Coiled-body Phosphoprotein Research Articles

Effects of the Nonstructural Protein-Nucleolar and Coiled-Body Phosphoprotein 1 Protein Interaction on rRNA Synthesis Through Telomeric Repeat-Binding Factor 2 Regulation Under Nucleolar Stress.

To investigate the effects and underlying molecular mechanisms of the interaction between the non-structural protein 1 (NS1) and nucleolar and coiled-body phosphoprotein 1 (NOLC1) on rRNA synthesis through nucleolar telomeric repeat-binding factor 2 (TRF2) under nucleolar stress in avian influenza A virus infection. The analysis of TRF2 ties into the exploration of ribosomal protein L11 (RPL11) and mouse double minute 2 (MDM2) because TRF2 has been found to interact with NOLC1, and the RPL11-MDM2 pathway plays an important role in nucleolar regulation and cellular processes. Both human embryonic kidney 293T cells and human lung adenocarcinoma A549 cells were transfected with the plasmids pCAGGS-HA and pCAGGS-HA-NS1, respectively. In addition, A549 cells were transfected with the plasmids pEGFP-N1, pEGFP-N1-NS1, and pDsRed2-N1-TRF2. The cell cycle was detected by flow cytometry, and coimmunoprecipitation was applied to examine the interactions between different proteins. The effect of NS1 on TRF2 was detected by immunoprecipitation, and the colocalization of NOLC1 and TRF2 or NS1 and TRF2 was visualized by immunofluorescence. Quantitative real-time PCR was conducted to detect the expression of the TRF2 and p21. There is a strong interaction between NOLC1 and TRF2, and the colocalization of NOLC1 and TRF2 in the nucleus. The protein expression of NOLC1 in A549-HA-NS1 cells was lower than that in A549-HA cells, which was accompanied by the upregulated protein expression of p53 in A549-HA-NS1 cells (all p < .05). TRF2 was scattered throughout the nucleus without clear nucleolar aggregation. RPL11 specifically interacted with MDM2 in the NS1 group, and expression of the p21 gene was significantly increased in the HA-NS1 group compared with the HA group (p < .01). NS1 protein can lead to the reduced aggregation of TRF2 in the nucleolus, inhibition of rRNA expression, and cell cycle blockade by interfering with the NOLC1 protein and generating nucleolar stress.

Dysregulated Ribosome Biogenesis Is a Targetable Vulnerability in Triple-Negative Breast Cancer: MRPS27 as a Key Mediator of the Stemness-inhibitory Effect of Lovastatin.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited effective therapeutic options readily available. We have previously demonstrated that lovastatin, an FDA-approved lipid-lowering drug, selectively inhibits the stemness properties of TNBC. However, the intracellular targets of lovastatin in TNBC remain largely unknown. Here, we unexpectedly uncovered ribosome biogenesis as the predominant pathway targeted by lovastatin in TNBC. Lovastatin induced the translocation of ribosome biogenesis-related proteins including nucleophosmin (NPM), nucleolar and coiled-body phosphoprotein 1 (NOLC1), and the ribosomal protein RPL3. Lovastatin also suppressed the transcript levels of rRNAs and increased the nuclear protein level and transcriptional activity of p53, a master mediator of nucleolar stress. A prognostic model generated from 10 ribosome biogenesis-related genes showed outstanding performance in predicting the survival of TNBC patients. Mitochondrial ribosomal protein S27 (MRPS27), the top-ranked risky model gene, was highly expressed and correlated with tumor stage and lymph node involvement in TNBC. Mechanistically, MRPS27 knockdown inhibited the stemness properties and the malignant phenotypes of TNBC. Overexpression of MRPS27 attenuated the stemness-inhibitory effect of lovastatin in TNBC cells. Our findings reveal that dysregulated ribosome biogenesis is a targetable vulnerability and targeting MRPS27 could be a novel therapeutic strategy for TNBC patients.

NOLC1 was identified as a tumor suppressor gene in thyroid cancer and correlated with prognosis by bioinformatics.

Thyroid cancer (THCA) is the most common endocrine malignancy, mainly affecting women's unilateral glandular lobes. However, for relapsed and distant metastasis of THCA patients, the existing early diagnosis and treatment methods were still insufficient, and a new method was urgently needed to diagnose and treat them. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) was one of the most phosphorylated proteins in the cell, which was located mainly in the nucleolus. In addition, more and more studies have confirmed that NOLC1 plays a crucial role in various pathological processes, such as the occurrence and progression of cancer and viral infection. A previous study showed that NOLC1, as a member of RNA-binding protein, was significantly correlated with the prognosis of THCA patients. However, further exploration of NOLC1 in THCA is limited. To further explore the role of NOLC1 in THCA, we conducted expression and survival prognosis analysis of NOLC1 using multiple databases. We also evaluated the correlation between NOLC1 gene expression and clinical characteristics of THCA patients. Furthermore, we analyzed the relationship between NOLC1 and other genes, followed by enrichment analysis to investigate its metabolic pathways and molecular metabolism processes. Additionally, we examined the association between immune cell infiltration in tumor microenvironment and NOLC1. Notably, through vitro experiments, we confirmed the tumor suppressive effect of NOLC1 on the proliferation and migration of human THCA cells, providing evidence for clinical diagnosis of THCA. Furthermore, we confirmed the tumor suppressive effect of NOLC1 in vivo xenograft assay. To sum up, our results suggest that NOLC1 is a tumor suppressor gene for THCA.

T7 phage display reveals NOLC1 as a GM3 binding partner in human breast cancer MCF-7 cells

Ganglioside GM3 is a simple monosialoganglioside (NeuAc-Gal-Glc-ceramide) that modulates cell adhesion, proliferation, and differentiation. Previously, we reported isolation of GM3-binding vascular endothelial growth factor receptor and transforming growth factor-β receptor by the T7 phage display method (Chung et al., 2009; Kim et al., 2013). To further identify novel proteins interacting with GM3, we extended the T7 phage display method in this study. After T7 phage display biopanning combined with immobilized biotin-labeled 3′-sialyllactose prepared on a streptavidin-coated microplate, we isolated 100 candidate sequences from the human lung cDNA library. The most frequently detected clones from the blast analysis were the human nucleolar and coiled-body phosphoprotein 1 (NOLC1) sequences. We initially identified NOLC1 as a molecule that possibly binds to GM3 and confirmed this binding ability using the glutathione S-transferase fusion protein. Herein, we report another GM3-interacting protein, NOLC1, that can be isolated by the T7 phage display method. These results are expected to be helpful for elucidating the functional roles of ganglioside GM3 with NOLC1. When human breast cancer MCF-7 cells were examined for subcellular localization of NOLC1, immunofluorescence of NOLC1 was observed in the intracellular region. In addition, NOLC1 expression was increased in the nucleolus after treatment with the anticancer drug doxorubicin. GM3 and NOLC1 levels in the doxorubicin-treated MCF-7 cells were correlated, indicating possible associations between GM3 and NOLC1. Therefore, direct interactions between carbohydrates and cellular proteins can pave the path for new signaling phenomena in biology.

MCM5 is an oncogene of colon adenocarcinoma and promotes progression through cell cycle control

Many patients with colon adenocarcinoma (COAD) are diagnosed at an advanced stage, and the molecular mechanism of COAD progression is intricate and controversial. Therefore, there is an urgent need to identify more novel prognosis biomarkers for COAD and elucidate the molecular mechanism of this disease. The present study aimed to screen out key genes correlated with COAD prognosis. In this study, a key module was identified and four hub genes (MCM5 (encoding minichromosome maintenance complex component 5), NOLC1 (encoding nucleolar and coiled-body phosphoprotein 1), MYC (encoding MYC proto-oncogene, BHLH transcription factor), and CDK4 (encoding cyclin dependent kinase 4)) were selected that correlated with COAD prognosis, based on the GSE9348 dataset in Gene Expression Omnibus database. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that MCM5 correlated with the cell cycle. Furthermore, MCM5 expression was upregulated in tumor tissues of patients with COAD compared with that in adjacent tissues, based on various databases, including The Cancer Genome Atlas, the Clinical Proteomic Tumor Analysis Consortium database, and the Human Protein Atlas database. Small interfering RNA-mediated knockdown of MCM5 inhibited the cell cycle and migration of colorectal cancer cells in vitro. And western blotting results indicated that factors correlated with cell cycle (CDK2/6, Cyclin D3, P21) were downregulated after knockdown of MCM5 in vitro. Besides, downregulation of MCM5 was demonstrated to inhibit lung metastasis of COAD in nude mice model. In conclusion, MCM5 is an oncogene of COAD that promotes COAD progression via cell cycle control.

Roles of NOLC1 in cancers and viral infection.

The nucleolus is considered the center of metabolic control and an important organelle for the biogenesis of ribosomal RNA (rRNA). Nucleolar and coiled-body phosphoprotein 1(NOLC1), which was originally identified as a nuclear localization signal-binding protein is a nucleolar protein responsible for nucleolus construction and rRNA synthesis, as well as chaperone shuttling between the nucleolus and cytoplasm. NOLC1 plays an important role in a variety of cellular life activities, including ribosome biosynthesis, DNA replication, transcription regulation, RNA processing, cell cycle regulation, apoptosis, and cell regeneration. In this review, we introduce the structure and function of NOLC1. Then we elaborate its upstream post-translational modification and downstream regulation. Meanwhile, we describe its role in cancer development and viral infection which provide a direction for future clinical applications. The relevant literatures from PubMed have been reviewed for this article. NOLC1 plays an important role in the progression of multiple cancers and viral infection. In-depth study of NOLC1 provides a new perspective for accurate diagnosis of patients and selection of therapeutic targets.

Identification of new telomere- and telomerase-associated autoantigens in systemic sclerosis

PurposeIn up to 20% of patients with systemic sclerosis (SSc) no known autoantibody specificity can be identified. Recently discovered autoantigens, such as telomeric repeat binding factor 1 (TERF1), as well as established autoantigens, like RuvBL1/2, are associated with telomere and telomerase biology. We aimed to identify new telomere- and telomerase-associated autoantigens in patients with SSc without known autoantibody specificity. MethodsUnlabelled protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS) was performed with sera of 106 patients with SSc from two tertiary referral centres that had a nuclear pattern on HEp-2 indirect immunofluorescence without previously identified autoantibody. Telomere- or telomerase-associated proteins or protein complexes precipitated by individual sera were identified. Candidate autoantigens were confirmed through immunoprecipitation-western blot (IP-WB). A custom Luminex xMAP assay for 5 proteins was evaluated with sera from persons with SSc (n = 467), other systemic autoimmune rheumatic diseases (n = 923), non-rheumatic disease controls (n = 187) and healthy controls (n = 199). ResultsEight telomere- and telomerase-associated autoantigens were identified in a total of 11 index patients, including the THO complex (n = 3, all with interstitial lung disease and two with cardiac involvement), telomeric repeat-binding factor 2 (TERF2, n = 1), homeobox-containing protein 1 (HMBOX1, n = 2), regulator of chromosome condensation 1 (RCC1, n = 1), nucleolar and coiled-body phosphoprotein 1 (NOLC1, n = 1), dyskerin (DKC1, n = 1), probable 28S rRNA (cytosine(4447)-C(5))-methyltransferase (NOP2, n = 1) and nuclear valosin-containing protein-like (NVL, n = 2). A Luminex xMAP assay for THO complex subunit 1 (THOC1), TERF2, NOLC1, NOP2 and NVL revealed high reactivity in all index patients, but also in other patients with SSc and disease controls. However, the reactivity by xMAP assay in these other patients was not confirmed by IP-WB. ConclusionIP-MS revealed key telomere- and telomerase-associated proteins and protein complexes as autoantigens in patients with SSc.

NOLC1 knockdown suppresses prostate cancer progressions by reducing AKT phosphorylation and β-catenin accumulation

Although several studies have focused on cancer diagnosis and therapy, prostate cancer (PC) remains an intractable disease. Androgen deprivation therapy (ADT), which is used to treat early stage PC can lead to the development of castration-resistant prostate cancer (CRPC), which is highly associated with androgen receptor (AR) mutations. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) is a chaperone that shuttles between the nucleus and the cytoplasm. Studies suggest that NOLC1 regulates PC progression; however, the underlying mechanisms remain unclear. Herein, we showed that NOLC1 knockdown suppresses PC cell proliferation by altering the signaling pathways and the expression of various proteins involved in DNA replication, amino acid metabolism, and RNA processing. Mechanistically, NOLC1 knockdown suppressed cell cycle progression by inhibiting AKT phosphorylation and β-catenin accumulation. Finally, we showed that NOLC1 expression is higher in human PC than in human hyperplastic prostate tissues. Altogether, we demonstrated that NOLC1 knockdown suppresses the progression of both AR-positive and AR-negative PC cells by inducing changes in the expression of several genes leading to cell cycle arrest. Thus, NOLC1 might be a novel and promising therapeutic target for PC.

Atypical chemokine receptor 3 induces colorectal tumorigenesis in mice by promoting β-arrestin-NOLC1-fibrillarin-dependent rRNA biogenesis.

Atypical chemokine receptor 3 (ACKR3) has emerged as a key player in various biological processes. Its atypical "intercepting receptor" properties have established ACKR3 as the major regulator in the pathophysiological processes in many diseases. In this study, we investigated the role of ACKR3 activation in promoting colorectal tumorigenesis. We showed that ACKR3 expression levels were significantly increased in human colon cancer tissues, and high levels of ACKR3 predicted the increased severity of cancer. In Villin-ACKR3 transgenic mice with a high expression level of CKR3 in their intestinal epithelial cells, administration of AOM/DSS induced more severe colorectal tumorigenesis than their WT littermates. Cancer cells of Villin-ACKR3 transgenic mice were characterised by the nuclear β-arrestin-1 (β-arr1)-activated perturbation of rRNA biogenesis. In HCT116 cells, cotreatment with CXCL12 and AMD3100 selectively activated ACKR3 and induced nuclear translocation of β-arr1, leading to an interaction of β-arr1 with nucleolar and coiled-body phosphoprotein 1 (NOLC1). NOLC1, as the phosphorylated protein, further interacted with fibrillarin, a conserved nucleolar methyltransferase responsible for ribosomal RNA methylation in the nucleolus, thereby increasing the methylation in histone H2A and promoting rRNA transcription in ribosome biogenesis. In conclusion, ACKR3 promotes colorectal tumorigenesis through the perturbation of rRNA biogenesis by the β-arr1-induced interaction of NOLC1 with fibrillarin.

Nucleolar and Coiled-Body Phosphoprotein 1 Is Associated With Stemness and Represents a Potential Therapeutic Target in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and lacks approved specific targeted therapies. One of the major reasons why TNBC is difficult to treat is the high proportion of cancer stem cells within the tumor tissue. Nucleolus is the location of ribosome biogenesis which is frequently overactivated in cancer cells and overactivation of ribosome biogenesis frequently drives the malignant transformation of cancer. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) is a nucleolar protein responsible for nucleolus organization and rRNA synthesis and plays an important role in ribosome biogenesis. However, the correlation of NOLC1 expression with patient prognosis and its value as a therapeutic target have not been evaluated in TNBC. In the current study, based on bioinformatics analysis of the online databases, we found that the expression of NOLC1 was higher in breast cancer tissues than normal tissues, and NOLC1 was expressed at a higher level in TNBC than other subtypes of breast cancer. GSEA analysis revealed that stemness-related pathways were significantly enriched in breast cancer with high NOLC1 gene expression. Further analyses using gene expression profiling interactive analysis 2 (GEPIA2), tumor immune estimation resource (TIMER) and search tool for retrieval of interacting genes/proteins (STRING) demonstrated that NOLC1 was significantly associated with stemness in both all breast cancer and basal-like breast cancer/TNBC patients at both gene and protein levels. Knockdown of NOLC1 by siRNA decreased the protein level of the key stemness regulators MYC and ALDH and inhibited the sphere-forming capacity in TNBC cell line MDA-MB-231. Univariate and multivariate Cox regression analyses demonstrated that NOLC1 was an independent risk factor for overall survival in breast cancer. PrognoScan and Kaplan-Meier plotter analyses revealed that high expression of NOLC1 was associated with poor prognosis in both all breast cancer and TNBC patients. Further immunohistochemical analysis of breast cancer patient samples revealed that TNBC cells had a lower level of NOLC1 in the nucleus compared with non-TNBC cells. These findings suggest that NOLC1 is closely associated with the stemness properties of TNBC and represents a potential therapeutic target for TNBC.

The inhibition of circular RNA circNOLC1 by propofol/STAT3 attenuates breast cancer stem cells function via miR-365a-3p/STAT3 signaling

BackgroundBreast cancer remains one of the most dreadful female malignancies globally, in which cancer stem cells (CSCs) play crucial functions. Circular RNAs have drawn great attention in cancer research area and propofol is a widely applied intravenous anesthetic agent. Methods: In the current study, we explored the function of circular RNA nucleolar and coiled-body phosphoprotein 1 (circNOLC1) in CSCs of breast cancer and the inhibitory impact of propofol on circNOLC1.ResultsThe expression of circNOLC1 was induced in breast cancer tissues compared with the non-tumor tissues. The silencing of circNOLC1 was able to repress the viability of breast cancer cells. Meanwhile, the numbers of colony formation were suppressed by circNOLC1 knockdown in breast cancer cells. The inhibition of circNOLC1 reduced the invasion and migration ability of breast cancer cells. The mRNA and protein levels of E-cadherin were enhanced but Vimentin levels were reduced by the silencing of circNOLC1. The repression of circNOLC1 decreased the side population (SP) ratio in breast cancer cells. Meanwhile, the sphere formation ability of breast cancer cells was attenuated by the silencing of circNOLC1. The levels of ATP-binding cassette (ABC) superfamily G member 2 (ABCG2), c-Myc, B cell-specific Moloney murine leukemia virus integration site 1 (Bmi1), and SRY-box transcription factor 2 (Sox2) were repressed by the depletion of circNOLC1 in the cells. Regarding to the mechanism, circNOLC1 functioned as a competing endogenous RNAs (ceRNAs) for microRNA-365a-3p (miR-365a-3p) and the inhibition of miR-365a-3p rescued circNOLC1 depletion-repressed proliferation and cancer stem cell activity of breast cancer. MiR-365a-3p targeted signal transducer and activator of transcription 3 (STAT3) in breast cancer cells and circNOLC1 enhanced STAT3 expression by sponging miR-365a-3p. The overexpression of STAT3 could reverse miR-365a-3p or circNOLC1 depletion-inhibited proliferation and cancer stem cell properties of breast cancer. Interestingly, the expression of circNOLC1 and STAT3 was repressed by the treatment of propofol. The enrichment of STAT3 on circNOLC1 promoter was inhibited by propofol. The expression of circNOLC1 was suppressed by the silencing of STAT3 in the cells. The inhibition of circNOLC1 expression by propofol was rescued under the co-treatment of STAT3 overexpression. The overexpression of circNOLC1 rescued propofol-attenuated proliferation and cancer stem cell functions in vitro and in vivo.ConclusionsThus, we concluded that circNOLC1 contributes to CSCs properties and progression of breast cancer by targeting miR-365a-3p /STAT3 axis and propofol inhibited circNOLC1 by repressing STAT3 in a feedback mechanism.

A Zic2/Runx2/NOLC1 signaling axis mediates tumor growth and metastasis in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is one of the most common malignancies with rapid growth and high metastasis, but lacks effective therapeutic targets. Here, using public sequencing data analyses, quantitative real-time PCR assay, western blotting, and IHC staining, we characterized that runt-related transcription factor 2 (Runx2) was significantly upregulated in ccRCC tissues than that in normal renal tissues, which was associated with the worse survival of ccRCC patients. Overexpression of Runx2 promoted malignant proliferation and migration of ccRCC cells, and inversely, interfering Runx2 with siRNA attenuates its oncogenic ability. RNA sequencing and functional studies revealed that Runx2 enhanced ccRCC cell growth and metastasis via downregulation of tumor suppressor nucleolar and coiled-body phosphoprotein 1 (NOLC1). Moreover, increased Zic family member 2 (Zic2) was responsible for the upregulation of Runx2 and its oncogenic functions in ccRCC. Kaplan–Meier survival analyses indicated that ccRCC patients with high Zic2/Runx2 and low NOLC1 had the worst outcome. Therefore, our study demonstrates that Zic2/Runx2/NOLC1 signaling axis promotes ccRCC progression, providing a set of potential targets and prognostic indicators for patients with ccRCC.

MiRNA 146b mediates the regulation of nucleolar size and activity in polyploid megakaryocytes.

Megakaryocytes (MKs) follow a unique cell cycle duplication process, called endomitosis, resulting in polyploidisation of cells. It is hypothesised that polyploidy, as well as an increment in cytoplasm volume, allow more efficient platelets generation from MKs. Although polyploidy leads to an increase in the DNA amount, which impacts gene expression, little is known about ribosomal biogenesis in these polylobulated polyploid cells. The nucleolus acts as a hub for ribosomal biogenesis, which in turn governs the protein synthesis rate of the cells. We therefore estimated the size and activity of the nucleolus in polyploid cells during megakaryopoiesis in vitro. Polyploid megakaryocytic cell lines and in vitro cultured MKs, which were obtained from human cord blood-derived CD 34+ cells, revealed that miRNA 146b regulated the activity of nucleolar and coiled-body phosphoprotein 1, which plays an integral role in determining nucleolar size and activity. Additionally, miRNA-146b was up-regulated during endomitosis and was found to promote megakaryopoiesis. We propose that miRNA 146b regulates not only nucleolar size and activity, but also megakaryopoiesis. This study highlights the importance of nucleolar activity and miRNA in the progression of megakaryopoiesis and thrombopoiesis.

Identification of Novel Prognosis and Prediction Markers in Advanced Prostate Cancer Tissues Based on Quantitative Proteomics.

Prostate cancer (PCa) is the most frequent cancer found in males worldwide, and its mortality rate is increasing every year. However, there are no known molecular markers for advanced or aggressive PCa, and there is an urgent clinical need for biomarkers that can be used for prognosis and prediction of PCa. Mass spectrometry-based proteomics was used to identify new biomarkers in tissues obtained from patients with PCa who were diagnosed with T2, T3, or metastatic PCa in regional lymph nodes. Among 1,904 proteins identified in the prostate tissues, 344 differentially expressed proteins were defined, of which 124 were up-regulated and 216 were down-regulated. Subsequently, based on the results of partial least squares discriminant analysis and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, we proposed that spermidine synthase (SRM), nucleolar and coiled-body phosphoprotein 1 (NOLC1), and prostacyclin synthase (PTGIS) represent new protein biomarkers for diagnosis of advanced PCa. These proteomics results were verified by immunoblot assays in metastatic PCa cell lines and by indirect enzyme-linked immunosorbent assay in prostate specimens. SRM was significantly increased depending on the cancer stage, confirming the possibility of using SRM as a biomarker for prognosis and prediction of advanced PCa.

Abstract 2606: Genetic contribution of MYC to the development of primary prostate cancer

Abstract Despite the predictive value of Gleason score (Gs), the molecular characteristics and associated biology that underpin specific Gleason patterns (Gp) remain understudied. Based on previous data showing increased MYC activity in higher-grade Gp4 versus lower-grade Gp3 tumor foci and addressing the paradox that prostate cancer is not hyper-proliferative, we sought to examine the genetic contribution of MYC to locally-advanced prostate cancer development. We screened a series of Gs7 cancers in which the Gp3 and Gp4 components were adjacent and represented contributing elements of the index lesion. Tumor foci were collected by laser capture microdissection based on MYC staining and purified for RNA; transcriptome profiling was performed. To further guide our bioinformatics analyses, we generated a LNCaP prostate cancer cell line harboring MYC knockdown. RNA-seq and ChIP-seq was performed to identify differentially bound regions of the cistrome that correlate with altered gene expression and elucidate potential mechanism(s) of action for MYC in primary prostate cancer. In contrast to other reports, MYC knockdown had the effect of increasing global transcription. Moreover, the publicly available Cancer Genome Atlas (TCGA) prostate cancer dataset was curated according to MYC status based on extreme case design using reverse phase protein assay data. Preliminary results show: (1) increased MYC protein levels, but not proliferation, in Gp4 tumor foci as compared to adjacent Gp3, (2) distinct histology and biology based on MYC tumor status, and (3) found recurrent enrichment for gene sets cross-referencing differentially expressed genes between TCGA, tissue, and our engineered cell line. Of note, we observed recurrent enrichment for the Acosta Proliferation Independent MYC Targets Up gene set, which correlated with MYC activity. When this gene set was cross-referenced between all three datasets, the gene NOLC1 was identified. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) plays an important role in rRNA synthesis and ribosome biosynthesis; its role in tumorigenesis appears context dependent. NOLC1 was found to be positively correlated with MYC expression and may thus contribute to MYC-driven prostate cancer. Although further cases are needed to determine the frequency at which NOLC1 drives MYC-driven prostate cancer, our results suggest a novel role for MYC in mediating development of locally-advanced prostate cancer. Citation Format: Nichelle C. Whitlock, Shana Trostel, Rayann Atway, Elizabeth Walton, Brian J. Capaldo, Huihui Ye, Adam G. Sowalsky. Genetic contribution of MYC to the development of primary prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2606.

Nucleolar TRF2 attenuated nucleolus stress-induced HCC cell-cycle arrest by altering rRNA synthesis

The nucleolus is an important organelle that is responsible for the biogenesis of ribosome RNA (rRNA) and ribosomal subunits assembly. It is also deemed to be the center of metabolic control, considering the critical role of ribosomes in protein translation. Perturbations of rRNA synthesis are closely related to cell proliferation and tumor progression. Telomeric repeat-binding factor 2 (TRF2) is a member of shelterin complex that is responsible for telomere DNA protection. Interestingly, it was recently reported to localize in the nucleolus of human cells in a cell-cycle-dependent manner, while the underlying mechanism and its role on the nucleolus remained unclear. In this study, we found that nucleolar and coiled-body phosphoprotein 1 (NOLC1), a nucleolar protein that is responsible for the nucleolus construction and rRNA synthesis, interacted with TRF2 and mediated the shuttle of TRF2 between the nucleolus and nucleus. Abating the expression of NOLC1 decreased the nucleolar-resident TRF2. Besides, the nucleolar TRF2 could bind rDNA and promoted rRNA transcription. Furthermore, in hepatocellular carcinoma (HCC) cell lines HepG2 and SMMC7721, TRF2 overexpression participated in the nucleolus stress-induced rRNA inhibition and cell-cycle arrest.

Nucleolar and coiled-body phosphoprotein 1 (NOLC1) regulates the nucleolar retention of TRF2

Telomeric repeat-binding factor 2 (TRF2) was reported to localize in the nucleolus of human cells in a cell cycle-dependent manner; however, the underlying mechanism remains unclear. Here, we found that nucleolar and coiled-body phosphoprotein 1 (NOLC1) interacted with TRF2 and mediated the shuttling of TRF2 between the nucleolus and nucleus in human 293T and HepG2 cells. Ablation of NOLC1 expression increased the number of nuclear TRF2 foci and decreased the nucleolar level of TRF2. Conversely, NOLC1 overexpression promoted the nucleolar accumulation of TRF2. NOLC1 overexpression also increased the number of 53BP1 foci and induced the DNA damage response. In addition, co-expression of TRF2 rescued NOLC1 overexpression-induced cell cycle arrest and apoptosis.

Identification of NS1 domains of avian H5N1 influenza virus which influence the interaction with the NOLC1 protein

Non-structural protein 1 (NS1) is an important virulence factor encoded by influenza A virus. NS1 can interact with a variety of host cell proteins to interfere with the host innate immune response and to promote effective viral replication. Our previous work has shown that only the effector domain of NS1 (amino acid residues 74-230/237) is sufficient to interact with nucleolar and coiled-body phosphoprotein 1 (NOLC1). To investigate the exact region of NS1 that interacts with NOLC1, we used only the effector domain of NS1 and constructed various mutants having different deletions, and then tested their ability to interact with NOLC1 via pull-down assay. Only the mutant containing amino acid residues 104–200 showed positive interaction with NOLC1. To further determine the key amino acids of the NS1 effector domain which are crucial for interaction with NOLC1, several mutants containing a single amino acid substitution were made and their interaction with NOLC1 was tested. Only the mutant D120A or R195A showed reduced binding with NOLC1, suggesting that D120 and R195 were crucial to the binding of NS1 to NOLC1. This study lays the foundation for further research aiming at furthering our understanding of the interaction between NS1 and host cells.

Identification of NS1 domains of avian H5N1 influenza virus which influence the interaction with the NOLC1 protein.

Non-structural protein 1 (NS1) is an important virulence factor encoded by influenza A virus. NS1 can interact with a variety of host cell proteins to interfere with the host innate immune response and to promote effective viral replication. Our previous work has shown that only the effector domain of NS1 (amino acid residues 74-230/237) is sufficient to interact with nucleolar and coiled-body phosphoprotein 1 (NOLC1). To investigate the exact region of NS1 that interacts with NOLC1, we used only the effector domain of NS1 and constructed various mutants having different deletions, and then tested their ability to interact with NOLC1 via pull-down assay. Only the mutant containing amino acid residues 104-200 showed positive interaction with NOLC1. To further determine the key amino acids of the NS1 effector domain which are crucial for interaction with NOLC1, several mutants containing a single amino acid substitution were made and their interaction with NOLC1 was tested. Only the mutant D120A or R195A showed reduced binding with NOLC1, suggesting that D120 and R195 were crucial to the binding of NS1 to NOLC1. This study lays the foundation for further research aiming at furthering our understanding of the interaction between NS1 and host cells.

Methylation of nucleolar and coiled-body phosphoprotein 1 is associated with the mechanism of tumorigenesis in hepatocellular carcinoma

Nucleolar and coiled-body phosphoprotein1 (NOLC1) plays an essential role in the synthesis of rRNA and the biosynthesis of ribosomes. Previous studies suggest that NOLC1 is crucial for normal cell growth, and plays a role in the regulation of tumorigenesis of nasopharyngeal carcinoma (NPC) and demonstrate that both NOLC1 and tumor protein53 work synergistically to activate the MDM2 promoter in NPC cells. Yet, the functioning of NOLC1 in liver cancer remains unknown. The aim of the present study was to understand how the NOLC1 gene is regulated in liver carcinogenesis. In this study, we showed that NOLC1 was silenced or downregulated in liver tumor tissues when compared with that in the matched non-cancer tissues. In addition, human hepatoma cells weakly expressed NOLC1, whereas cultured human normal liver cell lines expressed abundant levels. The hypermethylation status in the promoter CpG1 start region appeared to be correlated with the NOLC1 expression levels in liver cell lines or liver normal and tissue specimens. We found that four CpG dinucleotides were located at the CpG1 start region. Further molecular analysis of mutagenesis indicated that the four CpG dinucleotides play a role in the promoter activity of the NOLC1 gene. The expression of NOLC1 and DNA methylation of its promoter affected cell proliferation and apoptosis. The expression of NOLC1 in hepatoma cell lines was restored following exposure to the demethylation agent, 5-azacytidine. Low expression of NOLC1 in hepatoma cell lines and liver cancer tissues was associated with cyclinD3. In conclusion, our study demonstrated that DNA methylation is a key mechanism of silenced NOLC1 expression in human hepatocellular carcinoma cells, and NOLC1 gene hypermethylation of the four CpG dinucleotides is a potential biomarker for hepatocellular carcinoma.