Nucleolin Is a Functional Binding Protein for Salinomycin in

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Nucleolin is a functional binding protein for salinomycin in neuroblastoma stem cells Fengfei Wang, Shuang Zhou, Dan Qi, Shi-Hua Xiang, Eric T Wong, Xuejing Wang, Ekokobe Fonkem, Tze-chen Hsieh, Jianhua Yang, Batool Kirmani, John B. Shabb, Joseph M. Wu, Min Wu, Jason H. Huang, Wei-Hsuan Yu, and Erxi Wu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b12872 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 28, 2019

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Journal of the American Chemical Society

Nucleolin is a functional binding protein for salinomycin in neuroblastoma stem cells Fengfei Wang *#[1,2,3,4], Shuang Zhou #[1,2,5], Dan Qi# [1,2], Shi-Hua Xiang[6], Eric T. Wong[5], Xuejing Wang[7], Ekokobe Fonkem[1,2,3,4,8], Tze-chen Hsieh[9], Jianhua Yang[10], Batool Kirmani[3, 4], John B. Shabb[11], Joseph M. Wu[9], Min Wu[11], Jason H. Huang *[1,2,4], Wei-Hsuan Yu *[12], Erxi Wu *[1,2,4,8,13] 1Department

of Neurosurgery, Baylor Scott & White Health, Temple, TX 78508, USA

2Neuroscience

Institute, Baylor Scott & White Health, Temple, TX 76502, USA

3Department

of Neurology, Baylor Scott & White Health, Temple, TX 78508, USA

4Department

of Surgery, Texas A & M University College of Medicine, Temple, TX 76504, USA

5Cancer

Research Institute, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA 6Nebraska

Center for Virology, School of Veterinary Medicine and Biomedical Sciences, University of NebraskaLincoln, Lincoln, NE 68583, USA 7Department

of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China

8LIVESTRONG 9Department

Cancer Institutes, Dell Medical School, the University of Texas at Austin, Austin, TX 78712, USA

of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA

10Texas

Children's Cancer Center, Department of Pediatrics, Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA 11Department

of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND 58202, USA 12Institute

of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan, China 13Department

of Pharmaceutical Sciences, Texas A & M University College of Pharmacy, College Station, TX 77843,

USA #Equal

first authors

*Correspondence authors. E-mail: [email protected]; [email protected]; [email protected].

[email protected];

Disclosure of Conflicts of Interests The authors declare no competing financial interest. Keywords: Chemical proteomics • monocarboxylic polyether antibiotic • binding target• pediatric cancer • CD34 • cancer stem cell

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ABSTRACT The aim of this study is to illuminate a novel therapeutic approach by identifying a functional binding target of salinomycin, an emerging anti-cancer stem cell (CSC) agent, and to help dissecting the underlying action mechanisms. By utilizing integrated strategies, we identify that nucleolin (NCL) is likely a salinomycin binding target and a critical regulator involved in human neuroblastoma (NB) CSC activity. Salinomycin markedly suppresses NB CD34 expression and reduces CD34+ cell population in an NCL-dependent manner via disruption of the interaction of NCL with CD34 promoter. The elevated levels of NCL expression in NB tumors are associated with poor patient survival. Altogether, these results indicate that NCL is likely a novel functional salinomycin-binding target that exhibits the potential to be a prognostic marker for NB therapy.

INTRODUCTION Salinomycin is a monocarboxylic polyether chemical originally isolated from an actinobacteria species called Streptomyces albus and has been widely used as an anti-coccidial agent. In a screen of approximately 16,000 natural and commercially available chemical compounds, Weinberg and Lander’s groups identified salinomycin as a cancer stem cell (CSC) killer 1. Subsequent studies show that salinomycin is capable of overcoming drug resistance and enhancing the sensitivity of cancer cells to radiation and chemotherapeutic treatments 2-4. The efficacy of salinomycin on cancer patients has also been documented and appears to involve the elimination of CSCs, in parallel with the induction of partial clinical regression, even among patients previously treated with toxic doses of chemotherapeutic agents accompanied by the development of therapy-resistant cancers 5. Although growing evidence shows that salinomycin has efficacy on eliminating CSCs through a plethora of biological activities including: modulation of manifold signaling pathways in cancer cells, such as Wnt and p38 MAPK pathways 2, 67 , alteration of transport of cations as ionophore in the target organisms including protozoa and gram-positive bacteria 2, 8, release of calcium from the endoplasmic reticulum (ER), and induction of ER stress in breast cancer cells 9-12, the precise molecular mechanism of salinomycin’s suppression activity on CSCs, particularly the identity and mechanisms of its intracellular binding target, remains unclear. Neuroblastoma (NB) is the most frequently diagnosed solid extracranial malignant tumor in the pediatric population and accounts for 15% of malignancy-related death in early childhood 13-14. Treatment options remain limited for 60% of patients with high risk NB 15. The failure to respond to chemotherapeutic regimens and distant organ dissemination is correlated with the presence of CSCs that are not effectively eliminated by conventional chemotherapies 16. To find a more effective therapeutic strategy to treat patients with NB, in this study we first define the NB-CSC markers through analysis of the expression levels of reported potential NB-CSC markers in NB tumors. We then apply integrated strategies such as chemical proteomics and chromatin immunoprecipitation (ChIP)-qPCR assays, to evaluate the efficacy of salinomycin in NB as well as unravel the action mechanism of salinomycin by focusing on its binding protein target and the clinical relevance of salinomycin binding protein in NB.

RESULTS Salinomycin suppresses NB cells, NB CSCs, and reduces CD34+ NB cell population Based on the expression levels of CSC markers e.g. CD133, SH-SY5Y and IMR32 are considered as CSC rich cell lines, while SK-N-AS is a typical non-tumorigenic cell line 16-17. We analyzed the effects of salinomycin on these NB cell lines. We observed significantly inhibition of cell proliferation with the IC50 at 1 μM for the tumorigenic SH-SY5Y and IMR32 cells, and ~4 μM for the non-tumorigenic SK-N-AS cells (Figure 1A). We also detected marked suppression of tumorsphere formation in the tumorigenic SH-SY5Y and IMR32 cells (Figures 1B-C) as well as the induction of cell cycle arrest at G2 phase in NB cells in response to salinomycin treatment (Figures S1A-C). In comparison to SH-SY5Y and IMR32 cells, SKN-AS cells with significantly lower or undetectable levels of mRNA expression of CD133 and c-kit, two common CSC


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surface markers for NB 18 (Figures S1D-E), are less sensitive to salinomycin treatments 16-17, indicating that salinomycin preferentially suppresses the activities of CSC-like cells. We further analyzed the changes in CD133+ and c-kit+ cells in response to salinomycin treatments and observed a significant reduction of both CD133+ and c-kit+ NB cell populations (Figures 1D-F). We also examined the effects of salinomycin on the CD34+ NB population and found that the existence of CD34+ cell population in tumorigenic NB cells and salinomycin markedly reduced CD34+ cells in all tested NB cell lines (Figures 1D and G).

Identification of nucleolin (NCL) as salinomycin’s binding target and functional mediator To understand the molecular mechanism of salinomycin on NB cells, we postulate the existence of a specific target protein in the salinomycin responsive cells. A modified “drug affinity responsive target stability” (DARTS) method was applied to identify salinomycin’s binding proteins in NB cells 19-20. This approach is developed according to the observation that when a protein interacts with a molecule such as a drug to form drug-protein complex; this complex may result in their conformational changes. Consequently, it reduces the sensitivity to the enzyme digesting. SH-SY5Y cells treated with 0 μM, 1 μM, and 4 μM of salinomycin, respectively, were used for the DARTS experiment (Figure 2A). The protein bands on the SDS-PAGE gel with increased densities (lane 2 & lane 3) compared to the control (lane 1) were selected for mass spectrometry (MS) analysis 21 and were then identified as NCL. By using modified DARTS approach followed by Western blotting analysis with NCL specific antibody, we confirmed that NCL is a potential binding protein of salinomycin in both SH-SY5Y and IMR32 cell lines (Figure 2B). To verify this result, we then applied anti-salinomycin antibody beads and anti-NCL antibody beads to the total protein lysate of NB cells for the co-immunoprecipitation (Figure 2C). As shown in Figure 2C, salinomycin and NCL reciprocally were co-precipitated, suggesting that NCL is likely a binding protein target of salinomycin. To explore the possible relationship between the efficacy of salinomycin in NB and NCL expression, we analyzed the effects of siRNAs against NCL and the combination of NCL siRNA with salinomycin on NB cell proliferation and tumorsphere formation. Our data show that knockdown of NCL markedly reduces the cell proliferation by 30% (Figure 2D) and tumorsphere formation by 55% (Figure 2E), respectively. Notably, when NCL is knocked down, salinomycin has no additional effects on cell proliferation and tumorsphere formation (Figures 2D-E), indicating that NCL is required for salinomycin’s action in NB cells.

Elevated level of NCL is associated with a poor prognostic outcome for patients with NB We surmised that NCL expression in NBs should have a critical clinical value. We thus analyzed NCL expression and the correlation of NCL level in NB tumors with patients’ outcomes in a large cohort of NB samples previously investigated by DNA microarray or NGS (www.R2.amc.nl) 22-24. NB is known to have a feature of MYCN amplification in most cases 25. We first found that all NB tumors express NCL and an elevated level of NCL was observed in the NB tumors with MYCN amplification (Figures S2A, D). Meantime, we also analyzed NCL expression in NB cells and observed that NCL is detected in all cell lines tested though levels vary (Figure S2B-C). Next, as shown in Figure 3A-B & G-H, we revealed that an elevated level of NCL in NB is correlated with patients’ short survival. In addition, the rate of disease relapse and the International Neuroblastoma Staging System (INSS) tumor stages are also increased with higher level of NCL (Figures S2E-F). These results indicate that NCL level is correlated with NB patients’ outcome and may have the potential to be a prognostic marker of NB CSC as well.

Salinomycin suppresses CD34 expression via NCL To visualize the regulation of NCL and CD34 by salinomycin, we determined the expression levels of NCL and CD34 in NB cells using fluorescence labeled antibodies following exposure to salinomycin treatment. As shown in Figure 4A, CD34



is predominantly expressed on the surface area of SH-SY5Y cells, while NCL is mainly distributed in the nucleus in NB cells. The expression levels of the cell surface CD34 molecule are apparently reduced in response to salinomycin treatments. These results are further confirmed by Western blotting analysis (Figures 4B-F). Overexpression of NCL induced a markedly increase of CD34 protein expression (Figures 4B-C). Upon salinomycin treatment, cells with overexpressed NCL showed a more pronounced response to salinomycin treatment as evident by the reduction in CD34+ cells (Figures 4D and S3A). In addition, knockdown of NCL protein by using siRNAs decreased the expression levels of CD34 (Figures 4E-F), concomitant with reduction in the percentage of CD34+ cells (Figures 4G and S3B); the NB cells lacking NCL exhibited less response to salinomycin treatment on CD34 expression (Figures 4E-G). These results suggest that salinomycin modulates CD34 expression in an NCL dependent manner.

Salinomycin suppresses CD34 expression through disruption of the interaction of NCL with CD34 promoter NCL was previously shown to be recruited to the CD34 gene promoter region to modulate the transcription of CD34 and regulate gene expression in CD34+ hematopoietic cell 26-27. To determine how salinomycin regulates the expression of CD34, we analyzed the mRNA levels of CD34 in NB cells after salinomycin treatments and found that salinomycin significantly decreases the expression of the CD34 gene at mRNA level (Figure 5A). The postulated interaction of NCL with the CD34 promoter is depicted in Figure S4A. Furthermore, by using chromatin immuno-precipitation (ChIP) combined with quantitative PCR analysis (ChIP-qPCR), the CD34 promoter region is detected from the SH-SY5Y lysates of control cells but not the cells treated with salinomycin, indicating that the interaction between NCL and CD34 promoter in the salinomycin treated cells is abolished (Figures 5B). Transient transfected SH-SY5Y cells with NCL-YFP vectors with treatment of 0.5 μM salinomycin also induce the cytoplasmic redistribution of NCL from nucleolus (Figure 5C). The expression and the phosphorylation of NCL are not significantly affected by salinomycin (Figures S4B-D), which further supports our hypothesis that salinomycin mediates its anti-CSC effects by disrupting the interactions of NCL with its biological partners such as CD34 (Figure 5D). Altogether, these results indicate that salinomycin could suppress CD34 expression via disruption of the interaction of NCL with CD34 gene promoter.

DISCUSSION By using NB cells containing CSC-like cells as a model system, we demonstrate that salinomycin effectively inhibits NB growth with an IC50 significantly lower than that found with most currently used chemotherapeutic drugs for NB, e.g., carboplatin 28. Particularly, cells are more sensitive to salinomycin in the tumorsphere formation assay, a method for evaluating CSC activity, than in the MTS assay, indicating that salinomycin preferentially suppresses CSC like cells, which is consistent with a previous report that salinomycin is of highly selective inhibitory effect on breast CSCs 1. Furthermore, we determine that NCL is a binding protein for salinomycin and salinomycin discrupts CD34 expression in NB by salinomycin via its binding target NCL. The elevated levels of NCL gene in NB tumors are found to be associated with poor prognosis. Our findings have broadened the landscape of the mechanism of salinomycin’s functions. In 2009, Gupta et al. showed that salinomycin has CSC-specific inhibitory effect on breast cancer cells, and the potency is more than 100-fold higher than paclitaxel 1. Salinomycin is an ionophore for cations (e.g., K+, Na+) for phospholipid membrane bilayer toward most cell types 8, 29. It is possible that salinomycin, in one aspect, probably depends on its ion binding properties to exert its CSC inhibitory activity. Park et al. showed that there is high expression level of large conductance Ca2+-activated K+ channel related proteins in the subpopulation of CD133+ for SH-SY5Y NB cells 30. Also Mai et al. developed a synthetic derivative of salinomycin (ironomycin), and observed its potent inhibition on breast CSCs via sequestering iron in lysosome 31. Recently, the mechanism of salinomycin’s effect on CSCs has been partially attributed to its inhibitory effects in Wnt signaling through the block for the phosphorylation of lipoprotein receptor related protein 6 (LRP6), a Wnt co-receptor, and


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inducing its degradation 6 or by suppressing LRP6 expression and subsequently inhibits Wnt/β-catenin and mTORC1 signaling in breast and prostate cancer cells 7. Salinomycin is also be reported to be associated with the transport of polar alkali metals via lipophilic membranes 9-10 and have roles on inducing endoplasmic reticulum (ER) Ca2+ release, ER stress, etc. in breast cancer cells 11-12, 32. However, the knowledge of ion channel specific difference in CSCs is still limited; the precise mechanism of salinomycin impairing the viability of both CSCs and apoptosis-resistant cancer cells, and the reduction effect of salinomycin on CSC cell surface marker expression has been largely unclear. In the present study, we reveal that NCL, a primary binding protein target of salinomycin, plays critical roles in salinomycin’s anti-cancer and antiCSC activity. Cell surface NCL was found to exist as part of a highly stable 500 kDa protein complex consisting of two Wnt related proteins, and targeting the surface NCL could change the complex organization and thus lead to inhibitory effects of cancer cells 33. In addition, NCL was found to be a binding partner with urokinase-type plasminogen activator (uPA) which , in many human cancers, is over-expressed in the tumorstromal invasive microenvironment and plays a vital role in cancer stemness 34. Notably, the receptor (i.e, uPA receptor [uPAR]) for this NCL binding partner has been shown to regulate Wnt/β-catenin signaling and mediate induction of CSC-like population in medulloblastoma cells 35. Also, Bihatia et al. showed active β-catenin level was NCL-dependent in hematopoietic stem/progenitor cell (HSPC) 36. These studies suggest that NCL may be involved in salinomycin’s regulation in Wnt signaling. Although, further investigations are needed to delineate the functions of NCL in the regulation of Wnt signaling, unveiling of the link between NCL and salinomycin will prompt us to study their therapeutic potential further and contribute to the understanding of anti-CSC effect of salinomycin from another aspect as well. Here, we attempt to look for the salinomycin’s binding target through a unique aspect by utilizing integrated strategies DARTS experiment and Co-IP cross confirmation. We identify that NCL is likely a salinomycin binding target and a critical regulator involved in human NB CSC activity as well as uncover the promising role of NCL in anti-CSC therapeutics for NB patients. The next question we ask is that if salinomycin’s binding target-NCL is differentially expressed in CSCs cells. Indeed, a couple of previous reports showed that NCL plays a role in stemness maintenance of embryonic stem cells 37 and has its increasing relevance in cancer development 38 as well as cell surface NCL as a target protein in breast CSC 39-40. Moreover, in this very study, we found that NCL binds a CSC biomarker CD34’s promoter region and the elevated levels of NCL gene in NB tumors are associated with poor patient survival. Thus, our data may further explain why salinomycin can selectively inhibit CSCs. NCL is shown to be located on the surface of actively proliferating cancer cells, including CSCs, but not on non-tumor cells 40. NCL is ubiquitously expressed in various eukaryotic cell compartments, including the nucleolus, the nucleoplasm, the cytoplasm, and the cell membrane which implicates facilitating multiple cellular functions. It has been reported that camptothecin challenge can induce a dramatic redistribution of NCL, from the cell nucleolus to the cell nucleoplasm 41. The action of NCL redistribution from cell nucleolus to cell cytoplasm, such as mitochondria, induced by salinomycin prompts a great interest in investigating the molecular mechanism underlying NCL C terminal YFP tag traffic to mitochondria-like compartment facilitating CSC killing in the near future. Thus, salinomycins and anti-NCL could be used as attractive strategies for anti-CSC treatments. NCL is known as a nucleotides binding protein involved in multiple cell events 42. It is also an abundant nucleolar protein which plays an essential role in ribosome biogenesis 43. By using ChIP-qPCR analysis, we showed that salinomycin efficiently reduces CD34+ cells due to inhibition of the CD34 gene transcription by interfering with the binding of NCL with CD34. Though further study on how salinomycin binds to NCL affects CSC cell proliferation and does this binding disrupt only CD34 gene expression or more are still under investigation, the results shown in Figures 2,4-5 and Figures S4 suggest that NCL is likely a binding protein target of salinomycin and their interaction interrupt the binding of NCL and CD34 promoter region. Lu and colleagues recently reported that in contrary to cancer cells, normal human peripheral blood lymphocytes show resistance to salinomycin treatment 6, salinomycin may be used as an effective therapeutic agent for NB patients, especially for high risk NB patients receiving CD34+ autologous stem cells transplantation in order to prevent cancer relapse 44 and improve survival. Tumor staging and MYCN amplification are among the most clinically relevant and highly statistically significant risk factors for NB patients 25. Through analysis of the expression levels of salinomycin’s binding protein in NB patients, we reveal here that the expression of NCL in NB tumors is correlated with disease progression and the INSS tumor stages, e.g., the tumors in the patients at stage 4 or 4S have markedly higher levels of NCL compared with that of patients at stage 1 (vs.



stage 4: P=4.6e-08; vs. stage 4S: P=0.01) or 2 (vs. stage 4: P=1.8e-05; vs. stage 4S: P=0.042) (Figures S2A, F), and with the amplification of the oncogene MYCN at diagnosis. This study further demonstrates that IMR32 cells with MYCN amplification express higher levels of NCL than other tested cell lines (Figure S2B-C), and the cells with higher levels of NCL are more sensitive to salinomycin treatment (Figures 1, S2B-C). These data suggest that NCL could be a potential valuable prognostic marker for patients with NB. In summary, NCL, as an essential functional binding protein of salinomycin, is likely responsible for salinomycin’s anticancer and anti-CSC like cell activities. Furthermore, salinomycin’s binding target NCL is correlated with the clinical outcomes of NB patients. The suppressive effects of salinomycin on NB CSCs occur via suppression of CD34 expression and the disruption of the interaction of NCL with the CD34 promoter. Future work including clinical studies evaluating of NCL blockage and the efficacy of salinomycin in NB patients is warranted.

MATERIALS & METHODS Cell Culture, Transient Transfection, and Chemicals SH-SY5Y, IMR32, and SK-N-AS, were purchased from the American Type Culture Collection (ATCC). Cells were cultured in MEM (Cellgro) fortified with 4 mM L-glutamine, 10% FBS, 1% non-essential amino acids, 1% pyruvic acid sodium salt, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C with 5% CO2. NB cells were grown (1 x106 /well) in 6-well plates one day before transient transfection, and NCL siRNA (Qiagen, # GS4691), GFP plasmids (Addgene, #11153), NCL C-terminal Tag YFP or GFP-NCL plasmids (Addgene, #28176), were transfected into NB cells using LipofectAmine 2000 (Invitrogen) following the manufacturer's protocol. Salinomycin was obtained from Sigma.

Determination of Inhibition of Cell Proliferation NB cells (1 x105 per well) cultured in complete medium were seeded into 96-well plates the day before treatment. Control vehicle (dimethyl sulfoxide (DMSO)) or salinomycin at various concentrations was added to the seeded cells for 2 d and 20 µl MTS solution (Promega) was added to the cells. After 4 h incubation, cell proliferation was determined via recording the optical densities at 490 nm. The results were expressed as the percentages of actively proliferating cells compared to the control cultures.

Tumorsphere Formation Tumorsphere formation assay was conducted using a previous reported method 45. Briefly, the media for tumorsphere was prepared using a half : half mixture of F-12 and DMEM (Invitrogen), with the supply of 40 ng/ml bFGF (R&D Systems), 20 ng/ml EGF (R&D Systems), 2 µg/ml heparin (Sigma), 1% N2 and B27 (Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma), and 1× antibiotic / antimycotic (Mediatech). 200 cells / 200 µl medium were seeded into each well of Ultra-low attachment 96-well plates (Corning). The formed tumorspheres in various conditions were enumerated at day 7 using phase-contrast microscope under the 10x lens.

Analysis of Cell Surface Markers using Flow Cytometry Cells (1 x106 /well) cultured in complete medium were seeded into 6-well plates one day before treatment. DMSO control or 1µM Salinomycin was added to the appropriate wells. 24 h later, cells were stained with anti-CD133 (Biobyt), or antiCD34 (Biolegend), or anti-CD117 (c-kit) (Biolegend), and Anti-Mouse IgG FITC (Sigma). Mouse IgG (Santa Cruz) was used as isotype control. Cells were analyzed on BD AccuriTM C6 (BD Biosciences).

Quantitative Reverse Transcription PCR (RT-qPCR) RT-qPCR was performed using our previous reported method 46. Briefly, RNA was extracted from NB cells using the RNeasy Plus reagent (Qiagen) in accordance with the vendor’s instruction. The quantity and purity of RNA were detected using NanoDrop 1000 spectrophotometer (Thermo Scientific). Total RNAs (1 µg) were used for cDNA synthesis using


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SuperScript first-strand synthesis system (Invitrogen) according to manufacturer‘s protocol . The designed primers used in this work are purchased from Integrated DNA Technology (IDT) and shown in Table S1. 2 µl of the diluted cDNA synthesized from total RNAs was used as template of the quantitative PCR reaction. PCR was performed using SYBR GreenER qPCR SuperMix (Invitrogen) and 7500 Fast & 7500 Real-Time PCR System (Life technology).

Propidium Iodide (PI) Staining and Flow Cytometry Analysis Cells (1 x106 /well) cultured in complete medium were added to 6-well plates and grown overnight. DMSO control or salinomycin was added to the appropriate wells and incubated for 1 d. Cells were then stained with PI (Sigma) following the manufacturer’s protocol. Cell cycle data were acquired on the Cell Lab QuantaTM SC system (Beckman Coulter).

Western Blotting NB cells were lysed using Tris-Triton cell lysis buffer (TTL buffer, 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% Triton X100, 5% glycerol) with the supply of protease inhibitor cocktail (Roche Applied Science) and phosphatase inhibitor cocktail 100x (Cell Signaling) and total proteins were extracted. Samples with the same amount of cell total proteins were boiled in 6x SDS-PAGE sampling buffer, separated using 12% SDS-PAGE, and transferred onto a nitrocellulose membrane. Immunoblots were probed with antibodies specific for NCL (Abcam, # ab22758), purified anti-NCL-phosphorylated (Thr76/Thr84) antibody (Biolegend, #609403), or CD34 (Santa Cruz, # sc-9095). Anti--actin antibody (Sigma) was used for assessing protein loading in each membrane. Intensity of detected proteins were analyzed by the Immun-Star HRP peroxide Luminol/Enhancer (BIO–RAD) and exposed on BioBlue-XR Autoradiography Film (Alkali Scientific).

Binding Protein Identification Binding protein identification was performed by a modified method as previously described 19-20 . Briefly, SH-SY5Y NB cells (2x106) were treated with salinomycin or DMSO and 40 min later, cells were lysed with TTL buffer. After centrifugation (32,869 g) using Thermo Scientific Microfuge 11R with F14 rotor for 10 min, lysates were adjusted to the same protein concentration and final volume with cell lysis buffer. All steps of this experiment were carried out on ice or at 4 °C to prevent premature protein degradation. After quickly warmed to room temperature, 50 μg of lysate was then proteolysed using 100 ng thermolysin for 10 min. To terminate proteolysis reaction, 0.5 M of EDTA (pH 8.0) was applied to each sample at a ratio of 1 : 10, mixed well on ice. All samples were then boiled in 6x SDS-PAGE sampling buffer and separated by 12% Tris-HCl SDS-PAGE followed by EZBlueTM GEL STAINING process (Sigma). Protein bands enriched by salinomycin treatment were then subjected to in-gel trypsin digestion followed by mass spectrometry analysis.

Mass Spectrometry Analysis In-gel trypsin digestion was performed using a method similar to the published procedures 21. Gel bands were excised and destained. Proteins were reduced in-gel with 4 mM dithiothreitol (DTT) in 50 mM ammonium bicarbonate (NH4HCO3) for 15 min at 60°C. A final concentration of 16 mM iodoacetamide was added and alkylation was allowed for 30 min at room temperature in the dark. The reaction was terminated with the addition of 3 mM DTT. The gel slice was then equilibrated with NH4HCO3, dehydrated with 100% acetonitrile (ACN), and rehydrated using 0.02 µg Trypsin Gold (Promega) in 40 mM NH4HCO3 and digested overnight at 37°C. Approximately 50 µl peptides were extracted. The samples were acidified with formic acid to make 0.1% final concentration. Samples were analyzed by rpHPLC and spotted onto a MALDI target plate using a TEMPO-LC integrated nanoflow HPLC/spotter. A total of 8.8 μl sample was injected onto a ProteCol C18 0.3 x 10 mm trap (3 μm 300 Å pore size). The samples were desalted with Buffer A (2% ACN/0.1% formic acid v/v) for 10 min at 10 μl/min. Peptides were eluted in-line through a 0.1 x 100 mm Magic AQ C18 (5 μm) column with a 30 min gradient from 100 % Buffer A to 60% Buffer A, 60% Buffer B (98% ACN/0.1% formic acid v/v) at 1 μl/min. Eluate was mixed postcolumn with one volume of 10 mg/ml α-cyano-4-hydroxycinnamic acid (CHCA) in 75% ACN/0.1% formic acid v/v. The matrix/eluant mix was spotted at 18 sec/spot. Column was regenerated at 70% ACN/0.1% formic acid. Spot sets were analyzed on an AB4800 MALDI TOF/TOF mass analyzer. The m/z range was 800-4000. Top 10 precursors per spot were selected for MS/MS with the weakest precursor first.



Immunoprecipitation Assay Immunoprecipitation (IP) was conducted using the Pierce crosslink magnetic IP/Co-IP kit (Thermo Scientific, #88805) following the provided instruction from the manufacturer. Briefly, cells were lysed using Tris-Triton cell lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% Triton X-100, and 5% glycerol) supplemented with protease inhibitor cocktail (Roche Applied Science) and total proteins were extracted. Cell lysates were then treated with 10 μM salinomycin for 1 h at 4°C. 5 μg of antibody was used for each IP reaction and cross-linked to Protein A/G magnetic beads via disuccinimidyl suberate (DSS). For each IP reaction, 2 mg of protein lysates were reacted with specific antibody cross-linked beads at 4 °C overnight. The resulting beads were then harvested with a magnet stand and washed three times with washing buffer (0.2% Tween20 PBS). The bound proteins were eluted by heating at 95°C in 6x SDS-PAGE sampling buffer and subjected to immunoblotting analysis. Immunoblots were then probed with antibodies specific for NCL (Abcam, # ab22758). Antisalinomycin antibody was purchased from Abcam (# ab123955), which was produced through the immunogen of bovine thyroglobulin conjugated to the compound. Normal sheep IgG (# sc-2717) and rabbit IgG (# sc-2027) served as controls were purchased from Santa Cruz.

Immunofluorescence SH-SY5Y cells were fixed using 4% formaldehyde for 15 min, then treated with 0.1% Triton X-100 for 15 min and blocked by 5% BSA for 20 min at room temperature. Then the cells were incubated overnight at 4°C with the anti-NCL (Abcam, # ab22758) and anti-CD34 (R&D Systems, # MAB72271) primary antibodies. Cells were then exposed to goat anti-mouse IgG-R-Phycoerythrin (Sigma, # P9287) and goat anti-rabbit IgG-FITC (Santa Cruz, # sc-2012) secondary antibodies for 2 h at room temperature. 0.5 μg/ml Hoechst 33258 dye or DAPI (Invitrogen) was used to stain nuclei for 5 min. The cells were observed and photographed under Leica DMi 8 fluorescence microscope. Chromatin ImmunoPrecipitation (ChIP)-qPCR Assay Cells (5 x106 /well) cultured in complete medium were seeded in 10 cm cell culture dish overnight. Then salinomycin was added to the cells and incubated for 4 h followed by the cross-link using 10 ml 1% formaldehyde for 30 min at room temperature. The reaction was ceased by adding 1 ml 1.37 M glycine and mixed immediately. The plates were then transferred on ice and washed thrice with Buffer A (PBS supplemented with protease inhibitor cocktail). Cells were then trypsinized and harvested by centrifuging at 1,000 rpm for 5 min at 4°C. The cell pellet was resuspended using 500 µl Buffer B (0.625 mM Hepes, pH 7.8, 0.03M KCl, 1.5 mM MgCl2, 0.01% Igepal CA-630, 1 mM DTT) and left on ice for 10 min. The cells were dounced 10-15 times to release the nuclei. The released nuclei were then harvested by centrifuging at 2,000 rpm for 5 min. The cells were resuspended in 500 µl Buffer C (4 mM NaCl, 1 mM EDTA, 50 mM Hepes pH 7.9, 0.1% 7deoxycholic acid sodium salt, 1% Triton X-100, 0.1% SDS) supplemented with protease inhibitor cocktail. Sonication was performed (using Branson Digital sonifier ®) at 30% amplitude for 10 sec each three times. The resultant lysates were then centrifuged at 14,000 rpm for 15 min at 4°C. The supernatant was transferred to a fresh tube and pre-cleaned with 50 µl of the Protein-A sepharoseTM CL–4B (GE Healthcare) slurry (80 mg in Buffer C per 500 µl lysate) with constant rotation for 1 h in cold room. After centrifuged again at 2,000 rpm for 5 min and the supernatant containing the pre-cleaned chromatin was collected. 50 µl aliquot of each sample was saved to serve as the Input DNA. 5 µl primary antibody (Anti-NCL (Abcam) or control Rabbit IgG (Santa Cruz) were added to the rest of the sample and incubated in a cold room with constant rotation for overnight. Next day, each sample was added with 50 µl Protein-A sepharose slurry and incubated for 2 h with constant rotation in the cold room. Then, after centrifuging at 10,000 rpm for 3 min, the beads were then washed twice. For each time, the beads were washed with Buffer C, Buffer D (2.5 mM Hepes pH 7.9, 0.5 M NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% 7-deoxycholic acid sodium salt, protease inhibitor cocktail), Buffer E (2.5 mM Hepes pH 7.9, 12.5 mM NaCl, 1 mM EDTA, 0.1% 7-deoxycholic acid sodium salt, 1% Triton X-100, 0.1% SDS) and TE buffer (1 mM EDTA, 10 mM Tris, pH 8.0). 200 µl Elution buffer (0.05 M Tris pH 8.0, 1 mM EDTA, 0.05 M sodium bicarbonate, 0.05% SDS) was then added to the beads and incubated at 65°C for 10 min. Centrifugation was performed at 14000 rpm for 1 min and the supernatant was collected. Beads were eluted again to obtain a total 400 µl. In parallel, the saved input DNA was thawed


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and 350 µl elution buffer was added to adjust the total volume up to 400 µl. Each tube was added with 16.5 µl 5 M NaCl and incubated at 65°C overnight for reverse cross-linking. 2 µl RNase (Sigma) was added the next day and incubated for 1 h at 37°C. 4 µl 0.5 M EDTA and 2 µl Proteinase K (Qiagen) was added and samples were incubated for 2 h at 42°C. DNA were then extracted with chloroform/isoamyalcoholonce after centrifugation at 14,000 rpm for 10 min. The aqueous phase was collected to which 40 µl 3 M sodium acetate and 1 ml ethanol was added and was incubated overnight at -20°C for precipitation. The samples were then centrifuged at 14,000 rpm for 0.5 h the next day and the pellets were washed once with 80% ethanol. The immunoprecipitate (IP) and the Input sample pellets were resuspended using 50 µl Tris, pH 8.5. The chromatin precipitates were then obtained for qPCR. Primers used for analysis are shown in Table S1.

NB Tumor Datasets and Patients Outcome Analyses Three datasets of NB samples previously analyzed by microarray and next-generation sequencing were used for NCL expression in NB patients, including the studies containing 498 samples 24, 694 samples 23, and 88 samples 22, respectively. Through the R2 software (http://r2.amc.nl), we analyzed NCL gene expression levels in NB patients and determined the correlation of NCL levels in NBs with their clinical outcomes. The expression levels of NCL were also analyzed in different subgroups based on MYCN amplification, risk group and International Neuroblastoma Staging System (INSS) stage. The statistical analysis of NCL gene in different groups of NBs was conducted using one-way ANOVA. The correlations between the expression levels of NCL with patients’ survival were analyzed using chi-squared test and Kaplan-Meier curves. Survival outcome was determined from the date of initial diagnosis until the date of death or last follow up owing to progressive nature of the disease. The survival distribution was assessed using Kaplan-Meier curves, and optimal cut-off selection (chisquared test p-value).

Statistical Analysis Statistical analyses were performed using Minitab 16.1.1. All of the quantitative data are represented as mean ± SD (standard deviation) in the Figures. Comparison analyses between two groups were performed using paired student’s t test. P-value < 0.05 was regarded to be statistically significant.

ASSOCIATED CONTENT Supporting Information Supplementary Figure S1-4 and their legends. Supplemental table S1: primers and conditions for RT-qPCR and ChIP-qPCR.

AUTHOR INFORMATION Corresponding Authors *[email protected]; *[email protected]; *[email protected] *[email protected] Author Contributions All authors contributed to the preparation of this manuscript and have given approval to the final version of the manuscript. #These authors are equal first authors. *These are corresponding authors.

Funding Sources



This study was, in part, supported by grants provided by Baylor Scott & White Health Start-up fund, Plummer funds, the National Center for Research Resources (NCRR; P20 RR020151) and the National Institute of General Medical Sciences (NIGMS; P20 GM103505, P20GM103442, P20GM113123, P30 GM103332,), National Institute of Allergy and Infectious Diseases (NIAID; R01 AI109317-01A1, R01 AI138203-01) from the National Institutes of Health (NIH). Mass spectrometry was supported by ND INBRE with funding from the National Center for Research Resources (5P20RR01647112) and the National Institute of General Medical Sciences (8 P20 GM103442-12), NIH.

ACKNOWLEDGMENT We sincerely thank Dr. Bruce Beutler at UT Southwestern Medical Center for his valuable comments and inputs for this manuscript.

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FIGURE LEGENDS Figure 1. Salinomycin suppresses NB-CSC. (A) Salinomycin inhibits NB cell proliferation. Cell viability was assessed using MTS assay 48 h after DMSO control or salinomycin treatment (0.25, 0.5, 1, 2, and 4 μM). (B) All tested NB cell lines except non-tumorigenic SK-N-AS form tumorsphere and salinomycin suppresses NB cell tumorsphere formation. Cells treated with DMSO control or 1 μM salinomycin were grown in tumorsphere-media, and the number of tumorspheres was counted at day 7. (C) Salinomycin suppresses NB cell tumorsphere formation. Cells treated with DMSO control or 1 μM salinomycin were grown in tumorsphere-media. Images were captured at day 7. (D) Salinomycin reduces NB-CSC population. Cells were treated with DMSO control or 1 μM salinomycin in this experiment. (E-G) Quantification of the flow cytometry data. (E) Salinomycin inhibits CD133+ NB cell population. (F) Salinomycin inhibits c-kit+ NB cell population. (G) Salinomycin inhibits CD34+ NB cell population. Data represent the mean ± SD of triplicate experiments. The statistical differences were determined using paired student’s t-test and performed using Minitab. *p

Nucleolin Is a Functional Binding Protein for Salinomycin in

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