Regulation Of Centrosome Function Research Articles

Mutant p53 drives the loss of heterozygosity by the upregulation of Nek2 in breast cancer cells

BackgroundMutations in one allele of the TP53 gene in early stages are frequently followed by the loss of the remaining wild-type p53 (wtp53) allele (p53LOH) during tumor progression. Despite the strong notion of p53LOH as a critical step in tumor progression, its oncogenic outcomes that facilitate the selective pressure for p53LOH occurrence were not elucidated.MethodsUsing MMTV;ErbB2 mouse model of breast cancer carrying heterozygous R172H p53 mutation, we identified a novel gain-of-function (GOF) activity of mutant p53 (mutp53): the exacerbated loss of wtp53 allele in response to γ-irradiation.ResultsAs consequences of p53LOH in mutp53 heterozygous cells, we observed profound stabilization of mutp53 protein, the loss of p21 expression, the abrogation of G2/M checkpoint, chromosomal instability, centrosome amplification, and transcriptional upregulation of mitotic kinase Nek2 (a member of Never in Mitosis (NIMA) Kinases family) involved in the regulation of centrosome function. To avoid the mitotic catastrophe in the absence of G2/M checkpoint, cells with centrosome amplification adapt Nek2-mediated centrosomes clustering as pro-survival mutp53 GOF mechanism enabling unrestricted proliferation and clonal expansion of cells with p53LOH. Thus, the clonal dominance of mutp53 cells with p53LOH may represent the mechanism of irradiation-induced p53LOH. We show that pharmacological and genetic ablation of Nek2 decreases centrosome clustering and viability of specifically mutp53 cells with p53LOH.ConclusionIn a heterogeneous tumor population, Nek2 inhibition may alter the selective pressure for p53LOH by contraction of the mutp53 population with p53LOH, thus, preventing the outgrowth of genetically unstable, more aggressive cells.

NudC-like protein 2 restrains centriole amplification by stabilizing HERC2

Centriole duplication is tightly controlled to occur once per cell cycle, and disruption of this synchrony causes centriole amplification, which is frequently observed in many cancers. Our previous work showed that nuclear distribution gene C (NudC)-like protein 2 (NudCL2) localizes to centrosomes; however, little is known about the role of NudCL2 in the regulation of centrosome function. Here, we find that NudCL2 is required for accurate centriole duplication by stabilizing the E3 ligase HECT domain and RCC1-like domain-containing protein 2 (HERC2). Knockout (KO) of NudCL2 using CRISPR/Cas9-based genome editing or depletion of NudCL2 using small interfering RNA causes significant centriole amplification. Overexpression of NudCL2 significantly suppresses hydroxyurea-induced centriole overduplication. Quantitative proteomic analysis reveals that HERC2 is downregulated in NudCL2 KO cells. NudCL2 is shown to interact with and stabilize HERC2. Depletion of HERC2 leads to the similar defects to that in NudCL2-downregulated cells, and ectopic expression of HERC2 effectively rescues the centriole amplification caused by the loss of NudCL2, whereas the defects induced by HERC2 depletion cannot be reversed by exogenous expression of NudCL2. Either loss of NudCL2 or depletion of HERC2 leads to the accumulation of ubiquitin-specific peptidase 33 (USP33), a centrosomal protein that positively regulates centriole duplication. Moreover, knockdown of USP33 reverses centriole amplification in both NudCL2 KO and HERC2-depleted cells. Taken together, our data suggest that NudCL2 plays an important role in maintaining the fidelity of centriole duplication by stabilizing HERC2 to control USP33 protein levels, providing a previously undescribed mechanism restraining centriole amplification.

Abstract 989: NEDD9 expression promotes epithelial ovarian cancer growth and dissemination

Abstract NEDD9 (Neural precursor cell Expressed, Developmentally Downregulated 9) is a scaffolding protein with roles in focal adhesion signaling, cell motility and regulation of centrosome function. NEDD9 has been reported as an important player in development and progression of several different solid tumors; however, its role in gynecologic cancers is poorly understood. In this study we investigated the role of NEDD9 in development and progression of epithelial ovarian cancer (EOC) using murine models. Three mouse strains were used: MISIIR-TAg mice that develop spontaneous ovarian carcinomas; MISIIR-TAg-Low mice that express the TAg transgene, but do not develop tumors; and Nedd9 null mice. To determine the effects of loss of Nedd9 on ovarian carcinoma development, MISIIR-TAg and Nedd9-/- mice were crossed and spontaneous ovarian tumor growth in MISIIR-TAg;Nedd9-/- and MISIIR-TAg;Nedd9+/+ mice was monitored and quantified by longitudinal magnetic resonance imaging (MRI). MISIIR-TAg-Nedd9-/- mice, compared to MISIIR-TAg-Nedd9+/+ mice, exhibited delayed tumor development and decreased tumor burden. Correspondingly, microarray analysis of tumors showed that several key oncogenic signaling pathways were upregulated in MISIIR-TAg-Nedd9+/+ mice compared to MISIIR-TAg-Nedd9-/- mice. Murine ovarian carcinoma (MOVCAR) cell lines were established from MISIIR-TAg-Nedd9-/- and MISIIR-TAg-Nedd9+/+ mice and essential tumorigenic features compared using in vitro assays. Interestingly, although these cells displayed no differences in adhesion or proliferation, Nedd9-/- MOVCAR cells were more aggressive than Nedd9+/+ cells in assays of migration and invasion. However, when grown as orthotopic allografts in permissive MISIIR-TAg-Low;Nedd9+/+ hosts, in vivo tumor growth was delayed and the number of metastatic tumor nodules was decreased in mice engrafted with Nedd9-/- cells compared to those injected with Nedd9+/+ cells. To study the potential role of Nedd9 in the tumor microenvironment, immune cell populations were compared in: 1) orthotopic allografts of Nedd9+/+ MOVCAR cells grown in MISIIR-TAg-Low;Nedd9-/- and MISIIR-TAg-Low;Nedd9+/+ hosts; or 2) spontaneous ovarian tumors in MISIIR-TAg-Nedd9-/- and MISIIR-TAg-Nedd9+/+ mice. In the allograft model, the number and total volume of metastatic tumor nodules was not significantly different in Nedd9-/- or Nedd9+/+ hosts; however, genotype-associated differences in tumor infiltrating NK-cell numbers were observed. Comparison of the spontaneous tumor models showed that the number of tumor infiltrating macrophages and NK-cells were decreased in MISIIR-TAg-Nedd9-/- mice. Collectively, these data suggest that Nedd9 promotes development and progression of EOC, through induction of oncogenic signaling and by pro-tumorigenic alterations of the immune cell microenvironment. Citation Format: Rashid Gabbasov, Laura E. Bickel, Shane W. O'Brien, Samuel Litwin, Sachiko Seo, Erica A. Golemis, Denise C. Connolly. NEDD9 expression promotes epithelial ovarian cancer growth and dissemination. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 989. doi:10.1158/1538-7445.AM2014-989

Tankyrase 1 regulates centrosome function by controlling CPAP stability

CPAP--a gene mutated in primary microcephaly--is required for procentriole formation. Here we show that CPAP degradation and function is controlled by the poly(ADP-ribose) polymerase tankyrase 1. CPAP is PARsylated by tankyrase 1 in vitro and in vivo. Overexpression of tankyrase 1 leads to CPAP proteasomal degradation, preventing centriole duplication, whereas depletion of tankyrase 1 stabilizes CPAP in G1, generating elongated procentrioles and multipolarity. Tankyrase 1 localizes to centrosomes exclusively in G1, coinciding with CPAP degradation. Hence, tankyrase 1-mediated PARsylation regulates CPAP levels during the cell cycle to limit centriole elongation and ensure normal centrosome function.

Role of Focal Adhesion Kinase Ser-732 Phosphorylation in Centrosome Function during Mitosis

Focal adhesion kinase (FAK) is the major cytoplasmic tyrosine kinase in focal adhesions and a critical mediator of integrin signaling in a variety of cells, including endothelial cells (ECs). Here we describe a new function for FAK in the regulation of centrosome functions in a Ser-732 phosphorylation-dependent manner during mitosis. Deletion of FAK in primary ECs causes increases in centrosome numbers, multipolar and disorganized spindles, and unaligned chromosomes during mitosis. Re-expression of wild-type FAK, but not S732A mutant, rescued these mitotic defects, suggesting a role for Ser-732 phosphorylation in the regulation of centrosomal functions. Consistent with this possibility, Ser-732-phosphorylated FAK was found to co-localize in centrosomes in mitotic cells. FAK also associated with cytoplasmic dynein in a Ser-732 phosphorylation-dependent manner. Further analysis in FAK-null primary ECs showed that S732A mutant could rescue EC migration but not proliferation or tubulogenesis in vitro. Last, we showed that deletion of FAK in ECs reduced tumor angiogenesis in vivo, which could be restored by re-expression of wild-type FAK but not S732A mutant. Together, these studies demonstrated a novel role for Ser-732 phosphorylation of FAK in the regulation of centrosome function during mitosis, which may contribute to EC proliferation and angiogenesis.

ZFPL1, a novel ring finger protein required for cis-Golgi integrity and efficient ER-to-Golgi transport

The Golgi apparatus occupies a central position within the secretory pathway, but the molecular mechanisms responsible for its assembly and organization remain poorly understood. We report here the identification of zinc finger protein-like 1 (ZFPL1) as a novel structural component of the Golgi apparatus. ZFPL1 is a conserved and widely expressed integral membrane protein with two predicted zinc fingers at the N-terminus, the second of which is a likely ring domain. ZFPL1 directly interacts with the cis-Golgi matrix protein GM130. Depletion of ZFPL1 results in the accumulation of cis-Golgi matrix proteins in the intermediate compartment (IC) and the tubulation of cis-Golgi and IC membranes. Loss of ZFPL1 function also impairs cis-Golgi assembly following brefeldin A washout and slows the rate of cargo trafficking into the Golgi apparatus. Effects upon Golgi matrix protein localization and cis-Golgi structure can be rescued by wild-type ZFPL1 but not mutants defective in GM130 binding. Together, these data suggest that ZFPL1 has an important function in maintaining the integrity of the cis-Golgi and that it does so through interactions with GM130.

Potential existence of two independent centrosome-targeting domains in PP4

Protein phosphatase 4 (PP4) is an important member in the PPP family of protein Ser/Thr phosphatases. It has been proven to regulate a variety of cellular processes such as centrosome maturation, microtubule nucleation, splicesome assembly, and JNK pathway activation. Compared to the crystallized and structurally well defined phosphatase PP1 and PP2B, little is known about the structure of PP4. Besides the conserved motifs characteristic of the PPP family, no information is available on the other domains of PP4. PP4 is reported to localize to the centrosome in many species such as Drosophila, Caenorhabditis elegans and mammalian cells, which suggests a conserved role of PP4 in the regulation of centrosome function. Unlike several other centrosomal proteins, no sequence has been identified for PP4 that can target it to specific centrosomal localization. In this study, we used a combination of PCR mutagenesis and transient expression of GFP-tagged proteins in mammalian cells, and identified two PP4 centrosome-targeting domains of 68–136 and 134–220 aa. These two domains may be associated for appropriate localization to the centrosome. The findings are useful for further elucidating the function of the domains and other structural characteristics of PP4.

Involvement of Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribosyl)ation in Regulation of Centrosome Function

The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.

The Aurora kinases: role in cell transformation and tumorigenesis.

Aurora kinases representing a novel family of serine/threonine kinases have been identified as key regulators of the mitotic cell division process. The three members of this kinase family, identified so far, referred to as Aurora-A, Aurora-B and Aurora-C kinases, are close homologues of the prototypic yeast Ipll and Drosophila aurora kinases, which are known to be involved in the regulation of centrosome function, bipolar spindle assembly and chromosome segregation processes. All three members of the mammalian kinase family have a catalytic domain that is highly conserved with a short C-terminal domain and an N-terminal domain of varying sizes. Following their discovery about five years ago, extensive research has focused on understanding the biological roles of these kinases and elucidation of their pathways, which regulate cell proliferation and maintenance of normal cellular phenotypes. Significant interest in the subject was generated since all three Aurora kinases family members were reported to be overexpressed in many human cancers, and elevated expression has been correlated with chromosomal instability and clinically aggressive disease in some instances. Ectopic overexpression of one member of the family, Aurora-A, was shown to induce oncogenic transformation in cells. Unlike most other putative oncogenes identified, so far, members of this kinase family are expressed and active at the highest level during G2-M phase of the cell cycle. Aurora kinases are localized at the centrosomes of interphase cells, at the poles of the bipolar spindle and in the midbody of the mitotic apparatus. Substrates identified for the Aurora-A and Aurora-B kinases, include a kinesin-like motor protein, spindle apparatus proteins, histone H3 protein, kinetochore protein and the tumor suppressor protein p53. Identification of Aurora kinases as RasGAP Src homology 3 domain binding protein, also implicates these kinases as potential effectors in the Ras pathway relevant to oncogenesis. Abnormal elevated expression of Aurora kinases detected in human cancer cells could help explain the underlying biological mechanisms responsible for the development of many cellular phenotypes associated with malignant cells. Identification of these mechanisms offers the possibility of designing novel targeted therapies for cancer in the future.

Vaccinia virus infection disrupts microtubule organization and centrosome function

We examined the role of the microtubule cytoskeleton during vaccinia virus infection. We found that newly assembled virus particles accumulate in the vicinity of the microtubule-organizing centre in a microtubule- and dynein–dynactin complex-dependent fashion. Microtubules are required for efficient intracellular mature virus (IMV) formation and are essential for intracellular enveloped virus (IEV) assembly. As infection proceeds, the microtubule cytoskeleton becomes dramatically reorganized in a fashion reminiscent of overexpression of microtubule-associated proteins (MAPs). Consistent with this, we report that the vaccinia proteins A10L and L4R have MAP-like properties and mediate direct binding of viral cores to microtubules in vitro. In addition, vaccinia infection also results in severe reduction of proteins at the centrosome and loss of centrosomal microtubule nucleation efficiency. This represents the first example of viral-induced disruption of centrosome function. Further studies with vaccinia will provide insights into the role of microtubules during viral pathogenesis and regulation of centrosome function.

Foundations in Cancer Research p53 and ATM: Cell Cycle, Cell Death, and Cancer

The development of a normal cell into a tumor cell appears to depend in part on mutations in genes that normally control cell cycle and cell death, thereby resulting in inappropriate cellular survival and tumorigenesis. ATM ("mutated in ataxia-telangiectasia") and p53 are two gene products that are believed to play a major role in maintaining the integrity of the genome such that alterations in these gene products may contribute to increased incidence of genomic changes such as deletions, translocations, and amplifications, which are common during oncogenesis. p53 is a critical participant in a signal transduction pathway that mediates either a G1 arrest or apoptosis in response to DNA damage. In addition, p53 is believed to be involved in the mitotic spindle checkpoint and in the regulation of centrosome function. Following certain cytotoxic stresses, normal ATM function is required for p53-mediated G1 arrest. ATM is also involved in other cellular processes such as S phase and G2-M phase arrest and in radiosensitivity. The understanding of the roles that both p53 and ATM play in cell cycle progression and cell death in response to DNA damage may provide new insights into the molecular mechanisms of cellular transformation and may help identify potential targets for improved cancer therapies.