that replication origins are fundamental organizers and regulators of gene activity through embryonic development.

Deep Proteome Analysis Identifies Age-Related Processes in C. elegans.

Effective network analysis of protein data requires high-quality proteomic datasets. Here, we report a near doubling in coverage of the C. elegans adult proteome, identifying >11,000 proteins in total with ∼9,400 proteins reproducibly detected in three biological replicates. Using quantitative mass spectrometry, we identify proteins whose abundances vary with age, revealing a concerted downregulation of proteins involved in specific metabolic pathways and upregulation of cellular stress responses with advancing age. Among these are ∼30 peroxisomal proteins, including the PRX-5/PEX5 import protein. Functional experiments confirm that protein import into the peroxisome is compromised in vivo in old animals. We also studied the behavior of the set of age-variant proteins in chronologically age-matched, long-lived daf-2 insulin/IGF-1-pathway mutants. Unexpectedly, the levels of many of these age-variant proteins did not scale with extended lifespan. This indicates that, despite their youthful appearance and extended lifespans, not all aspects of aging are reset in these long-lived mutants.

Global Proteomics Analysis of the Response to Starvation in C. elegans.

Periodic starvation of animals induces large shifts in metabolism but may also influence many other cellular systems and can lead to adaption to prolonged starvation conditions. To date, there is limited understanding of how starvation affects gene expression, particularly at the protein level. Here, we have used mass-spectrometry-based quantitative proteomics to identify global changes in the Caenorhabditis elegans proteome due to acute starvation of young adult animals. Measuring changes in the abundance of over 5,000 proteins, we show that acute starvation rapidly alters the levels of hundreds of proteins, many involved in central metabolic pathways, highlighting key regulatory responses. Surprisingly, we also detect changes in the abundance of chromatin-associated proteins, including specific linker histones, histone variants, and histone posttranslational modifications associated with the epigenetic control of gene expression. To maximize community access to these data, they are presented in an online searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/).

Dynamic SUMO modification regulates mitotic chromosome assembly and cell cycle progression in Caenorhabditis elegans.

The small ubiquitin-like modifier (SUMO), initially characterized as a suppressor of a mutation in the gene encoding the centromeric protein MIF2, is involved in many aspects of cell cycle regulation. The dynamics of conjugation and deconjugation and the role of SUMO during the cell cycle remain unexplored. Here we used Caenorhabditis elegans to establish the contribution of SUMO to a timely and accurate cell division. Chromatin-associated SUMO conjugates increase during metaphase but decrease rapidly during anaphase. Accumulation of SUMO conjugates on the metaphase plate and proper chromosome alignment depend on the SUMO E2 conjugating enzyme UBC-9 and SUMO E3 ligase PIAS(GEI-17). Deconjugation is achieved by the SUMO protease ULP-4 and is crucial for correct progression through the cell cycle. Moreover, ULP-4 is necessary for Aurora B(AIR-2) extraction from chromatin and relocation to the spindle mid-zone. Our results show that dynamic SUMO conjugation plays a role in cell cycle progression.

NDK-1, the homolog of NM23-H1/H2 regulates cell migration and apoptotic engulfment in C. elegans.

Abnormal regulation of cell migration and altered rearrangement of cytoskeleton are characteristic of metastatic cells. The first described suppressor of metastatic processes is NM23-H1, which displays NDPK (nucleoside-diphosphate kinase) activity. To better understand the role of nm23 genes in cell migration, we investigated the function of NDK-1, the sole Caenorhabditis elegans homolog of group I NDPKs in distal tip cell (DTC) migration. Dorsal phase of DTC migration is regulated by integrin mediated signaling. We find that ndk-1 loss of function mutants show defects in this phase. Epistasis analysis using mutants of the α-integrin ina-1 and the downstream functioning motility-promoting signaling module (referred to as CED-10 pathway) placed NDK-1 downstream of CED-10/Rac. As DTC migration and engulfment of apoptotic corpses are analogous processes, both partially regulated by the CED-10 pathway, we investigated defects of apoptosis in ndk-1 mutants. Embryos and germ cells defective for NDK-1 showed an accumulation of apoptotic cell corpses. Furthermore, NDK-1::GFP is expressed in gonadal sheath cells, specialized cells for engulfment and clearence of apoptotic corpses in germ line, which indicates a role for NDK-1 in apoptotic corpse removal. In addition to the CED-10 pathway, engulfment in the worm is also mediated by the CED-1 pathway. abl-1/Abl and abi-1/Abi, which function in parallel to both CED-10/CED-1 pathways, also regulate engulfment and DTC migration. ndk-1(-);abi-1(-) double mutant embryos display an additive phenotype (e. g. enhanced number of apoptotic corpses) which suggests that ndk-1 acts in parallel to abi-1. Corpse number in ndk-1(-);ced-10(-) double mutants, however, is similar to ced-10(-) single mutants, suggesting that ndk-1 acts downstream of ced-10 during engulfment. In addition, NDK-1 shows a genetic interaction with DYN-1/dynamin, a downstream component of the CED-1 pathway. In summary, we propose that NDK-1/NDPK might represent a converging point of CED-10 and CED-1 pathways in the process of cytoskeleton rearrangement.

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans.

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.

The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans.

The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis.

If you don't want them shed them.

Seminal studies in C. elegans contributed to our general understanding of programmed cell death conferred by apoptosis. A recent study unravelled a new form of cell death in the worm and provided insights into its regulation. Affected cells are shed from intact tissues, a modality of death likely to be conserved and relevant to cancer.

The opportunistic pathogen Serratia marcescens utilizes type VI secretion to target bacterial competitors.

The type VI secretion system (T6SS) is the most recently described and least understood of the protein secretion systems of Gram-negative bacteria. It is widely distributed and has been implicated in the virulence of various pathogens, but its mechanism and exact mode of action remain to be defined. Additionally there have been several very recent reports that some T6SSs can target bacteria rather than eukaryotic cells. Serratia marcescens is an opportunistic enteric pathogen, a class of bacteria responsible for a significant proportion of hospital-acquired infections. We describe the identification of a functional T6SS in S. marcescens strain Db10, the first report of type VI secretion by an opportunist enteric bacterium. The T6SS of S. marcescens Db10 is active, with secretion of Hcp to the culture medium readily detected, and is expressed constitutively under normal growth conditions from a large transcriptional unit. Expression of the T6SS genes did not appear to be dependent on the integrity of the T6SS. The S. marcescens Db10 T6SS is not required for virulence in three nonmammalian virulence models. It does, however, exhibit dramatic antibacterial killing activity against several other bacterial species and is required for S. marcescens to persist in a mixed culture with another opportunist pathogen, Enterobacter cloacae. Importantly, this antibacterial killing activity is highly strain specific, with the S. marcescens Db10 T6SS being highly effective against another strain of S. marcescens with a very similar and active T6SS. We conclude that type VI secretion plays a crucial role in the competitiveness, and thus indirectly the virulence, of S. marcescens and other opportunistic bacterial pathogens.

Stable-isotope labeling with amino acids in nematodes.

We describe an approach for accurate quantitation of global protein dynamics in Caenorhabditis elegans. We adapted stable-isotope labeling with amino acids in cell culture (SILAC) for nematodes by feeding worms a heavy lysine- and heavy arginine-labeled Escherichia coli strain and report a genetic solution to elminate the problem of arginine-to-proline conversion. Combining our approach with quantitative proteomics methods, we characterized the heat-shock response in worms.