Stem cell pathways and Notch signaling an emerging role in the development of acquired Drug Resistance in Neuroblastoma

Stem cell pathways and Notch signaling an emerging role in the development of acquired Drug Resistance in Neuroblastoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Stem cell pathways and Notch signaling an emerging role in the development of acquired Drug Resistance in Neuroblastoma John Clark- Corrigal, Masood Zaka, Svetlana Myssina, Martin Michaelis, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2471355/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose : Neuroblastoma is a paediatric tumour that develops from embryonal neural crest cells that give rise to the sympathetic nervous system. Aggressive high-risk disease remains a clinical challenge and despite multi-modal therapy, survival rates are poor. Most neuroblastomas initially respond well to induction chemotherapy however, 50-60% of patients with high-risk disease will relapse with aggressive disease. A major obstacle in the successful treatment of this disease is the development of acquired resistance to chemotherapeutic agents. We hypothesize that aggressive neuroblastomas acquire a more immature phenotype in addition to increased expression of genes linked to stem cell pathways. Methods : In this study we investigated the role of stem cell related genes in the development of acquired drug resistance using Q- Real Time PCR and bioinformatics analysis on three-paired vincristine sensitive and resistant cell lines. Results : The study outlines 11 differentially expressed genes with several targets involved primarily in notch signalling, and stem cell development. Conclusions: The findings implicated in this study, which include comparative analysis against patient microarray data, warrants further study on the functional effects of these genes/pathways to elucidate their role in acquired drug resistance. neuroblastoma stem-cells drug resistance relapse bioinformatics Notch Figures Figure 1 Figure 2 1. Introduction Neuroblastoma (NB) is a childhood cancer which arises in the developing sympathetic nervous system, specifically the neuronal ganglia which derives from embryonic neural crest cells (Louis and Shohet 2015 ). In the United Kingdom around 98 new cases children under the age of 15 are diagnosed annually (Public Health England 2021 ). High risk patients, categorised by the presence of MYCN amplification or in children over 18 months with metastatic disease, have less than 40% chance of long term survival (Speleman et al. 2016 ). Tumour heterogeneity and clonal evolution are now established as characteristic hallmarks of NB, with emerging evidence suggesting that solid tumour cancer cells may not follow the hierarchical organisation of other cancer cells; instead, following clonal evolution from one subtype to another and generating distinctly recognisable tumours. This may explain the variation in clinical manifestations and the unpredictable nature in response to treatment (Ngan 2015 ). Whilst most high-risk patients do initially respond to induction multi-modal chemotherapy, unfortunately over 50% relapse, and in many cases the returning tumour cells possess a drug resistant phenotype (Keshelava et al. 2001 ; Owens et al. 2016 ). VCR is currently included in Rapid COJEC induction chemotherapy which comprises a combination of five agents (cisplatin, vincristine, carboplatin, etoposide and cyclophosphamide) and is currently used to treat NB patients in Europe (Smith and Foster 2018 ). Vincristine (VCR) targets cell microtubule formation (Kotchetkov et al. 2003 ; Piskareva et al. 2015 ), which exert integral roles in cell scaffolding as well as being regulated during cell cycle. Thus, microtubule inhibition leads to cell cycle arrest and eventual cell death by apoptosis (Kothari et al. 2016 ). A major obstacle in the successful treatment of neuroblastoma is the acquisition of resistance to chemotherapeutic agents and hence, is the most pressing factor leading to therapy failure and relapse. Drug resistance is a problem for many cancers including neuroblastoma, with a variety of potential mechanisms driving this resistance including cancer stem cells, epigenetics and genetic alterations(Hanahan and Weinberg 2011 ). Epithelial to mesenchymal transition (EMT), is a reversible process whereby cells undergo a functional change from a fixed epithelial cell to that of a more mobile nonspecialised cell which has the capacity to differentiate into other specialised cells or self-renew, has been highlighted as an early mechanism in the development of metastasis. Furthermore, emerging evidence in literature demonstrating EMT as a causative factor in drug resistance in most cancer types (Shibue and Weinberg 2017 ; Tanabe et al. 2020 ). Notch signalling is an evolutionarily conserved pathway which plays an essential role in embryo development and cell regulation, including proliferation, survival and self-renewal of stem cells. Recent research has implicated Notch signalling in promoting therapeutic resistance via cancer stem cells (CSCs)(Moore et al. 2020 ; Kumar et al. 2021 ). This small subset of the tumour population are nascent cells with innate therapeutic resistance and the capacity to restore the tumour population with resistant cells. From this concept comes the theory that the CSCs hold not only the key to drug resistance, but also tumour spread by metastases. Like their stem cell counterpart, CSCs enhance the stemness and dedifferentiation of the tumour and promote the transition between epithelial and mesenchymal phenotype (Venkatesh et al. 2018 ). In preclinical research across several cancer types, notch signalling activity is upregulated in brain(Cenciarelli et al. 2017 ; Sun et al. 2020 ), breast (McGowan et al. 2011 ; Miao et al. 2020 ) and pancreatic cancer(Yao and Qian 2010 ; Cui et al. 2019 ; Feng et al. 2020 ), conversely in colorectal cancers it is largely downregulated (Chu et al. 2011 ; Meisel et al. 2020 ). Many paediatric cancers have dysregulated signalling pathways which are involved in the development of their cells of origin, particularly notch, wnt and hedgehog (Filbin and Monje 2019 ). Identification of biological drivers that contribute towards drug resistance is important in order to develop more effective targeted therapeutics and essential to improving high risk patient survival (Housman et al. 2014 ) In this study we investigated the potential role of stem cell related genes and pathways, in the development of acquired drug resistance in three paired neuroblastoma cell line models of drug resistance. 2. Materials And Methods Methods Cell culture Three paired parental and drug-adapted neuroblastoma cell lines; SHSY5Y, IMR5 and IMR32 were obtained from the Resistant Cancer Cell Line (RCCL) collection ( www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html ). Cell lines were treated with 10ng/ml of VCR to generate resistant cells; SHSY5YrVCR, IMR5rVCR, IMR32rVCR. Cells were cultured using Iscove’s Dulbeco’s Modified Eagle Medium (DMEM) (Sigma, UK) with 10% fetal calf serum (FCS), L- glutamine and1% primocin. All cell lines were incubated at 37°C, 5% CO 2 . Cells were passaged once they reached 70–80% confluency level. RNA extraction RNA (Ribose Nucleic Acid) extraction was performed using the RNeasy mini kit (Qiagen, uk) according to manufacturer’s instructions. RNA quality and quantification were assessed using the Nanodrop Lite Spectrophotometer (Thermo Scientific, UK) and first strand DNA synthesized using the iScript™ cDNA synthesis kit (Bio-Rad, UK). Rt2 Profiler Array The Human Cancer Stem Cell RT2 profiler Array analysis (Qiagen, UK) which contains primers for 96 genes related to human cancer stem cells, was performed according to protocol format R in the manufacturer’s instructions on three paired parental and acquired drug resistant cell lines. IMR32 & IMR32-VCR, IMR5 & IMR5-VCR and SHSY5Y & SHSY5Y-VCR. Analysis of mRNA expression levels was performed by Real Time-QPCR (Qiagen Rotorgene 100) of 96 genes related to human cancer stem cells, against a panel of three paired parental and acquired drug resistant cell lines with an acquired drug resistance to the microtubule-inhibitor; Vincristine. Fold change was assessed using the 2 ΔΔct method, using GAPDH and β-actin as the respective housekeeping genes. Bioinformatics analysis The CtData for each cell line was further tested using Limma package from R Bioconductor for differential expression of genes by making linear contrast matrices for control and case cell lines. Linear models were then fitted for identification of dysregulation genes using lm() function from the package. Subsequent p-values were further tested for multiple testing method of false discovery rate. All genes showing p-values 1 and < -1 were picked for overexpression and down regulation. Significant genes from three contrasts were compared for identification of common genes among the cell lines. Functional analysis Functional enrichment analysis were performed on 11 genes using combination of different methods available. Curated gene sets from Molecular Signature Database (MSigDB), DAVID Bioinformatics Resource 6.8, GeneMania and STRING were extracted for the construction of metagene. Fgsea package of Bioconductor was used for the prediction of potential involvement of 11 genes in regulatory processes, functions and pathways. All the pathways with FDR < 0.05 were considered significant. In Silico Analysis All data was collected from the R2: Genomics Analysis and Visualization Platform 35 , using publicly available neuroblastoma patient data. Expression cut-off was determined by a log rank test, which determined an optimum cut off, rather than using an average value. R2: Genomics Analysis and Visualization Platform ( http://r2.amc.nl ) Kaplan-Meir plots were constructed to compare high and low expression of the 11 differentially expressed target genes with important clinical parameters stage, overall survival and event free survival. Statistical analysis was shown using a two-tailed, unpaired student t-test with a confidence interval of 95% ) (Appendix One) 3. Results This study has generated novel data and identified several genes that are up-regulated in drug resistant cell lines; SHSY5YrVCR, IMR5rVCR and IMR32rVCR, compared with the paired parental cell line. In addition, we have also identified an important subset of genes that are down-regulated in drug resistant cell lines. Many of these candidate genes have been shown to exert a role in aggressive disease in other common cancers such as breast, prostate and small cell lung cancers. Elevation of stem cell and EMT related genes in chemo-resistant cell lines Comparative gene expression analysis was undertaken following Real Time-qPCR on each parental and drug resistant cell line and showed deregulation of 11 genes across the three cell lines, ATP-binding cassette sub-family B member 5 (ABCB5), ATP-binding cassette super-family G member 2 (ABCG2), Delta like ligand 1 (DLL1), Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), Integrin alpha 2 (ITGA2), LIN 28 homolog A (LIN28A), LIN 28 homolog B (LIN28B), MYC, Zinc finger protein SNAI1 (SNAI1), SRY (sex determining region Y)-box 2 (SOX2) and WEE1 G2 Checkpoint Kinase (WEE1) (Fig. 1 A.). Subsequent unsupervised hierarchical clustering was conducted (Fig. 1 B.) which observed upregulation of LIN28A, ABCG5, LIN28B and DLL1 in IMR32, with downregulation of ERBB2, ABCG2, WEE1 and MYC. IMR5 displayed upregulation of ITGA2, MYC, SNAI1, WEE1 and ABCG2, with downregulation of LIN28B, ABCB5, LIN28A and SOX2. SHSY5Y highlighted upregulation of SOX2, ERBB2, ABCG2 and WEE1, downregulation of SNAI1, DLL1, ITGA2 and MYC (Fig. 1 B). Bioinformatics analysis shows a regulatory network highlighting EMT and stemness genes In addition to the pathway analysis, we also created a regulatory network composed of 8 of the genes highlighted within our study. This regulatory network developed from connections with previous literature illustrates a core role for MYC, showing direct interactions with WEE1 and SOX2, links through one other gene to ABCG2, ERRBB2, DLL1, ITGA2 and SNAI1 (Fig. 2 A). In further detail you can see how many genes are influenced by the genes within our network (Fig. 2 B). Gene enrichment analysis highlights NOTCH signalling related pathways Gene set enrichment analysis on the target genes; highlighted DLL1, MYC and NOTCH1 in particular, all with a highly significant false discovery rate. The Top regulatory pathways extracted from the gene set enrichment analysis were correlated with Significance FDR < 0.05 and this confirmed involvement of the NOTCH signalling pathway in acquired drug resistance (Table 1 ). In-silico classification further outlines roles of genes in drug resistance in established studies in other malignancies. In-silico analysis was performed to determine whether our 11 differentially expressed target genes correlated with important clinical outcomes using four large neuroblastoma patient data sets including disease stage, overall survival (OVR) and event free survival (EFS). Kaplan-Meier plots revealed that low expression of ABCB5, ABCG2, and MYC was associated with lower overall survival in patients with stage 4 disease (INSS). Furthermore, elevated expression of ERBB2, ITGA2, LIN28A, LIN28B, SNAI1, SOX2, DLL1 and WEE1 were associated with decreased overall survival in neuroblastoma patients with stage 4 disease (Supplementary Fig. 1 and Table 1 ). 4. Discussion Neuroblastoma is a paediatric tumour that develops from embryonic neural crest cells that give rise to the sympathetic nervous system. Whilst most neuroblastomas initially respond well to induction chemotherapy, 50–60% of patients with high-risk disease relapse with aggressive disease. Acquired drug resistance is a major therapeutic challenge in the treatment of such patients. Using an array of 96 genes related to cancer stem cells in three-paired parental and Vincristine drug resistant neuroblastoma cell lines, this study outlines 11 prominent genes across all cell lines; ABCG2, ERBB2, LIN28A, SOX2, WEE1 MYC, SNAI1, LIN28B, ABCB5, DLL1 and ITAG2 (Fig. 1 B). Differing expressions of the same genes across the cell lines, highlights the hallmark heterogeneity of disease. Using complex bioinformatics analysis, we created a regulatory network composed of 12 genes, 8 of which were highlighted within our study and relating genes discovered from the database (Fig. 2 A&B). The network displayed a core role for MYC directly influencing genes associated with oncogenic proteins (ERBB2), self-renewal (SOX2) and cell cycle (WEE1). Indirectly, repression of MYC appears to cause over expression of hallmark drug-resistance genes such as SNAI1 and ABCG2, whilst decreasing expression of NOTCH signalling protein DLL1 and integrin protein ITGA2 (Kang et al. 2009 ; Chen et al. 2012 ; Ongaratti et al. 2016 ). Interestingly MYC (C-MYC) was associated with lower overall survival across four data sets, in contrast to MYCN amplification which is strongly correlated to poor overall and event free survival in high risk neuroblastoma patients (Somasundaram et al. 2019 ) suggestive of a potential inverse correlation (Westermann et al. 2008 ). Whilst most other cancers display a positive correlation between C-MYC overexpression and a number of the genes highlighted in our study, a number of studies have implicated that c- and MYCN can compensate for loss of expression by one another in normal embryonic stem cells(ESCs), induced pluripotent stem cells(IPSCs) and neuroblastoma(Cotterman and Knoepfler 2009 ; Varlakhanova et al. 2010 ; Nakagawa et al. 2010 ). This may explain the findings we have discovered. Similar to our findings, a study in triple negative breast cancer, showed elevated SOX2 expression alongside overexpression of ABCG2 and the EMT marker Twist- related protein 1 (TWIST1) in drug resistant cells. However, when paclitaxel was administered to SOX2 inhibited mammospheres, ABCG2 and TWIST1 were downregulated, alongside decreased expression of other EMT markers (SNAI1) which induced arrested migration and reduced formation of spheres, indicative of loss of self-renewal (Mukherjee et al. 2017 ). This study highlights the potential of the EMT-Stemness axis in therapeutic resistance, where SOX2-dependent TWIST1 overexpression maintains stemness in addition to enabling migration. Subsequent inhibition of the axis causes regained sensitivity. Here we’ve highlighted deregulation of notch signalling pathway genes in drug resistance cell lines, recent research from clinical samples showed 60% (28/46) of clinical samples from neuroblastoma patients with overexpression of NOTCH1 linked to recurrence and lower overall survival (Metovic et al. 2022 ). EMT is a complex process, recent data has shown that Notch signalling directly influences overexpression of Snail and decreased expression of E-cadherin (Timmerman et al. 2004 ; Saad et al. 2010 ). Snai1 expression and loss of E-Cadherin are associated with several cancers and causing increased migration and invasion (Chen et al. 2010 ; Zhang et al. 2017 ), interestingly a recent study in osteosarcoma found that sub-lethal doxorubicin (Dox) treatments showed significant correlation between EMT and Notch signalling in doxorubicin resistant cell lines (Yang et al. 2017 ) which supports our findings within the context acquired drug resistance. Delta like 1 (DLL1) has also been associated with disease recurrence in other cancer types, including hepatocellular carcinoma (Ma et al. 2016 ) and with poor prognosis in non-small cell lung cancer (Pancewicz-Wojtkiewicz et al. 2017 ). A recent study in Neuroblastoma cell lines, showed that DLL1 was highly expressed in MYCN amplified cells, and could be inhibited by miRNAs(Bettinsoli et al. 2017 ). In aggressive medulloblastoma, NOTCH signalling pathway has shown to regulate self-renewal and metastasis, with TWIST1 and Polycomb complex protein BMI-1 ( BMI1) influential in NOTCH1 induced metastases (Kahn et al. 2018 ). Two genes, SOX2 and LIN28A are involved with stem cell pluripotency. Interestingly, Yamanaka’s seminal study on the generation of induced pluripotent stem cells (iPSCs) utilised both SOX2 and LIN28A to reprogram adult somatic cells to an undifferentiated state (Takahashi and Yamanaka 2006 ). Since then there has been an emerging interest in genes linked to stem cells and pluripotency and their role in tumorigenesis (Müller et al. 2016 ), however whether that role is extended to therapeutic resistance is understudied. The up-regulation of both SOX2 and LIN28A across all VCR-resistant cell lines may suggest that the acquisition of a stem cell phenotype is important for the acquisition of drug resistance, which occurs in 50% of patients with high risk tumours. This trend is not limited to VCR resistance alone however, in doxorubicin resistant gastric cancer stem cells, Sox2 and ABCG2 overexpression was associated with the dox-resistant phenotype (Tian et al. 2012 ) in addition to elevated LIN28B overexpression in gastric cancer cells with dox-resistance (Teng et al. 2015 ). DLL1 exerts a role in development of drug resistance in dox-resistant cell lines and biopsies in osteosarcoma (Pu et al. 2017 ). Interestingly, conflicting studies regarding Snai1 expression in breast adenocarcinoma cell lines in dox-resistant settings,(Lim et al. 2013 ; Tsou et al. 2015 ), however Lim et al. ‘s study highlights the importance of EMT-stemness interaction in drug resistance. Highlighting heterogeneity within the ‘same’ cancer. A recent study conducted into whether inhibition of autophagy paired with very intense chemotherapeutic treatments can prevent development of drug resistance in NB, the initial results seem positive (Chen et al. 2022 ). This is where the balance between vanquishing the tumour without the consequences of aggressive treatment on the patient. A recent study has highlighted an axis between BMP4-wnt-notch, whereby BMP has a tumour suppressor role, this same work showed that induction of notch proteins halted growth of NB in IMR32 and SH-SY5Y (Szemes et al. 2020 ). Low BMP4 and notch expression leads to an aggressive NB phenotype, perhaps there is a complexity within the signalling pathways that mean upregulation in notch signalling still leads to the aggressive phenotype. The research landscape has also associated wnt signalling in the development of therapeutic resistance across different cancer types(Forgham et al. 2015 ; Vieira et al. 2015 ; He et al. 2018 ; Fu et al. 2019 ; Yang et al. 2019 ; Clark-Corrigall et al. 2022 ). This underlines the necessity of a global transcriptome of the cell lines in our study in order to further understand the molecular changes in therapeutic resistance. There have been some studies investigating the relationship between CSC phenotype and EMT (Prieto-Vila et al. 2017 ), where activation of stem cell related pathways directly promote the transition to a mesenchymal profile. Previously, Notch signalling was widely understood as a regulator of cell fate decisions, self – renewal, maintenance of tissue stem cells and tissue wound healing but more recently as a regulator of survival and regeneration of CSCs (Capaccione and Pine 2013 ). However, a definitive molecular mechanism behind the CSC-EMT relationship still remains elusive (Shibue and Weinberg 2017 ). Data from in-silico analysis (Supplementary Fig. 1 &Table 1 ) showed that the worst probabilities for event free survival in late stage (INSS Stage 4) neuroblastoma were ERBB2 overexpression (10% after 3 years in the Versteeg data set – 88, 15% reach 5 year survival in the Kocak dataset (649)), ITGA2 overexpression (0% were predicted to reach 2 year follow up in both the primary NRC dataset – 283 and Versteeg dataset − 88), SOX2 overexpression (10% at 2 year follow up in the Versteeg dataset, 0% reached 4 year follow up Kocak dataset (649) and DLL1 overexpression (predicts 20% reach 2 year follow up in the Versteeg (88) and NRC (283). Confirmation of the link with important clinical parameters in four patient data sets further supports the potential role of these genes in the development of acquired therapeutic resistance in patients with neuroblastoma and potentially other childhood cancers. Furthermore, in support of our data a landmark study by van Groningen et al investigating the role of super-enhancers on intra-tumoral heterogeneity identified two distinct lineages; adrenergic (ADRN) and mesenchymal (MES) differentiation with the Notch pathway as a key driver of dedifferentiated mesenchymal identity. In both in vivo and in vitro studies MES cells were shown to have enhanced migration, be more resistant to common neuroblastoma treatments and increased prevalence of relapse disease, which reinforces the idea that treatment exerts selective pressure much like the cancer stem cell hypothesis postulates (Groningen et al. 2017 ). Since then it has been shown that Notch 3 signalling is a key driver in the reprogramming of ADRN cells to a more mesenchymal state (Groningen et al. 2019 ) and that MES cells are now known to utilise retinoic acid, an agent used in a maintenance dose to prevent relapse, to proliferate and mobilise (Groningen et al. 2021 ). A recent study from Newcastle has found that TOP2B expression was needed to maintain the ADRN state, and also lowered notch signalling factors like Notch 1–3 (Khazeem et al. 2022 ).These studies show that ADRN cells can reprogram themselves and become a more dedifferentiated cell type using NOTCH signalling. To further support this mouse models, have implicated Notch signaling in the maintenance of neural stem cells maintenance in the fetal brain. Such observations from developmental biology have shown that Notch exerts similar roles which are vital to the tumourigenicity of cancer stem cells particularly in solid tumors including glioblastoma, ovarian cancer, and breast cancer (Aster et al. 2017 ; Yi et al. 2019 ; Miao et al. 2020 ; Yang et al. 2021 ). Unfortunately, due to the complexity of NB there is no perfect mouse model but even with the advent of iPSC based practices researchers still trying to elucidate this in neuroblastoma(Gonzalez Malagon and Liu 2022 ). Our research data warrants further study into this panel of genes in acquired drug resistance in neuroblastoma. 5. Conclusions Our study highlights a core role for MYC and NOTCH signalling associated genes in the development of acquired Vincristine drug resistance in our cell line models. Alterations to individuals within these groups of genes may have further reaching consequences to the stem cell – EMT axis which drive a drug resistant phenotype. Our panel of three paired Vincristine resistant cell lines provide a unique in-vitro platform to perform studies to understand the molecular mechanisms underlying the development of drug resistance. With the addition of powerful large patient tumour data sets and bioinformatics analyses, we’ve further highlighted potential for a specific network of genes between niches which have a negative impact on survival and require further research. This could subsequently lead to the discovery of new therapeutic targets that improve the survival of those with the aggressive disease. The findings implicated in this study, which include comparative analysis against microarray data, warrants further studies on the functional effects of the genes and pathways identified in this study in childhood cancers. Declarations Author Contributions: Conceptualization, Jane Carr-Wilkinson and Shafiq Ahmed.; methodology, Jane Carr-Wilkinson, Martin Michaelis. Jindrich Cinatl, jr Masood Zaka.; software, Masood Zaka.; validation, Svetlana Myssina, Martin Michaelis and John Clark-Corrigall.; formal analysis, Masood Zaka and Svetlana Myssina .; investigation, Masood Zaka and Svetlana Myssina.; resources, , Jane Carr-Wilkinson, Martin Michaelis. Jindrich Cinatl, jr Masood Zaka.; data curation, Masood Zaka.; writing—original draft preparation, Svetlana Myssina, John Clark-Corrigall.; writing—review and editing, Jane Carr-Wilkinson, Shafiq Ahmed.; visualization, Svetlana Myssina, John Clark-Corrigall.; supervision, Jane Carr-Wilkinson, Shafiq Ahmed.; project administration, Jane Carr-Wilkinson.; funding acquisition, Jane Carr-Wilkinson, Shafiq Ahmed. All authors have read and agreed to the published version of the manuscript. Funding: Acknowledgments: We would like to thank Florian Rothweiler for the administration and shipping of the cell lines used in this study. Conflicts of Interest: The authors declare no conflict of interest. References Aster JC, Pear WS, Blacklow SC (2017) The Varied Roles of Notch in Cancer. Annu Rev Pathol Mech Dis 12:245–275. https://doi.org/10.1146/annurev-pathol-052016-100127 Bettinsoli P, Ferrari-Toninelli G, Bonini SA, et al (2017) Notch ligand Delta-like 1 as a novel molecular target in childhood neuroblastoma. BMC Cancer 17:. https://doi.org/10.1186/s12885-017-3340-3 Capaccione KM, Pine SR (2013) The Notch signaling pathway as a mediator of tumor survival. Carcinogenesis 34:1420–1430. https://doi.org/10.1093/carcin/bgt127 Cenciarelli C, Marei HE, Zonfrillo M, et al (2017) The interference of Notch1 target Hes1 affects cell growth, differentiation and invasiveness of glioblastoma stem cells through modulation of multiple oncogenic targets. Oncotarget 8:17873–17886. https://doi.org/10.18632/oncotarget.15013 Chen J, Imanaka N, Chen J, Griffin JD (2010) Hypoxia potentiates Notch signaling in breast cancer leading to decreased E-cadherin expression and increased cell migration and invasion. Br J Cancer 102:351–360. https://doi.org/10.1038/sj.bjc.6605486 Chen S, Xu Y, Chen Y, et al (2012) SOX2 Gene Regulates the Transcriptional Network of Oncogenes and Affects Tumorigenesis of Human Lung Cancer Cells. PLOS ONE 7:e36326. https://doi.org/10.1371/journal.pone.0036326 Chen T, Zeng C, Li Z, et al (2022) Investigation of chemoresistance to first-line chemotherapy and its possible association with autophagy in high-risk neuroblastoma. Front Oncol 12: Chu D, Zhang Z, Zhou Y, et al (2011) Notch1 and Notch2 have opposite prognostic effects on patients with colorectal cancer. Ann Oncol 22:2440–2447. https://doi.org/10.1093/annonc/mdq776 Clark-Corrigall J, Myssina S, Michaelis M, et al (2022) Elevated Expression of LGR5 and WNT Signaling Factors in Neuroblastoma Cells With Acquired Drug Resistance. Cancer Invest 1–10. https://doi.org/10.1080/07357907.2022.2136682 Cotterman R, Knoepfler PS (2009) N-Myc Regulates Expression of Pluripotency Genes in Neuroblastoma Including lif, klf2, klf4, and lin28b. PLoS ONE 4:. https://doi.org/10.1371/journal.pone.0005799 Cui L, Dong Y, Wang X, et al (2019) Downregulation of long noncoding RNA SNHG1 inhibits cell proliferation, metastasis, and invasion by suppressing the Notch-1 signaling pathway in pancreatic cancer. J Cell Biochem 120:6106–6112. https://doi.org/10.1002/jcb.27897 Feng L, Wang K, Tang P, et al (2020) Deubiquitinase USP18 promotes the progression of pancreatic cancer via enhancing the Notch1-c-Myc axis. Aging 12:19273–19292. https://doi.org/10.18632/aging.103760 Filbin M, Monje M (2019) Developmental origins and emerging therapeutic opportunities for childhood cancer. Nat Med 25:367–376. https://doi.org/10.1038/s41591-019-0383-9 Forgham H, Johnson D, Carter N, et al (2015) Stem Cell Markers in Neuroblastoma—An Emerging Role for LGR5. Mol Cell Oncol 77. https://doi.org/10.3389/fcell.2015.00077 Fu J, Si L, Zhuang Y, et al (2019) Wnt/β‑catenin inhibition reverses multidrug resistance in pediatric acute lymphoblastic leukemia. Oncol Rep 41:1387–1394. https://doi.org/10.3892/or.2018.6902 Gonzalez Malagon SG, Liu KJ (2022) Linking neural crest development to neuroblastoma pathology. Development 149:dev200331. https://doi.org/10.1242/dev.200331 Groningen T van, Akogul N, Westerhout EM, et al (2019) A NOTCH feed-forward loop drives reprogramming from adrenergic to mesenchymal state in neuroblastoma. Nat Commun 10:1530. https://doi.org/10.1038/s41467-019-09470-w Groningen T van, Koster J, Valentijn LJ, et al (2017) Neuroblastoma is composed of two super-enhancer-associated differentiation states. Nat Genet 49:1261–1266. https://doi.org/10.1038/ng.3899 Groningen T van, Niklasson CU, Chan A, et al (2021) An immature subset of neuroblastoma cells synthesizes retinoic acid and depends on this metabolite. bioRxiv 2021.05.18.444639. https://doi.org/10.1101/2021.05.18.444639 Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674. https://doi.org/10.1016/j.cell.2011.02.013 He L, Zhu H, Zhou S, et al (2018) Wnt pathway is involved in 5-FU drug resistance of colorectal cancer cells. Exp Mol Med 50:1–12. https://doi.org/10.1038/s12276-018-0128-8 Housman G, Byler S, Heerboth S, et al (2014) Drug resistance in cancer: an overview. Cancers 6:1769–1792. https://doi.org/10.3390/cancers6031769 Kahn SA, Wang X, Nitta RT, et al (2018) Notch1 regulates the initiation of metastasis and self-renewal of Group 3 medulloblastoma. Nat Commun 9:4121. https://doi.org/10.1038/s41467-018-06564-9 Kang KW, Im YB, Go W-J, Han H-K (2009) c-Myc Amplification Altered the Gene Expression of ABC- and SLC-Transporters in Human Breast Epithelial Cells. Mol Pharm 6:627–633. https://doi.org/10.1021/mp800116f Keshelava N, Zuo JJ, Chen P, et al (2001) Loss of p53 Function Confers High-Level Multidrug Resistance in Neuroblastoma Cell Lines. Cancer Res 61:6185–6193 Khazeem MM, Casement JW, Schlossmacher G, et al (2022) TOP2B Is Required to Maintain the Adrenergic Neural Phenotype and for ATRA-Induced Differentiation of SH-SY5Y Neuroblastoma Cells. Mol Neurobiol 59:5987–6008. https://doi.org/10.1007/s12035-022-02949-6 Kotchetkov R, Cinatl J, Blaheta R, et al (2003) Development of resistance to vincristine and doxorubicin in neuroblastoma alters malignant properties and induces additional karyotype changes: a preclinical model. Int J Cancer 104:36–43. https://doi.org/10.1002/ijc.10917 Kothari A, Hittelman WN, Chambers TC (2016) Cell cycle-dependent mechanisms underlie vincristine-induced death of primary acute lymphoblastic leukemia cells. Cancer Res canres.2104.2015. https://doi.org/10.1158/0008-5472.CAN-15-2104 Kumar V, Vashishta M, Kong L, et al (2021) The Role of Notch, Hedgehog, and Wnt Signaling Pathways in the Resistance of Tumors to Anticancer Therapies. Front Cell Dev Biol 9: Lim S, Becker A, Zimmer A, et al (2013) SNAI1-Mediated Epithelial-Mesenchymal Transition Confers Chemoresistance and Cellular Plasticity by Regulating Genes Involved in Cell Death and Stem Cell Maintenance. PLoS ONE 8:. https://doi.org/10.1371/journal.pone.0066558 Louis CU, Shohet JM (2015) Neuroblastoma: Molecular Pathogenesis and Therapy. Annu Rev Med 66:49–63. https://doi.org/10.1146/annurev-med-011514-023121 Ma L, Dong P, Liu L, et al (2016) Overexpression of protein O-fucosyltransferase 1 accelerates hepatocellular carcinoma progression via the Notch signaling pathway. Biochem Biophys Res Commun 473:503–510. https://doi.org/10.1016/j.bbrc.2016.03.062 McGowan PM, Simedrea C, Ribot EJ, et al (2011) Notch1 Inhibition Alters the CD44hi/CD24lo Population and Reduces the Formation of Brain Metastases from Breast Cancer. Mol Cancer Res 9:834–844. https://doi.org/10.1158/1541-7786.MCR-10-0457 Meisel CT, Porcheri C, Mitsiadis TA (2020) Cancer Stem Cells, Quo Vadis? The Notch Signaling Pathway in Tumor Initiation and Progression. Cells 9:1879. https://doi.org/10.3390/cells9081879 Metovic J, Napoli F, Osella-Abate S, et al (2022) Overexpression of INSM1, NOTCH1, NEUROD1, and YAP1 genes is associated with adverse clinical outcome in pediatric neuroblastoma. Virchows Arch. https://doi.org/10.1007/s00428-022-03406-4 Miao K, Lei JH, Valecha MV, et al (2020) NOTCH1 activation compensates BRCA1 deficiency and promotes triple-negative breast cancer formation. Nat Commun 11:3256. https://doi.org/10.1038/s41467-020-16936-9 Moore G, Annett S, McClements L, Robson T (2020) Top Notch Targeting Strategies in Cancer: A Detailed Overview of Recent Insights and Current Perspectives. Cells 9:1503. https://doi.org/10.3390/cells9061503 Mukherjee P, Gupta A, Chattopadhyay D, Chatterji U (2017) Modulation of SOX2 expression delineates an end-point for paclitaxel-effectiveness in breast cancer stem cells. Sci Rep 7:. https://doi.org/10.1038/s41598-017-08971-2 Müller M, Hermann PC, Liebau S, et al (2016) The role of pluripotency factors to drive stemness in gastrointestinal cancer. Stem Cell Res 16:349–357. https://doi.org/10.1016/j.scr.2016.02.005 Nakagawa M, Takizawa N, Narita M, et al (2010) Promotion of direct reprogramming by transformation-deficient Myc. Proc Natl Acad Sci U S A 107:14152–14157. https://doi.org/10.1073/pnas.1009374107 Ngan ES-W (2015) Heterogeneity of neuroblastoma. Oncoscience 2:837–838 Ongaratti BR, Silva CBO, Trott G, et al (2016) Expression of merlin, NDRG2, ERBB2, and c-MYC in meningiomas: relationship with tumor grade and recurrence. Braz J Med Biol Res 49:. https://doi.org/10.1590/1414-431X20155125 Owens C, Li BK, Thomas KE, Irwin MS (2016) Surveillance imaging and radiation exposure in the detection of relapsed neuroblastoma. Pediatr Blood Cancer 63:1786–1793. https://doi.org/10.1002/pbc.26099 Pancewicz-Wojtkiewicz J, Eljaszewicz A, Kowalczuk O, et al (2017) Prognostic significance of Notch ligands in patients with non-small cell lung cancer. Oncol Lett 13:506–510. https://doi.org/10.3892/ol.2016.5420 Piskareva O, Harvey H, Nolan J, et al (2015) The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro. Cancer Lett 364:142–155. https://doi.org/10.1016/j.canlet.2015.05.004 Prieto-Vila M, Takahashi R, Usuba W, et al (2017) Drug Resistance Driven by Cancer Stem Cells and Their Niche. Int J Mol Sci 18:2574. https://doi.org/10.3390/ijms18122574 Pu Y, Zhao F, Wang H, Cai S (2017) MiR-34a-5p promotes multi-chemoresistance of osteosarcoma through down-regulation of the DLL1 gene. Sci Rep 7:44218. https://doi.org/10.1038/srep44218 Public Health England (2021) Cancer in children, teenagers and young adults cancer statistics report 2021 Saad S, Stanners SR, Yong R, et al (2010) Notch mediated epithelial to mesenchymal transformation is associated with increased expression of the Snail transcription factor. Int J Biochem Cell Biol 42:1115–1122. https://doi.org/10.1016/j.biocel.2010.03.016 Shibue T, Weinberg RA (2017) EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol 14:611–629. https://doi.org/10.1038/nrclinonc.2017.44 Smith V, Foster J (2018) High-Risk Neuroblastoma Treatment Review. Child Basel Switz 5:. https://doi.org/10.3390/children5090114 Somasundaram DB, Aravindan S, Yu Z, et al (2019) Droplet digital PCR as an alternative to FISH for MYCN amplification detection in human neuroblastoma FFPE samples. BMC Cancer 19:106. https://doi.org/10.1186/s12885-019-5306-0 Speleman F, Park JR, Henderson TO (2016) Neuroblastoma: A Tough Nut to Crack. Am Soc Clin Oncol Educ Book Am Soc Clin Oncol Meet 35:e548-557. https://doi.org/10.14694/EDBK_159169 Sun Z, Wang L, Zhou Y, et al (2020) Glioblastoma Stem Cell-Derived Exosomes Enhance Stemness and Tumorigenicity of Glioma Cells by Transferring Notch1 Protein. Cell Mol Neurobiol 40:767–784. https://doi.org/10.1007/s10571-019-00771-8 Szemes M, Melegh Z, Bellamy J, et al (2020) A Wnt-BMP4 Signaling Axis Induces MSX and NOTCH Proteins and Promotes Growth Suppression and Differentiation in Neuroblastoma. Cells 9:783. https://doi.org/10.3390/cells9030783 Takahashi K, Yamanaka S (2006) Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126:663–676. https://doi.org/10.1016/j.cell.2006.07.024 Tanabe S, Quader S, Cabral H, Ono R (2020) Interplay of EMT and CSC in Cancer and the Potential Therapeutic Strategies. Front Pharmacol 11:. https://doi.org/10.3389/fphar.2020.00904 Teng R, Hu Y, Zhou J, et al (2015) Overexpression of Lin28 Decreases the Chemosensitivity of Gastric Cancer Cells to Oxaliplatin, Paclitaxel, Doxorubicin, and Fluorouracil in Part via microRNA-107. PLOS ONE 10:e0143716. https://doi.org/10.1371/journal.pone.0143716 Tian T, Zhang Y, Wang S, et al (2012) Sox2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer. J Biomed Res 26:336–345. https://doi.org/10.7555/JBR.26.20120045 Timmerman LA, Grego-Bessa J, Raya A, et al (2004) Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev 18:99–115. https://doi.org/10.1101/gad.276304 Tsou S-H, Chen T-M, Hsiao H-T, Chen Y-H (2015) A Critical Dose of Doxorubicin Is Required to Alter the Gene Expression Profiles in MCF-7 Cells Acquiring Multidrug Resistance. PLOS ONE 10:e0116747. https://doi.org/10.1371/journal.pone.0116747 Varlakhanova NV, Cotterman RF, deVries WN, et al (2010) myc maintains embryonic stem cell pluripotency and self-renewal. Differ Res Biol Divers 80:9–19. https://doi.org/10.1016/j.diff.2010.05.001 Venkatesh V, Nataraj R, Thangaraj GS, et al (2018) Targeting Notch signalling pathway of cancer stem cells. Stem Cell Investig 5: Vieira G, Chockalingam S, Melegh Z, et al (2015) LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma. In: Oncotarget. https://pubmed.ncbi.nlm.nih.gov/26517508/. Accessed 3 Dec 2020 Westermann F, Muth D, Benner A, et al (2008) Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas. Genome Biol 9:R150. https://doi.org/10.1186/gb-2008-9-10-r150 Yang J, Guo W, Wang L, et al (2017) Notch signaling is important for epithelial-mesenchymal transition induced by low concentrations of doxorubicin in osteosarcoma cell lines. Oncol Lett 13:2260–2268. https://doi.org/10.3892/ol.2017.5708 Yang L, Bai Y, Zhang C, et al (2021) Overexpression of BMP9 promotes ovarian cancer progression via Notch1 signaling. Neoplasma 68:1190–1200. https://doi.org/10.4149/neo_2021_210326N404 Yang W, Wu B, Ma N, et al (2019) BATF2 reverses multidrug resistance of human gastric cancer cells by suppressing Wnt/β-catenin signaling. Vitro Cell Dev Biol - Anim 55:445–452. https://doi.org/10.1007/s11626-019-00360-5 Yao J, Qian C (2010) Inhibition of Notch3 enhances sensitivity to gemcitabine in pancreatic cancer through an inactivation of PI3K/Akt-dependent pathway. Med Oncol 27:1017–1022. https://doi.org/10.1007/s12032-009-9326-5 Yi L, Zhou X, Li T, et al (2019) Notch1 signaling pathway promotes invasion, self-renewal and growth of glioma initiating cells via modulating chemokine system CXCL12/CXCR4. J Exp Clin Cancer Res 38:339. https://doi.org/10.1186/s13046-019-1319-4 Zhang L, Sha J, Yang G, et al (2017) Activation of Notch pathway is linked with epithelial-mesenchymal transition in prostate cancer cells. Cell Cycle Georget Tex 16:999–1007. https://doi.org/10.1080/15384101.2017.1312237 Table Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFiguresmergedJan2023.pdf Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Expression profiling of gene identified in three stem cells. Unsupervised hierarchical clustering heat map. Expression level is colour coded: red for over-expressed and green for under-expressed genes. (OVR – Overall survival and EFS – event free survival). Table S1: Supplementary Table 1: Table showing in-silico analysis of four relevant data sets from the R2: Genomics Analysis and Visualization Platform ( href="http://r2.amc.nl/">http://r2.amc.nl) confirmed that deregulated expression of our gene panel was associated with decreased overall survival in neuroblastoma patients with stage 4 disease. Table1.png Table 1: Top regulatory pathways of gene set enrichment analysis. Significance FDR < 0.05 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2471355","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":167368299,"identity":"d3bfdd99-e2d8-400b-967f-a91960997725","order_by":0,"name":"John Clark- Corrigal","email":"","orcid":"","institution":"School of Nursing and Health Sciences, University of Sunderland, UK","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"John","middleName":"Clark-","lastName":"Corrigal","suffix":""},{"id":167368300,"identity":"bd559343-e3dc-43be-ab45-16a6daf13fff","order_by":1,"name":"Masood Zaka","email":"","orcid":"","institution":"National Horizon Centre, Teesside University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masood","middleName":"","lastName":"Zaka","suffix":""},{"id":167368301,"identity":"d5d0d7c8-f252-4ba5-b1d1-a216949094f3","order_by":2,"name":"Svetlana Myssina","email":"","orcid":"","institution":"School of Nursing and Health Sciences, University of Sunderland, UK","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Svetlana","middleName":"","lastName":"Myssina","suffix":""},{"id":167368302,"identity":"89f09c78-5e07-4cc4-9869-082bd14e43aa","order_by":3,"name":"Martin Michaelis","email":"","orcid":"","institution":"School of Biosciences and Industrial Biotechnology Centre, University of Kent","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Michaelis","suffix":""},{"id":167368303,"identity":"045c5626-0655-4aa8-946f-0ba98b14a5a0","order_by":4,"name":"Jindrich Cinatl, Jr","email":"","orcid":"","institution":"UKInstitut fuer Medizinische Virologie, Klinikum der Goethe-Universitaet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jindrich","middleName":"","lastName":"Cinatl","suffix":"Jr"},{"id":167368304,"identity":"e6d37c12-cde1-4558-8366-f21dca308793","order_by":5,"name":"Shafiq Ahmed","email":"","orcid":"","institution":"School of Medicine, University of Sunderland","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shafiq","middleName":"","lastName":"Ahmed","suffix":""},{"id":167368305,"identity":"bcab040d-b11b-4ea0-becd-5d1b353b007e","order_by":6,"name":"Jane Carr-Wilkinson","email":"data:image/png;base64,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","orcid":"","institution":"School of Medicine, University of Sunderland","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jane","middleName":"","lastName":"Carr-Wilkinson","suffix":""}],"badges":[],"createdAt":"2023-01-12 13:29:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2471355/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2471355/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":31635877,"identity":"4c03ee73-978c-4684-a591-8fc59caeba41","added_by":"auto","created_at":"2023-01-16 15:30:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":240743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially expressed genes identified in three paired NB cell line models of drug resistance.\u003c/strong\u003e (A) Venn diagram showing 11 differentially expressed genes among the three cell lines. (B) Heatmap: showing elevated gene expression (Red) and decreased gene expression (green).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2471355/v1/5d602bf0085e6a9957adf4ee.png"},{"id":31635878,"identity":"bd535c2d-007b-4926-a5bc-9bd177aa213d","added_by":"auto","created_at":"2023-01-16 15:30:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":369896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) A regulatory network of shared genes.\u003c/strong\u003e MYC is highlighted in a central role with projections either directly or indirectly to other genes within the study including ABCG2, ERBB2, ITGA2, SNAI1, SOX2 and WEE1. Red colour indicating over-expression of genes and green colour represents under-expression. The grey colour are the connecting genes from database. (B) A regulatory network of shared genes. Red colour indicating over-expression of genes and green colour represents under-expression. The grey colour are the connecting genes from database.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2471355/v1/0c6ec7ea6e0743dcf27f6ac0.png"},{"id":31893915,"identity":"18a7a21d-6e15-4e60-85a5-7bfbe10191fa","added_by":"auto","created_at":"2023-01-21 08:29:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1047492,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2471355/v1/f4752fb6-631c-4827-b8e8-0f8d550220b8.pdf"},{"id":31635879,"identity":"a3453461-dba1-49cc-b66d-8a7dcf01629d","added_by":"auto","created_at":"2023-01-16 15:30:14","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":300050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u003c/strong\u003e The following are available online at www.mdpi.com/xxx/s1, Figure S1: Expression profiling of gene identified in three stem cells. Unsupervised hierarchical clustering heat map. Expression level is colour coded: red for over-expressed and green for under-expressed genes. (OVR – Overall survival and EFS – event free survival). Table S1: Supplementary Table 1: Table showing in-silico analysis of four relevant data sets from the R2: Genomics Analysis and Visualization Platform (\u003ca href=\"http://r2.amc.nl/\"\u003ehttp://r2.amc.nl\u003c/a\u003e) confirmed that deregulated expression of our gene panel was associated with decreased overall survival in neuroblastoma patients with stage 4 disease.\u003c/p\u003e","description":"","filename":"SupplementaryFiguresmergedJan2023.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2471355/v1/4c5a6265e3768b73f1e524a1.pdf"},{"id":31635876,"identity":"2a6b8ce6-48bf-4ae2-a06e-c1f1ef99ba83","added_by":"auto","created_at":"2023-01-16 15:30:14","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1: \u003c/strong\u003eTop regulatory pathways of gene set enrichment analysis. Significance FDR \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"Table1.png","url":"https://assets-eu.researchsquare.com/files/rs-2471355/v1/c22725e08ced823ea57b5ef9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stem cell pathways and Notch signaling an emerging role in the development of acquired Drug Resistance in Neuroblastoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNeuroblastoma (NB) is a childhood cancer which arises in the developing sympathetic nervous system, specifically the neuronal ganglia which derives from embryonic neural crest cells (Louis and Shohet \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the United Kingdom around 98 new cases children under the age of 15 are diagnosed annually (Public Health England \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). High risk patients, categorised by the presence of \u003cem\u003eMYCN\u003c/em\u003e amplification or in children over 18 months with metastatic disease, have less than 40% chance of long term survival (Speleman et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Tumour heterogeneity and clonal evolution are now established as characteristic hallmarks of NB, with emerging evidence suggesting that solid tumour cancer cells may not follow the hierarchical organisation of other cancer cells; instead, following clonal evolution from one subtype to another and generating distinctly recognisable tumours. This may explain the variation in clinical manifestations and the unpredictable nature in response to treatment (Ngan \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Whilst most high-risk patients do initially respond to induction multi-modal chemotherapy, unfortunately over 50% relapse, and in many cases the returning tumour cells possess a drug resistant phenotype (Keshelava et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Owens et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVCR is currently included in Rapid COJEC induction chemotherapy which comprises a combination of five agents (cisplatin, vincristine, carboplatin, etoposide and cyclophosphamide) and is currently used to treat NB patients in Europe (Smith and Foster \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Vincristine (VCR) targets cell microtubule formation (Kotchetkov et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Piskareva et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which exert integral roles in cell scaffolding as well as being regulated during cell cycle. Thus, microtubule inhibition leads to cell cycle arrest and eventual cell death by apoptosis (Kothari et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA major obstacle in the successful treatment of neuroblastoma is the acquisition of resistance to chemotherapeutic agents and hence, is the most pressing factor leading to therapy failure and relapse. Drug resistance is a problem for many cancers including neuroblastoma, with a variety of potential mechanisms driving this resistance including cancer stem cells, epigenetics and genetic alterations(Hanahan and Weinberg \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEpithelial to mesenchymal transition (EMT), is a reversible process whereby cells undergo a functional change from a fixed epithelial cell to that of a more mobile nonspecialised cell which has the capacity to differentiate into other specialised cells or self-renew, has been highlighted as an early mechanism in the development of metastasis. Furthermore, emerging evidence in literature demonstrating EMT as a causative factor in drug resistance in most cancer types (Shibue and Weinberg \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tanabe et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotch signalling is an evolutionarily conserved pathway which plays an essential role in embryo development and cell regulation, including proliferation, survival and self-renewal of stem cells. Recent research has implicated Notch signalling in promoting therapeutic resistance via cancer stem cells (CSCs)(Moore et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This small subset of the tumour population are nascent cells with innate therapeutic resistance and the capacity to restore the tumour population with resistant cells.\u003c/p\u003e \u003cp\u003eFrom this concept comes the theory that the CSCs hold not only the key to drug resistance, but also tumour spread by metastases. Like their stem cell counterpart, CSCs enhance the stemness and dedifferentiation of the tumour and promote the transition between epithelial and mesenchymal phenotype (Venkatesh et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In preclinical research across several cancer types, notch signalling activity is upregulated in brain(Cenciarelli et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), breast (McGowan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Miao et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and pancreatic cancer(Yao and Qian \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Cui et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Feng et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), conversely in colorectal cancers it is largely downregulated (Chu et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Meisel et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMany paediatric cancers have dysregulated signalling pathways which are involved in the development of their cells of origin, particularly notch, wnt and hedgehog (Filbin and Monje \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIdentification of biological drivers that contribute towards drug resistance \u003cb\u003eis\u003c/b\u003e important in order to develop more effective targeted therapeutics and essential to improving high risk patient survival (Housman et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn this study we investigated the potential role of stem cell related genes and pathways, in the development of acquired drug resistance in three paired neuroblastoma cell line models of drug resistance.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003eCell culture\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThree paired parental and drug-adapted neuroblastoma cell lines; SHSY5Y, IMR5 and IMR32 were obtained from the Resistant Cancer Cell Line (RCCL) collection (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html\u003c/span\u003e\u003cspan address=\"http://www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Cell lines were treated with 10ng/ml of VCR to generate resistant cells; SHSY5YrVCR, IMR5rVCR, IMR32rVCR. Cells were cultured using Iscove\u0026rsquo;s \u003cb\u003eDulbeco\u0026rsquo;s Modified Eagle Medium\u003c/b\u003e (DMEM) (Sigma, UK) with 10% fetal calf serum (FCS), L- glutamine and1% primocin. All cell lines were incubated at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were passaged once they reached 70\u0026ndash;80% confluency level.\u003c/p\u003e \u003cp\u003e\u003cb\u003eRNA extraction\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRNA (Ribose Nucleic Acid) extraction was performed using the RNeasy mini kit (Qiagen, uk) according to manufacturer\u0026rsquo;s instructions. RNA quality and quantification were assessed using the Nanodrop Lite Spectrophotometer (Thermo Scientific, UK) and first strand DNA synthesized using the iScript\u0026trade; cDNA synthesis kit (Bio-Rad, UK).\u003c/p\u003e \u003cp\u003e\u003cb\u003eRt2 Profiler Array\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe Human Cancer Stem Cell RT2 profiler Array analysis (Qiagen, UK) which contains primers for 96 genes related to human cancer stem cells, was performed according to protocol format R in the manufacturer\u0026rsquo;s instructions on three paired parental and acquired drug resistant cell lines. IMR32 \u0026amp; IMR32-VCR, IMR5 \u0026amp; IMR5-VCR and SHSY5Y \u0026amp; SHSY5Y-VCR. Analysis of mRNA expression levels was performed by Real Time-QPCR (Qiagen Rotorgene 100) of 96 genes related to human cancer stem cells, against a panel of three paired parental and acquired drug resistant cell lines with an acquired drug resistance to the microtubule-inhibitor; Vincristine. Fold change was assessed using the 2\u003csup\u003eΔΔct\u003c/sup\u003e method, using GAPDH and β-actin as the respective housekeeping genes.\u003c/p\u003e \u003cp\u003e\u003cb\u003eBioinformatics analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe CtData for each cell line was further tested using \u003cem\u003eLimma\u003c/em\u003e package from R Bioconductor for differential expression of genes by making linear contrast matrices for control and case cell lines. Linear models were then fitted for identification of dysregulation genes using \u003cem\u003elm()\u003c/em\u003e function from the package. Subsequent p-values were further tested for multiple testing method of false discovery rate. All genes showing p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log2FC\u0026thinsp;\u0026gt;\u0026thinsp;1 and \u0026lt; -1 were picked for overexpression and down regulation. Significant genes from three contrasts were compared for identification of common genes among the cell lines.\u003c/p\u003e \u003cp\u003e\u003cb\u003eFunctional analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFunctional enrichment analysis were performed on 11 genes using combination of different methods available. Curated gene sets from Molecular Signature Database (MSigDB), DAVID Bioinformatics Resource 6.8, GeneMania and STRING were extracted for the construction of metagene. Fgsea package of Bioconductor was used for the prediction of potential involvement of 11 genes in regulatory processes, functions and pathways. All the pathways with FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003cp\u003e\u003cb\u003eIn Silico Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll data was collected from the R2: Genomics Analysis and Visualization Platform \u003csup\u003e35\u003c/sup\u003e, using publicly available neuroblastoma patient data. Expression cut-off was determined by a log rank test, which determined an optimum cut off, rather than using an average value. R2: Genomics Analysis and Visualization Platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://r2.amc.nl\u003c/span\u003e\u003cspan address=\"http://r2.amc.nl\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eKaplan-Meir plots were constructed to compare high and low expression of the 11 differentially expressed target genes with important clinical parameters stage, overall survival and event free survival. Statistical analysis was shown using a two-tailed, unpaired student t-test with a confidence interval of 95% ) (Appendix One)\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003eThis study has generated novel data and identified several genes that are up-regulated in drug resistant cell lines; SHSY5YrVCR, IMR5rVCR and IMR32rVCR, compared with the paired parental cell line. In addition, we have also identified an important subset of genes that are down-regulated in drug resistant cell lines. Many of these candidate genes have been shown to exert a role in aggressive disease in other common cancers such as breast, prostate and small cell lung cancers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElevation of stem cell and EMT related genes in chemo-resistant cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparative gene expression analysis was undertaken following Real Time-qPCR on each parental and drug resistant cell line and showed deregulation of 11 genes across the three cell lines, \u003cem\u003eATP-binding\u003c/em\u003e cassette \u003cem\u003esub-family B member 5\u003c/em\u003e (ABCB5), \u003cem\u003eATP-binding cassette super-family G member 2\u003c/em\u003e (ABCG2), \u003cem\u003eDelta like ligand 1\u003c/em\u003e (DLL1), \u003cem\u003eErb-B2 Receptor Tyrosine Kinase 2\u003c/em\u003e (ERBB2), \u003cem\u003eIntegrin alpha 2\u003c/em\u003e (ITGA2), \u003cem\u003eLIN 28 homolog A\u003c/em\u003e (LIN28A), \u003cem\u003eLIN 28 homolog B\u003c/em\u003e (LIN28B), MYC, \u003cem\u003eZinc finger protein SNAI1\u003c/em\u003e (SNAI1), \u003cem\u003eSRY (sex determining region Y)-box 2\u003c/em\u003e (SOX2) and \u003cem\u003eWEE1 G2 Checkpoint Kinase\u003c/em\u003e (WEE1) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA.).\u003c/p\u003e\n\u003cp\u003eSubsequent unsupervised hierarchical clustering was conducted (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB.) which observed upregulation of LIN28A, ABCG5, LIN28B and DLL1 in IMR32, with downregulation of ERBB2, ABCG2, WEE1 and MYC. IMR5 displayed upregulation of ITGA2, MYC, SNAI1, WEE1 and ABCG2, with downregulation of LIN28B, ABCB5, LIN28A and SOX2. SHSY5Y highlighted upregulation of SOX2, ERBB2, ABCG2 and WEE1, downregulation of SNAI1, DLL1, ITGA2 and MYC (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics analysis shows a regulatory network highlighting EMT and stemness genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to the pathway analysis, we also created a regulatory network composed of 8 of the genes highlighted within our study. This regulatory network developed from connections with previous literature illustrates a core role for MYC, showing direct interactions with WEE1 and SOX2, links through one other gene to ABCG2, ERRBB2, DLL1, ITGA2 and SNAI1 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). In further detail you can see how many genes are influenced by the genes within our network (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene enrichment analysis highlights NOTCH signalling related pathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene set enrichment analysis on the target genes; highlighted DLL1, MYC and NOTCH1 in particular, all with a highly significant false discovery rate. The Top regulatory pathways extracted from the gene set enrichment analysis were correlated with Significance FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and this confirmed involvement of the NOTCH signalling pathway in acquired drug resistance (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ctable border=\"1\" id=\"Tab1\"\u003e\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn-silico classification further outlines roles of genes in drug resistance in established studies in other malignancies.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn-silico analysis was performed to determine whether our 11 differentially expressed target genes correlated with important clinical outcomes using four large neuroblastoma patient data sets including disease stage, overall survival (OVR) and event free survival (EFS). Kaplan-Meier plots revealed that low expression of ABCB5, ABCG2, and MYC was associated with lower overall survival in patients with stage 4 disease (INSS). Furthermore, elevated expression of ERBB2, ITGA2, LIN28A, LIN28B, SNAI1, SOX2, DLL1 and WEE1 were associated with decreased overall survival in neuroblastoma patients with stage 4 disease (Supplementary Fig. 1 and Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNeuroblastoma is a paediatric tumour that develops from embryonic neural crest cells that give rise to the sympathetic nervous system. Whilst most neuroblastomas initially respond well to induction chemotherapy, 50\u0026ndash;60% of patients with high-risk disease relapse with aggressive disease. Acquired drug resistance is a major therapeutic challenge in the treatment of such patients.\u003c/p\u003e \u003cp\u003eUsing an array of 96 genes related to cancer stem cells in three-paired parental and Vincristine drug resistant neuroblastoma cell lines, this study outlines 11 prominent genes across all cell lines; \u003cem\u003eABCG2, ERBB2, LIN28A, SOX2, WEE1 MYC, SNAI1, LIN28B, ABCB5, DLL1\u003c/em\u003e and \u003cem\u003eITAG2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Differing expressions of the same genes across the cell lines, highlights the hallmark heterogeneity of disease.\u003c/p\u003e \u003cp\u003eUsing complex bioinformatics analysis, we created a regulatory network composed of 12 genes, 8 of which were highlighted within our study and relating genes discovered from the database (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026amp;B). The network displayed a core role for MYC directly influencing genes associated with oncogenic proteins (ERBB2), self-renewal (SOX2) and cell cycle (WEE1). Indirectly, repression of MYC appears to cause over expression of hallmark drug-resistance genes such as SNAI1 and ABCG2, whilst decreasing expression of NOTCH signalling protein DLL1 and integrin protein ITGA2 (Kang et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ongaratti et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Interestingly \u003cem\u003eMYC (C-MYC)\u003c/em\u003e was associated with lower overall survival across four data sets, in contrast to \u003cem\u003eMYCN\u003c/em\u003e amplification which is strongly correlated to poor overall and event free survival in high risk neuroblastoma patients (Somasundaram et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) suggestive of a potential inverse correlation (Westermann et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhilst most other cancers display a positive correlation between \u003cem\u003eC-MYC\u003c/em\u003e overexpression and a number of the genes highlighted in our study, a number of studies have implicated that c- and \u003cem\u003eMYCN\u003c/em\u003e can compensate for loss of expression by one another in normal embryonic stem cells(ESCs), induced pluripotent stem cells(IPSCs) and neuroblastoma(Cotterman and Knoepfler \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Varlakhanova et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Nakagawa et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This may explain the findings we have discovered.\u003c/p\u003e \u003cp\u003eSimilar to our findings, a study in triple negative breast cancer, showed elevated SOX2 expression alongside overexpression of ABCG2 and the EMT marker \u003cb\u003eTwist-\u003c/b\u003erelated \u003cb\u003eprotein 1\u003c/b\u003e (TWIST1) in drug resistant cells. However, when paclitaxel was administered to SOX2 inhibited mammospheres, ABCG2 and TWIST1 were downregulated, alongside decreased expression of other EMT markers (SNAI1) which induced arrested migration and reduced formation of spheres, indicative of loss of self-renewal (Mukherjee et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This study highlights the potential of the EMT-Stemness axis in therapeutic resistance, where SOX2-dependent TWIST1 overexpression maintains stemness in addition to enabling migration. Subsequent inhibition of the axis causes regained sensitivity.\u003c/p\u003e \u003cp\u003eHere we\u0026rsquo;ve highlighted deregulation of notch signalling pathway genes in drug resistance cell lines, recent research from clinical samples showed 60% (28/46) of clinical samples from neuroblastoma patients with overexpression of NOTCH1 linked to recurrence and lower overall survival (Metovic et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEMT is a complex process, recent data has shown that Notch signalling directly influences overexpression of Snail and decreased expression of E-cadherin (Timmerman et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Saad et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Snai1 expression and loss of E-Cadherin are associated with several cancers and causing increased migration and invasion (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), interestingly a recent study in osteosarcoma found that sub-lethal doxorubicin (Dox) treatments showed significant correlation between EMT and Notch signalling in doxorubicin resistant cell lines (Yang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) which supports our findings within the context acquired drug resistance. Delta like 1 (DLL1) has also been associated with disease recurrence in other cancer types, including hepatocellular carcinoma (Ma et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and with poor prognosis in non-small cell lung cancer (Pancewicz-Wojtkiewicz et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A recent study in Neuroblastoma cell lines, showed that DLL1 was highly expressed in \u003cem\u003eMYCN\u003c/em\u003e amplified cells, and could be inhibited by miRNAs(Bettinsoli et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In aggressive medulloblastoma, NOTCH signalling pathway has shown to regulate self-renewal and metastasis, with TWIST1 and \u003cb\u003ePolycomb complex protein BMI-1 (\u003c/b\u003eBMI1) influential in NOTCH1 induced metastases (Kahn et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTwo genes, SOX2 and LIN28A are involved with stem cell pluripotency. Interestingly, Yamanaka\u0026rsquo;s seminal study on the generation of induced pluripotent stem cells (iPSCs) utilised both SOX2 and LIN28A to reprogram adult somatic cells to an undifferentiated state (Takahashi and Yamanaka \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Since then there has been an emerging interest in genes linked to stem cells and pluripotency and their role in tumorigenesis (M\u0026uuml;ller et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), however whether that role is extended to therapeutic resistance is understudied. The up-regulation of both SOX2 and LIN28A across all VCR-resistant cell lines may suggest that the acquisition of a stem cell phenotype is important for the acquisition of drug resistance, which occurs in 50% of patients with high risk tumours.\u003c/p\u003e \u003cp\u003eThis trend is not limited to VCR resistance alone however, in doxorubicin resistant gastric cancer stem cells, Sox2 and ABCG2 overexpression was associated with the dox-resistant phenotype (Tian et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) in addition to elevated LIN28B overexpression in gastric cancer cells with dox-resistance (Teng et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). DLL1 exerts a role in development of drug resistance in dox-resistant cell lines and biopsies in osteosarcoma (Pu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Interestingly, conflicting studies regarding Snai1 expression in breast adenocarcinoma cell lines in dox-resistant settings,(Lim et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tsou et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), however Lim et al. \u0026lsquo;s study highlights the importance of EMT-stemness interaction in drug resistance. Highlighting heterogeneity within the \u0026lsquo;same\u0026rsquo; cancer. A recent study conducted into whether inhibition of autophagy paired with very intense chemotherapeutic treatments can prevent development of drug resistance in NB, the initial results seem positive (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This is where the balance between vanquishing the tumour without the consequences of aggressive treatment on the patient.\u003c/p\u003e \u003cp\u003eA recent study has highlighted an axis between BMP4-wnt-notch, whereby BMP has a tumour suppressor role, this same work showed that induction of notch proteins halted growth of NB in IMR32 and SH-SY5Y (Szemes et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Low BMP4 and notch expression leads to an aggressive NB phenotype, perhaps there is a complexity within the signalling pathways that mean upregulation in notch signalling still leads to the aggressive phenotype. The research landscape has also associated wnt signalling in the development of therapeutic resistance across different cancer types(Forgham et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Vieira et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; He et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Clark-Corrigall et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This underlines the necessity of a global transcriptome of the cell lines in our study in order to further understand the molecular changes in therapeutic resistance.\u003c/p\u003e \u003cp\u003eThere have been some studies investigating the relationship between CSC phenotype and EMT (Prieto-Vila et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), where activation of stem cell related pathways directly promote the transition to a mesenchymal profile. Previously, Notch signalling was widely understood as a regulator of cell fate decisions, self \u0026ndash; renewal, maintenance of tissue stem cells and tissue wound healing but more recently as a regulator of survival and regeneration of CSCs (Capaccione and Pine \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, a definitive molecular mechanism behind the CSC-EMT relationship still remains elusive (Shibue and Weinberg \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eData from \u003cem\u003ein-silico\u003c/em\u003e analysis (Supplementary Fig.\u0026nbsp;1 \u0026amp;Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) showed that the worst probabilities for event free survival in late stage (INSS Stage 4) neuroblastoma were ERBB2 overexpression (10% after 3 years in the Versteeg data set \u0026ndash; 88, 15% reach 5 year survival in the Kocak dataset (649)), ITGA2 overexpression (0% were predicted to reach 2 year follow up in both the primary NRC dataset \u0026ndash; 283 and Versteeg dataset \u0026minus;\u0026thinsp;88), SOX2 overexpression (10% at 2 year follow up in the Versteeg dataset, 0% reached 4 year follow up Kocak dataset (649) and DLL1 overexpression (predicts 20% reach 2 year follow up in the Versteeg (88) and NRC (283). Confirmation of the link with important clinical parameters in four patient data sets further supports the potential role of these genes in the development of acquired therapeutic resistance in patients with neuroblastoma and potentially other childhood cancers.\u003c/p\u003e \u003cp\u003eFurthermore, in support of our data a landmark study by van Groningen et al investigating the role of super-enhancers on intra-tumoral heterogeneity identified two distinct lineages; adrenergic (ADRN) and mesenchymal (MES) differentiation with the Notch pathway as a key driver of dedifferentiated mesenchymal identity. In both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies MES cells were shown to have enhanced migration, be more resistant to common neuroblastoma treatments and increased prevalence of relapse disease, which reinforces the idea that treatment exerts selective pressure much like the cancer stem cell hypothesis postulates (Groningen et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Since then it has been shown that Notch 3 signalling is a key driver in the reprogramming of ADRN cells to a more mesenchymal state (Groningen et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and that MES cells are now known to utilise retinoic acid, an agent used in a maintenance dose to prevent relapse, to proliferate and mobilise (Groningen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A recent study from Newcastle has found that TOP2B expression was needed to maintain the ADRN state, and also lowered notch signalling factors like Notch 1\u0026ndash;3 (Khazeem et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).These studies show that ADRN cells can reprogram themselves and become a more dedifferentiated cell type using NOTCH signalling.\u003c/p\u003e \u003cp\u003eTo further support this mouse models, have implicated Notch signaling in the maintenance of neural stem cells maintenance in the fetal brain. Such observations from developmental biology have shown that Notch exerts similar roles which are vital to the tumourigenicity of cancer stem cells particularly in solid tumors including glioblastoma, ovarian cancer, and breast cancer (Aster et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yi et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Miao et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Unfortunately, due to the complexity of NB there is no perfect mouse model but even with the advent of iPSC based practices researchers still trying to elucidate this in neuroblastoma(Gonzalez Malagon and Liu \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur research data warrants further study into this panel of genes in acquired drug resistance in neuroblastoma.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOur study highlights a core role for MYC and NOTCH signalling associated genes in the development of acquired Vincristine drug resistance in our cell line models. Alterations to individuals within these groups of genes may have further reaching consequences to the stem cell \u0026ndash; EMT axis which drive a drug resistant phenotype.\u003c/p\u003e \u003cp\u003eOur panel of three paired Vincristine resistant cell lines provide a unique in-vitro platform to perform studies to understand the molecular mechanisms underlying the development of drug resistance. With the addition of powerful large patient tumour data sets and bioinformatics analyses, we\u0026rsquo;ve further highlighted potential for a specific network of genes between niches which have a negative impact on survival and require further research. This could subsequently lead to the discovery of new therapeutic targets that improve the survival of those with the aggressive disease. The findings implicated in this study, which include comparative analysis against microarray data, warrants further studies on the functional effects of the genes and pathways identified in this study in childhood cancers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, Jane Carr-Wilkinson and Shafiq Ahmed.; methodology, Jane Carr-Wilkinson, Martin Michaelis.\u0026nbsp;Jindrich Cinatl, jr Masood Zaka.; software, Masood Zaka.; validation, Svetlana Myssina, Martin Michaelis and John Clark-Corrigall.; formal analysis, Masood Zaka and Svetlana Myssina .; investigation, Masood Zaka and Svetlana Myssina.; resources, , Jane Carr-Wilkinson, Martin Michaelis.\u0026nbsp;Jindrich Cinatl, jr Masood Zaka.; data curation,\u0026nbsp;Masood Zaka.; writing\u0026mdash;original draft preparation, Svetlana Myssina, John Clark-Corrigall.; writing\u0026mdash;review and editing, Jane Carr-Wilkinson, Shafiq Ahmed.; visualization, Svetlana Myssina, John Clark-Corrigall.; supervision, Jane Carr-Wilkinson, Shafiq Ahmed.; project administration, Jane Carr-Wilkinson.; funding acquisition, Jane Carr-Wilkinson, Shafiq Ahmed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We would like to thank Florian Rothweiler for the administration and shipping of the cell lines used in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAster JC, Pear WS, Blacklow SC (2017) The Varied Roles of Notch in Cancer. Annu Rev Pathol Mech Dis 12:245\u0026ndash;275. https://doi.org/10.1146/annurev-pathol-052016-100127\u003c/li\u003e\n \u003cli\u003eBettinsoli P, Ferrari-Toninelli G, Bonini SA, et al (2017) Notch ligand Delta-like 1 as a novel molecular target in childhood neuroblastoma. BMC Cancer 17:. https://doi.org/10.1186/s12885-017-3340-3\u003c/li\u003e\n \u003cli\u003eCapaccione KM, Pine SR (2013) The Notch signaling pathway as a mediator of tumor survival. Carcinogenesis 34:1420\u0026ndash;1430. https://doi.org/10.1093/carcin/bgt127\u003c/li\u003e\n \u003cli\u003eCenciarelli C, Marei HE, Zonfrillo M, et al (2017) The interference of Notch1 target Hes1 affects cell growth, differentiation and invasiveness of glioblastoma stem cells through modulation of multiple oncogenic targets. Oncotarget 8:17873\u0026ndash;17886. https://doi.org/10.18632/oncotarget.15013\u003c/li\u003e\n \u003cli\u003eChen J, Imanaka N, Chen J, Griffin JD (2010) Hypoxia potentiates Notch signaling in breast cancer leading to decreased E-cadherin expression and increased cell migration and invasion. Br J Cancer 102:351\u0026ndash;360. https://doi.org/10.1038/sj.bjc.6605486\u003c/li\u003e\n \u003cli\u003eChen S, Xu Y, Chen Y, et al (2012) SOX2 Gene Regulates the Transcriptional Network of Oncogenes and Affects Tumorigenesis of Human Lung Cancer Cells. PLOS ONE 7:e36326. https://doi.org/10.1371/journal.pone.0036326\u003c/li\u003e\n \u003cli\u003eChen T, Zeng C, Li Z, et al (2022) Investigation of chemoresistance to first-line chemotherapy and its possible association with autophagy in high-risk neuroblastoma. Front Oncol 12:\u003c/li\u003e\n \u003cli\u003eChu D, Zhang Z, Zhou Y, et al (2011) Notch1 and Notch2 have opposite prognostic effects on patients with colorectal cancer. Ann Oncol 22:2440\u0026ndash;2447. https://doi.org/10.1093/annonc/mdq776\u003c/li\u003e\n \u003cli\u003eClark-Corrigall J, Myssina S, Michaelis M, et al (2022) Elevated Expression of LGR5 and WNT Signaling Factors in Neuroblastoma Cells With Acquired Drug Resistance. Cancer Invest 1\u0026ndash;10. https://doi.org/10.1080/07357907.2022.2136682\u003c/li\u003e\n \u003cli\u003eCotterman R, Knoepfler PS (2009) N-Myc Regulates Expression of Pluripotency Genes in Neuroblastoma Including lif, klf2, klf4, and lin28b. PLoS ONE 4:. https://doi.org/10.1371/journal.pone.0005799\u003c/li\u003e\n \u003cli\u003eCui L, Dong Y, Wang X, et al (2019) Downregulation of long noncoding RNA SNHG1 inhibits cell proliferation, metastasis, and invasion by suppressing the Notch-1 signaling pathway in pancreatic cancer. J Cell Biochem 120:6106\u0026ndash;6112. https://doi.org/10.1002/jcb.27897\u003c/li\u003e\n \u003cli\u003eFeng L, Wang K, Tang P, et al (2020) Deubiquitinase USP18 promotes the progression of pancreatic cancer via enhancing the Notch1-c-Myc axis. Aging 12:19273\u0026ndash;19292. https://doi.org/10.18632/aging.103760\u003c/li\u003e\n \u003cli\u003eFilbin M, Monje M (2019) Developmental origins and emerging therapeutic opportunities for childhood cancer. Nat Med 25:367\u0026ndash;376. https://doi.org/10.1038/s41591-019-0383-9\u003c/li\u003e\n \u003cli\u003eForgham H, Johnson D, Carter N, et al (2015) Stem Cell Markers in Neuroblastoma\u0026mdash;An Emerging Role for LGR5. Mol Cell Oncol 77. https://doi.org/10.3389/fcell.2015.00077\u003c/li\u003e\n \u003cli\u003eFu J, Si L, Zhuang Y, et al (2019) Wnt/\u0026beta;‑catenin inhibition reverses multidrug resistance in pediatric acute lymphoblastic leukemia. Oncol Rep 41:1387\u0026ndash;1394. https://doi.org/10.3892/or.2018.6902\u003c/li\u003e\n \u003cli\u003eGonzalez Malagon SG, Liu KJ (2022) Linking neural crest development to neuroblastoma pathology. Development 149:dev200331. https://doi.org/10.1242/dev.200331\u003c/li\u003e\n \u003cli\u003eGroningen T van, Akogul N, Westerhout EM, et al (2019) A NOTCH feed-forward loop drives reprogramming from adrenergic to mesenchymal state in neuroblastoma. Nat Commun 10:1530. https://doi.org/10.1038/s41467-019-09470-w\u003c/li\u003e\n \u003cli\u003eGroningen T van, Koster J, Valentijn LJ, et al (2017) Neuroblastoma is composed of two super-enhancer-associated differentiation states. Nat Genet 49:1261\u0026ndash;1266. https://doi.org/10.1038/ng.3899\u003c/li\u003e\n \u003cli\u003eGroningen T van, Niklasson CU, Chan A, et al (2021) An immature subset of neuroblastoma cells synthesizes retinoic acid and depends on this metabolite. bioRxiv 2021.05.18.444639. https://doi.org/10.1101/2021.05.18.444639\u003c/li\u003e\n \u003cli\u003eHanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646\u0026ndash;674. https://doi.org/10.1016/j.cell.2011.02.013\u003c/li\u003e\n \u003cli\u003eHe L, Zhu H, Zhou S, et al (2018) Wnt pathway is involved in 5-FU drug resistance of colorectal cancer cells. Exp Mol Med 50:1\u0026ndash;12. https://doi.org/10.1038/s12276-018-0128-8\u003c/li\u003e\n \u003cli\u003eHousman G, Byler S, Heerboth S, et al (2014) Drug resistance in cancer: an overview. Cancers 6:1769\u0026ndash;1792. https://doi.org/10.3390/cancers6031769\u003c/li\u003e\n \u003cli\u003eKahn SA, Wang X, Nitta RT, et al (2018) Notch1 regulates the initiation of metastasis and self-renewal of Group 3 medulloblastoma. Nat Commun 9:4121. https://doi.org/10.1038/s41467-018-06564-9\u003c/li\u003e\n \u003cli\u003eKang KW, Im YB, Go W-J, Han H-K (2009) c-Myc Amplification Altered the Gene Expression of ABC- and SLC-Transporters in Human Breast Epithelial Cells. Mol Pharm 6:627\u0026ndash;633. https://doi.org/10.1021/mp800116f\u003c/li\u003e\n \u003cli\u003eKeshelava N, Zuo JJ, Chen P, et al (2001) Loss of p53 Function Confers High-Level Multidrug Resistance in Neuroblastoma Cell Lines. Cancer Res 61:6185\u0026ndash;6193\u003c/li\u003e\n \u003cli\u003eKhazeem MM, Casement JW, Schlossmacher G, et al (2022) TOP2B Is Required to Maintain the Adrenergic Neural Phenotype and for ATRA-Induced Differentiation of SH-SY5Y Neuroblastoma Cells. Mol Neurobiol 59:5987\u0026ndash;6008. https://doi.org/10.1007/s12035-022-02949-6\u003c/li\u003e\n \u003cli\u003eKotchetkov R, Cinatl J, Blaheta R, et al (2003) Development of resistance to vincristine and doxorubicin in neuroblastoma alters malignant properties and induces additional karyotype changes: a preclinical model. Int J Cancer 104:36\u0026ndash;43. https://doi.org/10.1002/ijc.10917\u003c/li\u003e\n \u003cli\u003eKothari A, Hittelman WN, Chambers TC (2016) Cell cycle-dependent mechanisms underlie vincristine-induced death of primary acute lymphoblastic leukemia cells. Cancer Res canres.2104.2015. https://doi.org/10.1158/0008-5472.CAN-15-2104\u003c/li\u003e\n \u003cli\u003eKumar V, Vashishta M, Kong L, et al (2021) The Role of Notch, Hedgehog, and Wnt Signaling Pathways in the Resistance of Tumors to Anticancer Therapies. Front Cell Dev Biol 9:\u003c/li\u003e\n \u003cli\u003eLim S, Becker A, Zimmer A, et al (2013) SNAI1-Mediated Epithelial-Mesenchymal Transition Confers Chemoresistance and Cellular Plasticity by Regulating Genes Involved in Cell Death and Stem Cell Maintenance. PLoS ONE 8:. https://doi.org/10.1371/journal.pone.0066558\u003c/li\u003e\n \u003cli\u003eLouis CU, Shohet JM (2015) Neuroblastoma: Molecular Pathogenesis and Therapy. Annu Rev Med 66:49\u0026ndash;63. https://doi.org/10.1146/annurev-med-011514-023121\u003c/li\u003e\n \u003cli\u003eMa L, Dong P, Liu L, et al (2016) Overexpression of protein O-fucosyltransferase 1 accelerates hepatocellular carcinoma progression via the Notch signaling pathway. Biochem Biophys Res Commun 473:503\u0026ndash;510. https://doi.org/10.1016/j.bbrc.2016.03.062\u003c/li\u003e\n \u003cli\u003eMcGowan PM, Simedrea C, Ribot EJ, et al (2011) Notch1 Inhibition Alters the CD44hi/CD24lo Population and Reduces the Formation of Brain Metastases from Breast Cancer. Mol Cancer Res 9:834\u0026ndash;844. https://doi.org/10.1158/1541-7786.MCR-10-0457\u003c/li\u003e\n \u003cli\u003eMeisel CT, Porcheri C, Mitsiadis TA (2020) Cancer Stem Cells, Quo Vadis? The Notch Signaling Pathway in Tumor Initiation and Progression. Cells 9:1879. https://doi.org/10.3390/cells9081879\u003c/li\u003e\n \u003cli\u003eMetovic J, Napoli F, Osella-Abate S, et al (2022) Overexpression of INSM1, NOTCH1, NEUROD1, and YAP1 genes is associated with adverse clinical outcome in pediatric neuroblastoma. Virchows Arch. https://doi.org/10.1007/s00428-022-03406-4\u003c/li\u003e\n \u003cli\u003eMiao K, Lei JH, Valecha MV, et al (2020) NOTCH1 activation compensates BRCA1 deficiency and promotes triple-negative breast cancer formation. Nat Commun 11:3256. https://doi.org/10.1038/s41467-020-16936-9\u003c/li\u003e\n \u003cli\u003eMoore G, Annett S, McClements L, Robson T (2020) Top Notch Targeting Strategies in Cancer: A Detailed Overview of Recent Insights and Current Perspectives. Cells 9:1503. https://doi.org/10.3390/cells9061503\u003c/li\u003e\n \u003cli\u003eMukherjee P, Gupta A, Chattopadhyay D, Chatterji U (2017) Modulation of SOX2 expression delineates an end-point for paclitaxel-effectiveness in breast cancer stem cells. Sci Rep 7:. https://doi.org/10.1038/s41598-017-08971-2\u003c/li\u003e\n \u003cli\u003eM\u0026uuml;ller M, Hermann PC, Liebau S, et al (2016) The role of pluripotency factors to drive stemness in gastrointestinal cancer. Stem Cell Res 16:349\u0026ndash;357. https://doi.org/10.1016/j.scr.2016.02.005\u003c/li\u003e\n \u003cli\u003eNakagawa M, Takizawa N, Narita M, et al (2010) Promotion of direct reprogramming by transformation-deficient Myc. Proc Natl Acad Sci U S A 107:14152\u0026ndash;14157. https://doi.org/10.1073/pnas.1009374107\u003c/li\u003e\n \u003cli\u003eNgan ES-W (2015) Heterogeneity of neuroblastoma. Oncoscience 2:837\u0026ndash;838\u003c/li\u003e\n \u003cli\u003eOngaratti BR, Silva CBO, Trott G, et al (2016) Expression of merlin, NDRG2, ERBB2, and c-MYC in meningiomas: relationship with tumor grade and recurrence. Braz J Med Biol Res 49:. https://doi.org/10.1590/1414-431X20155125\u003c/li\u003e\n \u003cli\u003eOwens C, Li BK, Thomas KE, Irwin MS (2016) Surveillance imaging and radiation exposure in the detection of relapsed neuroblastoma. Pediatr Blood Cancer 63:1786\u0026ndash;1793. https://doi.org/10.1002/pbc.26099\u003c/li\u003e\n \u003cli\u003ePancewicz-Wojtkiewicz J, Eljaszewicz A, Kowalczuk O, et al (2017) Prognostic significance of Notch ligands in patients with non-small cell lung cancer. Oncol Lett 13:506\u0026ndash;510. https://doi.org/10.3892/ol.2016.5420\u003c/li\u003e\n \u003cli\u003ePiskareva O, Harvey H, Nolan J, et al (2015) The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro. Cancer Lett 364:142\u0026ndash;155. https://doi.org/10.1016/j.canlet.2015.05.004\u003c/li\u003e\n \u003cli\u003ePrieto-Vila M, Takahashi R, Usuba W, et al (2017) Drug Resistance Driven by Cancer Stem Cells and Their Niche. Int J Mol Sci 18:2574. https://doi.org/10.3390/ijms18122574\u003c/li\u003e\n \u003cli\u003ePu Y, Zhao F, Wang H, Cai S (2017) MiR-34a-5p promotes multi-chemoresistance of osteosarcoma through down-regulation of the DLL1 gene. Sci Rep 7:44218. https://doi.org/10.1038/srep44218\u003c/li\u003e\n \u003cli\u003ePublic Health England (2021) Cancer in children, teenagers and young adults cancer statistics report 2021\u003c/li\u003e\n \u003cli\u003eSaad S, Stanners SR, Yong R, et al (2010) Notch mediated epithelial to mesenchymal transformation is associated with increased expression of the Snail transcription factor. Int J Biochem Cell Biol 42:1115\u0026ndash;1122. https://doi.org/10.1016/j.biocel.2010.03.016\u003c/li\u003e\n \u003cli\u003eShibue T, Weinberg RA (2017) EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nat Rev Clin Oncol 14:611\u0026ndash;629. https://doi.org/10.1038/nrclinonc.2017.44\u003c/li\u003e\n \u003cli\u003eSmith V, Foster J (2018) High-Risk Neuroblastoma Treatment Review. Child Basel Switz 5:. https://doi.org/10.3390/children5090114\u003c/li\u003e\n \u003cli\u003eSomasundaram DB, Aravindan S, Yu Z, et al (2019) Droplet digital PCR as an alternative to FISH for MYCN amplification detection in human neuroblastoma FFPE samples. BMC Cancer 19:106. https://doi.org/10.1186/s12885-019-5306-0\u003c/li\u003e\n \u003cli\u003eSpeleman F, Park JR, Henderson TO (2016) Neuroblastoma: A Tough Nut to Crack. Am Soc Clin Oncol Educ Book Am Soc Clin Oncol Meet 35:e548-557. https://doi.org/10.14694/EDBK_159169\u003c/li\u003e\n \u003cli\u003eSun Z, Wang L, Zhou Y, et al (2020) Glioblastoma Stem Cell-Derived Exosomes Enhance Stemness and Tumorigenicity of Glioma Cells by Transferring Notch1 Protein. Cell Mol Neurobiol 40:767\u0026ndash;784. https://doi.org/10.1007/s10571-019-00771-8\u003c/li\u003e\n \u003cli\u003eSzemes M, Melegh Z, Bellamy J, et al (2020) A Wnt-BMP4 Signaling Axis Induces MSX and NOTCH Proteins and Promotes Growth Suppression and Differentiation in Neuroblastoma. Cells 9:783. https://doi.org/10.3390/cells9030783\u003c/li\u003e\n \u003cli\u003eTakahashi K, Yamanaka S (2006) Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126:663\u0026ndash;676. https://doi.org/10.1016/j.cell.2006.07.024\u003c/li\u003e\n \u003cli\u003eTanabe S, Quader S, Cabral H, Ono R (2020) Interplay of EMT and CSC in Cancer and the Potential Therapeutic Strategies. Front Pharmacol 11:. https://doi.org/10.3389/fphar.2020.00904\u003c/li\u003e\n \u003cli\u003eTeng R, Hu Y, Zhou J, et al (2015) Overexpression of Lin28 Decreases the Chemosensitivity of Gastric Cancer Cells to Oxaliplatin, Paclitaxel, Doxorubicin, and Fluorouracil in Part via microRNA-107. PLOS ONE 10:e0143716. https://doi.org/10.1371/journal.pone.0143716\u003c/li\u003e\n \u003cli\u003eTian T, Zhang Y, Wang S, et al (2012) Sox2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer. J Biomed Res 26:336\u0026ndash;345. https://doi.org/10.7555/JBR.26.20120045\u003c/li\u003e\n \u003cli\u003eTimmerman LA, Grego-Bessa J, Raya A, et al (2004) Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev 18:99\u0026ndash;115. https://doi.org/10.1101/gad.276304\u003c/li\u003e\n \u003cli\u003eTsou S-H, Chen T-M, Hsiao H-T, Chen Y-H (2015) A Critical Dose of Doxorubicin Is Required to Alter the Gene Expression Profiles in MCF-7 Cells Acquiring Multidrug Resistance. PLOS ONE 10:e0116747. https://doi.org/10.1371/journal.pone.0116747\u003c/li\u003e\n \u003cli\u003eVarlakhanova NV, Cotterman RF, deVries WN, et al (2010) myc maintains embryonic stem cell pluripotency and self-renewal. Differ Res Biol Divers 80:9\u0026ndash;19. https://doi.org/10.1016/j.diff.2010.05.001\u003c/li\u003e\n \u003cli\u003eVenkatesh V, Nataraj R, Thangaraj GS, et al (2018) Targeting Notch signalling pathway of cancer stem cells. Stem Cell Investig 5:\u003c/li\u003e\n \u003cli\u003eVieira G, Chockalingam S, Melegh Z, et al (2015) LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/\u0026beta;-catenin signalling in neuroblastoma. In: Oncotarget. https://pubmed.ncbi.nlm.nih.gov/26517508/. Accessed 3 Dec 2020\u003c/li\u003e\n \u003cli\u003eWestermann F, Muth D, Benner A, et al (2008) Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas. Genome Biol 9:R150. https://doi.org/10.1186/gb-2008-9-10-r150\u003c/li\u003e\n \u003cli\u003eYang J, Guo W, Wang L, et al (2017) Notch signaling is important for epithelial-mesenchymal transition induced by low concentrations of doxorubicin in osteosarcoma cell lines. Oncol Lett 13:2260\u0026ndash;2268. https://doi.org/10.3892/ol.2017.5708\u003c/li\u003e\n \u003cli\u003eYang L, Bai Y, Zhang C, et al (2021) Overexpression of BMP9 promotes ovarian cancer progression via Notch1 signaling. Neoplasma 68:1190\u0026ndash;1200. https://doi.org/10.4149/neo_2021_210326N404\u003c/li\u003e\n \u003cli\u003eYang W, Wu B, Ma N, et al (2019) BATF2 reverses multidrug resistance of human gastric cancer cells by suppressing Wnt/\u0026beta;-catenin signaling. Vitro Cell Dev Biol - Anim 55:445\u0026ndash;452. https://doi.org/10.1007/s11626-019-00360-5\u003c/li\u003e\n \u003cli\u003eYao J, Qian C (2010) Inhibition of Notch3 enhances sensitivity to gemcitabine in pancreatic cancer through an inactivation of PI3K/Akt-dependent pathway. Med Oncol 27:1017\u0026ndash;1022. https://doi.org/10.1007/s12032-009-9326-5\u003c/li\u003e\n \u003cli\u003eYi L, Zhou X, Li T, et al (2019) Notch1 signaling pathway promotes invasion, self-renewal and growth of glioma initiating cells via modulating chemokine system CXCL12/CXCR4. J Exp Clin Cancer Res 38:339. https://doi.org/10.1186/s13046-019-1319-4\u003c/li\u003e\n \u003cli\u003eZhang L, Sha J, Yang G, et al (2017) Activation of Notch pathway is linked with epithelial-mesenchymal transition in prostate cancer cells. Cell Cycle Georget Tex 16:999\u0026ndash;1007. https://doi.org/10.1080/15384101.2017.1312237 \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"neuroblastoma, stem-cells, drug resistance, relapse, bioinformatics, Notch","lastPublishedDoi":"10.21203/rs.3.rs-2471355/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2471355/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eNeuroblastoma is a paediatric tumour that develops from embryonal neural crest cells that give rise to the sympathetic nervous system. Aggressive high-risk disease remains a clinical challenge and despite multi-modal therapy, survival rates are poor. Most neuroblastomas initially respond well to induction chemotherapy however, 50-60% of patients with high-risk disease will relapse with aggressive disease. A major obstacle in the successful treatment of this disease is the development of acquired resistance to chemotherapeutic agents. We hypothesize that aggressive neuroblastomas acquire a more immature phenotype in addition to increased expression of genes linked to stem cell pathways.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: In this study we investigated the role of stem cell related genes in the development of acquired drug resistance using Q- Real Time PCR and bioinformatics analysis on three-paired vincristine sensitive and resistant cell lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The study outlines 11 differentially expressed genes with several targets involved primarily in notch signalling, and stem cell development.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe findings implicated in this study, which include comparative analysis against patient microarray data, warrants further study on the functional effects of these genes/pathways to elucidate their role in acquired drug resistance.\u003c/p\u003e","manuscriptTitle":"Stem cell pathways and Notch signaling an emerging role in the development of acquired Drug Resistance in Neuroblastoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-16 15:30:09","doi":"10.21203/rs.3.rs-2471355/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c367f5b4-794c-486a-9440-5bab8df51e5f","owner":[],"postedDate":"January 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-01-21T08:29:18+00:00","versionOfRecord":[],"versionCreatedAt":"2023-01-16 15:30:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2471355","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2471355","identity":"rs-2471355","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}