Formation Of Centrosomes Research Articles

Caspase-mediated AURKA cleavage at Asp132 is essential for paclitaxel to elicit cell apoptosis.

Background: Aurora kinase A (AURKA) is a potent oncogene that is often aberrantly expressed during tumorigenesis, and is associated with chemo-resistance in various malignancies. However, the role of AURKA in chemo-resistance remains largely elusive. Methods: The cleavage of AURKA upon viral infection or apoptosis stimuli was assesed by immunoblotting assays in several cancer cells or caspase deficient cell line models. The effect of AURKA cleavage at Asp132 on mitosis was explored by live cell imaging and immunofluorescence staining experiments. The role of Asp132-cleavage of AURKA induced by the chemotherapy drug paclitaxel was investigated using TUNEL, immunohistochemistry assay in mouse tumor xenograft model and patient tissues. Results: The proteolytic cleavage of AURKA at Asp132 commonly occurs in several cancer cell types, regardless of viral infection or apoptosis stimuli. Mechanistically, caspase 3/7/8 cleave AURKA at Asp132, and the Asp132-cleaved forms of AURKA promote cell apoptosis by disrupting centrosome formation and bipolar spindle assembly in metaphase during mitosis. The AURKAD132A mutation blocks the expression of cleaved caspase 3 and EGR1, which leads to reduced therapeutic effects of paclitaxel on colony formation and malignant growth of tumor cells in vitro and in vivo using a murine xenograft model and cancer patients. Conclusions: This study reveals that caspase-mediated AURKAD132 proteolysis is essential for paclitaxel to elicit cell apoptosis and indicates that AURKAD132 is a potential key target for chemotherapy.

The AP-2 complex interacts with γ-TuRC and regulates the proliferative capacity of neural progenitors.

Centrosomes are organelles that nucleate microtubules via the activity of gamma-tubulin ring complexes (γ-TuRC). In the developing brain, centrosome integrity is central to the progression of the neural progenitor cell cycle, and its loss leads to microcephaly. We show that NPCs maintain centrosome integrity via the endocytic adaptor protein complex-2 (AP-2). NPCs lacking AP-2 exhibit defects in centrosome formation and mitotic progression, accompanied by DNA damage and accumulation of p53. This function of AP-2 in regulating the proliferative capacity of NPCs is independent of its role in clathrin-mediated endocytosis and is coupled to its association with the GCP2, GCP3, and GCP4 components of γ-TuRC. We find that AP-2 maintains γ-TuRC organization and regulates centrosome function at the level of MT nucleation. Taken together, our data reveal a novel, noncanonical function of AP-2 in regulating the proliferative capacity of NPCs and open new avenues for the identification of novel therapeutic strategies for the treatment of neurodevelopmental and neurodegenerative disorders with AP-2 complex dysfunction.

Intracellular IL-23 Receptor (IL-23R) Is a Regulator of Mitotic Spindle and Centrosome Formation and Is Essential for AML Viability

IL-23 receptor (IL-23R) is a heterodimeric cytokine cell surface receptor that is traditionally expressed on T cells. The cytokine, IL-23, is secreted by dendritic cells and macrophages in response to inflammatory stimuli. Here, we identify a novel function and localization for IL-23R in which intracellular IL-23R is necessary for AML viability and acts as a critical regulator of the mitotic spindle. We analyzed gene ontologies that were upregulated in AML samples compared to normal hematopoietic cells. Amongst upregulated gene ontologies, the mitotic spindle ontology was enriched in AML samples. Further analysis of all protein coding genes and their correlation to the mitotic spindle ontology surprisingly returned IL-23R as a top hit. Given that IL-23R is not known to be expressed in AML, we confirmed the presence of IL-23R protein by immunoblotting in 14 of 20 primary AML patient samples, while it was undetectable in bulk (n=5) and CD34+ sorted (n=3) normal hematopoietic cells. Consistent with previous investigations, we confirmed cell surface localization of IL-23R in double negative T cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were present on the cell surface of AML cells. Instead, IL-23R was detected intracellularly in the cytoplasm and nucleus of AML cell lines, as well as primary AML cells (including the stem cell fraction). We demonstrated intracellular localization using flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. To ensure we were detecting genuine IL-23R protein, we probed for IL-23R using 4 different antibodies targeting 4 different epitopes of the receptor whilst using 4 methods of detection (immunoblotting, flow cytometry, confocal microscopy, and immunoprecipitation). We also demonstrated intracellular localization of the IL-23R heterodimer subunit, IL12Rβ1, which is known to bind the IL-23R subunit to form the fully functional IL-23 receptor. To elucidate the function of intracellular IL-23R, we performed BioID mass spectrometry to identify proteins that interact with IL-23R. Compared to controls, we identified 61 proteins that preferentially interacted with IL-23R. 36 of those 61 proteins are known cytoplasmic or nuclear localized proteins. Pathway analysis of those interacting proteins identified the mitotic spindle as a top pathway corroborating with our bioinformatics analysis. Proximity Ligation Assay (PLA) and confocal microscopy verified that endogenous IL-23R protein interacted with mitotic spindle associated proteins, NUMA, TMEM201, TACC1, and BAG6 in OCI-AML2 cells and primary AML samples. Consistent with our PLA results, we demonstrated IL-23R co-localized with the mitotic spindle and centrosomes in AML cell lines and primary patient samples. Knockdown and knockout of IL-23R in AML cells led to the dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and spindle orientation errors. Knockdown of IL-23R reduced cell proliferation and viability in OCI-AML2, TEX, K562, NB4, and U937 cells compared to non-targeting controls. IL-23R knockdown also reduced the engraftment efficiency of TEX leukemia cells in murine bone marrow. Subsequent knockdowns of IL-23R in primary AML cells demonstrated decreased clonogenic growth and reduced engraftment into the marrow of immune deficient mice. In contrast, knockdown of IL-23R in normal human cord blood cells did not impair their engraftment into murine bone marrow. Finally, we analyzed hematopoietic cells and stem cells in IL-23R -/- mice and found that knockout of IL-23R did not decrease blood counts or the abundance and function of normal hematopoietic cells. In summary, we discovered a novel intracellular localization and function for IL-23R in AML. IL-23R regulates mitotic spindle and centrosome formation and is necessary for AML cell viability. We have thus identified a new biological function for IL-23R and a potential therapeutic target for AML.

A genetic basis for facultative parthenogenesis in Drosophila

Facultative parthenogenesis enables sexually reproducing organisms to switch between sexual and asexual parthenogenetic reproduction. To gain insights into this phenomenon, we sequenced the genomes of sexually reproducing and parthenogenetic strains of Drosophila mercatorum and identified differences in the gene expression in their eggs. We then tested whether manipulating the expression of candidate gene homologs identified in Drosophila mercatorum could lead to facultative parthenogenesis in the non-parthenogenetic species Drosophila melanogaster. This identified a polygenic system whereby increased expression of the mitotic protein kinase polo and decreased expression of a desaturase, Desat2, caused facultative parthenogenesis in the non-parthenogenetic species that was enhanced by increased expression of Myc. The genetically induced parthenogenetic Drosophila melanogaster eggs exhibit de novo centrosome formation, fusion of the meiotic products, and the onset of development to generate predominantly triploid offspring. Thus, we demonstrate a genetic basis for sporadic facultative parthenogenesis in an animal.

DIS3 depletion in multiple myeloma causes extensive perturbation in cell cycle progression and centrosome amplification.

DIS3 gene mutations occur in approximately 10% of patients with multiple myeloma (MM); furthermore, DIS3 expression can be affected by monosomy 13 and del(13q), found in roughly 40% of MM cases. Despite the high incidence of DIS3 mutations and deletions, the biological significance of DIS3 and its contribution to MM pathogenesis remain poorly understood. In this study we investigated the functional role of DIS3 in MM, by exploiting a loss-of-function approach in human MM cell lines. We found that DIS3 knockdown inhibits proliferation in MM cell lines and largely affects cell cycle progression of MM plasma cells, ultimately inducing a significant increase in the percentage of cells in the G0/G1 phase and a decrease in the S and G2/M phases. DIS3 plays an important role not only in the control of the MM plasma cell cycle, but also in the centrosome duplication cycle, which are strictly co-regulated in physiological conditions in the G1 phase. Indeed, DIS3 silencing leads to the formation of supernumerary centrosomes accompanied by the assembly of multipolar spindles during mitosis. In MM, centrosome amplification is present in about a third of patients and may represent a mechanism leading to genomic instability. These findings strongly prompt further studies investigating the relevance of DIS3 in the centrosome duplication process. Indeed, a combination of DIS3 defects and deficient spindle-assembly checkpoint can allow cells to progress through the cell cycle without proper chromosome segregation, generating aneuploid cells which ultimately lead to the development of MM.

Centrosome Formation in the Bovine Early Embryo.

Centrosome formation during early development in mice and rats occurs due to the appearance of centrioles de novo. In contrast, in humans and other non-rodent mammals, centrioles are thought to be derived from spermatozoa. Ultrastructural study of zygotes and early embryos of cattle at full series of ultrathin sections show that the proximal centriole of the spermatozoon disappears by the end of the first cleavage division. Centrioles appear in two to four cell embryos in fertilized oocytes and in parthenogenetic embryos. Centriole formation includes the appearance of atypical centrioles with randomly arranged triplets and centrioles with microtubule triplets of various lengths. After the third cleavage, four centriolar cylinders appear for the first time in the blastomeres while each embryo still has two atypical centrioles. Our results showed that the mechanisms of centriole formation in different groups of mammals are universal, differing only in the stage of development in which they occur.

Abstract 333: Interleukin 23 receptor is a critical regulator of the mitotic spindle and necessary for AML viability

Abstract Classically, the interleukin-23 receptor (IL-23R) is expressed on the surface of T cells where activation by its ligand, interleukin-23 (IL-23) leads to T cell activation through the JAK/STAT pathway. Here, we identify a novel localization and function for IL-23R in which intracellular IL-23R is necessary for AML viability by regulating mitotic spindle and centrosome formation. IL-23R was identified as a potential target through InterLinked Therapeutics’ ITX machine learning platform. IL-23R was identified through this unbiased in silico screen as a top hit and a potential regulator of the G2/M checkpoint and mitotic spindle. We confirmed that IL-23R protein was overexpressed >2-fold in 6 of 8 primary AML patient samples compared to mean IL-23R expression in normal hematopoietic cells. Consistent with prior studies, we confirmed cell surface localization of IL-23R in double negative T cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were detected on the surface of AML cells and primary AML patient samples. Rather, IL-23R was located intracellularly as demonstrated by flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. We confirmed intracellular localization of IL-23R in AML cells using 4 different antibodies targeted against 4 different epitopes of the receptor. We used BioID mass spectrometry to identify novel protein interactors with intracellular IL-23R and identified 61 interacting proteins. 36 of those 61 were nuclear or cytoplasmic in localization and the largest subset was associated with mitotic spindle regulation. By Proximity Ligation Assay, we confirmed the interactions between endogenous IL-23R with known mitotic spindle proteins NUMA, TMEM201, BAG6, and TACC1 in OCI-AML2 cells and primary AML samples. By confocal microscopy, we discovered that IL-23R co-localized with the mitotic spindle and centrosomes in AML cells. Genetic knockout of IL-23R increased spindle defects with multipolarity, lagging chromosomes, and spindle orientation errors. Interestingly, heterodimeric partner of IL-23R, IL12Rβ1 was also detected to be co-localized with mitotic spindles but the cytokine, IL-23, could not be detected. Knockdown of IL-23R also reduced cell growth in OCI-AML2, TEX, K562, NB4, and U937 cells. IL-23R knockdown also reduced the clonogenic growth and engraftment efficiency of TEX leukemia cells into murine bone marrow. In summary, intracellular IL-23R is a critical regulator of mitosis in AML and is necessary for AML viability. This study highlights a novel function and localization of IL-23R in AML and identifies a potential therapeutic target. Citation Format: Nathan Duong, Dilshad H. Khan, Geethu E. Thomas, Rose Hurren, Jong Bok Lee, Jonathan St-Germain, Lily Drimmer, Yongran Yan, Neil MacLean, Marcela Gronda, Brandon D. Brown, Brian Raught, Andrea Arruda, Hong Chang, Mark D. Minden, Li Zhang, Steven M. Kornblau, Vito Spadavecchio, Aaron D. Schimmer. Interleukin 23 receptor is a critical regulator of the mitotic spindle and necessary for AML viability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 333.

Intracellular IL-23 Receptor (IL-23R) Is Necessary for AML Viability and Regulates Mitotic Spindle and Centrosome Formation

The IL-23 receptor (IL-23R) is a cell surface receptor classically expressed on T cells. In response to inflammatory stimuli, such as microbial infection, dendritic cells and macrophages secrete the cytokine IL-23, which stimulates IL-23R leading to increased T cell activation and proliferation. Here, we identify a novel function for IL-23R in which intracellular IL-23R is necessary for AML viability by regulating mitotic spindle and centrosome formation. An unbiased in silico screen was performed using the Interlinked Therapeutics ITX machine learning platform to identify novel targets and mechanisms in AML. This analysis identified IL-23R as a top hit and a potential regulator of the G2/M checkpoint and mitotic spindle. We confirmed IL-23R protein was over-expressed >2-fold in 6 of 8 primary AML patient samples compared to mean IL-23R expression (n=3) in normal hematopoietic cells. IL-23R is classically a cell surface receptor, and we confirmed cell surface localization in DNT (Double Negative T) cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were present on the cell surface of AML cells and primary AML patient samples. Rather, IL-23R was located intracellularly in the cytoplasm and nucleus as demonstrated by flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. To validate that we were indeed detecting IL-23R, we confirmed intracellular localization in AML cells using 4 different antibodies directed against 4 different epitopes of the receptor. To understand the function of intracellular IL-23R, we used BioID mass spectrometry to identify proteins that interact with IL-23R. Compared to the BioID controls, we identified 61 proteins that preferentially interacted with IL-23R. Of the 61 proteins, 36 are known cytoplasmic or nuclear proteins. Pathway analysis of interacting proteins identified mitotic spindle formation as the top pathway. Using a Proximity Ligation Assay and confocal microscopy, we validated the interaction of endogenous IL-23R with the known mitotic spindle proteins, NUMA, TMEM201, TACC1, and BAG6 in OCI-AML2 cells. In addition, by confocal microscopy, we demonstrated that IL-23R co-localizes with the mitotic spindle and centrosomes in AML. Knockdown and knockout of IL-23R in AML cells led to dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and spindle orientation errors. We then evaluated the effects of IL-23R knockdown on AML growth and viability. Knockdown or knockout of IL-23R reduced AML growth in OCI-AML2, TEX, K562, NB4, and U937 cells. In addition, knockdown of IL-23R led to a significant reduction in engraftment efficiency of TEX cells into murine bone marrow. To evaluate the effects of IL-23R depletion on normal hematopoiesis, we evaluated constitutive homozygote IL-23R knockout mice. IL23R-/- mice were bred in normal ratios and grew similar to wild type mice. IL-23R was associated with the mitotic spindle and centrosomes in hematopoietic cells of wild type mice and no IL-23R was detected in the knockout mice. Complete blood counts did not differ compared to wild type mice. The abundance of HSC (hematopoietic stem cell) and LSK (Lin−Sca-1+c-Kit+) hematopoietic stem cells were also similar between IL-23R -/- and wild type mice. In summary, we discovered that IL-23R is necessary for AML growth and viability by regulating the formation of the mitotic spindle and centrosome. Thus, we identified a new function and localization for IL-23R and highlight a new therapeutic target for this disease.

Analysis of Ser/Thr Kinase HASPIN-Interacting Proteins in the Spermatids.

HASPIN is predominantly expressed in spermatids, and plays an important role in cell division in somatic and meiotic cells through histone H3 phosphorylation. The literature published to date has suggested that HASPIN may play multiple roles in cells. Here, 10 gene products from the mouse testis cDNA library that interact with HASPIN were isolated using the two-hybrid system. Among them, CENPJ/CPAP, KPNA6/importin alpha 6, and C1QBP/HABP1 were analyzed in detail for their interactions with HASPIN, with HASPIN phosphorylated C1QBP as the substrate. The results indicated that HASPIN is involved in spermatogenesis through the phosphorylation of C1QBP in spermatids, and also may be involved in the formation of centrosomes.

Triploid-induced complete sterility in the scallop Nodipecten subnodosus might be triggered by an early and sustained DNA damage response

Triploid Nodipecten subnodosus scallops, unlike other mollusks, are completely sterile. In this study we focused on understanding the underlying molecular changes of triploid sterility using a transcriptomic approach. Total RNA from the gonad of diploid scallops in inactive and initial gametogenic stages and triploid scallops of the same cohort and ages were sequenced employing Illumina RNA-Seq. From 68,244 assembled and annotated transcripts, 1120 had terms associated with meiosis checkpoint or arrest, DNA damage response, or recombination. Differential gene expression analyses were conducted by contrasting initial vs. inactive stages of gametogenesis in each ploidy. In diploids, genes participating in homologous recombination during meiosis (msh5 and kdm8), spindle organization (nup62), centrosome formation (cenp-T), and sex differentiation (Ns-dmta2 and pum3), were up-regulated during initial gametogenesis. In triploids, a different set of genes were up-regulated during initial gametogenesis, and included genes involved in the DNA damage response and double strand break repair (rad51-C, xpc, myoVI), in the transition of metaphase/anaphase of mitosis (slp1 and nuf2), as well as genes that trigger both the intrinsic and extrinsic (caspase-3, icad, bmcc1) and extrinsic apoptosis pathways only (tnfr1, dab2ip). The results suggest significant DNA damage in triploids initial gametogenesis, possibly as a consequence of failing to repair double-strand breaks during DNA replication. This coincides with previous observations in which few triploid scallops showed gametic stages more advanced than oogonia or spermatogonia, and when present they were few.

Kinesin-14 KIFC1 modulates spindle assembly and chromosome segregation in mouse spermatocytes

Kinesin-14 KIFC1 regulates spindle assembly and centrosome clustering in diverse organisms during cell division. KIFC1 proteins are essential for spindle assembly and chromosome alignment in mitosis. However, the roles and mechanisms of KIFC1 proteins in male spermatocytes remain largely unknown. In this study, we reveal that KIFC1 proteins mainly accumulate at the centrosomes and central spindle in mouse spermatocytes both in vitro and in vivo. We utilize two KIFC1 specific inhibitors, AZ82 and CW069, for the inhibition of KIFC1 in mouse spermatogenic cells and cultured GC-2 spd(ts) cells. We find that KIFC1 inhibition results in the increase of spermatocytes with micronuclei, the disorganization of the meiotic spindles, and the formation of multiple centrosomes. Furthermore, we demonstrate that KIFC1 inhibition leads to spindle defects, chromosome misalignment and the formation of aneuploidy in cultured GC-2 spd(ts) cells. In this study, we reveal that KIFC1 proteins are critical for centrosome maintenance and chromosome stability in mouse spermatocytes.

Abstract 2039: The role of HORMAD1 in DNA damage repair in squamous cell carcinomas

Abstract Genomic instability is a prominent hallmark of cancer, however the dynamic mechanisms that drive and sustain genomic instability remain elusive. Research demonstrates that numerous cancers with increased levels of genomic instability express meiosis genes and undergo a process called meiomitosis. Cancer meiomitosis is the orchestrated activation of both mitotic and meiotic machineries in neoplastic cells that confer a selective advantage. Specifically, meiCT (meiosis-specific cancer/testis) genes that specialize in the reductional division of germ cells in meiosis I are ectopically expressed in several cancers, particularly in squamous cell carcinomas. The meiCT gene, HORMAD1 is a meiosis specific protein that functions to ensure that a sufficient number of DSBs are formed for appropriate meiotic progression and to maintain genome integrity throughout homologous recombination. Studies show that HORMAD1 is significantly upregulated in several cancers and is regarded as a potentially important oncogene that plays a role in sustaining increased genomic instability. With the use of shRNA mediated knockdown of HORMAD1, we evaluated the effects ofHORMAD1 on genomic instability and survival in squamous cell carcinomas (SCCs). Our results demonstrate that HORMAD1 knockdown in SCC cancer cell lines leads to an increase in genomic instability denoted by an increase in double strand breaks, micronuclei formation and ploidy. Furthermore, shHORMAD1 results in increased sensitivity to etoposide treatment leading to increased centrosome formation and reduced cell survival. We also demonstrate thatHORMAD1 modulates a functional and balanced level of genomic instability by eliciting an DNA damage response through homologous recombination mechanisms that allows SCCs to thrive following DNA damage. Citation Format: Jennifer Gantchev, Amelia Martinez Villarreal, Brandon Ramchatesingh, Ivan V. Litvinov. The role of HORMAD1 in DNA damage repair in squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2039.

BAP1-inactivated melanocytic tumors show prominent centrosome amplification and associated loss of primary cilia.

BRCA1-associated protein (BAP1) is a tumor suppressor whose loss is associated with various malignancies. The primary cilium is an organelle involved in signal transduction and cell cycle progression. Primary cilia have been shown to be absent in melanoma but retained to some extent in melanocytic nevi, and the severity of dysplasia influences the degree of cilia loss. Additionally, studies have revealed roles for BAP1 in centrosome and mitotic spindle formation. Because the primary cilium is nucleated on the mother centriole, we examined the connection between the presence of primary cilia and the formation of centrosomes in BAP1-inactivated melanocytic tumors (BIMTs). We evaluated the cilia and centrosomes in 11 BIMTs and five conventional melanocytic nevi using immunofluorescence staining of acetylated alpha-tubulin and gamma-tubulin. We found that, compared to nevi, BIMTs show loss of primary cilia and amplification of centrosomes. Occasional nevi also showed increased centrioles; however, these foci of amplification were more likely to be ciliated than those in BIMTs. Although centrosome amplification does not absolutely correlate with loss of primary cilia in melanocytic neoplasms, absence of BAP1 exacerbates the phenotype. Moreover, aberrant centrosome and cilia formation are likely critical in the pathogenesis of other BAP1-inactivated tumors.

SAPs as a new model to probe the pathway of centriole and centrosome assembly.

Centrioles are important cellular organelles involved in the formation of both cilia and centrosomes. It is therefore not surprising that their dysfunction may lead to a variety of human pathologies. Studies have identified a conserved pathway of proteins required for centriole formation, and investigations using the embryo of the fruit fly Drosophila melanogaster have been crucial in elucidating their dynamics. However, a full understanding of how these components interact has been hampered by the total absence of centrioles in null mutant backgrounds for any of these core centriole factors. Here, I review our recent work describing a new model for investigating these interactions in the absence of bona fide centrioles. Sas-6 Ana2 Particles (SAPs) form when two core centriole factors, Sas-6 and Ana2, are co-over-expressed in fruit fly eggs. Crucially, they form even in eggs lacking other core centriole proteins. I review our characterisation of SAPs, and provide one example of how they have been used to investigate the role of a core centriole protein in PCM formation. I then consider some of the strengths and weaknesses of the SAP model, and discuss them in the context of other models for centriole study in Drosophila. Similar aggregates have been seen in other systems upon expression of centriole factors, so SAPs may also be a useful approach to study centriole proteins in other organisms.

Involvement of AGN-1 protein, which binds to protein phosphatase 6, in centrosome formation during the process of cell division

Involvement of AGN-1 protein, which binds to protein phosphatase 6, in centrosome formation during the process of cell division

SASS6 Promotes Proliferation of Esophageal Squamous Carcinoma Cells by Inhibiting the p53 Signaling Pathway

SASS6 encodes for the Homo sapiens SAS-6 centriolar assembly protein and is important for proper centrosome formation. Although centrosomes are amplified in a wide variety of tumor types, abnormally high SASS6 expression had previously only been identified in colon cancer. However, the role of SASS6 in esophageal squamous cell carcinoma (ESCC) pathogenesis has not yet been elucidated. The aim of this study was to investigate the role and mechanisms of SASS6 in ESCC. The expression level of PNCK mRNA was detected in specimens of ESCC (n = 20) and paired paracancerous tissues (n = 20) by qPCR, and that of PNCK protein was detected in specimens of ESCC paraffin tissue (n = 316) and corresponding paraffin paracancerous tissue (n = 94) by immunohistochemistry. The correlations of PNCK protein expression level with clinical stage and survival prognosis in ESCC patients were also investigated, respectively. Celigo Cell Counting and MTT assay were used to measure cell viability. Apoptosis and colony formation were detected by flow cytometric analysis and colony assay. The bioluminescence imaging was used to evaluate the effects of SASS6 knockdown on tumor growth using a xenograft animal model. The global gene expression profile was determined in wild type and SASS6-depleted ESCC cells via transcriptomics analysis. Mechanically, the changes of p53 signaling pathway were detected by Western blotting. We found that the mRNA and protein levels of SASS6 were increased in human ESCC samples. In addition, SASS6 protein expression was associated with the esophageal cancer stage and negatively impacted survival of patients with ESCC. Furthermore, silencing of SASS6 inhibited cell growth and promoted apoptosis of ESCC cells in vitro and inhibited xenograft tumor formation in vivo. A genetic cluster and pathway analysis showed that SASS6 regulates the p53 signaling pathway. Western blot analysis demonstrated that CCND2, GADD45A, and EIF4EBP1 protein expression decreased and that TP53 protein expression increased in the shSASS group when compared with the control group. SASS6 promoted the proliferation of esophageal cancer by inhibiting the p53 signaling pathway. Thus, SASS6 has potential as a novel tumor marker and therapeutic target for ESCC.

SASS6 promotes proliferation of esophageal squamous carcinoma cells by inhibiting the p53 signaling pathway.

SASS6 encodes for the Homo sapiens SAS-6 centriolar assembly protein and is important for proper centrosome formation. Although centrosomes are amplified in a wide variety of tumor types, abnormally high SASS6 expression had previously only been identified in colon cancer. Moreover, the role of SASS6 in esophageal squamous cell carcinoma (ESCC) pathogenesis has not yet been elucidated. The aim of this study was to investigate the role and mechanisms of SASS6 in ESCC. In this study, we found that the mRNA and protein levels of SASS6 were increased in human ESCC samples. In addition, SASS6 protein expression was associated with the esophageal cancer stage and negatively affected survival of patients with ESCC. Furthermore, silencing of SASS6 inhibited cell growth and promoted apoptosis of ESCC cells in vitro and inhibited xenograft tumor formation in vivo. A genetic cluster and pathway analysis showed that SASS6 regulated the p53 signaling pathway. Western blot demonstrated that CCND2, GADD45A and EIF4EBP1 protein expression decreased and that TP53 protein expression increased after the knockdown of SASS6 in ESCC cells. Therefore, SASS6 promoted the proliferation of esophageal cancer by inhibiting the p53 signaling pathway. SASS6 has potential as a novel tumor marker and a therapeutic target for ESCC.

Protein phosphatase 1 regulatory subunit 35 is required for ciliogenesis, notochord morphogenesis, and cell-cycle progression during murine development

Protein phosphatases regulate a wide array of proteins through post-translational modification and are required for a plethora of intracellular events in eukaryotes. While some core components of the protein phosphatase complexes are well characterized, many subunits of these large complexes remain unstudied. Here we characterize a loss-of-function allele of the protein phosphatase 1 regulatory subunit 35 (Ppp1r35) gene. Homozygous mouse embryos lacking Ppp1r35 are developmental delayed beginning at embryonic day (E) 7.5 and have obvious morphological defects at later stages. Mutants fail to initiate turning and do not progress beyond the size or staging of normal E8.5 embryos. Consistent with recent in vitro studies linking PPP1R35 with the microcephaly protein Rotatin and with a role in centrosome formation, we show that Ppp1r35 mutant embryos lack primary cilia. Histological and molecular analysis of Ppp1r35 mutants revealed that notochord development is irregular and discontinuous and consistent with a role in primary cilia, that the floor plate of the neural tube is not specified. Similar to other mutant embryos with defects in centriole function, Ppp1r35 mutants displayed increased cell death that is prevalent in the neural tube and an increased number of proliferative cells in prometaphase. We hypothesize that loss of Ppp1r35 function abrogates centriole homeostasis, resulting in a failure to produce functional primary cilia, cell death and cell cycle delay/stalling that leads to developmental failure. Taken together, these results highlight the essential function of Ppp1r35 during early mammalian development and implicate this gene as a candidate for human microcephaly.

Cep97 Is Required for Centriole Structural Integrity and Cilia Formation in Drosophila

Centrioles are highly elaborate microtubule-based structures responsible for the formation of centrosomes and cilia. Despite considerable variation across species and tissues within any given tissue, their size is essentially constant [1, 2]. While the diameter of the centriole cylinder is set by the dimensions of the inner scaffolding structure of the cartwheel [3], how centriole length is set so precisely and stably maintained over many cell divisions is not well understood. Cep97 and CP110 are conserved proteins that localize to the distal end of centrioles and have been reported to limit centriole elongation in vertebrates [4, 5]. Here, we examine Cep97 function in Drosophila melanogaster. We show that Cep97 is essential for formation of full-length centrioles in multiple tissues of the fly. We further identify the microtubule deacetylase Sirt2 as a Cep97 interactor. Deletion of Sirt2 likewise affects centriole size. Interestingly, so does deletion of the acetylase Atat1, indicating that loss of stabilizing acetyl marks impairs centriole integrity. Cep97 and CP110 were originally identified as inhibitors of cilia formation in vertebrate cultured cells [6], and loss of CP110 is a widely used marker of basal body maturation. In contrast, in Drosophila, Cep97 appears to be only transiently removed from basal bodies and loss of Cep97 strongly impairs ciliogenesis. Collectively, our results support a model whereby Cep97 functions as part of a protective cap that acts together with the microtubule acetylation machinery to maintain centriole stability, essential for proper function in cilium biogenesis.

Besnoitia besnoiti\u2013driven endothelial host cell cycle alteration

Besnoitia besnoiti is an important obligate intracellular parasite of cattle which primarily infects host endothelial cells of blood vessels during the acute phase of infection. Similar to the closely related parasite Toxoplasma gondii, B. besnoiti has fast proliferating properties leading to rapid host cell lysis within 24–30 h p.i. in vitro. Some apicomplexan parasites were demonstrated to modulate the host cellular cell cycle to successfully perform their intracellular development. As such, we recently demonstrated that T. gondii tachyzoites induce G2/M arrest accompanied by chromosome missegregation, cell spindle alteration, formation of supernumerary centrosomes, and cytokinesis impairment when infecting primary bovine umbilical vein endothelial cells (BUVEC). Here, we follow a comparative approach by using the same host endothelial cell system for B. besnoiti infections. The current data showed that—in terms of host cell cycle modulation—infections of BUVEC by B. besnoiti tachyzoites indeed differ significantly from those by T. gondii. As such, cyclin expression patterns demonstrated a significant upregulation of cyclin E1 in B. besnoiti–infected BUVEC, thereby indicating parasite-driven host cell stasis at G1-to-S phase transition. In line, the mitotic phase of host cell cycle was not influenced since alterations of chromosome segregation, mitotic spindle formation, and cytokinesis were not observed. In contrast to respective T. gondii–related data, we furthermore found a significant upregulation of histone H3 (S10) phosphorylation in B. besnoiti–infected BUVEC, thereby indicating enhanced chromosome condensation to occur in these cells. In line to altered G1/S-transition, we here additionally showed that subcellular abundance of proliferating cell nuclear antigen (PCNA), a marker for G1 and S phase sub-stages, was affected by B. besnoiti since infected cells showed increased nuclear PCNA levels when compared with that of control cells.