STIL Overexpression Is Associated with Chromosomal Numerical Abnormalities in Non-Small-Cell Lung Carcinoma Through Centrosome Amplification
Shunsuke OhtsukaHisami KatoRei IshikawaHirofumi WatanabeRyosuke MiyazakiShin-ya KatsuragiKatsuhiro YoshimuraHidetaka YamadaYasuhiro SakaiYusuke InoueYusuke TakanashiKeigo SekiharaKazuhito FunaiHaruhiko SugimuraKazuya Shinmura
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Abstract:
STIL is a regulatory protein essential for centriole biogenesis, and its dysregulation has been implicated in various diseases, including malignancies. However, its role in non-small-cell lung carcinoma (NSCLC) remains unclear. In this study, we examined STIL expression and its potential association with chromosomal numerical abnormalities (CNAs) in NSCLC using The Cancer Genome Atlas (TCGA) dataset, immunohistochemical analysis, and in vitro experiments with NSCLC cell lines designed to overexpress STIL. TCGA data revealed upregulated STIL mRNA expression in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), the two major subtypes of NSCLC. Immunohistochemical analysis of cases from our hospital (LUAD, n = 268; LUSC, n = 98) revealed STIL protein overexpression. To elucidate the functional role of STIL, an inducible STIL-overexpressing H1299 NSCLC cell line was generated. Overexpression of STIL in these cells promoted centrosome amplification, leading to chromosomal instability. Finally, analysis of arm-level chromosomal copy number alterations from the TCGA dataset revealed that elevated STIL mRNA expression was associated with CNAs in both LUAD and LUSC. These findings suggest that STIL overexpression is associated with CNAs in NSCLC, likely through centrosome amplification, which is linked to chromosomal instability and might represent a potential therapeutic target for NSCLC treatment.Keywords:
Chromosome instability
Centriole
Giving an old organelle the old heave-ho Centrioles are ancient cellular organelles that build centrosomes, the major microtubule-organizing centers in animal cells. Duplication of centrioles is tightly controlled to ensure that each dividing cell has precisely two centrosomes. Human cancer cells often have extra centrosomes, which has been hypothesized to confer a proliferative advantage. Wong et al. developed small molecules (centrinones) that allowed them to reversibly “delete” centrioles from cells (see the Perspective by Stearns). Surprisingly, cancer cells continued to divide in the absence of centrosomes, whereas normal cells stopped dividing. Science , this issue p. 1155 ; see also p. 1091
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ABSTRACT Centriole assembly requires a small number of conserved proteins. The precise pathway of centriole assembly has been difficult to study, as the lack of any one of the core assembly proteins [Plk4, Ana2 (the homologue of mammalian STIL), Sas-6, Sas-4 (mammalian CPAP) or Asl (mammalian Cep152)] leads to the absence of centrioles. Here, we use Sas-6 and Ana2 particles (SAPs) as a new model to probe the pathway of centriole and centrosome assembly. SAPs form in Drosophila eggs or embryos when Sas-6 and Ana2 are overexpressed. SAP assembly requires Sas-4, but not Plk4, whereas Asl helps to initiate SAP assembly but is not required for SAP growth. Although not centrioles, SAPs recruit and organise many centriole and centrosome components, nucleate microtubules, organise actin structures and compete with endogenous centrosomes to form mitotic spindle poles. SAPs require Asl to efficiently recruit pericentriolar material (PCM), but Spd-2 (the homologue of mammalian Cep192) can promote some PCM assembly independently of Asl. These observations provide new insights into the pathways of centriole and centrosome assembly.
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Centrioles are symmetrical, barrel-shaped, microtubule-based structures, that serve as building platforms for the formation of centrosomes and cilia. In dividing cells, they recruit a matrix of proteins called pericentriolar material (PCM) to form the centrosome, the major microtubule-organizing centre in animal cells. In differentiating cells, centrioles also function as basal bodies for the formation of flagella and cilia. To ensure the proper segregation of centrioles during cell division, centriole duplication is tightly controlled and coordinated with the cell cycle. To date, several factors have been identified in human cells that are recruited in a consecutive fashion to nascent centrioles to template the outgrowth of one procentriole orthogonally to the pre-existing one during each cell cycle. In addition to structural components, these duplication factors include the kinase Plk4 (polo-like kinase 4), which is pivotal for the initiation of centriole duplication.
Here, we have taken two approaches to investigate centrosomes in more detail: first, the human centrosomal proteins Cep192 and Cep152 were functionally characterized; second, the localization of key centrosomal and centriolar components was analyzed using super-resolution three-dimensional structured illumination microscopy (3D-SIM).
1. Previously, Cep192 and Cep152 had been identified as novel centrosomal proteins in a proteomic screen. Homologues of Cep152 in flies and zebrafish had been implicated in PCM recruitment and centrosome duplication, but Cep152 had not been investigated in humans. Likewise, the worm homologue of Cep192 also functions in PCM recruitment and centrosome duplication. However, in humans its role in centriole duplication remained controversial.
Here, we have established that Cep152 is dispensable, whereas Cep192 is essential for PCM recruitment in mitotic cells. This functional difference is further illustrated by their differential subcellular localizations during interphase and mitosis. The stable centrosomal integration of Cep152 depended on Cep192, whereas Cep192 localized independently of Cep152.
Furthermore, both Cep152 and Cep192 were required for proper centriole duplication, thereby clarifying the controversy about the implication of Cep192 in this process. We also show that Cep192 and Cep152 co-operate in the centriolar recruitment of the kinase Plk4. Concomitantly, centriole duplication was only inhibited to a similar extent as in Plk4- or Sas-6-depleted cells, if both Cep152 and Cep192 were depleted. In agreement, we have identified and characterized interactions of Plk4 with the N termini of both Cep152 and Cep192. Finally, not only the recruitment of Plk4, but also of other duplication factors such as CPAP and Sas-6 was impaired in Cep152- and/ or Cep192-depleted cells.
We have also addressed the regulation of Cep152 and Cep192. Centrosomal levels of both proteins increased towards mitosis. Similarly, cytoplasmic Cep152 levels peaked when cells approached mitosis, whereas Cep192 levels were stable.
Hence, we show that both Cep152 and Cep192 function as centriole duplication factors. Both proteins exert a partly redundant function and their co-operation orchestrates recruitment of Plk4 as well as other centriole duplication factors and thus canonical centriole duplication.
2. Using 3D-SIM we have analysed the spatial relationship of 18 centriole and PCM components of human centrosomes at different cell cycle stages.
During mitosis, PCM proteins formed extended networks with interspersed gamma-Tubulin. In interphase, most proteins were arranged at specific and defined distances from the walls of centrioles, resulting in ring-like staining. Additionally, orientation of Cep152 with its C-terminus close to centriole walls and its N-terminus facing outwards was visualised using site-specific antibodies against either terminus of the protein. At the distal end of centrioles, appendage proteins formed rings with several density masses, usually with a multiplicity below that expected from the 9-fold symmetry of centrioles. Although Cep164 remained centriolar throughout the cell cycle, the number of discernible density masses was clearly reduced during mitosis. At the proximal end, Sas-6 formed a dot at the site of daughter centriole formation, consistent with its role in cartwheel formation. Plk4 and STIL co-localized with Sas-6, but the bulk of the cartwheel protein Cep135 was associated with mother centrioles. Remarkably, Plk4 formed a dot on the surface of the mother centriole even before Sas-6 staining became detectable, indicating that Plk4 constitutes an early marker for the site of nascent centriole formation.
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Centrioles organise centrosomes and cilia, and these organelles have an important role in many cell processes. In flies, the centriole protein Ana1 is required for the assembly of functional centrosomes and cilia. It has recently been shown that Cep135 (also known as Bld10) initially recruits Ana1 to newly formed centrioles, and that Ana1 then recruits Asl (known as Cep152 in mammals) to promote the conversion of these centrioles into centrosomes. Here, we show that ana1 mutants lack detectable centrosomes in vivo, that Ana1 is irreversibly incorporated into centrioles during their assembly and appears to play a more important role in maintaining Asl at centrioles than in initially recruiting Asl to centrioles. Unexpectedly, we also find that Ana1 promotes centriole elongation in a dose-dependent manner: centrioles are shorter when Ana1 dosage is reduced and are longer when Ana1 is overexpressed. This latter function of Ana1 appears to be distinct from its role in centrosome and cilium function, as a GFP-Ana1 fusion lacking the N-terminal 639 amino acids of the protein can support centrosome assembly and cilium function but cannot promote centriole over-elongation when overexpressed.
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Centrioles are microtubule-based cellular structures present in most human cells that build centrosomes and cilia. Proliferating cells have only two centrosomes and this number is stringently maintained through the temporally and spatially controlled processes of centriole assembly and segregation. The assembly of new centrioles begins in early S phase and ends in the third G1 phase from their initiation. This lengthy process of centriole assembly from their initiation to their maturation is characterized by numerous structural and still poorly understood biochemical changes, which occur in synchrony with the progression of cells through three consecutive cell cycles. As a result, proliferating cells contain three structurally, biochemically, and functionally distinct types of centrioles: procentrioles, daughter centrioles, and mother centrioles. This age difference is critical for proper centrosome and cilia function. Here we discuss the centriole assembly process as it occurs in somatic cycling human cells with a focus on the structural, biochemical, and functional characteristics of centrioles of different ages.
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After duplication of the centriole pair during S phase, the centrosome functions as a single microtubule-organizing center until the onset of mitosis, when the duplicated centrosomes separate for bipolar spindle formation. The mechanisms regulating centrosome cohesion and separation during the cell cycle are not well understood. In this study, we analyze the protein rootletin as a candidate centrosome linker component. As shown by immunoelectron microscopy, endogenous rootletin forms striking fibers emanating from the proximal ends of centrioles. Moreover, rootletin interacts with C-Nap1, a protein previously implicated in centrosome cohesion. Similar to C-Nap1, rootletin is phosphorylated by Nek2 kinase and is displaced from centrosomes at the onset of mitosis. Whereas the overexpression of rootletin results in the formation of extensive fibers, small interfering RNA-mediated depletion of either rootletin or C-Nap1 causes centrosome splitting, suggesting that both proteins contribute to maintaining centrosome cohesion. The ability of rootletin to form centriole-associated fibers suggests a dynamic model for centrosome cohesion based on entangling filaments rather than continuous polymeric linkers.
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