Centromere/kinetochore is assembled through CENP-C oligomerization.

Kinetochore is an essential protein complex required for accurate chromosome segregation. The constitutive centromere-associated network (CCAN), a subcomplex of the kinetochore, associates with centromeric chromatin and provides a platform for the kinetochore assembly. The CCAN protein CENP-C is thought to be a central hub for the centromere/kinetochore organization. However, the role of CENP-C in CCAN assembly needs to be elucidated. Here, we demonstrate that both the CCAN-binding domain and the C-terminal region that includes the Cupin domain of CENP-C are necessary and sufficient for chicken CENP-C function. Structural and biochemical analyses reveal self-oligomerization of the Cupin domains of chicken and human CENP-C. We find that the CENP-C Cupin domain oligomerization is vital for CENP-C function, centromeric localization of CCAN, and centromeric chromatin organization. These results suggest that CENP-C facilitates the centromere/kinetochore assembly through its oligomerization.

Pericentromeric noncoding RNA changes DNA binding of CTCF and inflammatory gene expression in senescence and cancer.

Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and non-cell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.

Condensin I-mediated mitotic chromosome assembly requires association with chromokinesin KIF4A.

The chromokinesin KIF4A has been implicated in shaping mitotic chromosomes, but its functional relationship to condensin complexes remains controversial. Here, we found that, in mitosis, KIF4A associates with condensin I but not with condensin II. Mutational analyses indicated that the enrichment of condensin I to chromosomal axes depends on its association with KIF4A in a way that likely involves its motor activity. Remarkably, this interaction is required for condensin I to confer physiological properties to chromosomes. These observations provide an insight into how condensin I is enriched at chromosomal axes and underscore the significance of axial structure in organizing mitotic chromosomes.

Profiling chromosomal-level variations in gastric malignancies.

Aneuploidy arises from persistent chromosome segregation errors, or chromosomal instability. Although it has long been known as a hallmark of cancer cells, reduced cellular fitness upon induced ploidy alterations hinders the understanding of how aneuploidy relates to cancer development in the body. In this study, we used FISH analysis targeting centromeres to indicate ploidy changes, and quantitatively evaluated the ploidy statuses of gastric tumors derived from a total of 214 patients, ranging from early to advanced disease. We found that cancer cells reveal a marked elevation of aneuploid population, increasingly in cases diagnosed in advanced stages. The expansion of the aneuploid population is well associated with p53 deficiency, consistent with its essential role in genome maintenance. Comparisons among multiple locations within the tumor, or between the primary and metastatic tumors, indicated that cancer cells mostly retain their ploidy alterations throughout primary tumors, but metastatic tumors may consist of cells with either increased or decreased levels of aneuploidy. We also found that a notable proportion of polyploid cells are often already present in chronic gastritis epithelia. These observations underscore that chromosome-level variations are widespread in gastric cancers, shaping their genetic heterogeneity and malignant properties.

Identification of a novel nucleolar protein complex required for mitotic chromosome segregation through centromeric accumulation of Aurora B.

The nucleolus is a membrane-less nuclear structure that disassembles when cells undergo mitosis. During mitosis, nucleolar factors are thus released from the nucleolus and dynamically change their subcellular localization; however, their functions remain largely uncharacterised. Here, we found that a nucleolar factor called nucleolar protein 11 (NOL11) forms a protein complex with two tryptophan-aspartic acid (WD) repeat proteins named WD-repeat protein 43 (WDR43) and Cirhin in mitotic cells. This complex, referred to here as the NWC (NOL11-WDR43-Cirhin) complex, exists in nucleoli during interphase and translocates to the periphery of mitotic chromosomes, i.e., perichromosomal regions. During mitotic progression, both the congression of chromosomes to the metaphase plate and sister chromatid cohesion are impaired in the absence of the NWC complex, as it is required for the centromeric enrichment of Aurora B and the associating phosphorylation of histone H3 at threonine 3. These results reveal the characteristics of a novel protein complex consisting of nucleolar proteins, which is required for regulating kinetochores and centromeres to ensure faithful chromosome segregation.

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.

Pathogenicity assessment of variants for breast cancer susceptibility genes based on BRCAness of tumor sample.

Genes involved in the homologous recombination repair pathway-as exemplified by BRCA1, BRCA2, PALB2, ATM, and CHEK2-are frequently associated with hereditary breast and ovarian cancer syndrome. Germline mutations in the loci of these genes with loss of heterozygosity or additional somatic truncation at the WT allele lead to the development of breast cancers with characteristic clinicopathological features and prominent genomic features of homologous recombination deficiency, otherwise referred to as "BRCAness." Although clinical genetic testing for these and other genes has increased the chances of identifying pathogenic variants, there has also been an increase in the prevalence of variants of uncertain significance, which poses a challenge to patient care because of the difficulties associated with making further clinical decisions. To overcome this challenge, we sought to develop a methodology to reclassify the pathogenicity of these unknown variants using statistical modeling of BRCAness. The model was developed with Lasso logistic regression by comparing 116 genomic attributes derived from 37 BRCA1/2 biallelic mutant and 32 homologous recombination-quiescent breast cancer exomes. The model showed 95.8% and 86.7% accuracies in the training cohort and The Cancer Genome Atlas validation cohort, respectively. Through application of the model for variant reclassification of homologous recombination-associated hereditary breast and ovarian cancer causal genes and further assessment with clinicopathological features, we finally identified one likely pathogenic and five likely benign variants. As such, the BRCAness model developed from the tumor exome was robust and provided a reasonable basis for variant reclassification.

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.

Nuclear microenvironment in cancer: Control through liquid-liquid phase separation.

The eukaryotic nucleus is not a homogenous single-spaced but a highly compartmentalized organelle, partitioned by various types of membraneless structures, including nucleoli, PML bodies, paraspeckles, DNA damage foci and RNA clouds. Over the past few decades, these nuclear structures have been implicated in biological reactions such as gene regulation and DNA damage response and repair, and are thought to provide "microenvironments," facilitating these reactions in the nucleus. Notably, an altered morphology of these nuclear structures is found in many cancers, which may relate to so-called "nuclear atypia" in histological examinations. While the diagnostic significance of nuclear atypia has been established, its nature has remained largely enigmatic and awaits characterization. Here, we review the emerging biophysical principles that govern biomolecular condensate assembly in the nucleus, namely, liquid-liquid phase separation (LLPS), to investigate the nature of the nuclear microenvironment. In the nucleus, LLPS is typically driven by multivalent interactions between proteins with intrinsically disordered regions, and is also facilitated by protein interaction with nucleic acids, including nuclear non-coding RNAs. Importantly, an altered LLPS leads to dysregulation of nuclear events and epigenetics, and often to tumorigenesis and tumor progression. We further note the possibility that LLPS could represent a new therapeutic target for cancer intervention.

Enrichment of CLDN18-ARHGAP fusion gene in gastric cancers in young adults.

Gastric cancer in young adults has been pointed out to comprise a subgroup associated with distinctive clinicopathological features, including an equal gender distribution, advanced disease, and diffuse-type histology. Comprehensive molecular analyses of gastric cancers have led to molecular-based classifications and to specific and effective treatment options. The molecular traits of gastric cancers in young adults await investigations, which should provide a clue to explore therapeutic strategies. Here, we studied 146 gastric cancer patients diagnosed at the age of 40 years or younger at the Cancer Institute Hospital (Tokyo, Japan). Tumor specimens were examined for Helicobacter pylori infection, Epstein-Barr virus positivity, and for the expression of mismatch repair genes to indicate microsatellite instability. Overexpression, gene amplifications, and rearrangements of 18 candidate driver genes were examined by immunohistochemistry and FISH. Although only a small number of cases were positive for Epstein-Barr virus and microsatellite instability (n = 2 each), we repeatedly found tumors with gene fusion between a tight-junction protein claudin, CLDN18, and a regulator of small G proteins, ARHGAP, in as many as 22 cases (15.1%), and RNA sequencing identified 2 novel types of the fusion. Notably, patients with the CLDN18-ARHGAP fusion revealed associations between aggressive disease and poor prognosis, even when grouped by their clinical stage. These observations indicate that a fusion gene between CLDN18 and ARHGAP is enriched in younger age-onset gastric cancers, and its presence could contribute to their aggressive characteristics.

The initial phase of chromosome condensation requires Cdk1-mediated phosphorylation of the CAP-D3 subunit of condensin II.

The cell cycle transition from interphase into mitosis is best characterized by the appearance of condensed chromosomes that become microscopically visible as thread-like structures in nuclei. Biochemically, launching the mitotic program requires the activation of the mitotic cyclin-dependent kinase Cdk1 (cyclin-dependent kinase 1), but whether and how Cdk1 triggers chromosome assembly at mitotic entry are not well understood. Here we report that mitotic chromosome assembly in prophase depends on Cdk1-mediated phosphorylation of the condensin II complex. We identified Thr 1415 of the CAP-D3 subunit as a Cdk1 phosphorylation site, which proved crucial as it was required for the Polo kinase Plk1 (Polo-like kinase 1) to localize to chromosome axes through binding to CAP-D3 and thereby hyperphosphorylate the condensin II complex. Live-cell imaging analysis of cells carrying nonphosphorylatable CAP-D3 mutants in place of endogenous protein suggested that phosphorylation of Thr 1415 is required for timely chromosome condensation during prophase, and that the Plk1-mediated phosphorylation of condensin II facilitates its ability to assemble chromosomes properly. These observations provide an explanation for how Cdk1 induces chromosome assembly in cells entering mitosis, and underscore the significance of the cooperative action of Plk1 with Cdk1.

HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle.

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the 'used' cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

Chromosomal instability: A common feature and a therapeutic target of cancer.

Most cancer cells are aneuploid, containing abnormal numbers of chromosomes, mainly caused by elevated levels of chromosome missegregation, known as chromosomal instability (CIN). These well-recognized, but poorly understood, features of cancers have recently been studied extensively, unraveling causal relationships between CIN and cancer. Here we review recent findings regarding how CIN and aneuploidy occur, how they affect cellular functions, how cells respond to them, and their relevance to diseases, especially cancer. Aneuploid cells are under various kinds of stresses that result in reduced cellular fitness. Nevertheless, genetic heterogeneity derived from CIN allows the selection of cells better adapted to their environment, which supposedly facilitates generation and progression of cancer. We also discuss how we can exploit the properties of cancer cells exhibiting CIN for effective cancer therapy.

CAMP (C13orf8, ZNF828) is a novel regulator of kinetochore-microtubule attachment.

Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore-microtubule attachment, chromosome alignment-maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc-finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1-dependent phosphorylation at multiple sites during mitosis. CAMP-depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K-fibres to the tension exerted upon establishment of sister kinetochore bi-orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C-terminal region containing the zinc-finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP-E and CENP-F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP-E and CENP-F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore-microtubule attachment during bi-orientation.

Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1.

Cyclin-dependent kinase 1 (Cdk1) is thought to trigger centrosome separation in late G2 phase by phosphorylating the motor protein Eg5 at Thr927. However, the precise control mechanism of centrosome separation remains to be understood. Here, we report that in G2 phase polo-like kinase 1 (Plk1) can trigger centrosome separation independently of Cdk1. We find that Plk1 is required for both C-Nap1 displacement and for Eg5 localization on the centrosome. Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927. Nevertheless, Plk1-driven centrosome separation is slow and staggering, while Cdk1 triggers fast movement of the centrosomes. We find that actin-dependent Eg5-opposing forces slow down separation in G2 phase. Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation. Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement. Our findings implicate the modulation of MT stability in G2 and M phase as a regulatory element in the control of centrosome separation.

Bipartite binding interface recruiting HP1 to chromosomal passenger complex at inner centromeres.

Maintenance of ploidy depends on the mitotic kinase Aurora B, the catalytic subunit of the chromosomal passenger complex (CPC) whose proficient activity is supported by HP1 enriched at inner centromeres. HP1 is known to associate with INCENP of the CPC in a manner that depends on the PVI motif conserved across HP1 interactors. Here, we found that the interaction of INCENP with HP1 requires not only the PVI motif but also its C-terminally juxtaposed domain. Remarkably, these domains conditionally fold the ß-strand (PVI motif) and the α-helix from a disordered sequence upon HP1 binding and render INCENP with high affinity to HP1. This bipartite binding domain termed SSH domain (Structure composed of Strand and Helix) is necessary and sufficient to attain a predominant interaction of HP1 with INCENP. These results identify a unique HP1-binding module in INCENP that ensures enrichment of HP1 at inner centromeres, Aurora B activity, and thereby mitotic fidelity.

Nucleoporin Nup188 is required for chromosome alignment in mitosis.

Most cancer cells are aneuploid, which could be caused by defects in chromosome segregation machinery. Nucleoporins (Nup) are components of the nuclear pore complex, which is essential for nuclear transport during interphase, but several nucleoporins are also known to be involved in chromosome segregation. Here we report a novel function of Nup188, one of the nucleoporins regulating chromosome segregation. Nup188 localizes to spindle poles during mitosis, through the C-terminal region of Nup188. In Nup188-depleted mitotic cells, chromosomes fail to align to the metaphase plate, which causes mitotic arrest due to the spindle assembly checkpoint. Both the middle and the C-terminal regions were required for chromosome alignment. Robust K-fibers, microtubule bundles attaching to kinetochores, were hardly formed in Nup188-depleted cells. Significantly, we found that Nup188 interacts with NuMA, which plays an instrumental role in focusing microtubules at centrosomes, and NuMA localization to spindle poles is perturbed in Nup188-depleted cells. These data suggest that Nup188 promotes chromosome alignment through K-fiber formation and recruitment of NuMA to spindle poles.

Current Strategy to Treat Immunogenic Gastrointestinal Cancers: Perspectives for a New Era.

Since pembrolizumab, an anti-programmed death-1 (PD-1) antibody, showed a dramatic response to immunogenic cancers with microsatellite instability-high (MSI-H) and/or deficient mismatch repair (dMMR) in the pilot clinical trial KEYNOTE-016, subsequent studies have confirmed durable responses of anti-PD-1 inhibitors for MSI-H/dMMR solid tumors. As immunotherapy is described as a "game changer," the therapeutic landscape for MSI-H/dMMR solid tumors including gastrointestinal cancers has changed considerably in the last decade. An MSI/MMR status has been established as the predictive biomarker for immune checkpoint blockades, playing an indispensable role in the clinical practice of patients with MSI-H/dMMR tumors. Immunotherapy is also now investigated for locally advanced MSI-H/dMMR gastrointestinal cancers. Despite this great success, a few populations with MSI-H/dMMR gastrointestinal cancers do not respond to immunotherapy, possibly due to the existence of intrinsic or acquired resistance mechanisms. Clarifying the underlying mechanisms of resistance remains a future task, whereas attempts to overcome resistance and improve the efficacy of immunotherapy are currently ongoing. Herein, we review recent clinical trials with special attention to MSI-H/dMMR gastrointestinal cancers together with basic/translational findings, which provide their rationale, and discuss perspectives for the further therapeutic development of treatment in this field.

A single starfish Aurora kinase performs the combined functions of Aurora-A and Aurora-B in human cells.

Aurora, an essential mitotic kinase, is highly conserved during evolution. Most vertebrates have at least two Aurora kinases, Aurora-A and Aurora-B, which have distinct functions in the centrosome-spindle and inner centromere-midbody, respectively. However, some non-vertebrate deuterostomes have only a single Aurora. It remains to be verified whether the single Aurora performs the same functions as vertebrate Auroras A and B combined. We have isolated a cDNA of a single Aurora (ApAurora) from the echinoderm starfish, Asterina pectinifera, and show that ApAurora displays most features of both Aurora-A and Aurora-B in starfish oocytes and early embryos. Furthermore, ApAurora that is stably expressed in HeLa cells can substitute for both human Aurora-A and Aurora-B when either is reduced by RNAi. A single ApAurora thus has properties of both Aurora-A and Aurora-B in starfish eggs and HeLa cells. Together with phylogenetic analysis indicating that ApAurora forms a clade with all types of vertebrate Auroras and single Auroras of non-vertebrate deuterostomes, our observations support the idea that the single Aurora found in non-vertebrate deuterostomes represents the ancestor that gave rise to various types of vertebrate Auroras. This study thus provides functional evidence for phylogenetic considerations.

Chromosome arm length, and a species-specific determinant, define chromosome arm width.

Mitotic chromosomes in different organisms adopt various dimensions. What defines these dimensions is scarcely understood. Here, we compare mitotic chromosomes in budding and fission yeasts harboring similarly sized genomes distributed among 16 or 3 chromosomes, respectively. Hi-C analyses and superresolution microscopy reveal that budding yeast chromosomes are characterized by shorter-ranging mitotic chromatin contacts and are thinner compared with the thicker fission yeast chromosomes that contain longer-ranging mitotic contacts. These distinctions persist even after budding yeast chromosomes are fused to form three fission-yeast-length entities, revealing a species-specific organizing principle. Species-specific widths correlate with the known binding site intervals of the chromosomal condensin complex. Unexpectedly, within each species, we find that longer chromosome arms are always thicker and harbor longer-ranging contacts, a trend that we also observe with human chromosomes. Arm length as a chromosome width determinant informs mitotic chromosome formation models.