Upregulation of ESPL1 is associated with poor prognostic outcomes in endometrial cancer.

BACKGROUND: Extra spindle pole bodies-like 1 (ESPL1) is known to play a crucial role in the segregation of sister chromatids during mitosis. Overexpression of ESPL1 is considered to have oncogenic effects in various human cancers. However, the specific biological function of ESPL1 in endometrial cancer (EC) remains unclear. METHODS: The TCGA and GEO databases were utilized to assess the expression of ESPL1 in EC. Immunohistochemistry was utilized to detect separase expression in EC samples. Kaplan-Meier survival analysis and Cox regression analysis were performed to evaluate the diagnostic and prognostic significance of ESPL1 in EC. Gene Set Enrichment Analysis (GSEA) was employed to explore the potential signaling pathway of ESPL1 in EC. Cell proliferation and colony formation ability were analyzed using CCK-8 and colony formation assay. RESULTS: Our analysis revealed that ESPL1 is significantly upregulated in EC, and its overexpression is associated with advanced clinical characteristics and unfavourable prognostic outcomes. Suppression of ESPL1 attenuated proliferation of EC cell line. CONCLUSION: The upregulation of ESPL1 is associated with advanced disease and poor prognosis in EC patients. These findings suggest that ESPL1 has the potential to serve as a diagnostic and prognostic biomarker in EC, highlighting its significance in the management of EC patients.

The expression of ESPL1 was higher in EC tissue than normal endometrial tissue.ESPL1 could be a potential prognostic marker for EC.

Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric FRET-Based Molecular Sensor upon Cell Cycle Progression.

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.

Securin acetylation prevents precocious separase activation and premature sister chromatid separation.

Lysine acetylation of non-histone proteins plays crucial roles in many cellular processes. In this study, we examine the role of lysine acetylation during sister chromatid separation in mitosis. We investigate the acetylation of securin at K21 by cell-cycle-dependent acetylome analysis and uncover its role in separase-triggered chromosome segregation during mitosis. Prior to the onset of anaphase, the acetylated securin via TIP60 prevents its degradation by the APC/CCDC20-mediated ubiquitin-proteasome system. This, in turn, restrains precocious activation of separase and premature separation of sister chromatids. Additionally, the acetylation-dependent stability of securin is also enhanced by its dephosphorylation. As anaphase approaches, HDAC1-mediated deacetylation of securin promotes its degradation, allowing released separase to cleave centromeric cohesin. Blocking securin deacetylation leads to longer anaphase duration and errors in chromosome segregation. Thus, this study illustrates the emerging role of securin acetylation dynamics in mitotic progression and genetic stability.

Role of chromosomal cohesion and separation in aneuploidy and tumorigenesis.

Cell division is a crucial process, and one of its essential steps involves copying the genetic material, which is organized into structures called chromosomes. Before a cell can divide into two, it needs to ensure that each newly copied chromosome is paired tightly with its identical twin. This pairing is maintained by a protein complex known as cohesin, which is conserved in various organisms, from single-celled ones to humans. Cohesin essentially encircles the DNA, creating a ring-like structure to handcuff, to keep the newly synthesized sister chromosomes together in pairs. Therefore, chromosomal cohesion and separation are fundamental processes governing the attachment and segregation of sister chromatids during cell division. Metaphase-to-anaphase transition requires dissolution of cohesins by the enzyme Separase. The tight regulation of these processes is vital for safeguarding genomic stability. Dysregulation in chromosomal cohesion and separation resulting in aneuploidy, a condition characterized by an abnormal chromosome count in a cell, is strongly associated with cancer. Aneuploidy is a recurring hallmark in many cancer types, and abnormalities in chromosomal cohesion and separation have been identified as significant contributors to various cancers, such as acute myeloid leukemia, myelodysplastic syndrome, colorectal, bladder, and other solid cancers. Mutations within the cohesin complex have been associated with these cancers, as they interfere with chromosomal segregation, genome organization, and gene expression, promoting aneuploidy and contributing to the initiation of malignancy. In summary, chromosomal cohesion and separation processes play a pivotal role in preserving genomic stability, and aberrations in these mechanisms can lead to aneuploidy and cancer. Gaining a deeper understanding of the molecular intricacies of chromosomal cohesion and separation offers promising prospects for the development of innovative therapeutic approaches in the battle against cancer.

Gene Expression Pattern of ESPL1, PTTG1 and PTTG1IP Can Potentially Predict Response to TKI First-Line Treatment of Patients with Newly Diagnosed CML.

The achievement of major molecular response (MMR, BCR::ABL1 ≤ 0.1% IS) within the first year of treatment with tyrosine kinase inhibitors (TKI) is a milestone in the therapeutic management of patients with newly diagnosed chronic myeloid leukemia (CML). We analyzed the predictive value of gene expression levels of ESPL1/Separase, PTTG1/Securin and PTTG1IP/Securin interacting protein for MMR achievement within 12 months. Relative expression levels (normalized to GUSB) of ESPL1, PTTG1 and PTTG1IP in white blood cells of patients (responders n = 46, non-responders n = 51) at the time of diagnosis were comparatively analyzed by qRT-PCR. 3D scatter plot analysis combined with a distance analysis performed with respect to a commonly calculated centroid center resulted in a trend to larger distances for non-responders compared to the responder cohort (p = 0.0187). Logistic regression and analysis of maximum likelihood estimates revealed a positive correlation of distance (cut-off) with non-achieving MMR within 12 months (p = 0.0388, odds ratio 1.479, 95%CI: 1.020 to 2.143). Thus, 10% of the tested non-responders (cut-off ≥ 5.9) could have been predicted already at the time of diagnosis. Future scoring of ESPL1, PTTG1 and PTTG1IP transcript levels may be a helpful tool in risk stratification of CML patients before initiation of TKI first = line treatment.

Separase and Roads to Disengage Sister Chromatids during Anaphase.

Receiving complete and undamaged genetic information is vital for the survival of daughter cells after chromosome segregation. The most critical steps in this process are accurate DNA replication during S phase and a faithful chromosome segregation during anaphase. Any errors in DNA replication or chromosome segregation have dire consequences, since cells arising after division might have either changed or incomplete genetic information. Accurate chromosome segregation during anaphase requires a protein complex called cohesin, which holds together sister chromatids. This complex unifies sister chromatids from their synthesis during S phase, until separation in anaphase. Upon entry into mitosis, the spindle apparatus is assembled, which eventually engages kinetochores of all chromosomes. Additionally, when kinetochores of sister chromatids assume amphitelic attachment to the spindle microtubules, cells are finally ready for the separation of sister chromatids. This is achieved by the enzymatic cleavage of cohesin subunits Scc1 or Rec8 by an enzyme called Separase. After cohesin cleavage, sister chromatids remain attached to the spindle apparatus and their poleward movement on the spindle is initiated. The removal of cohesion between sister chromatids is an irreversible step and therefore it must be synchronized with assembly of the spindle apparatus, since precocious separation of sister chromatids might lead into aneuploidy and tumorigenesis. In this review, we focus on recent discoveries concerning the regulation of Separase activity during the cell cycle.

Cohesin organization, dynamics, and subdomain functions revealed by genetic suppressor screening.

Cohesin is a heteropentameric protein complex that contributes to various aspects of chromosome structure and function, such as sister chromatid cohesion, genome compaction, and DNA damage response. Previous studies have provided abundant information on architecture and regional structures of the cohesin complex, but the configuration and structural dynamics of the whole cohesin complex are still largely unknown, partly due to flexibility of its coiled coils. We studied cohesin organization and dynamics using in vivo functional mutation compensation. Specifically, we developed and applied genetic suppressor screening methods to identify second mutations in cohesin complex genes that rescue lethality caused by various site-specific abnormalities in the cohesin complex. Functional analysis of these missense suppressor mutations revealed novel features of cohesin. Here, we summarize recent genetic suppressor screening results and insights into: 1) cohesin's structural organization when holding chromosomal DNAs; 2) interaction between cohesin head-kleisin and hinge; 3) ATP-driven cohesin conformational changes for genome packaging.

Separase Control and Cohesin Cleavage in Oocytes: Should I Stay or Should I Go?

The key to gametogenesis is the proper execution of a specialized form of cell division named meiosis. Prior to the meiotic divisions, the recombination of maternal and paternal chromosomes creates new genetic combinations necessary for fitness and adaptation to an ever-changing environment. Two rounds of chromosome segregation -meiosis I and II- have to take place without intermediate S-phase and lead to the creation of haploid gametes harboring only half of the genetic material. Importantly, the segregation patterns of the two divisions are fundamentally different and require adaptation of the mitotic cell cycle machinery to the specificities of meiosis. Separase, the enzyme that cleaves Rec8, a subunit of the cohesin complex constituting the physical connection between sister chromatids, has to be activated twice: once in meiosis I and immediately afterwards, in meiosis II. Rec8 is cleaved on chromosome arms in meiosis I and in the centromere region in meiosis II. This step-wise cohesin removal is essential to generate gametes of the correct ploidy and thus, embryo viability. Hence, separase control and Rec8 cleavage must be perfectly controlled in time and space. Focusing on mammalian oocytes, this review lays out what we know and what we still ignore about this fascinating mechanism.

Autocleavage of separase suppresses its premature activation by promoting binding to cyclin B1.

Accurate chromosome segregation requires timely activation of separase, a protease that cleaves cohesin during the metaphase-to-anaphase transition. However, the mechanism that maintains the inactivity of separase prior to this event remains unclear. We provide evidence that separase autocleavage plays an essential role in this process. We show that the inhibition of separase autocleavage results in premature activity before the onset of anaphase, accompanied by the formation of chromosomal bridges and spindle rocking. This deregulation is attributed to the reduced binding of cyclin B1 to separase that occurs during the metaphase-to-anaphase transition. Furthermore, when separase is mutated to render the regulation by cyclin B1 irrelevant, which keeps separase in securin-binding form, the deregulation induced by autocleavage inhibition is rescued. Our results reveal a physiological role of separase autocleavage in regulating separase, which ensures faithful chromosome segregation.

Identification of a novel substrate motif of yeast separase and deciphering the recognition specificity using AlphaFold2 and molecular dynamics simulation.

Separase is a giant cysteine protease and has multiple crucial functions. The most well-known substrate of separase is the kleisin subunit of cohesin, the cleavage of which triggers chromosome segregation during cell division (Uhlmann et al., 1999; Kamenz and Hauf, 2016) [1,2]. Recently, separase has also been found to cleave MCL-1 or BCL-XL proteins to trigger apoptosis (Hellmuth and Stemmann, 2020) [3]. Although substrate recognition through a short sequence right upstream of the cleavage site is well established, recent studies suggested that sequence elements outside this minimum cleavage site are required for optimal cleavage activity and specificity (Rosen et al., 2019; Uhlmann et al., 2000) [4,5]. However, the sequences and their underlying mechanism are largely unknown. To further explore the substrate determinants and recognition mechanism, we carried out sequence alignments and found a conserved motif downstream of the cleavage site in budding yeast. Using Alphafold2 and molecular dynamics simulations, we found this motif is recognized by separase in a conserved cleft near the binding groove of its inhibitor securin. Their binding is mutually exclusive and requires conformation changes of separase. These findings provide deeper insights into substrate recognition and activation of separase, and paved the way for discovering more substrates of separase.

MicroRNA-202 safeguards meiotic progression by preventing premature SEPARASE-mediated REC8 cleavage.

MicroRNAs (miRNAs) are believed to play important roles in mammalian spermatogenesis but the in vivo functions of single miRNAs in this highly complex developmental process remain unclear. Here, we report that miR-202, a member of the let-7 family, plays an important role in spermatogenesis by phenotypic evaluation of miR-202 knockout (KO) mice. Loss of miR-202 results in spermatocyte apoptosis and perturbation of the zygonema-to-pachynema transition. Multiple processes during meiosis prophase I including synapsis and crossover formation are disrupted, and inter-sister chromatid synapses are detected. Moreover, we demonstrate that Separase mRNA is a miR-202 direct target and provides evidence that miR-202 upregulates REC8 by repressing Separase expression. Therefore, we have identified miR-202 as a new regulating noncoding gene that acts on the established SEPARASE-REC8 axis in meiosis.

Aurora B/C-dependent phosphorylation promotes Rec8 cleavage in mammalian oocytes.

To generate haploid gametes, cohesin is removed in a stepwise manner from chromosome arms in meiosis I and the centromere region in meiosis II to segregate chromosomes and sister chromatids, respectively. Meiotic cohesin removal requires cleavage of the meiosis-specific kleisin subunit Rec8 by the protease separase.1,2 In yeast and C. elegans, Rec8 on chromosome arms has to be phosphorylated to be cleaved in meiosis I,3-7 whereas Rec8 at the centromere is protected from cleavage by the action of PP2A-B56.8-10 However, in mammalian meiosis, it is unknown whether Rec8 has to be equally phosphorylated for cleavage, and if so, the identity of the relevant kinase(s). This is due to technical challenges, as Rec8 is poorly conserved, preventing a direct translation of the knowledge gained from model systems such as yeast and C. elegans to mammals. Additionally, there is no turnover of Rec8 after cohesion establishment, preventing phosphomutant analysis of functional Rec8. To address the very basic question of whether Rec8 cleavage requires its phosphorylation in mammals, we adapted a biosensor that detects separase activity to study Rec8 cleavage in single mouse oocytes by live imaging. Crucially, through phosphomutant analysis, we identified phosphorylation sites in Rec8 promoting cleavage. We found that Rec8 cleavage depends on Aurora B/C kinase activities and identified an aminoacid residue that is phosphorylated in vivo. Accordingly, inhibition of Aurora B/C kinases during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation.

Cdc48 influence on separase levels is independent of mitosis and suggests translational sensitivity of separase.

Cdc48 (p97/VCP) is a AAA-ATPase that can extract ubiquitinated proteins from their binding partners and can cooperate with the proteasome for their degradation. A fission yeast cdc48 mutant (cdc48-353) shows low levels of the cohesin protease, separase, and pronounced chromosome segregation defects in mitosis. Separase initiates chromosome segregation when its binding partner securin is ubiquitinated and degraded. The low separase levels in the cdc48-353 mutant have been attributed to a failure to extract ubiquitinated securin from separase, resulting in co-degradation of separase along with securin. If true, Cdc48 would be important in mitosis. In contrast, we show here that low separase levels in the cdc48-353 mutant are independent of mitosis. Moreover, we find no evidence of enhanced separase degradation in the mutant. Instead, we suggest that the cdc48-353 mutant uncovers specific requirements for separase translation. Our results highlight a need to better understand how this key mitotic enzyme is synthesized.

Loss of sister kinetochore co-orientation and peri-centromeric cohesin protection after meiosis I depends on cleavage of centromeric REC8.

Protection of peri-centromeric (periCEN) REC8 cohesin from Separase and sister kinetochore (KT) attachment to microtubules emanating from the same spindle pole (co-orientation) ensures that sister chromatids remain associated after meiosis I. Both features are lost during meiosis II, resulting in sister chromatid disjunction and the production of haploid gametes. By transferring spindle-chromosome complexes (SCCs) between meiosis I and II in mouse oocytes, we discovered that both sister KT co-orientation and periCEN cohesin protection depend on the SCC, and not the cytoplasm. Moreover, the catalytic activity of Separase at meiosis I is necessary not only for converting KTs from a co- to a bi-oriented state but also for deprotection of periCEN cohesion, and cleavage of REC8 may be the key event. Crucially, selective cleavage of REC8 in the vicinity of KTs is sufficient to destroy co-orientation in univalent chromosomes, albeit not in bivalents where resolution of chiasmata may also be required.

Accumulation of Securin on Spindle During Female Meiosis I.

Chromosome segregation during female meiosis is frequently incorrect with severe consequences including termination of further development or severe disorders, such as Down syndrome. Accurate chromosome segregation requires tight control of a protease called separase, which facilitates the separation of sister chromatids by cohesin cleavage. There are several control mechanisms in place, including the binding of specific protein inhibitor securin, phosphorylation by cyclin-dependent kinase 1 (CDK1), and complex with SGO2 and MAD2 proteins. All these mechanisms restrict the activation of separase for the time when all chromosomes are properly attached to the spindle. In our study, we focused on securin and compared the expression profile of endogenous protein with exogenous securin, which is widely used to study chromosome segregation. We also compared the dynamics of securin proteolysis in meiosis I and meiosis II. Our study revealed that the expression of both endogenous and exogenous securin in oocytes is compartmentalized and that this protein accumulates on the spindle during meiosis I. We believe that this might have a direct impact on the regulation of separase activity in the vicinity of the chromosomes.

Separase cleaves the kinetochore protein Meikin at the meiosis I/II transition.

To generate haploid gametes, germ cells undergo two consecutive meiotic divisions requiring key changes to the cell division machinery. Here, we demonstrate that the protease separase rewires key cell division processes at the meiosis I/II transition by cleaving the meiosis-specific protein Meikin. Separase proteolysis does not inactivate Meikin but instead alters its function to create a distinct activity state. Full-length Meikin and the C-terminal Meikin separase cleavage product both localize to kinetochores, bind to Plk1 kinase, and promote Rec8 cleavage, but our results reveal distinct roles for these proteins in controlling meiosis. Mutations that prevent Meikin cleavage or that conditionally inactivate Meikin at anaphase I result in defective meiosis II chromosome alignment in mouse oocytes. Finally, as oocytes exit meiosis, C-Meikin is eliminated by APC/C-mediated degradation prior to the first mitotic division. Thus, multiple regulatory events irreversibly modulate Meikin activity during successive meiotic divisions to rewire the cell division machinery at two distinct transitions.

Functional analysis of LAT3 in prostate cancer: Its downstream target and relationship with androgen receptor.

L-type amino acid transporter 3 (LAT3, SLC43A1) is abundantly expressed in prostate cancer (PC) and is thought to play an essential role in PC progression through the cellular uptake of essential amino acids. Here, we analyzed the expression, function, and downstream target of LAT3 in PC. LAT3 was highly expressed in PC cells expressing androgen receptor (AR), and its expression was increased by dihydrotestosterone treatment and decreased by bicalutamide treatment. In chromatin immunoprecipitation sequencing of AR, binding of AR to the SLC43A1 region was increased by dihydrotestosterone stimulation. Knockdown of LAT3 inhibited cell proliferation, migration, and invasion, and the phosphorylation of p70S6K and 4EBP-1. Separase (ESPL1) was identified as a downstream target of LAT3 by RNA sequencing analysis. In addition, immunostaining of prostatectomy specimens was performed. In the multivariate analysis, high expression of LAT3 was an independent prognostic factor for recurrence-free survival (hazard ratio: 3.24; P = .0018). High LAT3 expression was correlated with the pathological T stage and a high International Society of Urological Pathology grade. In summary, our results suggest that LAT3 plays an important role in the progression of PC.

Unraveling pathologies underlying chromosomal instability in cancers.

Aneuploidy is a widespread feature of malignant tumors that arises through persistent chromosome mis-segregation in mitosis associated with a pathological condition called chromosomal instability, or CIN. Since CIN is known to have a causal relationship with poor prognosis accompanying by multi-drug resistance, tumor relapse, and metastasis, many research groups have endeavored to understand the mechanisms underlying CIN. In this review, we overview possible etiologies of CIN. The key processes to achieve faithful chromosome segregation include the regulation of sister chromatid cohesion, kinetochore-microtubule attachment, bipolar spindle formation, spindle-assembly checkpoint, and the activity of separase. Aberrant chromosome structures during DNA replication might also be a potential cause of CIN. Defective regulation in these processes can lead to chromosome mis-segregation, manifested by lagging chromosomes, and DNA bridges in anaphase, leading to gross chromosome rearrangements. Investigation into the molecular etiologies of CIN should allow us to explore novel strategies to intervene in CIN to control cancers.

The Cyclin B2/CDK1 Complex Conservatively Inhibits Separase Activity in Oocyte Meiosis II.

Recently, we have reported that the cyclin B2/CDK1 complex regulates homologous chromosome segregation through inhibiting separase activity in oocyte meiosis I, which further elucidates the compensation of cyclin B2 on cyclin B1's function in meiosis I. However, whether cyclin B2/CDK1 complex also negatively regulates separase activity during oocyte meiosis II remains unknown. In the present study, we investigated the function of cyclin B2 in meiosis II of oocyte. We found that stable cyclin B2 expression impeded segregation of sister chromatids after oocyte parthenogenetic activation. Consistently, stable cyclin B2 inhibited separase activation, while introduction of non-phosphorylatable separase mutant rescued chromatid separation in the stable cyclin B2-expressed oocytes. Therefore, the cyclin B2/CDK1 complex conservatively regulates separase activity via inhibitory phosphorylation of separase in both meiosis I and meiosis II of mouse oocyte.

Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II.

Partitioning of the genome in meiosis occurs through two highly specialized cell divisions, named meiosis I and meiosis II. Step-wise cohesin removal is required for chromosome segregation in meiosis I, and sister chromatid segregation in meiosis II. In meiosis I, mono-oriented sister kinetochores appear as fused together when examined by high-resolution confocal microscopy, whereas they are clearly separated in meiosis II, when attachments are bipolar. It has been proposed that bipolar tension applied by the spindle is responsible for the physical separation of sister kinetochores, removal of cohesin protection, and chromatid separation in meiosis II. We show here that this is not the case, and initial separation of sister kinetochores occurs already in anaphase I independently of bipolar spindle forces applied on sister kinetochores, in mouse oocytes. This kinetochore individualization depends on separase cleavage activity. Crucially, without kinetochore individualization in meiosis I, bivalents when present in meiosis II oocytes separate into chromosomes and not sister chromatids. This shows that whether centromeric cohesin is removed or not is determined by the kinetochore structure prior to meiosis II.