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postgraduate thesis: Functional roles of NFATc2 in regulating tumor-initiating cells of non-small-cell lung cancer

TitleFunctional roles of NFATc2 in regulating tumor-initiating cells of non-small-cell lung cancer
Authors
Issue Date2016
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Xiao, Z. [肖芷洁]. (2016). Functional roles of NFATc2 in regulating tumor-initiating cells of non-small-cell lung cancer. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractLung cancer is the most common cause of cancer deaths worldwide due to ineffective therapy and drug resistance. Within the bulk of heterogeneous tumor cell populations, tumor initiating cells (TIC) are those capable of self-renewal and drug resistance, contributing to aggressive cancer phenotypes. Stem cell-like properties can be acquired through stress-induced cell plasticity while calcium signaling integrates cellular responses to intra- and extracellular stresses. Being a major calcium signaling mediator, calcineurin/NFAT pathway involvement in lung TIC perpetuation remains unknown. This study aims at investigating the role and mechanisms of the calcineurin/NFAT pathway in regulating lung cancer TIC phenotypes. Participation of the calcineurin/NFAT axis in TIC regulation was first demonstrated using multiple pharmacological and genetic approaches, where disruption of the axis led to suppressed TIC characteristics. Analysis of NFAT family member expressions in non-small cell lung cancer (NSCLC) cell lines then identified NFATc2 as a likely candidate, while the statistically significant association of high NFATc2 expression with poor differentiation and adverse survival of human NSCLC supports its clinical relevance. Subsequently, NFATc2 overexpression and activation in TIC surrogates including tumor spheres and the ALDH+/CD44+ population was confirmed by q-PCR, western blot and luciferase reporter assays. Using lentivirus-based stable knockdown or overexpression experiments, the roles of NFATc2 in regulating pluripotency genes and TIC markers, sphere formation and cell mobility were shown in vitro. In vivo, xenograft models and limiting dilution assays revealed NFATc2 is involved in tumorigenesis. Together, results indicate NFATc2 supports lung TIC perpetuation. To elucidate the molecular mechanisms, the relation with pluripotency genes was investigated. SOX2 expression was identified to be significantly correlated with that of NFATc2 in both NSCLC cells and clinical lung adenocarcinomas. Using luciferase reporter assay, site-directed mutagenesis and ChIP-qPCR assay, it was found NFATc2 transcriptionally regulates SOX2 through enhancer binding. Its involvement in TIC phenotypes was further demonstrated using knockdown experiments in vitro and in vivo. As a known stem cell marker, ALDH1A1 was investigated as a downstream candidate of NFATc2/SOX2 coupling. Indeed, it was observed ALDH1A1 is upregulated by NFATc2 through SOX2. Immunohistochemistry (IHC) further revealed SOX2 expression positively correlates with that of ALDH1A1 in human lung cancer. The role of NFATc2/SOX2/ALDH1A1 axis in drug resistance was next studied, and demonstrated by simultaneous cytotoxic and targeted drugs treatment both in vitro and in vivo. In stable drug resistant cell lines, NFATc2 is activated and required for maintaining drug resistance. Since reactive oxygen species (ROS) homeostasis is a major mechanism of drug resistance and ALDH is known to alleviate oxidative stress, the role of NFATc2 /SOX2/ALDH1A1 in ROS regulation was studied. As measured by flow cytometry, down-regulation of NFATc2 induced, but overexpression of NFATc2 suppressed ROS level, which was reversed by down-regulation of SOX2 and ALDH1A1. Treatment of ROS scavenger preserved the TIC phenotypes that were inhibited by NFATc2 knockdown. In conclusion, NFATc2 is overexpressed and active in lung cancers, in which SOX2 enhancer binding and upregulation facilitates TIC phenotypes and contributes to drug resistance through ROS scavenging by involving the SOX2/ALDH1A1 axis.
DegreeDoctor of Philosophy
SubjectLungs - Cancer
Transcription factors
Dept/ProgramPathology
Persistent Identifierhttp://hdl.handle.net/10722/270269

 

DC FieldValueLanguage
dc.contributor.authorXiao, Zhijie-
dc.contributor.author肖芷洁-
dc.date.accessioned2019-05-23T02:26:25Z-
dc.date.available2019-05-23T02:26:25Z-
dc.date.issued2016-
dc.identifier.citationXiao, Z. [肖芷洁]. (2016). Functional roles of NFATc2 in regulating tumor-initiating cells of non-small-cell lung cancer. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/270269-
dc.description.abstractLung cancer is the most common cause of cancer deaths worldwide due to ineffective therapy and drug resistance. Within the bulk of heterogeneous tumor cell populations, tumor initiating cells (TIC) are those capable of self-renewal and drug resistance, contributing to aggressive cancer phenotypes. Stem cell-like properties can be acquired through stress-induced cell plasticity while calcium signaling integrates cellular responses to intra- and extracellular stresses. Being a major calcium signaling mediator, calcineurin/NFAT pathway involvement in lung TIC perpetuation remains unknown. This study aims at investigating the role and mechanisms of the calcineurin/NFAT pathway in regulating lung cancer TIC phenotypes. Participation of the calcineurin/NFAT axis in TIC regulation was first demonstrated using multiple pharmacological and genetic approaches, where disruption of the axis led to suppressed TIC characteristics. Analysis of NFAT family member expressions in non-small cell lung cancer (NSCLC) cell lines then identified NFATc2 as a likely candidate, while the statistically significant association of high NFATc2 expression with poor differentiation and adverse survival of human NSCLC supports its clinical relevance. Subsequently, NFATc2 overexpression and activation in TIC surrogates including tumor spheres and the ALDH+/CD44+ population was confirmed by q-PCR, western blot and luciferase reporter assays. Using lentivirus-based stable knockdown or overexpression experiments, the roles of NFATc2 in regulating pluripotency genes and TIC markers, sphere formation and cell mobility were shown in vitro. In vivo, xenograft models and limiting dilution assays revealed NFATc2 is involved in tumorigenesis. Together, results indicate NFATc2 supports lung TIC perpetuation. To elucidate the molecular mechanisms, the relation with pluripotency genes was investigated. SOX2 expression was identified to be significantly correlated with that of NFATc2 in both NSCLC cells and clinical lung adenocarcinomas. Using luciferase reporter assay, site-directed mutagenesis and ChIP-qPCR assay, it was found NFATc2 transcriptionally regulates SOX2 through enhancer binding. Its involvement in TIC phenotypes was further demonstrated using knockdown experiments in vitro and in vivo. As a known stem cell marker, ALDH1A1 was investigated as a downstream candidate of NFATc2/SOX2 coupling. Indeed, it was observed ALDH1A1 is upregulated by NFATc2 through SOX2. Immunohistochemistry (IHC) further revealed SOX2 expression positively correlates with that of ALDH1A1 in human lung cancer. The role of NFATc2/SOX2/ALDH1A1 axis in drug resistance was next studied, and demonstrated by simultaneous cytotoxic and targeted drugs treatment both in vitro and in vivo. In stable drug resistant cell lines, NFATc2 is activated and required for maintaining drug resistance. Since reactive oxygen species (ROS) homeostasis is a major mechanism of drug resistance and ALDH is known to alleviate oxidative stress, the role of NFATc2 /SOX2/ALDH1A1 in ROS regulation was studied. As measured by flow cytometry, down-regulation of NFATc2 induced, but overexpression of NFATc2 suppressed ROS level, which was reversed by down-regulation of SOX2 and ALDH1A1. Treatment of ROS scavenger preserved the TIC phenotypes that were inhibited by NFATc2 knockdown. In conclusion, NFATc2 is overexpressed and active in lung cancers, in which SOX2 enhancer binding and upregulation facilitates TIC phenotypes and contributes to drug resistance through ROS scavenging by involving the SOX2/ALDH1A1 axis. -
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.subject.lcshLungs - Cancer-
dc.subject.lcshTranscription factors-
dc.titleFunctional roles of NFATc2 in regulating tumor-initiating cells of non-small-cell lung cancer-
dc.typePG_Thesis-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplinePathology-
dc.description.naturepublished_or_final_version-
dc.date.hkucongregation2017-
dc.identifier.mmsid991044104198303414-

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