title: Transcriptomic analyses of the radiation response in head and neck squamous cell carcinoma subclones with different radiation sensitivity: time-course gene expression profiles and gene association networks.
abstract: BACKGROUND
  Acquired and inherent radioresistance of tumor cells is related to tumor relapse and poor prognosis - not only in head and neck squamous cell carcinoma (HNSCC). The underlying molecular mechanisms are largely unknown. Therefore, systemic in-depth analyses are needed to identify key regulators of radioresistance. In the present study, subclones of the CAL-33 HNSCC cell line with different radiosensitivity were analyzed to identify signaling pathways related to the different phenotypes.
  
  
  METHODS
  Subclones with altered radiosensitivity were generated by fractionated irradiation of the parental CAL-33 cells. Differences in radiosensitivity were confirmed in colony formation assays. Selected subclones were characterized at the genomic and transcriptomic level by SKY, array CGH, and mRNA-microarray analyses. Time-course gene expression analyses upon irradiation using a natural cubic spline regression model identified temporally differentially expressed genes. Moreover, early and late responding genes were identified. Gene association networks were reconstructed using partial correlation. The Reactome pathway database was employed to conduct pathway enrichment analyses.
  
  
  RESULTS
  The characterization of two subclones with enhanced radiation resistance (RP) and enhanced radiosensitivity (SP) revealed distinct genomic and transcriptomic changes compared to the parental cells. Differentially expressed genes after irradiation shared by both subclones pointed to important pathways of the early and late radiation response, including senescence, apoptosis, DNA repair, Wnt, PI3K/AKT, and Rho GTPase signaling. The analysis of the most important nodes of the gene association networks revealed pathways specific to the radiation response in different phenotypes of radiosensitivity. Exemplarily, for the RP subclone the senescence-associated secretory phenotype (SASP) together with GPCR ligand binding were considered as crucial. Also, the expression of endogenous retrovirus ERV3-1in response to irradiation has been observed, and the related gene association networks have been identified.
  
  
  CONCLUSIONS
  Our study presents comprehensive gene expression data of CAL-33 subclones with different radiation sensitivity. The resulting networks and pathways associated with the resistant phenotype are of special interest and include the SASP. The radiation-associated expression of ERV3-1 also appears highly attractive for further studies of the molecular mechanisms underlying acquired radioresistance. The identified pathways may represent key players of radioresistance, which could serve as potential targets for molecularly designed, therapeutical intervention.
authors.affiliation: Research Unit Radiation Cytogenetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, 85764, Neuherberg, Germany.
authors.forename: Agata
authors.initials: A
authors.lastname: Michna
authors.affiliation: Department of Radiotherapy and Radiation Oncology, Ludwig-Maximilians-University, 81377, Munich, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Schötz
authors.affiliation: Research Unit Radiation Cytogenetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, 85764, Neuherberg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Selmansberger
authors.affiliation: Research Unit Radiation Cytogenetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, 85764, Neuherberg, Germany.|Clinical Cooperation Group "Personalized Radiotherapy in Head and Neck Cancer", Helmholtz Zentrum München, 85764, Neuherberg, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: Zitzelsberger
authors.affiliation: Department of Radiotherapy and Radiation Oncology, Ludwig-Maximilians-University, 81377, Munich, Germany.|Clinical Cooperation Group "Personalized Radiotherapy in Head and Neck Cancer", Helmholtz Zentrum München, 85764, Neuherberg, Germany.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Lauber
authors.affiliation: Research Unit Radiation Cytogenetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, 85764, Neuherberg, Germany.|Clinical Cooperation Group "Personalized Radiotherapy in Head and Neck Cancer", Helmholtz Zentrum München, 85764, Neuherberg, Germany.
authors.forename: Kristian
authors.initials: K
authors.lastname: Unger
authors.affiliation: Research Unit Radiation Cytogenetics, Helmholtz Zentrum München, German Research Center for Environmental Health GmbH, 85764, Neuherberg, Germany. julia.hess@helmholtz-muenchen.de.|Clinical Cooperation Group "Personalized Radiotherapy in Head and Neck Cancer", Helmholtz Zentrum München, 85764, Neuherberg, Germany. julia.hess@helmholtz-muenchen.de.
authors.forename: Julia
authors.initials: J
authors.lastname: Hess
mesh_terms: D017209:Apoptosis / Q000528:radiation effects; D014408:Biomarkers, Tumor / Q000235:genetics*; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology / Q000532:radiotherapy; D002453:Cell Cycle / Q000528:radiation effects; D049109:Cell Proliferation / Q000528:radiation effects; D005720:Gamma Rays; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D053263:Gene Regulatory Networks; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000473:pathology / Q000532:radiotherapy; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics; D011836:Radiation Tolerance / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D059467:Transcriptome / Q000528:radiation effects*; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor; D012333:RNA, Messenger
keywords: Differentially expressed genes; Endogenous retrovirus; Gene association network; HNSCC; Head and neck cancer; Radioresistance; Signaling pathway; Time-course gene expression