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• W2858779744 abstract "Cancer stem cells (CSCs) are unique populations of cells that can self-renew and generate different cancer cell lineages. Although CSCs are believed to be a promising target for novel therapies, the specific mechanisms by which these putative therapeutics could intervene are less clear. Nitric oxide (NO) is a biological mediator frequently up-regulated in tumors and has been linked to cancer aggressiveness. Here, we search for targets of NO that could explain its activity. We find that it directly affects the stability and function of octamer-binding transcription factor 4 (Oct4), known to drive the stemness of lung cancer cells. We demonstrated that NO promotes the CSC-regulatory activity of Oct4 through a mechanism that involves complex formation between Oct4 and the scaffolding protein caveolin-1 (Cav-1). In the absence of NO, Oct4 forms a molecular complex with Cav-1, which promotes the ubiquitin-mediated proteasomal degradation of Oct4. NO promotes Akt-dependent phosphorylation of Cav-1 at tyrosine 14, disrupting the Cav-1:Oct4 complex. Site-directed mutagenesis and computational modeling studies revealed that the hydroxyl moiety at tyrosine 14 of Cav-1 is crucial for its interaction with Oct4. Both removal of the hydroxyl via mutation to phenylalanine and phosphorylation lead to an increase in binding free energy (ΔGbind) between Oct4 and Cav-1, destabilizing the complex. Together, these results unveiled a novel mechanism of CSC regulation through NO-mediated stabilization of Oct4, a key stem cell transcription factor, and point to new opportunities to design CSC-related therapeutics. Cancer stem cells (CSCs) are unique populations of cells that can self-renew and generate different cancer cell lineages. Although CSCs are believed to be a promising target for novel therapies, the specific mechanisms by which these putative therapeutics could intervene are less clear. Nitric oxide (NO) is a biological mediator frequently up-regulated in tumors and has been linked to cancer aggressiveness. Here, we search for targets of NO that could explain its activity. We find that it directly affects the stability and function of octamer-binding transcription factor 4 (Oct4), known to drive the stemness of lung cancer cells. We demonstrated that NO promotes the CSC-regulatory activity of Oct4 through a mechanism that involves complex formation between Oct4 and the scaffolding protein caveolin-1 (Cav-1). In the absence of NO, Oct4 forms a molecular complex with Cav-1, which promotes the ubiquitin-mediated proteasomal degradation of Oct4. NO promotes Akt-dependent phosphorylation of Cav-1 at tyrosine 14, disrupting the Cav-1:Oct4 complex. Site-directed mutagenesis and computational modeling studies revealed that the hydroxyl moiety at tyrosine 14 of Cav-1 is crucial for its interaction with Oct4. Both removal of the hydroxyl via mutation to phenylalanine and phosphorylation lead to an increase in binding free energy (ΔGbind) between Oct4 and Cav-1, destabilizing the complex. Together, these results unveiled a novel mechanism of CSC regulation through NO-mediated stabilization of Oct4, a key stem cell transcription factor, and point to new opportunities to design CSC-related therapeutics. Cancer stem cells (CSCs) 2The abbreviations used are: CSCcancer stem cellDPTAdipropylenetriamineNOSnitric-oxide synthasePLAproximity ligation assayMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidePIpropidium iodideNSCLCnon-small cell lung cancerFBSfetal bovine serumHRPhorseradish peroxidaseCBMcaveolin-binding motifNOSNO synthaseTFtranscription factor-seq-sequencingCHXcycloheximideMM/PBSAmolecular modeling/Poisson-Boltzmann surface areaPDBProtein Data BankMDmolecular dynamics. or cancer stem-like cells are specific cell populations that can differentiate and generate cancer cells in various types of cancer, including lung cancer (1Bonnet D. Dick J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat. Med. 1997; 3 (9212098): 730-73710.1038/nm0797-730Crossref PubMed Scopus (5460) Google Scholar, 2Salcido C.D. Larochelle A. Taylor B.J. Dunbar C.E. Varticovski L. Molecular characterisation of side population cells with cancer stem cell-like characteristics in small-cell lung cancer.Br. J. Cancer. 2010; 102 (20424609): 1636-164410.1038/sj.bjc.6605668Crossref PubMed Scopus (122) Google Scholar). These cells have characteristics similar to those of normal stem cells, in particular, an ability to self-renew and generate different cell lineages (3Shackleton M. Normal stem cells and cancer stem cells: similar and different.Semin. Cancer Biol. 2010; 20 (20435143): 85-9210.1016/j.semcancer.2010.04.002Crossref PubMed Scopus (112) Google Scholar). Such ability is believed to facilitate the progression of cancers and their aggressive behaviors (4Lobo N.A. Shimono Y. Qian D. Clarke M.F. The biology of cancer stem cells.Annu. Rev. Cell Dev. Biol. 2007; 23 (17645413): 675-69910.1146/annurev.cellbio.22.010305.104154Crossref PubMed Scopus (870) Google Scholar). Increasing evidence also suggests that CSCs possess several virulent features that make them invasive and resistant to chemo/radiotherapy (5Chang J.C. Cancer stem cells: Role in tumor growth, recurrence, metastasis, and treatment resistance.Medicine. 2016; 95 (27611935): S20-S2510.1097/MD.0000000000004766Crossref PubMed Scopus (234) Google Scholar). cancer stem cell dipropylenetriamine nitric-oxide synthase proximity ligation assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide propidium iodide non-small cell lung cancer fetal bovine serum horseradish peroxidase caveolin-binding motif NO synthase transcription factor -sequencing cycloheximide molecular modeling/Poisson-Boltzmann surface area Protein Data Bank molecular dynamics. The successful establishment of induced pluripotent stem cells that form fully differentiated cells has revealed that the differentiated cells retain the capacity to revert to stem cells, and certain regulatory transcription factors, such as octamer-binding transcription factor 4 (Oct4), Nanog, c-Myc, Klf4, Sox2, and Lin28, are essential for somatic cell reprogramming (6Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131 (18035408): 861-87210.1016/j.cell.2007.11.019Abstract Full Text Full Text PDF PubMed Scopus (14964) Google Scholar, 7Yu J. Hu K. Smuga-Otto K. Tian S. Stewart R. Slukvin I.I. Thomson J.A. Human induced pluripotent stem cells free of vector and transgene sequences.Science. 2009; 324 (19325077): 797-80110.1126/science.1172482Crossref PubMed Scopus (1784) Google Scholar). Likewise, cancer dedifferentiation is evident and has been linked to cancer progression and aggressive behaviors (8Gabbert H. Wagner R. Moll R. Gerharz C.-D. Tumor dedifferentiation: An important step in tumor invasion.Clin. Exp. Metastasis. 1985; 3 (3907917): 257-27910.1007/BF01585081Crossref PubMed Scopus (148) Google Scholar, 9Friedmann-Morvinski D. Verma I.M. Dedifferentiation and reprogramming: origins of cancer stem cells.EMBO Rep. 2014; 15 (24531722): 244-25310.1002/embr.201338254Crossref PubMed Scopus (301) Google Scholar). Similar to somatic cell reprogramming, tumor dedifferentiation is a reversal of cell development to a more immature state. Such discovery has led to an interest in uncovering the origin of CSCs and targeting these cells for therapy. In cancer, Oct4, Sox2, and Nanog have been shown to associate with the stemness of cells by sustaining the level of stem cell–related proteins, such as CD133, ALDH1A1, and ABCG2 (10Liu A. Yu X. Liu S. Pluripotency transcription factors and cancer stem cells: small genes make a big difference.Chin. J. Cancer. 2013; 32 (23419197): 483-487PubMed Google Scholar). Induced expression of the Oct4 gene and transmembrane delivery of the Oct4 protein were shown to induce the dedifferentiation of melanoma cells to CSC-like cells with a strong reduction in melanocytic markers and acquisition of spheroid-forming ability (11Kumar S.M. Liu S. Lu H. Zhang H. Zhang P.J. Gimotty P.A. Guerra M. Guo W. Xu X. Acquired cancer stem cell phenotypes through Oct4-mediated dedifferentiation.Oncogene. 2012; 31 (22286766): 4898-491110.1038/onc.2011.656Crossref PubMed Scopus (242) Google Scholar). In lung cancer, the expression of Oct4 correlates with aggressive behaviors and CSC properties (12Karoubi G. Gugger M. Schmid R. Dutly A. OCT4 expression in human non-small cell lung cancer: implications for therapeutic intervention.Interact. Cardiovasc. Thorac. Surg. 2009; 8 (19126554): 393-39710.1510/icvts.2008.193995Crossref PubMed Scopus (56) Google Scholar, 13Kobayashi I. Takahashi F. Nurwidya F. Nara T. Hashimoto M. Murakami A. Yagishita S. Tajima K. Hidayat M. Shimada N. Suina K. Yoshioka Y. Sasaki S. Moriyama M. Moriyama H. Takahashi K. Oct4 plays a crucial role in the maintenance of gefitinib-resistant lung cancer stem cells.Biochem. Biophys. Res. Commun. 2016; 473 (26996130): 125-13210.1016/j.bbrc.2016.03.064Crossref PubMed Scopus (41) Google Scholar14Chen Y.-C. Hsu H.-S. Chen Y.-W. Tsai T.-H. How C.-K. Wang C.-Y. Hung S.-C. Chang Y.-L. Tsai M.-L. Lee Y.-Y. Ku H.-H. Chiou S.-H. Oct-4 expression maintained cancer stem-like properties in lung cancer-derived CD133-positive cells.PLoS ONE. 2008; 3 (18612434): e263710.1371/journal.pone.0002637Crossref PubMed Scopus (405) Google Scholar). Moreover, in various cancers, the overexpression of Oct4 and its downstream targets is associated with CSCs and the progression of disease (15Liu C.G. Lu Y. Wang B.B. Zhang Y.J. Zhang R.S. Lu Y. Chen B. Xu H. Jin F. Lu P. Clinical implications of stem cell gene Oct-4 expression in breast cancer.Ann. Surg. 2011; 253 (21394007): 1165-117110.1097/SLA.0b013e318214c54eCrossref PubMed Scopus (68) Google Scholar, 16Hatefi N. Nouraee N. Parvin M. Ziaee S.-A. Mowla S.J. Evaluating the expression of Oct4 as a prognostic tumor marker in bladder cancer.Iran. J. Basic Med. Sci. 2012; 15 (23653844): 1154-1161PubMed Google Scholar17Kosaka T. Mikami S. Yoshimine S. Miyazaki Y. Daimon T. Kikuchi E. Miyajima A. Oya M. The prognostic significance of OCT4 expression in patients with prostate cancer.Hum. Pathol. 2016; 51 (27067776): 1-810.1016/j.humpath.2015.12.008Crossref PubMed Scopus (19) Google Scholar), although the underlying mechanisms remain obscure. In recent years, the role of the microenvironment in cancer progression has increasingly been recognized (18Quail D.F. Joyce J.A. Microenvironmental regulation of tumor progression and metastasis.Nat. Med. 2013; 19 (24202395): 1423-143710.1038/nm.3394Crossref PubMed Scopus (4440) Google Scholar). Nitric oxide (NO) is a key mediator frequently up-regulated in lung cancer microenvironment (19Liu P.F. Zhao D.H. Qi Y. Wang J.G. Zhao M. Xiao K. Xie L.X. The clinical value of exhaled nitric oxide in patients with lung cancer.Clin. Respir. J. 2018; 12 (DOI not found, 26934059): 23-30Crossref PubMed Scopus (16) Google Scholar). It is also known to play a pivotal role in controlling several cellular processes in both normal and pathological conditions (20Hirst D.G. Robson T. Nitric oxide physiology and pathology.Methods Mol. Biol. 2011; 704 (21161625): 1-1310.1007/978-1-61737-964-2_1Crossref PubMed Scopus (76) Google Scholar). In lung cancer, NO and its producing enzymes, nitric-oxide synthases (NOSs), are highly up-regulated (19Liu P.F. Zhao D.H. Qi Y. Wang J.G. Zhao M. Xiao K. Xie L.X. The clinical value of exhaled nitric oxide in patients with lung cancer.Clin. Respir. J. 2018; 12 (DOI not found, 26934059): 23-30Crossref PubMed Scopus (16) Google Scholar, 21Masri F. Role of nitric oxide and its metabolites as potential markers in lung cancer.Ann. Thorac. Med. 2010; 5 (20835304): 123-12710.4103/1817-1737.65036Crossref PubMed Scopus (37) Google Scholar). NO has also been shown to promote anoikis or cell survival after detachment (22Powan P. Chanvorachote P. Nitric oxide mediates cell aggregation and mesenchymal to epithelial transition in anoikis-resistant lung cancer cells.Mol. Cell. Biochem. 2014; 393 (24771070): 237-24510.1007/s11010-014-2066-7Crossref PubMed Scopus (14) Google Scholar), motility (23Saisongkorh V. Maiuthed A. Chanvorachote P. Nitric oxide increases the migratory activity of non-small cell lung cancer cells via AKT-mediated integrin αv and β1 upregulation.Cell Oncol. 2016; 39: 449-46210.1007/s13402-016-0287-3Crossref Scopus (16) Google Scholar), CSC-like phenotypes (24Yongsanguanchai N. Pongrakhananon V. Mutirangura A. Rojanasakul Y. Chanvorachote P. Nitric oxide induces cancer stem cell-like phenotypes in human lung cancer cells.Am. J. Physiol. Cell Physiol. 2015; 308 (25411331): C89-C10010.1152/ajpcell.00187.2014Crossref PubMed Scopus (48) Google Scholar), and chemotherapeutic resistance (25Chanvorachote P. Nimmannit U. Stehlik C. Wang L. Jiang B.H. Ongpipatanakul B. Rojanasakul Y. Nitric oxide regulates cell sensitivity to cisplatin-induced apoptosis through S-nitrosylation and inhibition of Bcl-2 ubiquitination.Cancer Res. 2006; 66 (16778213): 6353-636010.1158/0008-5472.CAN-05-4533Crossref PubMed Scopus (110) Google Scholar, 26Matsunaga T. Yamaji Y. Tomokuni T. Morita H. Morikawa Y. Suzuki A. Yonezawa A. Endo S. Ikari A. Iguchi K. El-Kabbani O. Tajima K. Hara A. Nitric oxide confers cisplatin resistance in human lung cancer cells through upregulation of aldo-keto reductase 1B10 and proteasome.Free Radic. Res. 2014; 48 (25156503): 1371-138510.3109/10715762.2014.957694Crossref PubMed Scopus (31) Google Scholar). Caveolin-1 (Cav-1) is a 21-kDa scaffolding protein found in caveolae (27Williams T.M. Lisanti M.P. The caveolin proteins.Genome Biol. 2004; 5 (15003112): 21410.1186/gb-2004-5-3-214Crossref PubMed Scopus (363) Google Scholar). Cav-1 is required for the formation of caveolae as well as its function, including endocytosis, lipid homeostasis, and signal transduction (28Liu P. Rudick M. Anderson R.G. Multiple functions of caveolin-1.J. Biol. Chem. 2002; 277 (12189159): 41295-4129810.1074/jbc.R200020200Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). The tumor suppressor function and scaffolding capacity of Cav-1 have been extensively studied in various cancers (29Bélanger M.M. Roussel É. Couet J. Caveolin-1 is down-regulated in human lung carcinoma and acts as a candidate tumor suppressor gene.Chest. 2004; 125 (15136443): 106S-107S10.1378/chest.125.5_suppl.106SAbstract Full Text Full Text PDF PubMed Google Scholar30Wiechen K. Diatchenko L. Agoulnik A. Scharff K.M. Schober H. Arlt K. Zhumabayeva B. Siebert P.D. Dietel M. Schäfer R. Sers C. Caveolin-1 is down-regulated in human ovarian carcinoma and acts as a candidate tumor suppressor gene.Am. J. Pathol. 2001; 159 (11696424): 1635-164310.1016/S0002-9440(10)63010-6Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 31Shyamsunder P. Vidyasekar P. Shukla A.R. Mohan S. Verma R.S. Lowered expression levels of a tumor suppressor gene–caveolin-1 within dysregulated gene networks of Fanconi anemia.Gene. 2013; 527 (23845778): 521-52810.1016/j.gene.2013.06.051Crossref PubMed Scopus (11) Google Scholar32Sun M.-Z. Guan Z. Liu S. Zhou X. Wang N. Shao S. Lin D. Caveolin-1 interferes cell growth of lung cancer NCI-H446 cell through the interactions with phospho-ERK1/2, estrogen receptor and progestin receptor.Biomed. Pharmacother. 2012; 66 (22564243): 242-24810.1016/j.biopha.2011.11.003Crossref PubMed Scopus (8) Google Scholar), but its role in the regulation of CSCs remains obscure. Furthermore, the co-expression of Cav-1 and NOS in certain cancers has been reported (33Yokomori H. Oda M. Yoshimura K. Nomura M. Wakabayashi G. Kitajima M. Ishii H. Elevated expression of caveolin-1 at protein and mRNA level in human cirrhotic liver: relation with nitric oxide.J. Gastroenterol. 2003; 38 (14564631): 854-86010.1007/s00535-003-1161-4Crossref PubMed Scopus (27) Google Scholar). In addition, the caveolin-scaffolding domain of Cav-1 exhibits protein–protein interactions with NOS, which resulted in a decrease of the cellular function of NOS (34Ju H. Zou R. Venema V.J. Venema R.C. Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity.J. Biol. Chem. 1997; 272 (9228013): 18522-1852510.1074/jbc.272.30.18522Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, 35Felley-Bosco E. Bender F.C. Courjault-Gautier F. Bron C. Quest A.F. Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells.Proc. Natl. Acad. Sci. U.S.A. 2000; 97 (11114180): 14334-1433910.1073/pnas.250406797Crossref PubMed Scopus (131) Google Scholar). These findings suggest that Cav-1 and NO may play an opposite role in regulating CSCs. However, detailed mechanisms and their cross-talk remain to be investigated. Oct4 is a key CSC mediator that promotes the expression of other CSC factors, and its introduction into cancer cells induces cancer stemness (36Chen Y.C. Hsu H.S. Chen Y.W. Tsai T.H. How C.K. Wang C.Y. Hung S.C. Chang Y.L. Tsai M.L. Lee Y.Y. Ku H.H. Chiou S.H. Oct-4 expression maintained cancer stem-like properties in lung cancer-derived CD133-positive cells.PLoS ONE. 2008; 3 (18612434): e263710.1371/journal.pone.0002637Crossref PubMed Scopus (430) Google Scholar). We found that Oct4 interacts with Cav-1 and that such interaction is under the regulation of NO. In this study, we hypothesized that Cav-1 regulates the cellular level of Oct4, and therefore it influences CSCs in lung cancer via a NO-dependent mechanism. Using molecular techniques and computer modeling approaches, we demonstrated for the first time that Cav-1 forms a molecular complex with Oct4 and regulates its expression through ubiquitin–proteasome-mediated degradation. We also investigated the roles of NO in complex formation and stem properties of lung cancer cells, and unveiled a novel mechanism of CSC regulation that may lead to a better understanding of cancer cell biology under nitrosative oxidative stress conditions, which may be exploited for CSC-targeted therapy. To study the potential regulation of CSCs by NO, we first characterized the optimal dose response to NO in human lung cancer cells. Subconfluent monolayers of human lung cancer H460 cells were exposed to various concentrations of DPTA NONOate, a well-established NO donor, for 24 h, and cell viability was determined by MTT assay. At the concentration range of 0–40 μm, the NO donor was found to have no significant effect on cell viability; however, at 80 μm or higher, a significant decrease in cell viability was observed in the treated cells (Fig. 1A). Apoptosis and necrosis studies by Hoechst 33342/propidium iodide (PI) assays also showed that the low-dose (<40 μm) DPTA NONOate had no significant effect on DNA condensation/fragmentation or nuclear PI fluorescence (Fig. 1, B and C), indicating the lack of apoptotic and necrotic cell death under the test conditions. To determine the effect of NO on the spheroid-forming ability of lung cancer cells under nonattachment conditions, H460, H23, and H292 cells were treated with 0–40 μm DPTA NONOate for 5 days, and their spheroid-forming capacity was evaluated by seeding them at low density on ultralow attached plates. Time courses of spheroid formation for H460, H23, and H292 cells at 10, 20, and 40 days post-seeding are depicted in Fig. 1, D, G, and J, whereas quantitative analyses of the spheroid number and size are shown in Fig. 1, E/F, H/I, and K/L, respectively. A significant increase in the number of spheroids formed at 20 days was recorded for the H460 and H292 cells treated with 10 μm or higher concentrations of DPTA NONOate, whereas a similar increase was observed for H23 cells at 5 μm or higher concentrations. At 40 days post-seeding, a significant increase in spheroid number was observed in H460, H23, and H292 cells treated with 5 μm or higher concentrations of NONOate. However, none of the treated cells showed a significant change in the spheroid number at 10 days. Fig. 1, F, I, and L, shows the relative spheroid size of the treated and untreated H460, H23, and H292 cells, respectively. Although the spheroid size of the treated cells was smaller than that of the control cells at 10 days, a significant increase in the spheroid size was observed at 20 and 40 days for all cell lines tested. At 20 days post-seeding, the increase in spheroid size was observed at the treatment dose of 10 μm or higher concentration for H460 and H292 cells and at 5 μm or higher concentration for H23 cells. At 40 days post-seeding, all cell lines exhibited a significant increase in spheroid size at the treatment dose of 5 μm or higher concentration. These results indicate that nontoxic concentrations of DPTA NONOate promote spheroid formation of lung cancer H460, H23, and H292 cells. Stem cell markers such as CD133, ALDH1A1, and ABCG2 and stemness transcription factors such as Oct4, Sox2, and Nanog are commonly accepted as key drivers or markers of CSCs (10Liu A. Yu X. Liu S. Pluripotency transcription factors and cancer stem cells: small genes make a big difference.Chin. J. Cancer. 2013; 32 (23419197): 483-487PubMed Google Scholar). We used these proteins to verify the CSC-inducing effect of NO in the tested lung cells. H460 cells were cultivated in the presence or absence of DPTA NONOate (5–40 μm) for 1, 3, and 5 days, and the expression level of these markers was assessed by Western blotting. Fig. 2, A–C, shows a dose- and time-dependent expression of CD133, ABCG2, ALDH1A1, and Oct4 in response to the NO treatment. No significant changes in the expression level of these proteins were observed 1 day after the treatment (Fig. 2D). At 3 days, an increased expression of Oct4 was seen at the treatment dose of 10 μm, whereas CD133 expression was up-regulated at 20 and 40 μm (Fig. 2E). Surprisingly, we observed a reduction in Sox2 expression at 3 days after the treatment with 20 and 40 μm DPTA NONOate. Fig. 2F further shows the up-regulation of CD133, ALDH1A1, and Oct4 at 5 days post-treatment with 10 μm or higher concentrations of DPTA NONOate and a decrease in Sox2 expression at the dose of 10 μm or higher. To confirm the above finding, we performed immunofluorescence experiments assessing the expression of CSC markers CD133 and Oct4 in H460 cells following NO exposure. The cells were treated with 40 μm DPTA NONOate for 5 days and analyzed for CD133 and Oct4 expression by immunofluorescence staining. Consistent with the Western blotting results, the immunofluorescence results indicate the up-regulation of CD133 and Oct4 expression in the treated lung cancer cells (Fig. 2G). The expression levels of stemness-associated proteins in response to the NO donor treatment were also assessed in H23 and H292 cells. Fig. 2, H and J, shows that treatment of H23 cells with DPTA NONOate for 5 days induced an up-regulation of CD133 and ABCG2 at the treatment dose of 5 μm or higher, whereas an increase in Oct4 and ALDH1A1 was observed at the dose of 20 μm or higher. Fig. 2, I and K, shows the results of NO treatment in H292 cells. A significant increase in Oct4 expression was seen at the treatment dose of 5 μm or higher and at 20 μm or higher for CD133, ABCG2, and ALDH1A1. These results along with the spheroid formation results support the regulatory role of NO in controlling the stemness of lung cancer cells. Two different passages of H460 cells treated with DPTA NONOate and their nontreated counterparts were subjected to microarray analysis as mentioned under “Experimental procedures.” The gene probe list of the chip is shown in Table S1. The CU-DREAM (37Aporntewan C. Mutirangura A. Connection up- and down-regulation expression analysis of microarrays (CU-DREAM): a physiogenomic discovery tool.Asian Biomed. 2011; 5: 257-26210.5372/1905-7415.0502.034Crossref Scopus (20) Google Scholar) program was used to analyze the raw data, and gene probes with significant differences (p < 0.05) were chosen for further evaluation using the Bioconductor R statistic program. The heat map generated from the Bioconductor R statistics program (Fig. 3A) illustrates the intensity detected for different gene probes. H460 control cell samples were labeled control 1 and control 2, and the DPTA NONOate–treated cells were labeled as DPTA NONOate 1 and DPTA NONOate 2. The high-resolution microarray heat map image is available in Fig. S1. The computer program bracket links two samples that have similar genotypes. As expected, control 1 and control 2 displayed expression profiles distinct from those of NO-treated 1 and 2. The probed gene with significant differences (p < 0.01) (Table S2) was selected as the next interpretation. Although the mRNA level of Oct4 analyzed by microarray in NO-treated cells was not significantly altered (Table S2), the Oct4 protein expression was significantly up-regulated (Fig. 2), suggesting that the NO may not regulate Oct4 up-regulation via transcriptional means but may affect the protein stability. The genes displayed significant differences (p < 0.01) (Table S2) between treatments, and control groups were subjected for Gene Ontology (GO) using Enrichr (an integrative web-based and mobile software application; http://amp.pharm.mssm.edu/Enrichr/) 3Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site. (38Chen E.Y. Tan C.M. Kou Y. Duan Q. Wang Z. Meirelles G.V. Clark N.R. Ma'ayan A. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool.BMC Bioinformatics. 2013; 14 (23586463): 12810.1186/1471-2105-14-128Crossref PubMed Scopus (3021) Google Scholar, 39Kuleshov M.V. Jones M.R. Rouillard A.D. Fernandez N.F. Duan Q. Wang Z. Koplev S. Jenkins S.L. Jagodnik K.M. Lachmann A. McDermott M.G. Monteiro C.D. Gundersen G.W. Ma'ayan A. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update.Nucleic Acids Res. 2016; 44 (27141961): W90-W9710.1093/nar/gkw377Crossref PubMed Scopus (3951) Google Scholar), providing the gene set enrichment analysis. We used the Enrichr-EhEA-2016 feature (39Kuleshov M.V. Jones M.R. Rouillard A.D. Fernandez N.F. Duan Q. Wang Z. Koplev S. Jenkins S.L. Jagodnik K.M. Lachmann A. McDermott M.G. Monteiro C.D. Gundersen G.W. Ma'ayan A. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update.Nucleic Acids Res. 2016; 44 (27141961): W90-W9710.1093/nar/gkw377Crossref PubMed Scopus (3951) Google Scholar), which analyzed and indicated the responsible transcription factors (TFs) for the set of gene expression changed with ChIP databases (PMID: 20709693) (40Lachmann A. Xu H. Krishnan J. Berger S.I. Mazloom A.R. Ma'ayan A. ChEA: transcription factor regulation inferred from integrating genome-wide ChIP-X experiments.Bioinformatics. 2010; 26 (20709693): 2438-244410.1093/bioinformatics/btq466Crossref PubMed Scopus (536) Google Scholar). Fig. 3B shows the analyzed results by Enrichr-EhEA-2016 indicating the TF names and PubMed IDs of ChIP-seq experiments; a list of NO-mediated gene expression changes that matched the gene list in each ChIP-seq; and the p value calculated from matched and unmatched genes in each ChIP-seq. The results showed that genes altered by NO treatment are the regulated target genes of various TFs, including Ap2-α, Ets1, Jar1d1A, Oct4, Myc, Sall4, Klf4, E2A, Top2B, Maf, and Stat3 (Fig. 3B). To verify the effect of NO in regulation of CSCs through Oct4 activity, the Oct4-targeted genes were analyzed by RT-PCR (Fig. 3C). The mRNA expression of COLEC12, LAMP1, MYH3, PER3, ROS26, and UBE2S genes were up-regulated after treatment with NO in a concentration-dependent manner. The RT-PCR results were consistent with the results from the microarray analysis. Fig. 3D indicates the selected NO-mediated gene alteration (only genes of which their mRNA expression is controlled by Oct4) and its primer sequences. Based on previous studies showing the role of Oct4 in controlling stem cell properties and expression of CSC markers CD133 and ALDH1A1 (41Jin G.P. Chang Z.Y. Schöler H.R. Pei D. Stem cell pluripotency and transcription factor Oct4.Cell Res. 2002; 12 (12528890): 321-32910.1038/sj.cr.7290134Crossref PubMed Scopus (280) Google Scholar, 42Zeineddine D. Hammoud A.A. Mortada M. Boeuf H. The Oct4 protein: more than a magic stemness marker.Am. J. Stem Cells. 2014; 3 (25232507): 74-82PubMed Google Scholar), our finding demonstrates that the effects of NO on Oct4 were not involved in the transcription process of Oct4. Thus, we investigated the potential regulation of NO on Oct4 post-translational modifications. Because NO has previously been shown to promote the stability of various proteins (43Li F. Sonveaux P. Rabbani Z.N. Liu S. Yan B. Huang Q. Vujaskovic Z. Dewhirst M.W. Li C.-Y. Regulation of HIF-1α stability through S-nitrosylation.Mol. Cell. 2007; 26 (17434127): 63-7410.1016/j.molcel.2007.02.024Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 44Matsumoto A. Comatas K.E. Liu L. Stamler J.S. Screening for nitric oxide-dependent protein–protein interactions.Science. 2003; 301 (12893946): 657-66110.1126/science.1079319Crossref PubMed Scopus (130) Google Scholar), we tested whether NO could stabilize Oct4, which may contribute to its up-regulation after the NO donor treatment. The cells were treated with 40 μm DPTA NONOate at various times in the presence or absence of cycloheximide (CHX), a protein synthesis inhibitor, and the expression of Oct4 was monitored by Western blotting (Fig. 4A). The results showed that treatment of the cells with DPTA NONOate significantly increased the expression of Oct4 over time as compared with the untreated control (Fig. 4B), suggesting the stabilizing effect of NO on Oct4. Because previous studies have shown that the cellul" @default.
• W2858779744 created "2018-07-19" @default.
• W2858779744 creator A5018505480 @default.
• W2858779744 creator A5019794851 @default.
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• W2858779744 creator A5091182994 @default.
• W2858779744 date "2018-08-01" @default.
• W2858779744 modified "2023-10-11" @default.
• W2858779744 title "Nitric oxide promotes cancer cell dedifferentiation by disrupting an Oct4:caveolin-1 complex: A new regulatory mechanism for cancer stem cell formation" @default.
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