Utility of gene expression studies in relation to radiation exposure and clinical outcomes: thyroid cancer in the Ukrainian-American cohort and late health effects in a MAYAK worker cohort.

PURPOSE: We herein report on changes in gene expression after radiation exposure to iodine-131 from the Chernobyl accident in the Ukrainian-American thyroid cohort and to external gamma ray or internal plutonium exposure in the Mayak Production Association radiation workers. MATERIALS AND METHODS: Taking advantage of access to tissue samples from the thyroid cancer cases in the Ukrainian-American cohort, our group tried to identify candidate genes to discriminate spontaneously occurring thyroid cancers from thyroid cancers caused by radiation exposure. We also examined gene expression changes in normal and cancerous thyroid tissue in relation to iodine-131 dose separately. Gene expression changes in the peripheral blood of radiation exposed Mayak workers were examined to elucidate the dose-to-gene and gene-to-health (e.g. cardiovascular disease) relationships. CONCLUSIONS: Results of both projects are discussed under the aspect of dose-response relationships (dose-to-gene) and clinical outcome relationships (gene-to-effect) in light of how mechanistic data can be translated into actionable knowledge for radiation protection or clinical purposes.

Transcriptome network of the papillary thyroid carcinoma radiation marker CLIP2.

BACKGROUND: We present a functional gene association network of the CLIP2 gene, generated by de-novo reconstruction from transcriptomic microarray data. CLIP2 was previously identified as a potential marker for radiation induced papillary thyroid carcinoma (PTC) of young patients in the aftermath of the Chernobyl reactor accident. Considering the rising thyroid cancer incidence rates in western societies, potentially related to medical radiation exposure, the functional characterization of CLIP2 is of relevance and contributes to the knowledge about radiation-induced thyroid malignancies. METHODS: We generated a transcriptomic mRNA expression data set from a CLIP2-perturbed thyroid cancer cell line (TPC-1) with induced CLIP2 mRNA overexpression and siRNA knockdown, respectively, followed by gene-association network reconstruction using the partial correlation-based approach GeneNet. Furthermore, we investigated different approaches for prioritizing differentially expressed genes for network reconstruction and compared the resulting networks with existing functional interaction networks from the Reactome, Biogrid and STRING databases. The derived CLIP2 interaction partners were validated on transcript and protein level. RESULTS: The best reconstructed network with regard to selection parameters contained a set of 20 genes in the 1st neighborhood of CLIP2 and suggests involvement of CLIP2 in the biological processes DNA repair/maintenance, chromosomal instability, promotion of proliferation and metastasis. Peptidylprolyl Isomerase Like 3 (PPIL3), previously identified as a potential direct interaction partner of CLIP2, was confirmed in this study by co-expression at the transcript and protein level. CONCLUSION: In our study we present an optimized preselection approach for genes subjected to gene-association network reconstruction, which was applied to CLIP2 perturbation transcriptome data of a thyroid cancer cell culture model. Our data support the potential carcinogenic role of CLIP2 overexpression in radiation-induced PTC and further suggest potential interaction partners of the gene.

Single-center versus multi-center data sets for molecular prognostic modeling: a simulation study.

BACKGROUND: Prognostic models based on high-dimensional omics data generated from clinical patient samples, such as tumor tissues or biopsies, are increasingly used for prognosis of radio-therapeutic success. The model development process requires two independent discovery and validation data sets. Each of them may contain samples collected in a single center or a collection of samples from multiple centers. Multi-center data tend to be more heterogeneous than single-center data but are less affected by potential site-specific biases. Optimal use of limited data resources for discovery and validation with respect to the expected success of a study requires dispassionate, objective decision-making. In this work, we addressed the impact of the choice of single-center and multi-center data as discovery and validation data sets, and assessed how this impact depends on the three data characteristics signal strength, number of informative features and sample size. METHODS: We set up a simulation study to quantify the predictive performance of a model trained and validated on different combinations of in silico single-center and multi-center data. The standard bioinformatical analysis workflow of batch correction, feature selection and parameter estimation was emulated. For the determination of model quality, four measures were used: false discovery rate, prediction error, chance of successful validation (significant correlation of predicted and true validation data outcome) and model calibration. RESULTS: In agreement with literature about generalizability of signatures, prognostic models fitted to multi-center data consistently outperformed their single-center counterparts when the prediction error was the quality criterion of interest. However, for low signal strengths and small sample sizes, single-center discovery sets showed superior performance with respect to false discovery rate and chance of successful validation. CONCLUSIONS: With regard to decision making, this simulation study underlines the importance of study aims being defined precisely a priori. Minimization of the prediction error requires multi-center discovery data, whereas single-center data are preferable with respect to false discovery rate and chance of successful validation when the expected signal or sample size is low. In contrast, the choice of validation data solely affects the quality of the estimator of the prediction error, which was more precise on multi-center validation data.

Epithelial-type systemic breast carcinoma cells with a restricted mesenchymal transition are a major source of metastasis.

Carcinoma cells undergo epithelial-mesenchymal transition (EMT); however, contributions of EMT heterogeneity to disease progression remain a matter of debate. Here, we addressed the EMT status of ex vivo cultured circulating and disseminated tumor cells (CTCs/DTCs) in a syngeneic mouse model of metastatic breast cancer (MBC). Epithelial-type CTCs with a restricted mesenchymal transition had the strongest lung metastases formation ability, whereas mesenchymal-type CTCs showed limited metastatic ability. EpCAM expression served as a surrogate marker to evaluate the EMT heterogeneity of clinical samples from MBC, including metastases, CTCs, and DTCs. The proportion of epithelial-type CTCs, and especially DTCs, correlated with distant metastases and poorer outcome of patients with MBC. This study fosters our understanding of EMT in metastasis and underpins heterogeneous EMT phenotypes as important parameters for tumor prognosis and treatment. We further suggest that EpCAM-dependent CTC isolation systems will underestimate CTC numbers but will quantify clinically relevant metastatic cells.

A Five-MicroRNA Signature Predicts Survival and Disease Control of Patients with Head and Neck Cancer Negative for HPV Infection.

PURPOSE: Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is associated with unfavorable prognosis, while independent prognostic markers remain to be defined. EXPERIMENTAL DESIGN: We retrospectively performed miRNA expression profiling. Patients were operated for locally advanced HPV-negative HNSCC and had received radiochemotherapy in eight different hospitals (DKTK-ROG; n = 85). Selection fulfilled comparable demographic, treatment, and follow-up characteristics. Findings were validated in an independent single-center patient sample (LMU-KKG; n = 77). A prognostic miRNA signature was developed for freedom from recurrence and tested for other endpoints. Recursive-partitioning analysis was performed on the miRNA signature, tumor and nodal stage, and extracapsular nodal spread. Technical validation used qRT-PCR. An miRNA-mRNA target network was generated and analyzed. RESULTS: For DKTK-ROG and LMU-KKG patients, the median follow-up was 5.1 and 5.3 years, and the 5-year freedom from recurrence rate was 63.5% and 75.3%, respectively. A five-miRNA signature (hsa-let-7g-3p, hsa-miR-6508-5p, hsa-miR-210-5p, hsa-miR-4306, and hsa-miR-7161-3p) predicted freedom from recurrence in DKTK-ROG [hazard ratio (HR) 4.42; 95% confidence interval (CI), 1.98-9.88, P < 0.001], which was confirmed in LMU-KKG (HR 4.24; 95% CI, 1.40-12.81, P = 0.005). The signature also predicted overall survival (HR 3.03; 95% CI, 1.50-6.12, P = 0.001), recurrence-free survival (HR 3.16; 95% CI, 1.65-6.04, P < 0.001), and disease-specific survival (HR 5.12; 95% CI, 1.88-13.92, P < 0.001), all confirmed in LMU-KKG data. Adjustment for relevant covariates maintained the miRNA signature predicting all endpoints. Recursive-partitioning analysis of both samples combined classified patients into low (n = 17), low-intermediate (n = 80), high-intermediate (n = 48), or high risk (n = 17) for recurrence (P < 0.001). CONCLUSIONS: The five-miRNA signature is a strong and independent prognostic factor for disease recurrence and survival of patients with HPV-negative HNSCC.See related commentary by Clump et al., p. 1441.

A genomic copy number signature predicts radiation exposure in post-Chernobyl breast cancer.

Breast cancer is the second leading cause of cancer death among women worldwide and besides life style, age and genetic risk factors, exposure to ionizing radiation is known to increase the risk for breast cancer. Further, DNA copy number alterations (CNAs), which can result from radiation-induced double-strand breaks, are frequently occurring in breast cancer cells. We set out to identify a signature of CNAs discriminating breast cancers from radiation-exposed and non-exposed female patients. We analyzed resected breast cancer tissues from 68 exposed female Chernobyl clean-up workers and evacuees and 68 matched non-exposed control patients for CNAs by array comparative genomic hybridization analysis (aCGH). Using a stepwise forward-backward selection approach a non-complex CNA signature, that is, less than ten features, was identified in the training data set, which could be subsequently validated in the validation data set (p value < 0.05). The signature consisted of nine copy number regions located on chromosomal bands 7q11.22-11.23, 7q21.3, 16q24.3, 17q21.31, 20p11.23-11.21, 1p21.1, 2q35, 2q35, 6p22.2. The signature was independent of any clinical characteristics of the patients. In all, we identified a CNA signature that has the potential to allow identification of radiation-associated breast cancer at the individual level.

Gain-of-function analysis of cis-acting diversification elements in DT40 cells.

Activation-induced cytidine deaminase (AID) is required for the immunoglobulin diversification processes of somatic hypermutation, gene conversion and class-switch recombination. The targeting of AID's deamination activity is thought to be a combination of cis- and trans-acting elements, but has not been fully elucidated. Deletion analysis of putative proximal cis-regulatory motifs, while helpful, fails to identify additive versus cumulative effects, redundancy, and may create new motifs where none previously existed. In contrast, gain-of-function analysis can be more insightful with fewer of the same drawbacks and the output is a positive result. Here, we show five defined DNA regions of the avian Igλ locus that are sufficient to confer events of hypermutation to a target gene. In our analysis, the essential cis-targeting elements fully reconstituted diversification of a transgene under heterologous promotion in the avian B-cell line DT40. Furthermore, to the best of our knowledge two of the five regions we report on here have not previously been described as individually having an influence on somatic hypermutation.

Lifetime study in mice after acute low-dose ionizing radiation: a multifactorial study with special focus on cataract risk.

Because of the increasing application of ionizing radiation in medicine, quantitative data on effects of low-dose radiation are needed to optimize radiation protection, particularly with respect to cataract development. Using mice as mammalian animal model, we applied a single dose of 0, 0.063, 0.125 and 0.5 Gy at 10 weeks of age, determined lens opacities for up to 2 years and compared it with overall survival, cytogenetic alterations and cancer development. The highest dose was significantly associated with increased body weight and reduced survival rate. Chromosomal aberrations in bone marrow cells showed a dose-dependent increase 12 months after irradiation. Pathological screening indicated a dose-dependent risk for several types of tumors. Scheimpflug imaging of the lens revealed a significant dose-dependent effect of 1% of lens opacity. Comparison of different biological end points demonstrated long-term effects of low-dose irradiation for several biological end points.

Expression of miRNA-26b-5p and its target TRPS1 is associated with radiation exposure in post-Chernobyl breast cancer.

Ionizing radiation is a well-recognized risk factor for the development of breast cancer. However, it is unknown whether radiation-specific molecular oncogenic mechanisms exist. We investigated post-Chernobyl breast cancers from radiation-exposed female clean-up workers and nonexposed controls for molecular changes. Radiation-associated alterations identified in the discovery cohort (n = 38) were subsequently validated in a second cohort (n = 39). Increased expression of hsa-miR-26b-5p was associated with radiation exposure in both of the cohorts. Moreover, downregulation of the TRPS1 protein, which is a transcriptional target of hsa-miR-26b-5p, was associated with radiation exposure. As TRPS1 overexpression is common in sporadic breast cancer, its observed downregulation in radiation-associated breast cancer warrants clarification of the specific functional role of TRPS1 in the radiation context. For this purpose, the impact of TRPS1 on the transcriptome was characterized in two radiation-transformed breast cell culture models after siRNA-knockdown. Deregulated genes upon TRPS1 knockdown were associated with DNA-repair, cell cycle, mitosis, cell migration, angiogenesis and EMT pathways. Furthermore, we identified the interaction partners of TRPS1 from the transcriptomic correlation networks derived from gene expression data on radiation-transformed breast cell culture models and sporadic breast cancer tissues provided by the TCGA database. The genes correlating with TRPS1 in the radiation-transformed breast cell lines were primarily linked to DNA damage response and chromosome segregation, while the transcriptional interaction partners in the sporadic breast cancers were mostly associated with apoptosis. Thus, upregulation of hsa-miR-26b-5p and downregulation of TRPS1 in radiation-associated breast cancer tissue samples suggests these molecules representing radiation markers in breast cancer.

Genomic amplification of Fanconi anemia complementation group A (FancA) in head and neck squamous cell carcinoma (HNSCC): Cellular mechanisms of radioresistance and clinical relevance.

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.

Natural Cubic Spline Regression Modeling Followed by Dynamic Network Reconstruction for the Identification of Radiation-Sensitivity Gene Association Networks from Time-Course Transcriptome Data.

Gene expression time-course experiments allow to study the dynamics of transcriptomic changes in cells exposed to different stimuli. However, most approaches for the reconstruction of gene association networks (GANs) do not propose prior-selection approaches tailored to time-course transcriptome data. Here, we present a workflow for the identification of GANs from time-course data using prior selection of genes differentially expressed over time identified by natural cubic spline regression modeling (NCSRM). The workflow comprises three major steps: 1) the identification of differentially expressed genes from time-course expression data by employing NCSRM, 2) the use of regularized dynamic partial correlation as implemented in GeneNet to infer GANs from differentially expressed genes and 3) the identification and functional characterization of the key nodes in the reconstructed networks. The approach was applied on a time-resolved transcriptome data set of radiation-perturbed cell culture models of non-tumor cells with normal and increased radiation sensitivity. NCSRM detected significantly more genes than another commonly used method for time-course transcriptome analysis (BETR). While most genes detected with BETR were also detected with NCSRM the false-detection rate of NCSRM was low (3%). The GANs reconstructed from genes detected with NCSRM showed a better overlap with the interactome network Reactome compared to GANs derived from BETR detected genes. After exposure to 1 Gy the normal sensitive cells showed only sparse response compared to cells with increased sensitivity, which exhibited a strong response mainly of genes related to the senescence pathway. After exposure to 10 Gy the response of the normal sensitive cells was mainly associated with senescence and that of cells with increased sensitivity with apoptosis. We discuss these results in a clinical context and underline the impact of senescence-associated pathways in acute radiation response of normal cells. The workflow of this novel approach is implemented in the open-source Bioconductor R-package splineTimeR.

uPAR enhances malignant potential of triple-negative breast cancer by directly interacting with uPA and IGF1R.

BACKGROUND: Due to lack of a targeted therapy for the triple-negative breast cancer (TNBC) patients, it is important to explore this aggressive breast cancer type in more detail and to establish novel therapeutic approaches. TNBC is defined negative for the protein expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). One prominent feature of this cancer type is the frequent overexpression of major components of the urokinase-type plasminogen activator system (uPAS) including uPA, its receptor uPAR and the inhibitor PAI-1, which may be valuable as therapeutic targets. METHODS: Direct interactions of uPAR with interactors were demonstrated by immunoprecipitations and proximity ligation assays. For stable knockdowns of target proteins, lentiviral vectors were used and the effects were analysed by immunoblottings and using in vitro cell viability, migration and invasion assays. Immunohistochemical and statistical analyses of biomarkers and clinical parameters were conducted in a TNBC cohort (n = 174). RESULTS: Direct tumour-promoting interactions of uPAR with uPA and the insulin-like growth factor receptor 1 (IGF1R) were shown in TNBC cells and these interactions were significantly reduced (p = 0.001) when uPAR was downregulated. The combined knockdown of uPAR and uPA or IGF1R additively and significantly reduced cell viability, migration and invasion of the model cell lines. In TNBC tissue, the complexes formed by uPAR with uPA or with IGF1R significantly correlated with the histological grade (p = 0.0019) as well as with cathepsin B and D (p ≤ 0.0001) that are implicated in cell invasion and metastasis. CONCLUSIONS: Our outcomes show that not only overexpressed biomarkers promote tumourigenesis, but rather their interactions further potentiate tumour progression. This study emphasises the potential of combined approaches targeting uPAR and its interactors with regard to an improved therapy of TNBC.

Positive Cofactor 4 (PC4) is critical for DNA repair pathway re-routing in DT40 cells.

PC4 is an abundant single-strand DNA binding protein that has been implicated in transcription and DNA repair. Here, we show that PC4 is involved in the cellular DNA damage response. To elucidate the role, we used the DT40 chicken B cell model, which produces clustered DNA lesions at Ig loci via the action of activation-induced deaminase. Our results help resolve key aspects of immunoglobulin diversification and suggest an essential role of PC4 in repair pathway choice. We show that PC4 ablation in gene conversion (GC)-active cells significantly disrupts GC but has little to no effect on targeted homologous recombination. In agreement, the global double-strand break repair response, as measured by γH2AX foci analysis, is unperturbed 16 hours post irradiation. In cells with the pseudo-genes removed (GC inactive), PC4 ablation reduced the overall mutation rate while simultaneously increasing the transversion mutation ratio. By tagging the N-terminus of PC4, gene conversion and somatic hypermutation are all but abolished even when native non-tagged PC4 is present, indicating a dominant negative effect. Our data point to a very early and deterministic role for PC4 in DNA repair pathway re-routing.

Depletion of Histone Demethylase Jarid1A Resulting in Histone Hyperacetylation and Radiation Sensitivity Does Not Affect DNA Double-Strand Break Repair.

Histone demethylases have recently gained interest as potential targets in cancer treatment and several histone demethylases have been implicated in the DNA damage response. We investigated the effects of siRNA-mediated depletion of histone demethylase Jarid1A (KDM5A, RBP2), which demethylates transcription activating tri- and dimethylated lysine 4 at histone H3 (H3K4me3/me2), on growth characteristics and cellular response to radiation in several cancer cell lines. In unirradiated cells Jarid1A depletion lead to histone hyperacetylation while not affecting cell growth. In irradiated cells, depletion of Jarid1A significantly increased cellular radiosensitivity. Unexpectedly, the hyperacetylation phenotype did not lead to disturbed accumulation of DNA damage response and repair factors 53BP1, BRCA1, or Rad51 at damage sites, nor did it influence resolution of radiation-induced foci or rejoining of reporter constructs. We conclude that the radiation sensitivity observed following depletion of Jarid1A is not caused by a deficiency in repair of DNA double-strand breaks.

Cyr61 and YB-1 are novel interacting partners of uPAR and elevate the malignancy of triple-negative breast cancer.

The triple-negative breast cancer (TNBC) is a very aggressive tumor type often occurring in young women and is associated with a bad prognosis for the patients. TNBC lacks established targets for breast cancer therapy, such as the estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). Therefore, novel therapeutic targets and strategies are needed for an improved treatment of this breast cancer subtype. TNBC and respective cell lines often overexpress proteins of the urokinase plasminogen activator system (uPAS) including uPA, its receptor uPAR and inhibitor PAI-1, which together with co-factors contribute to the malignancy of TNBC. Here, two novel interacting partners of uPAR, the cysteine-rich angiogenic inducer 61 (Cyr61) and the Y-box-binding protein 1 (YB-1) were identified and their differential expression demonstrated in TNBC cells as well as in tumors. In the TNBC cohort, both interactors significantly correlated with expression levels of cathepsin B, c-Met and the tumor grade. In addition, expression levels of Cyr61 significantly correlated with cathepsin D (p=0.03), insulin receptor (p≤0.001), insulin-like growth factor receptor 1 (IGF1R, p=0.015) and also with YB-1 (p=0.0004) levels. The interactions of uPAR with Cyr61 significantly correlated with expression levels of tumor-promoting biomarkers including plasminogen (p=0.0014), cathepsin B (p=0.032), c-Met (p=0.0192) as well as with the tumor grade (p=0.02). In multivariate survival analysis, YB-1 showed independent prognostic value (p=0.01). As the novel interacting partners, also together with uPAR, contribute to tumor progression and metastasis, both may be potential therapeutic targets in breast cancer.

CFAssay: statistical analysis of the colony formation assay.

BACKGROUND: Colony formation assay is the gold standard to determine cell reproductive death after treatment with ionizing radiation, applied for different cell lines or in combination with other treatment modalities. Associated linear-quadratic cell survival curves can be calculated with different methods. For easy code exchange and methodological standardisation among collaborating laboratories a software package CFAssay for R (R Core Team, R: A Language and Environment for Statistical Computing, 2014) was established to perform thorough statistical analysis of linear-quadratic cell survival curves after treatment with ionizing radiation and of two-way designs of experiments with chemical treatments only. METHODS: CFAssay offers maximum likelihood and related methods by default and the least squares or weighted least squares method can be optionally chosen. A test for comparision of cell survival curves and an ANOVA test for experimental two-way designs are provided. RESULTS: For the two presented examples estimated parameters do not differ much between maximum-likelihood and least squares. However the dispersion parameter of the quasi-likelihood method is much more sensitive for statistical variation in the data than the multiple R (2) coefficient of determination from the least squares method. CONCLUSION: The dispersion parameter for goodness of fit and different plot functions in CFAssay help to evaluate experimental data quality. As open source software interlaboratory code sharing between users is facilitated. AVAILABILITY: The package is available at http://www.bioconductor.org/packages/release/bioc/html/CFAssay.html .

Genomic copy number analysis of Chernobyl papillary thyroid carcinoma in the Ukrainian-American Cohort.

One of the major consequences of the 1986 Chernobyl reactor accident was a dramatic increase in papillary thyroid carcinoma (PTC) incidence, predominantly in patients exposed to the radioiodine fallout at young age. The present study is the first on genomic copy number alterations (CNAs) of PTCs of the Ukrainian-American cohort (UkrAm) generated by array comparative genomic hybridization (aCGH). Unsupervised hierarchical clustering of CNA profiles revealed a significant enrichment of a subgroup of patients with female gender, long latency (>17 years) and negative lymph node status. Further, we identified single CNAs that were significantly associated with latency, gender, radiation dose and BRAF V600E mutation status. Multivariate analysis revealed no interactions but additive effects of parameters gender, latency and dose on CNAs. The previously identified radiation-associated gain of the chromosomal bands 7q11.22-11.23 was present in 29% of cases. Moreover, comparison of our radiation-associated PTC data set with the TCGA data set on sporadic PTCs revealed altered copy numbers of the tumor driver genes NF2 and CHEK2. Further, we integrated the CNA data with transcriptomic data that were available on a subset of the herein analyzed cohort and did not find statistically significant associations between the two molecular layers. However, applying hierarchical clustering on a 'BRAF-like/RAS-like' transcriptome signature split the cases into four groups, one of which containing all BRAF-positive cases validating the signature in an independent data set.

Association of radiation-induced genes with noncancer chronic diseases in Mayak workers occupationally exposed to prolonged radiation.

We examined the association of gene expression with noncancer chronic disease outcomes in Mayak nuclear weapons plant workers who were exposed to radiation due to their occupation. We conducted a cross-sectional study with selection based on radiation exposure status of Mayak plant workers living in Ozyorsk who were alive in 2011 and either exposed to: combined incorporated Plutonium-239 ((239)Pu) and external gamma-ray exposure (n = 82); external gamma-ray exposure alone (n = 18); or were unexposed (n = 50) of Ozyorsk residents who provided community-based professional support for plant personnel and who were alive in 2011. Peripheral blood was taken and RNA was isolated and then converted into cDNA and stored at -20°C. In a previous analysis we screened the whole genome for radiation-associated candidate genes, and validated 15 mRNAs and 15 microRNAs using qRT-PCR. In the current analysis we examined the association of these genes with 15 different chronic diseases on 92 samples (47 males, 45 females). We examined the radiation-to-gene and gene-to-disease associations in statistical models stratified by gender and separately for each disease and exposure. We modeled radiation exposure as gamma or (239)Pu on both the continuous and categorical scales. Unconditional logistic regression was used to calculate odds ratios (OR), 95% confidence intervals (CI), and the concordance for genes that were significantly associated with radiation exposure and a specific disease outcome were identified. Altogether 12 mRNAs and 9 microRNAs appeared to be significantly associated with 6 diseases, including thyroid diseases (3 genes, OR: 1.2-5.1, concordance: 71-78%), atherosclerotic diseases (4 genes, OR: 2.5-10, concordance: 70-75%), kidney diseases (6 genes, OR: 1.3-8.6, concordance: 69-85%), cholelithiasis (3 genes, OR: 0.2-0.3, concordance: 74-75%), benign tumors [1 gene (AGAP4), OR: 3.7, concordance: 81%] and chronic radiation syndrome (4 genes, OR: 2.5-4.3, concordance: 70-99%). Further associations were found for systolic blood pressure (6 genes, OR: 3.7-10.6, concordance: 81-88%) and body mass index [1 gene (miR-484), OR: 3.7, concordance: 81%]. All associations were gender and exposure dependent. These findings suggest that gene expression changes observed after occupational prolonged radiation exposures may increase the risk for certain noncancer chronic diseases.

The impact of cysteine-rich intestinal protein 1 (CRIP1) in human breast cancer.

BACKGROUND: CRIP1 (cysteine-rich intestinal protein 1) has been found in several tumor types, its prognostic impact and its role in cellular processes, particularly in breast cancer, are still unclear. METHODS: To elucidate the prognostic impact of CRIP1, we analyzed tissues from 113 primary invasive ductal breast carcinomas using immunohistochemistry. For the functional characterization of CRIP1, its endogenous expression was transiently downregulated in T47D and BT474 breast cancer cells and the effects analyzed by immunoblotting, WST-1 proliferation assay and invasion assay. RESULTS: We found a significant correlation between CRIP1 and HER2 (human epidermal growth factor receptor 2) expression levels (p = 0.016) in tumor tissues. In Kaplan Meier analyses, CRIP1 expression was significantly associated with the distant metastases-free survival of patients, revealing a better prognosis for high CRIP1 expression (p = 0.039). Moreover, in multivariate survival analyses, the expression of CRIP1 was an independent negative prognostic factor, along with the positive prognosticators nodal status and tumor size (p = 0.029). CRIP1 knockdown in the T47D and BT474 breast cancer cell lines led to the increased phosphorylation of MAPK and Akt, to the reduced phosphorylation of cdc2, and to a significantly elevated cell proliferation in vitro (p < 0.001). These results indicate that reduced CRIP1 levels may increase cell proliferation and activate cell growth. In addition, CRIP1 knockdown increased cell invasion in vitro. CONCLUSIONS: Because the lack of CRIP1 expression in breast cancer tissue is significantly associated with a worse prognosis for patients and low endogenous CRIP1 levels in vitro increased the malignant potential of breast cancer cells, we hypothesize that CRIP1 may act as a tumor suppressor in proliferation and invasion processes. Therefore, CRIP1 may be an independent prognostic marker with significant predictive power for use in breast cancer therapy.

Clinical response to chemotherapy in oesophageal adenocarcinoma patients is linked to defects in mitochondria.

Chemotherapeutic drugs kill cancer cells, but it is unclear why this happens in responding patients but not in non-responders. Proteomic profiles of patients with oesophageal adenocarcinoma may be helpful in predicting response and selecting more effective treatment strategies. In this study, pretherapeutic oesophageal adenocarcinoma biopsies were analysed for proteomic changes associated with response to chemotherapy by MALDI imaging mass spectrometry. Resulting candidate proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and investigated for functional relevance in vitro. Clinical impact was validated in pretherapeutic biopsies from an independent patient cohort. Studies on the incidence of these defects in other solid tumours were included. We discovered that clinical response to cisplatin correlated with pre-existing defects in the mitochondrial respiratory chain complexes of cancer cells, caused by loss of specific cytochrome c oxidase (COX) subunits. Knockdown of a COX protein altered chemosensitivity in vitro, increasing the propensity of cancer cells to undergo cell death following cisplatin treatment. In an independent validation, patients with reduced COX protein expression prior to treatment exhibited favourable clinical outcomes to chemotherapy, whereas tumours with unchanged COX expression were chemoresistant. In conclusion, previously undiscovered pre-existing defects in mitochondrial respiratory complexes cause cancer cells to become chemosensitive: mitochondrial defects lower the cells' threshold for undergoing cell death in response to cisplatin. By contrast, cancer cells with intact mitochondrial respiratory complexes are chemoresistant and have a high threshold for cisplatin-induced cell death. This connection between mitochondrial respiration and chemosensitivity is relevant to anticancer therapeutics that target the mitochondrial electron transport chain.