Role Of Nucleolin Research Articles

Nucleolin acute degradation reveals novel functions in cell cycle progression and division in TNBC.

Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.

Exploring structural determinants and the role of nucleolin in formation of the long-range interactions between untranslated regions of p53 mRNA.

p53 protein is a key regulator of cellular homeostasis by coordinating the framework of antiproliferative pathways as a response to various stress factors. Although the main mechanism of stress-dependent induction of p53 protein relies on post-translational modifications influencing its stability and activity, a growing amount of evidence suggests that complex regulation of p53 expression occurs also at the mRNA level. This study explores structural determinants of long-range RNA-RNA interactions in p53 mRNA, crucial for stress-dependent regulation of p53 protein translation. We demonstrate that the 8-nt bulge motif plays a key structural role in base-pairing of complementary sequences from the 5' and 3' untranslated regions of p53 mRNA. We also show that one of the p53 translation regulators, nucleolin, displays an RNA chaperone activity and facilitates the association of sequences involved in the formation of long-range interactions in p53 mRNA. Nucleolin promotes base-pairing of complementary sequences through the bulge motif, because mutations of this region reduce or inhibit pairing while compensatory mutations restore this interaction. Mutational analysis of nucleolin reveals that all four RNA recognition motifs are indispensable for optimal RNA chaperone activity of nucleolin. These observations help to decipher the unique mechanism of p53 protein translation regulation pointing to bulge motif and nucleolin as the critical factors during intramolecular RNA-RNA recognition in p53 mRNA.

Role of Nucleolin in Endometrial Precancerous Hyperplasia and Carcinogenesis: Ex Vivo and In Silico Study.

Endometrial cancer (EC) is the most common gynaecological malignancy. Nucleolin (NCL) is involved in rDNA transcription, cell proliferation, and apoptosis, with high expression associated with worse overall survival (OS) in other adenocarcinomas. Our aims were to assess NCL gene and protein expression and explore the differential expression of NCL-associated genes (NAGs) in endometrial carcinogenesis. Endometrial samples were obtained from 157 women to include healthy, hyperplastic (EH), EC, and metastatic groups. RT-qPCR and immunohistochemistry were employed to assess NCL gene and protein levels. In silico analysis of NAGs in TCGA and GEO datasets was performed, with the prognostic value determined via Human Protein Atlas. NCL mRNA level of EC was lower than in healthy post-menopausal endometrium (p < 0.01). EH samples had lower NCL immuno-expression scores than healthy pre-menopausal (p < 0.001), benign post-menopausal (p < 0.01), and EC (p < 0.0001) samples. Metastatic lesions demonstrated higher NCL quick scores than primary tissue (p = 0.04). Higher NCL Immuno quick scores carried a worse OS in high-grade EC (p = 0.01). Interrogating Uterine Corpus Endometrial Carcinoma (TCGA-UCEC) and Uterine Carcinosarcoma (TCGA-UCS) cohorts revealed NCL to be the most highly upregulated gene in carcinosarcoma, with S100A11, LMNB2, RERG, E2F1 and CCNA2 representing key dysregulated NAGs in EC. Since NCL is implicated in transforming hyperplastic glands into cancer, with further involvement in metastasis, it is suggested to be a promising target for better-informed diagnosis, risk stratification, and management of EC.

A novel role for nucleolin in splice site selection

ABSTRACT Latent 5ʹ splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of initiator-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs.

Nucleolin As a Survival Predictor in Acute Lymphoblastic Leukemia and Its Promotion to Drug Resistance By ERK Pathway

Nucleolin (NCL) is an important nucleocytoplasmic multifunctional protein.It plays multiple functions in cell proliferation, apoptosis and angiogenesis. NCL displays oncogenic roles in a large variety of cancer cells, which was reported as high expression in most cancers. In these cancer cells, NCL overexpression influences cell survival, proliferation and invasion through its action on different cellular pathways. However, less research on the roles of NCL in hematologic malignancies was noted. Our previous studies showed that overexpression of NCL is associated with worse prognosis in the patients with acute leukemia and NCL expressed higher in resistant HL-60/ADR than in sensitive HL-60 cells. The potential mechanisms of NCL in chemo-resistance have yet to be investigated. Here we presented the association of NCL with clinical prognosis of acute lymphoblastic leukemia (ALL) and the role of NCL in drug resistance in T-ALL lines. Clinically, the results showed that overexpressed NCL at both mRNA and protein level was associated with a poorer overall survival (OS) and relapse free survival (RFS), as an independent prognostic marker in ALL. mRNA level of NCL in de novo ALL was quantitatively higher than in complete remission(CR) status, and refractory/relapse ALL had the highest level, which relative levels using 2-∆∆Ct method were 1.386, 0.682, and 2.159 respectively (Median(QR), P <0.05) . Expression of NCL protein was observed in 58.3% (35/60) de novo samples, 8.0% (7/87) CR samples and 27.7% (13/47) refractory/relapse samples (P <0.05). Two-year OS rates (Figure 1 ) were respectively 33.3%±8.6% vs 4.0%±1.0% for patients with NCL- negativity and NCL-positivity (P <0.05). Two-year RFS rates were respectively 16.3%±7.2% vs 3.0%±1.0% for patients with NCL-negativity and NCL-positivity (P <0.05). Upon above clinical data, we farther investigated the mechanism(s) by which NCL regulates cellular drug resistance in T-ALL Jurkat cell line. We established cell models with NCL different expression which was transfectedby lentivirus-mediated NCL overexpression vector or siRNA vector, in order to produce NCL overexpression and NCL knockdown models for comparison drug sensitivity. The IC50 of adriamycin on Jurkat cells with NCL-upregulation and NCL- downregulation were 1.362±0.271μg/ml and 0.077±0.010μg/ml respectively (P <0.05). The results of intracellular drug accumulation and effluxion analysis further strengthened the unambiguous promotion impact of NCL on insusceptibility to anticancer agent. Furthermore, we found that Ras/Raf/MEK/ERK signaling and its downstream cell apoptosis factors including c-Myc, Bcl-2 and drug resistance-associated molecules BCRP and MRP1 were all significantly up-regulated after NCL overexpression, conversely, down-regulated after NCL knockdown. Overexpressed NCL up-regulated c-Myc, Bcl-2, BCRP, LRP, MRP1 at mRNA levels, with 2-∆∆Ct values were 7.40±0.40, 1.43±0.11, 2.88±0.07, 2.13±0.02, and 3.35±15, respectively. On the contrary, knockdown of NCL led to reversed results, 2-∆∆Ct values of them as following: 0.24±0.03, 0.32±0.09, 0.03±0.01, 0.69±0.04, 0.55±0.08. Protein levels were in accordance with mRNA level. Notably, co-IP assays confirmed the NCL-Ras, NCL-ERK, NCL-BCRP and NCL-MRP1 interaction. When Ras/EKR signaling was inhibited, levels of BCRP, MRP1 were both decreased, but did not affect to NCL expression. In summary, our findings revealed the novel understanding role of NCL in ALL treatment unresponsiveness and the emerging marker of NCL as a survival predictor and treatment target in ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nucleolin Promotes Cisplatin Resistance in Cervical Cancer by the YB1-MDR1 Pathway.

Purpose Cervical cancer is the fourth most common cancer in women worldwide and is the main cause of cancer-related deaths in women. Cisplatin (DDP) is one of the major chemotherapeutic drugs for cervical cancer patients. But, drug resistance limits the effectiveness of cancer therapy. Nucleolin (NCL) is a nucleocytoplasmic multifunctional protein involved in the development of cancer. It has been reported that NCL may be a potential target for modulation of drug resistance. However, the precise molecular mechanisms are poorly understood. Materials and Methods Human cervical cancer Hela cells and their cisplatin-resistant cell line Hela/DDP were used in this study. The protein level of NCL in cervical cancer cells was measured by western blot analysis. Hela cells and Hela/DDP cells were transfected with NCL overexpression plasmid or NCL siRNA separately. MTT and EdU assay were performed to evaluate the cell viability and sensitivity to cisplatin. The drug efflux function of MDR1 protein was assessed by intracellular rhodamine-123 accumulation assay.The promoter activity of MDR1 was assessed by using a dual-luciferase reporter assay. Results We found that the protein level of NCL was elevated in Hela/DDP cells. Overexpression of NCL increased cervical cancer cell proliferation and attenuated the sensitivity to cisplatin. Overexpression of NCL increased Multidrug resistance (MDR1) gene expression and drug efflux. Our results demonstrated that NCL was highly related with cisplatin resistance in cervical cancer. NCL played an important role in MDR1 gene transcription through regulation of the transcription factor YB1. Conclusion Our findings revealed the novel role of NCL in cisplatin-resistant cervical cancer and NCL may be a potential therapeutic target for chemoresistance.

Corneal neovascularization is inhibited with nucleolin-binding aptamer, AS1411

Corneal neovascularization (CNV) is a common sight-threatening pathology that can be induced by a variety of inflammatory and angiogenic stimuli. Current CNV treatments include anti-inflammatory drugs and antibody-based inhibitors of vascular endothelial growth factor (VEGF). However, these are not always effective and novel therapeutic approaches are needed. Previous work has indicated a role for nucleolin (NCL) in VEGF-mediated neoangiogenesis in a suture-induced CNV model. The major goal for this current study is to test the effect of AS1411, a NCL-binding DNA aptamer that has reached human clinical trials, on neovascularization in a murine model of VEGF-mediated CNV. Our results show that topical administration of AS1411 can significantly inhibit corneal neovascularization in this model. Mechanistic studies indicate that AS1411 reduces the VEGF-stimulated proliferation, migration, and tube formation of primary cells obtained from human limbus stroma (HLSC). AS1411 treatment also significantly reduced VEGF-stimulated induction of miR-21 and miR-221 in HLSC, suggesting a role for these pro-angiogenic miRNAs in mediating the effects of AS1411 in this system. In sum, this new research further supports a role for NCL in the molecular etiology of CNV and identifies AS1411 as a potential anti-angiogenic CNV treatment that works by a novel mechanism of action.

Targeting Nucleolin for Reversal of Chemotherapy Resistance in Acute Lymphoblastic Leukemia

Chemotherapy resistance is considered to be the principal cause of ineffective treatment in acute lymphoblastic leukemia (ALL). Nucleolin (NCL) is high expression andplays oncogenic roles in most cancers. However, less research on the role of NCL in hematologic malignancies was noted. Our previous studies have showed that overexpression of NCL was associated with worse prognosis in the patients with acute leukemia and NCL expressionwashigher in resistant HL-60/ADR than in sensitive HL-60 cells. The potential mechanisms of NCL in chemotherapy resistance have yet to be revealed. Here we presented that expression of NCL was associated positively with chemotherapy resistance and poor prognosis in ALL. Overexpressed NCL at both mRNA and protein level was relevant to a poorer overall survival (OS) and relapse free survival (RFS), indicating NCL as an independent prognostic marker in ALL. mRNA level of NCL in de novo ALL was quantitatively higher than in complete remission(CR) status, and refractory/relapse ALL had the highest level. Upon above clinical data, we further investigated the mechanism(s) by which NCL regulated drug resistance in ALL cells. Remarkably, NCL expression was higher in resistant ALL cells relative to sensitive parental cells. When treated with ADM, NCL level was decreased in sensitive parental cells while unchanged in resistant cells. Overexpressing NCL suppressed drug sensitivity, altered drug effluxion and decreased intracellular drug accumulation, while inhibition of NCL led to a completely reversed appearance, more intracellular Adriamycin(ADM) mean fluorescence intensity (MFI) and percentage of ADM accumulated cells population. Overexpression of NCL increased significantly the IC50 of ADM. The IC50 of ADM on Jurkat-NCL-overexpression(OE), Jurkat-NCL-knockdown(KD), Molt-4-NCL-OE, Molt-4-NCL-KD, Nalm-6-NCL-OE, Nalm-6-NCL-KD were 1.362±0.271μg/ml, 0.077±0.010μg/ml, 4.863±0.733μg/ml, 0.081±0.018μg/ml, 0.237±0.042μg/ml and 0.046±0.002μg/ml, respectively (P &lt;0.05). Involvement of ATP-binding cassette (ABC) transporters was proved in NCL mediated drug resistance. Silencing NCL resulted in a decrease of P-gp, MRP1, LRP and BCRP in ALL cells, and NCL overexpression increased the MRP1, LRP and BCRP. The Akt/mTOR and ERK signaling pathways were involved in this procedure. Notably, co-IP assays confirmed the NCL-Ras, NCL-ERK and NCL-BCRP interaction. For intervention study, aptamer AS1411, a NCL inhibitor, could reduce drug resistance in ALL cell lines and primary ALL cells.Moreover, AS1411 treatment decreased BCRP protein expression. Furthermore, the ALL leukemia models that nude mice engrafted with Nalm-6 cells and NCG mice engrafted with Luc+ Nalm-6 cells were established, then treated with ADM plus AS1411 or control CRO for comparison drug sensitivity and survival. Growth of subcutaneous xenograft tumors was inhibited in those treated with AS1411 or ADM, compared to their respective controls treated with CRO or PBS. The stronger inhibition effect was observed in those treated with AS1411 combined with ADM. For Luc+Nalm-6 derived ALL model, leukemia progression was suppressed in mice treated with AS1411 and AS1411 combined with ADM. AS1411and ADM, especially combination of AS1411 and ADM, could improve survival of the leukemic mice compared to those treated with PBS. The results showed that NCL targeted by AS1411 sensitized ADM treatment and prolonged survival in vivo. In summary, our findings revealed NCL as a survival predictor and the novel role of NCL in ALL chemo-resistance. NCL may be a potential target for improving outcome in ALL. Disclosures No relevant conflicts of interest to declare.

PB2104 INHIBITION OF NUCLEOLIN REVERSED BORTEZOMIB RESISTANCE VIA THE REGULATION OF NF‐κB PATHWAY IN MULTIPLE MYELOMA CELLS

Background:The proteasome inhibitor bortezomib has remarkable efficacy against multiple myeloma (MM). However, intrinsic and acquired resistance to bortezomib could limit its efficacy. The potential resistance mechanisms have yet to be clarified. The nucleo‐cytoplasmic protein nucleolin (NCL) is high expression in several tumors, especially in resistance cells, that may act in the drug resistance. Previously we first observed the higher expression of NCL in KM3 bortezomib resistant cells (KBTZ) than its parental KM3 MM cells, indicating the role of NCL in bortezomib resistance and needing further research to elucidate.Aims:To investigate the potential mechanism(s) by which NCL regulates bortezomib resistance in MM cell line.MethodsKM3 MM cells and its bortezomib resistant cells KBTZ were transfected with lentiviruses carrying the shRNA sequences that target the endogenous NCL gene and control scrambled gene respectively. Post‐transfected cells were named as followed: KM3‐NCL‐Knockdown(KM3‐NCL‐KD), KM3‐NCL‐Scramble(KM3‐NCL‐Scr), KBTZ‐NCL‐Knockdown(KBTZ‐NCL‐KD), KBTZ‐NCL‐Scramble(KBTZ‐NCL‐Scr). Effect of NCL on cell viability and cells drug sensitivity were measured. Cell cycle related proteins and drug resistance related molecules expression level were evaluated. Related signaling pathways were also examined by western blot.Results:NCL silencing significantly inhibited cell proliferation and colony formation ability in KM3 and KBTZ cells. Colony sizes and numbers were both decreased in NCL silenced cells. Colony formation rates in KM3‐NCL‐KD, KM3‐NCL‐Scr, KBTZ‐NCL‐KD, KBTZ‐NCL‐Scr were 9.33% ± 0.47%, 27.22% ± 1.85%, 7.16% ± 0.24% and 28.22% ± 3.57%, respectively (P &lt; 0.05). Moreover, NCL silencing arrested cell cycle transition in G0/G1, resulted in an increasing trend in cell apoptosis. CyclinD, cyclinE, CDK2 proteins were downregulated and P21 protein was upregulated after knockdown of NCL. Notably, NCL silencing increased the sensitivity to bortezomib in KM3 cells and prominently reversed the resistance to bortezomib in KBTZ cells. IC50of bortezomib were 17.29 ± 3.35 ng/mlin KM3‐NCL‐Scr cells and 10.13 ± 1.5 ng/mlin KM3‐NCL‐KD cells (P &lt; 0.05), indicating a 1.7‐fold increase of bortezomib sensitivity after NCL knockdown. IC50of bortezomib were 590.78 ± 147.92 ng/mlin KBTZ‐NCL Scr cells and 63.48 ± 13.16 ng/mlin KBTZ‐NCL KD cells (P &lt; 0.05), meaning a 10.8‐fold reversal of bortezomib resistance after NCL knockdown (Figure 1). Besides, compared with Scr cells, both KM3‐ and KBTZ‐NCL‐KD cells displayed lessrhodamine‐123 effluxion, suggesting that NCL silencing suppressed drug efflux activity. Knockdown of NCL declined ATP‐binding cassette transporter proteins MRP1, LRP, BCRP at mRNA levels and protein levels. Defined mRNA expression level of KM3‐NCL‐Scr and KBTZ‐NCL‐Scr cells equal to 1, relative mRNA expression of MRP1, LRP, BCRPwere 0.56 ± 0.26, 0.13 ± 0.03, 0.13 ± 0.09 in KM3‐NCL‐KD cells and 0.69 ± 0.11, 0.35 ± 0.07, 0.53 ± 0.20 in KBTZ‐NCL‐KD cells, respectively. Furthermore,downregulated expression of P‐NF‐κB, P‐IκBα, IKKα and P‐IKKα protein, decreased intranuclear NF‐κB level and upregulated expression of IκBα protein were observed in both KM3‐ and KBTZ–NCL‐KD cells, indicating that NCL may act by regulating activity of NF‐κB signaling pathway.Summary/Conclusion:In conclusion, our resultssuggested that inhibition of NCL reversed bortezomib resistance via the regulation of NF‐κB pathway in multiple myeloma cells, indicatingthe novel molecular understanding of bortezomib resistance and the role of NCL in MM resistance, and the emerging marker of NCL as a potential target.image

Study on the expression and roles of nucleolin in cardiac injury in septic mice

Objective: To explore the expression and roles of nucleolin in cardiac injury in septic mice. Methods: C57BL/6 mice (WT mice) and myocardium-specific expression of nucleolin transgenic mice (TG mice) were randomly divided into sham group (n=10, sham-operated) and sepsis group (n=15, animal model). Cecal ligation and puncture (CLP) was adopted to produce animal models of sepsis. The expression of nucleolin was detected by Western blotting analysis at 0, 12, 24, 48 hours after the operation. The 7-day survival rate, haemodynamic measurement, levels of isoenzyme of creatinekinase-MB (CK-MB) and cardiac troponin I (cTnI) in serum and levels of reactive oxygen species (ROS) and malondlaldehyde (MDA) in myocardium were evaluated 24 hours after the operation. The data were compared between groups with t test. Results: The expression of nucleolin in myocardium up-regulated significantly in WT+CLP group when compared with that in the WT+Sham group(2.57±0.34 vs 1.00±0.15, t=7.468, P<0.01). Compared with those in the WT+Sham group, the survival rate decreased (33.3% vs 100%, χ(2)=13.375, P<0.01), maximal rate of pressure development (+dp/dtmax) declined (t=4.993, P<0.01), but the serum levels of CK-MB and cTnI and the levels of ROS and MDA in myocardium increased in the WT+CLP group(t=5.031, 4.335, 3.365, 2.375, all P<0.05). Compared with that in WT+CLP group, the 7-day survival rate of mice increased in TG+CLP group (60.0% vs 33.3%, χ(2)=8.227, P=0.004), and the cardiac function improved (t=2.337, P=0.019), but the serum levels of CK-MB and cTnI and the levels of ROS and MDA in myocardium in TG+CLP group reduced significantly (t=2.127, 3.347, 2.115, 2.224,P<0.05). Conclusion: The expression of nucleolin is up-regulated in the myocardium of septic mice, and the overexpression of nucleolin can inhibit oxidative stress injury, attenuate the cardiac injury and dysfunction, and reduce mortality in septic mice.

Nucleolin in Neurons of the Human Substantia Nigra

Using immunocytochemistry and confocal laser microscopy, the distribution of nucleolin (protein C23) in neurons of the human substantia nigra was investigated. Fragments of the human midbrain (n = 8) with the verified lack of neurodegeneration were used in the study. The material was fixed in zinc ethanol formaldehyde, a special fixative that provides high preservation of antigenic determinants. New morphological data on the distribution of nucleolin in neurons of the human substantia nigra were obtained. Nucleolin was found in the nucleolus and nucleoplasm of dopaminergic neurons but not in the cytoplasm or cell surface. In the nucleolus and nucleoplasm, protein C23 is distributed irregularly in the form of some accumulations (clumps) with fuzzy borders. Moreover, nucleolin distribution varied in structurally similar neurons containing cytoplasmic neuromelanin. The Marinesco bodies typically not exhibiting nucleolin expression were identified in the nucleus, in addition to nucleoli, by confocal laser microscopy with simultaneous use of transmitted light detector. The findings can contribute to elucidation of the role of nucleolin in the specific functions of normal dopaminergic neurons in the human substantia nigra and assessment of probable involvement of C23 in the development of nucleolar stress and neurodegeneration.

Abstract 497: Higher nucleolar index of nucleolin as an indicator of aberrant cellular DNA damage response (DDR)

Abstract Nucleoli provide a survival advantage for various tumor types, exemplified in prostate neoplasia, leukemia, lung, and breast carcinomas. Unregulated expression and increased nucleolar localization of many stress-responsive factors often correlate with hyper-proliferative status. Therefore, nucleolar changes in cancer cells are the first cytological indicators that can serve as an index for diagnostic and prognostic purposes. In this study, we examine an abundant nucleolar stress factor, nucleolin (NCL) for its sub-nuclear localization under normal and DNA damage conditions. NCL is a multifunctional RNA-binding phosphoprotein that plays critical role/s in many cellular processes including regulation of gene expression during normal cell cycle as well as in the cellular DNA damage response (DDR). NCL-mediated DDR involves changes in its phosphorylation status, sub-nuclear translocation and a multi-level regulation of p53-checkpoint signaling. However, the role/s of p53 status and the signaling pathways triggered during NCL-mediated stress response remain elusive, and forms the focus of this study. Taking advantage of two human ductal breast epithelial tumor cell lines with different genetic backgrounds (wild type, wt-p53 and mutant, mut-p53 L194F), we demonstrate that NCL is predominantly nucleolar in unstressed conditions irrespective of the p53 status. Upon genotoxic stress, however, wt-p53 is required for NCL to mobilize from nucleoli to the nucleoplasmic region. We observed a significant increase in nucleoplasmic index for NCL that is wt-p53 dependent and specific to the type of DNA damage. In contrast, in the presence of mut-p53, NCL failed to translocate even under various DNA damage conditions, resulting in a higher nucleolar index. Interestingly, another nucleolar factor, nucleophosmin (NPM) remained unaltered upon stress conditions even in the presence of wt-p53. This study corroborates the previous research that suggests NCL translocation requires wt-p53 and provides new insights into the role of p53-mutations (e.g. the zinc finger mutation in the central DNA binding region-L194F) in NCL-mediated DDR. Further, we also assessed the role of NCL and p53 (wt vs. mut) in regulating stress signaling (ATM/ATR and p38-kinase) pathways and gene expression in apoptosis. Together, our study elaborates a novel approach that correlates NCL sub-nuclear localization, downstream signaling pathways and gene expression during the cellular DDR. Citation Format: Iqra Nadeem, Amna Aslam, Jingyuan Wang, Anna Kozlova, Danielle Gordon, Rumsha Javed, Ruchama Steinberg, Rachele Dolce Rameau, Xinyin Jiang, Anjana D. Saxena. Higher nucleolar index of nucleolin as an indicator of aberrant cellular DNA damage response (DDR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 497. doi:10.1158/1538-7445.AM2017-497

MDM2 Degrades Deacetylated Nucleolin Through Ubiquitination to Promote Glioma Stem-Like Cell Enrichment for Chemotherapeutic Resistance.

Glioblastoma multiforme (GBM) is the most fatal of all brain cancers, and the standard care protocol for GBM patients is surgical tumor resection followed by radiotherapy and temozolomide (TMZ)-mediated chemotherapy. However, tumor recurrence frequently occurs, and recurrent GBM exhibits more malignancy and less sensitivity in response to chemotherapy. The malignancy and drug resistance primarily reflect the small population of glioma stem-like cells (GSC). Therefore, understanding the mechanism that controls GSC enrichment is important to benefit the prognosis of GBM patients. Nucleolin (NCL), which is responsible for ribosome biogenesis and RNA maturation, is overexpressed in gliomas. However, the role of NCL in GSC development and drug resistance is still unclear. In this study, we demonstrate that NCL attenuated GSC enrichment to enhance the sensitivity of GBM cells in response to TMZ. In GSC enrichment, NCL was significantly reduced at the protein level as a result of decreased protein stability. In particular, the inhibition of HDAC activity by suberoylanilide hydroxamic acid rescued NCL acetylation accompanied by the loss of mouse double minute 2 homolog (MDM2)-mediated ubiquitination. In addition, we found that NCL ubiquitination resulted from the activation of STAT3- and JNK-mediated signaling in GSC. Moreover, NCL inhibited the formation of stem-like spheres by attenuating the expression of Sox2, Oct4, and Bmi1. Furthermore, NCL sensitized the response of GBM cells to TMZ. Based on these findings, NCL expression is a potential indicator to predict chemotherapeutic efficiency in GBM patients.

Roles of nucleolin. Focus on cancer and anti-cancer therapy.

Nucleolin, a multifunctional protein distributed in the nucleolus, participates in many modulations including rDNA transcription, RNA metabolism, and ribosome assembly. Nucleolin is also found in the cytoplasm and on the cell membrane, and surface nucleolin can bind to various ligands to affect many physiological functions. The expression and localization of nucleolin is often abnormal in cancers, as the differential distribution of nucleolin in cancer can influence the carcinogenesis, proliferation, survival, and metastasis of cancer cells, leading to the cancer progression. Thus, nucleolin may be a novel and promising target for anti-cancer treatment. Here, we describe how nucleolin act functions in cancer development and describe nucleolin-dependent anti-cancer therapies.

Plant nucleolar DNA: Green light shed on the role of Nucleolin in genome organization

ABSTRACTThe nucleolus forms as a consequence of ribosome biogenesis, but it is also implicated in other cell functions. The identification of nucleolus-associated chromatin domains (NADs) in animal and plant cells revealed the presence of DNA sequences other than rRNA genes in and around the nucleolus. NADs display repressive chromatin signatures and harbour repetitive DNA, but also tRNA genes and RNA polymerase II-transcribed genes. Furthermore, the identification of NADs revealed a specific function of the nucleolus and the protein Nucleolin 1 (NUC1) in telomere biology. Here, we discuss the significance of these data with regard to nucleolar structure and to the role of the nucleolus and NUC1 in global genome organization and stability.

Tissue and cellular characterisation of nucleolin in a murine model of corneal angiogenesis.

Corneal neovascularisation (CNV), with consequent loss of transparency, is due to an imbalance of proangiogenic factors. Cell-surface nucleolin (NCL) has been associated with neo-angiogenesis. There are studies identifying NCL translocation from nucleus to the cell surface, which is essential for endothelial cell proliferation. To find the possible role of NCL in the generation of corneal neovessels, the aim of this study is to characterise the NCL presence and cell-localisation in non-injured corneas, as well as to describe the changes in NCL cell and tissue localisation in CNV, and to analyse the effect of bevacizumab on NCL cellular and tissular distribution. Suture-induced CNV was performed in mice. The corneal tissues were obtained and the histological and co-immunofluorescence assays were performed using different proteins, such as CD31, cadherin and isolectin B4. To determine the possible role of VEGF in NCL presence and localisation in our CNV model, bevacizumab was concomitantly used. Nucleolin was principally observed in the nucleus of the basal epithelial cells of normal corneas. Interestingly, angiogenesis-induced changes were observed in the localisation of NCL, not only in tissue but also at the cellular level where NCL was extranuclear in epithelial cells, stromal cells and neovessels. In contrast, these changes were reverted when bevacizumab was used. Besides, NCL was able to stain only aberrant corneal neovessels in comparison with retinal vessels. NCL mobilisation outside the nucleus during angiogenesis could have a possible role as a proangiogenic molecule in the corneal tissue.

Hemagglutinin of influenza A virus binds specifically to cell surface nucleolin and plays a role in virus internalization

The hemagglutinin (HA) protein of influenza A virus initiates cell entry by binding to sialic acids on target cells. In the current study, we demonstrated that in addition to sialic acids, influenza A/Puerto Rico/8/34 H1N1 (PR8) virus HA specifically binds to cell surface nucleolin (NCL). The interaction between HA and NCL was initially revealed with virus overlay protein binding assay (VOPBA) and subsequently verified with co-immunoprecipitation. Importantly, inhibiting cell surface NCL with NCL antibody, blocking PR8 viruses with purified NCL protein, or depleting endogenous NCL with siRNA all substantially reduced influenza virus internalization. We further demonstrated that NCL was a conserved cellular factor required for the entry of multiple influenza A viruses, including H1N1, H3N2, H5N1, and H7N9. Overall, our findings identified a novel role of NCL in influenza virus life cycle and established NCL as one of the host cell surface proteins for the entry of influenza A virus.

Nucleolin and nucleophosmin expression in seminomas and non-seminomatous testicular tumors.

Testicular tumors are heterogeneous group of neoplasms divided mainly into two types: seminomas and non-seminomas. Nucleolin (NCL) and nucleophosmin (NPM) are abundant nucleolar proteins involved in many physiologic and pathologic processes including cancer. Their overexpression was found in many tumors but it was not studied in testicular cancer. The study was performed on tissue microarrays of 19 seminomas, 21 embryonal carcinomas and 11 yolk sac tumors. The expression of NCL and NPM was detected with monoclonal antibodies and visualized with EnVision FLEX/HRP technique. Immunohistochemical reactions were measured with Aperio ImageScope Software and analyzed as means of percentages of all immunopositive cells in three groups of reaction intensity, i.e. 3+, 2+, and 1+ as well as of H-score. Seminomas showed higher expression of nucleolin indicated by higher H-score and higher percentage of positive cells than non-seminomas. The differences in subpopulations of NCL-positive cells were also found. Embryonal carcinomas and yolk sac tumors showed lower expression of NCL than seminomas indicated by H-score. The percentage of NCL-positive cells did not differ between embryonal carcinomas and seminomas while there were significant differences in subpopulations of cells. The percentage of NCL-positive cells in yolk sac tumors was lower than in seminomas. The results show different heterogeneity of subpopulations of NCL-positive cells in embryonal carcinomas and yolk sac tumors compared to seminomas. The analysis of nucleolin expression in embryonal carcinomas and yolk sac tumors showed no differences between these two tumor types. No differences in nucleophosmin expression between seminomas and non-seminomas were found. The differences in the expression of nucleolin between two groups of germ cell testicular tumors found in the current study indicate a new aspect of biology of these neoplasms and require further studies on the role of nucleolin in germ cell tumorigenesis.

A nucleolin-DNMT1 regulatory axis in acute myeloid leukemogenesis.

Nucleolin overexpression and DNA hypermethylation have been implicated in cancer pathogenesis, but whether and how these aberrations cooperate in controlling leukemia cell fate remains elusive. Here, we provide the first mechanistic insights into the role of nucleolin in leukemogenesis through creating a DNA hypermethylation profile in leukemia cells. We found that, in leukemia patients, nucleolin levels are significantly elevated and nucleolin overexpression strongly associates with DNMT upregulation and shorter survival. Enforced nucleolin expression augmented leukemia cell proliferation, whereas nucleolin dysfunction by RNA interference and inhibitory molecule AS1411 blocked leukemia cell clonogenic potential in vitro and impaired tumorigenesis in vivo. Mechanistic investigations showed that nucleolin directly activates NFκB signaling, and NFκB activates its downstream effector, DNA methylation machinery. Indeed, nucleolin overexpression increased NFκB phosphorylation and upregulated DNMT1 that is followed by DNA demethylation; by contrast, nucleolin dysfunction dephosphorylated NFκB and abrogated DNMT1 expression, which resulted in decreased global DNA methylation, restored p15INK4B expression and DNA hypomethylation on p15INK4B promoter. Notably, NFκB inactivation diminished, whereas NFκB overexpression enhanced DNMT1 promoter activity and endogenous DNMT1 expression. Collectively, our studies identify nucleolin as an unconventional epigenetic regulator in leukemia cells and demonstrate nucleolin-NFκB-DNMT1 axis as a new molecular pathway underlying AML leukemogenesis.

Overexpression of a partial fragment of the salt-responsive gene OsNUC1 enhances salt adaptation in transgenic Arabidopsis thaliana and rice (Oryza sativa L.) during salt stress

The rice (Oryza sativa L.) nucleolin gene, OsNUC1, transcripts were expressed in rice leaves, flowers, seeds and roots but differentially expressed within and between two pairs of salt-sensitive and salt-resistant rice lines when subjected to salt stress. Salt-resistant lines exhibited higher OsNUC1 transcript expression levels than salt-sensitive lines during 0.5% (w/v) NaCl salt stress for 6d. Two sizes of OsNUC1 full-length cDNA were found in the rice genome database and northern blot analysis confirmed their existence in rice tissues. The longer transcript (OsNUC1-L) putatively encodes for a protein with a serine rich N-terminal, RNA recognition motifs in the central domain and a glycine- and arginine-rich repeat in the C-terminal domain, while the shorter one (OsNUC1-S) putatively encodes for the similar protein without the N-terminus. Without salt stress, OsNUC1-L expressing Arabidopsis thaliana Atnuc1-L1 plants displayed a substantial but incomplete revertant phenotype, whereas OsNUC1-S expression only induced a weak effect. However, under 0.5% (w/v) NaCl salt stress they displayed a higher relative growth rate, longer root length and a lower H2O2 level than the wild type plants, suggesting a higher salt resistance. Moreover, they displayed elevated AtSOS1 and AtP5CS1 transcript levels. We propose that OsNUC1-S plays an important role in salt resistance during salt stress, a new role for nucleolin in plants.