ABSTRACT
Radio (chemo) therapy is a crucial treatment modality for head and neck squamous cell carcinoma (HNSCC), but relapse is frequent, and the underlying mechanisms remain largely elusive. Therefore, novel biomarkers are urgently needed. Previously, we identified gains on 16q23-24 to be associated with amplification of the Fanconi anemia A (FancA) gene and to correlate with reduced progression-free survival after radiotherapy. Here, we analyzed the effects of FancA on radiation sensitivity in vitro, characterized the underlying mechanisms, and evaluated their clinical relevance. Silencing of FancA expression in HNSCC cell lines with genomic gains on 16q23-24 resulted in significantly impaired clonogenic survival upon irradiation. Conversely, overexpression of FancA in immortalized keratinocytes conferred increased survival accompanied by improved DNA repair, reduced accumulation of chromosomal translocations, but no hyperactivation of the FA/BRCA-pathway. Downregulation of interferon signaling as identified by microarray analyses, enforced irradiation-induced senescence, and elevated production of the senescence-associated secretory phenotype (SASP) appeared to be candidate mechanisms contributing to FancA-mediated radioresistance. Data of the TCGA HNSCC cohort confirmed the association of gains on 16q24.3 with FancA overexpression and impaired overall survival. Importantly, transcriptomic alterations similar to those observed upon FancA overexpression in vitro strengthened the clinical relevance. Overall, FancA amplification and overexpression appear to be crucial for radiotherapeutic failure in HNSCC.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/radiotherapy , Fanconi Anemia Complementation Group A Protein/genetics , Gene Amplification , Head and Neck Neoplasms/radiotherapy , Radiation Tolerance/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cellular Senescence/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Disease-Free Survival , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Genotype , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/mortality , Head and Neck Neoplasms/pathology , Humans , Kaplan-Meier Estimate , Keratinocytes/pathology , Keratinocytes/radiation effects , Oligonucleotide Array Sequence Analysis , Phenotype , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Squamous Cell Carcinoma of Head and Neck , Time Factors , Transfection , Treatment Failure , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a very heterogeneous disease resulting in huge differences in the treatment response. New individualized therapy strategies including molecular targeting might help to improve treatment success. In order to identify potential targets, we developed a HNSCC radiochemotherapy cell culture model of primary HNSCC cells derived from two different patients (HN1957 and HN2092) and applied an integrative microRNA (miRNA) and mRNA analysis in order to gain information on the biological networks and processes of the cellular therapy response. We further identified potential target genes of four therapy-responsive miRNAs detected previously in the circulation of HNSCC patients by pathway enrichment analysis. RESULTS: The two primary cell cultures differ in global copy number alterations and P53 mutational status, thus reflecting heterogeneity of HNSCC. However, they also share many copy number alterations and chromosomal rearrangements as well as deregulated therapy-responsive miRNAs and mRNAs. Accordingly, six common therapy-responsive pathways (direct P53 effectors, apoptotic execution phase, DNA damage/telomere stress induced senescence, cholesterol biosynthesis, unfolded protein response, dissolution of fibrin clot) were identified in both cell cultures based on deregulated mRNAs. However, inflammatory pathways represented an important part of the treatment response only in HN1957, pointing to differences in the treatment responses of the two primary cultures. Focused analysis of target genes of four therapy-responsive circulating miRNAs, identified in a previous study on HNSCC patients, revealed a major impact on the pathways direct P53 effectors, the E2F transcription factor network and pathways in cancer (mainly represented by the PTEN/AKT signaling pathway). CONCLUSIONS: The integrative analysis combining miRNA expression, mRNA expression and the related cellular pathways revealed that the majority of radiochemotherapy-responsive pathways in primary HNSCC cells are related to cell cycle, proliferation, cell death and stress response (including inflammation). Despite the heterogeneity of HNSCC, the two primary cell cultures exhibited strong similarities in the treatment response. The findings of our study suggest potential therapeutic targets in the E2F transcription factor network and the PTEN/AKT signaling pathway.
Subject(s)
Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/therapy , Chemoradiotherapy , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/therapy , MicroRNAs/genetics , Aged , Aged, 80 and over , Cell Line, Tumor , Cluster Analysis , Female , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Male , MicroRNAs/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Signal Transduction/genetics , Squamous Cell Carcinoma of Head and NeckABSTRACT
Previous quantitative proteomic studies on the actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells, a cell model frequently used for investigating the mechanisms of TCDD toxicity, had indicated that dioxin exposure reduced the abundance of numerous proteins which are regulated at the level of protein synthesis initiation. In the present study, we have analysed the mechanism mediating this inhibition. TCDD treatment of the cells largely prevented the activation of eukaryotic translation initiation factor 4E-binding protein 1, a regulator of translation initiation and substrate of the mammalian target of rapamycin (mTOR). By "working upwards" from mTOR, we observed that TCDD inhibited endogenous and IGF-I-induced AKT and ERK activation by interfering with tyrosine phosphorylation of insulin receptor substrate 1. This inhibition was mediated by a TCDD-induced secreted factor which was identified as insulin-like growth factor binding protein 4 (IGFBP-4). The induction of IGFBP-4 protein was dependent on a functional aryl hydrocarbon receptor and was preceded by a rapid increase in the level of IGFBP-4 mRNA indicating that IGFBP-4 is a previously unknown transcriptional target of TCDD in 5L cells. IGFBP-4 was not induced by TCDD in the parental cell line of 5L cells, Fao, and in various closely related rat hepatoma cell lines as well as in other unrelated cell types. Analysis of 5L cell chromosomes by multicolour spectral karyotyping (SKY) revealed that the cells carry several hitherto uncharacterised chromosomal translocations. The observations suggest that in 5L cells the Igfbp-4 gene may have got under the control of a promoter containing dioxin responsive element(s) leading to the induction of IGFBP-4 by TCDD. These findings emphasise a particular caution when interpreting and extrapolating results on the action mechanisms of TCDD obtained in studies using 5L cells as a model system.
Subject(s)
Gene Expression Regulation, Neoplastic , Insulin-Like Growth Factor Binding Protein 4/biosynthesis , Liver Neoplasms, Experimental/metabolism , Polychlorinated Dibenzodioxins/toxicity , Animals , Cell Line, Tumor , Dioxins/toxicity , Gene Expression Regulation, Neoplastic/drug effects , Insulin-Like Growth Factor Binding Protein 4/genetics , Liver Neoplasms, Experimental/genetics , RatsABSTRACT
Epidemiological data show that ionising radiation increases the risk of cardiovascular disease. The endothelium is one of the main targets of radiation-induced damage. Rapid radiation-induced alterations in the biological processes were investigated after exposure to a clinically relevant radiation dose (2.5 Gy gamma radiation). The changes in protein expression were determined using the human endothelial cell line EA.hy926 as a model. Two complementary proteomic approaches, SILAC (Stable Isotope Labelling with Amino acids in Cell culture) and 2D-DIGE (Two Dimensional Difference-in-Gel-Electrophoresis) were used. The proteomes of the endothelial cells were analysed 4h and 24h after irradiation. Differentially expressed proteins were identified and quantified by MALDI-TOF/TOF and LTQ Orbitrap tandem mass spectrometry. The deregulated proteins were mainly categorised in four key pathways: (i) glycolysis/gluconeogenesis and synthesis/degradation of ketone bodies, (ii) oxidative phosphorylation, (iii) Rho-mediated cell motility and (iv) non-homologous end joining. We suggest that these alterations facilitate the repair processes needed to overcome the stress caused by irradiation and are indicative of the vascular damage leading to radiation-induced cardio- and cerebrovascular impairment.
Subject(s)
Endothelial Cells/chemistry , Endothelial Cells/radiation effects , Isotope Labeling/methods , Proteome/analysis , Two-Dimensional Difference Gel Electrophoresis/methods , Amino Acids , Cell Culture Techniques , Cell Proliferation/radiation effects , Cells, Cultured , Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/chemistry , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/radiation effects , Humans , Metabolic Networks and Pathways/physiology , Metabolic Networks and Pathways/radiation effects , Models, Biological , Proteome/metabolism , Proteomics/methods , Radiation Injuries/metabolism , Signal Transduction/radiation effects , Validation Studies as TopicABSTRACT
Recurrent translocations are well known hallmarks of many human solid tumors and hematological disorders, where patient- and breakpoint-specific information may facilitate prognostication and individualized therapy. In thyroid carcinomas, the proto-oncogenes RET and NTRK1 are often found to be activated through chromosomal rearrangements. However, many sporadic tumors and papillary thyroid carcinomas (PTCs) arising in patients with a history of exposure to elevated levels of ionizing irradiation do not carry these known abnormalities. We developed a rapid scheme to screen tumor cell metaphase spreads and identify candidate genes of tumorigenesis and neoplastic progression for subsequent functional studies. Using a series of overnight fluorescence in situ hybridization (FISH) experiments with pools comprised of bacterial artificial chromosome (BAC) clones, it now becomes possible to rapidly refine breakpoint maps and, within one week, progress from the low resolution Spectral Karyotyping (SKY) maps or Giemsa-banding (G-banding) karyotypes to fully integrated, high resolution physical maps including a list of candiate genes in the critical regions.
ABSTRACT
Structural genomic rearrangements are frequent findings in human cancers. Therefore, papillary thyroid carcinomas (PTCs) were investigated for chromosomal aberrations and rearrangements of the RET proto-oncogene. For this purpose, primary cultures from 23 PTC have been established and metaphase preparations were analysed by spectral karyotyping (SKY). In addition, interphase cell preparations of the same cases were investigated by fluorescence in situ hybridisation (FISH) for the presence of RET/PTC rearrangements using RET-specific DNA probes. SKY analysis of PTC revealed structural aberrations of chromosome 11 and several numerical aberrations with frequent loss of chromosomes 20, 21, and 22. FISH analysis for RET/PTC rearrangements showed prevalence of this rearrangement in 72% (16 out of 22) of cases. However, only subpopulations of tumour cells exhibited this rearrangement indicating genetic heterogeneity. The comparison of visual and automated scoring of FISH signals revealed concordant results in 19 out of 22 cases (87%) indicating reliable scoring results using the optimised scoring parameter for RET/PTC with the automated Metafer4 system. It can be concluded from this study that genomic rearrangements are frequent in PTC and therefore important events in thyroid carcinogenesis.
Subject(s)
Chernobyl Nuclear Accident , Chromosome Aberrations , Gene Rearrangement , Neoplasms, Radiation-Induced/genetics , Adolescent , Adult , Carcinoma , Carcinoma, Papillary , Chi-Square Distribution , Child , Female , Humans , Image Processing, Computer-Assisted , In Situ Hybridization, Fluorescence , Male , Proto-Oncogene Mas , Proto-Oncogene Proteins c-ret/genetics , Spectral Karyotyping , Thyroid Cancer, Papillary , Thyroid Neoplasms/genetics , Tumor Cells, CulturedABSTRACT
Epithelial cell lines were established from the transition and peripheral zones of human prostate by transduction with cdk4 and hTERT. The properties of these lines were investigated using immunocytochemical markers, ability to generate anchorage-independent colonies and by spectral karyotyping (SKY). Cells were exposed to fractionated doses of gamma irradiation to investigate their ability to transform. Cell lines were established from the transition and peripheral zones of human prostate. The expression of CD133, CK5, CK14, CK18, p16, PSCA, p63 and c-myc varied between the lines from the two regions. The line derived from the peripheral zone exhibited properties of a tumour line. A similar pattern was observed in two separate transductions. It was thus unlikely to be an in vitro transformation event, which is very rarely observed with human cells in vitro, and thus more likely to be derived from the immortalisation of a quiescent tumour clone. Fractionated irradiation of the transition zone cell line resulted in forming of transformed colonies. The transformed and tumour line had marked chromosomal rearrangements as demonstrated by SKY analysis. Cell lines have been derived from different zones of human prostate for studies on radiation carcinogenesis. The unirradiated cell line derived from the peripheral zone exhibited chromosomal rearrangements similar to those observed in prostate carcinoma. The cell line derived from the transitional zone exhibited a near diploid karyotype and could be transformed following exposure to fractionated doses of gamma irradiation.
Subject(s)
Cell Line , Cell Transformation, Neoplastic/genetics , Prostate/cytology , Cell Line/cytology , Cell Line/metabolism , Chromosome Aberrations , Cyclin-Dependent Kinase Inhibitor p16/genetics , Humans , Immunohistochemistry , Male , Spectral Karyotyping , Telomerase/genetics , Transduction, GeneticABSTRACT
Cytogenetic analysis of head and neck squamous cell carcinoma (HNSCC) established several biomarkers that have been correlated to clinical parameters during the past years. Adequate cell culture model systems are required for functional studies investigating those potential prognostic markers in HNSCC. We have used a cell line, CAL 33, for the establishment of a cell culture model in order to perform functional analyses of interesting candidate genes and proteins. The cell line was cytogenetically characterized using array CGH, spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH). As a starting point for the investigation of genetic markers predicting radiosensitivity in tumor cells, irradiation experiments were carried out and radiation responses of CAL 33 have been determined. Radiosensitivity of CAL 33 cells was intermediate when compared to published data on tumor cell lines.
ABSTRACT
Chromosomal copy number alterations and chromosomal rearrangements are frequent mutations in human cancer. Unlike copy number alterations, little is known about the role and occurrence of chromosomal rearrangements in breast cancer. This may be due to the fact that chromosome-based breakpoint analysis is widely restricted to cultured cells. In order to identify gene rearrangements in breast cancer, we studied the chromosomal breakpoints in radiation-transformed epithelial breast cell lines using a high-resolution array-based approach using 1 Mb bacterial artificial chromosome (BAC) arrays. The breakpoints were further narrowed down by fluorescence in situ hybridisation (FISH) with clones from the 32 k BAC library. The analysis of the cell lines B42-11 and B42-16 revealed rearrangements of chromosomes 7, 8, 10 and 12. We identified the genes Has2, Grid1, Ret, Cpm, Tbx3, Tbx5, Tuba1a, Wnt1 and Arf3 within the breakpoint regions. Quantitative RT-PCR showed a deregulated expression of all of these candidate genes except for Tbx5 and Tbx3. This is the first study demonstrating gene rearrangements and their deregulated mRNA expression in radiation-transformed breast cells. Since the gene rearrangements occurred in the transformed and tumourigenic cell lines only, it is likely that these were generated in conjunction with malignant transformation of the epithelial breast cells and therefore might reflect early molecular events in breast carcinogenesis. Initial studies indicate that these gene alterations are also found in sporadic breast cancers.
Subject(s)
Breast Neoplasms/genetics , Breast/ultrastructure , Cell Transformation, Neoplastic/genetics , Chromosome Aberrations , Chromosomes, Human/radiation effects , Genes, Neoplasm , Genetic Association Studies , Oncogene Proteins, Fusion/genetics , Animals , Breast/radiation effects , Breast Neoplasms/ultrastructure , Cell Line, Transformed/ultrastructure , Cell Transformation, Neoplastic/radiation effects , Chromosome Painting , Chromosomes, Artificial, Bacterial/genetics , Chromosomes, Human/ultrastructure , Comparative Genomic Hybridization , Epithelial Cells/metabolism , Epithelial Cells/radiation effects , Epithelial Cells/ultrastructure , Female , Gamma Rays/adverse effects , Gene Dosage , Gene Library , Humans , Mice , Mice, Nude , Oligonucleotide Array Sequence Analysis , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/isolation & purification , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Spectral KaryotypingABSTRACT
Neoplastic transformation induced by ionizing radiation was studied using a human retinal pigment epithelial cell line immortalized by telomerase. Radiation-transformed cell clones were tumorigenic in athymic mice and were analyzed by G-banding and comparative genomic hybridization (CGH). Radiation-transformed cloned cell lines and cell lines derived from tumors produced in athymic nude mice following transplantation exhibited a recurrent karyotype:45,XX,der(10),-13. CGH showed an amplification of 10p11.2 and a deletion of the remaining 10p. Positional cloning of the amplified region by FISH analysis and subsequent sequence analysis of BAC clones showing amplified FISH signals identified the candidate gene PARD3. This gene also was found to be transcriptionally expressed at an increased level. The findings indicate that PARD3 may play an important role in radiation-induced carcinogenesis of RPE cells. This is the first evidence for PARD3 amplification in human cancer cells.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Gene Amplification/genetics , Neoplasms, Radiation-Induced/genetics , Pigment Epithelium of Eye/pathology , Protein Kinase C/metabolism , Retinal Neoplasms/etiology , Retinal Neoplasms/genetics , Animals , Caenorhabditis elegans Proteins/physiology , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Chromosome Deletion , Chromosome Mapping/methods , Chromosomes, Human, Pair 10/genetics , Cytogenetic Analysis/methods , Humans , Mice , Mice, Nude , Nucleic Acid Hybridization , Protein Binding/physiology , Protein Serine-Threonine Kinases , Sequence Homology, Nucleic AcidABSTRACT
Tissue samples from 60 post-Chernobyl childhood thyroid tumors have been investigated. We used comparative genomic hybridization (CGH) to detect chromosomal gains and losses within the tumor DNA. This is the first CGH study on childhood thyroid tumors. The post-Chernobyl tumors showed chromosomal imbalances in 30% of tumors. The most frequent DNA copy number changes in post-Chernobyl tumors involved chromosomes 2, 7q11.2-21, 13q21-22, 21 (DNA gains), and chromosomes 16p/q, 20q, 22q (DNA losses). Some of these specific alterations detected in post-Chernobyl thyroid tumors (deletions on chromosomes 16p/q and 22q) have previously been reported in thyroid tumors as associated with an aggressive biologic behavior and may therefore also account for the more aggressive phenotype of papillary thyroid carcinoma (PTC) found in post- Chernobyl tumors. Eighteen percent of post-Chernobyl PTC that exhibit RET rearrangements also showed chromosomal imbalances indicating that either additional genetic events are involved in this subset of tumors, or that intratumoral genetic heterogeneity exists in these tumors, suggesting a oligoclonal pattern to tumor development.