Radiation Response Pathways Research Articles

Transcriptomes of Wet Skin Biopsies Predict Outcomes after Ionizing Radiation Exposure with Potential Dosimetric Applications in a Mouse Model.

Countermeasures for radiation diagnosis, prognosis, and treatment are trailing behind the proliferation of nuclear energy and weaponry. Radiation injury mechanisms at the systems biology level are not fully understood. Here, mice skin biopsies at h2, d4, d7, d21, and d28 after exposure to 1, 3, 6, or 20 Gy whole-body ionizing radiation were evaluated for the potential application of transcriptional alterations in radiation diagnosis and prognosis. Exposure to 20 Gy was lethal by d7, while mice who received 1, 3, or 6 Gy survived the 28-day time course. A Sammon plot separated samples based on survival and time points (TPs) within lethal (20 Gy) and sublethal doses. The differences in the numbers, regulation mode, and fold change of significantly differentially transcribed genes (SDTGs, p < 0.05 and FC > 2) were identified between lethal and sublethal doses, and down and upregulation dominated transcriptomes during the first post-exposure week, respectively. The numbers of SDTGs and the percentages of upregulated ones revealed stationary downregulation post-lethal dose in contrast to responses to sublethal doses which were dynamic and largely upregulated. Longitudinal up/downregulated SDTGs ratios suggested delayed and extended responses with increasing IR doses in the sublethal range and lethal-like responses in late TPs. This was supported by the distributions of common and unique genes across TPs within each dose. Several genes with potential dosimetric marker applications were identified. Immune, fibrosis, detoxification, hematological, neurological, gastric, cell survival, migration, and proliferation radiation response pathways were identified, with the majority predicted to be activated after sublethal and inactivated after lethal exposures, particularly during the first post-exposure week.

Dynamic Tumor Mutational Profiling Using a Novel Non-Invasive Swab Technique for Serial Whole Exome Sequencing of Cervical Tumors During Chemoradiation Therapy

Clinically relevant genetic predictors of radiation response for cervical cancer are understudied due to the morbidity of repeat invasive biopsies required to obtain genetic material. We have developed a novel noninvasive cervical swab technique to collect tumor DNA with adequate throughput to perform whole exome sequencing (WES) at serial timepoints over the course of chemoradiation therapy (CRT). We hypothesize that characterization of dynamic changes in the mutational landscape of cervical tumors during the course of CRT will allow for identification of molecular signatures correlating with therapy response.A total of 69 patients with FIGO stage IB-IVA cervical cancer who underwent definitive CRT were prospectively enrolled on an IRB approved clinical trial. Cervical tumor DNA samples were collected by a noninvasive swab technique at baseline, and after 1, 3, and 5 weeks of CRT. Matched buccal DNA swabs were obtained to filter somatic mutations for each patient. An initial cohort included 24 patients (81 samples) and a second cohort included 45 patients (135 samples). WES was performed on the Illumina HiSeq 4000 series. Raw sequencing data were aligned to a human reference genome and MuTect was applied to detect somatic single nucleotide variants (SNV). Tumor purity (TP) was estimated from the distribution of variant allelic fractions of SNVs. In patients with pelvic MRIs at baseline and week 5 of CRT, 3D tumor volumes (TV) were generated (n = 6,8). Regression analysis was used to evaluate the goodness of fit between TP and TV.Estimated TP was highly correlated with TV in both cohorts (R2 = 0.969 and 0.858). Between the two cohorts, there was overlap in 4 of the top 5 genes (80%) containing the greatest number of exonic mutations across all timepoints (PIK3CA, MUC4, TPRXL, TTN). The number of altered genes present at baseline decreased over time in both cohorts. When normalized to TP, relative mutational burden decreased among standard and exceptional responders (< 20% TV remaining at week 5 of CRT) and increased among poor responders (> 20% TV remaining at week 5). The top ranked genes by magnitude of change at week 5 included MUC2, PARP10 and PIK3CA. Pathway analysis of these top ranked genes mapped to key radiation response pathways including regulation of the G1/S transition.We validated this method of performing serial WES over the course of CRT in 2 independent prospective patient cohorts, highlighting the robustness of our novel noninvasive technique. This allowed for the identification of a panel of potential genes involved in CRT resistance. Future directions include examination of dynamic changes in copy number variation, indels, and tumor mutational burden towards a comprehensive analysis of clinically relevant drivers of CRT resistance. This may allow future targeted treatment for patients with a radioresistant genetic profile, or concurrent targeted therapy for tumors with actionable mutations.

A gene expression-based immune content predictor for survival and postoperative radiotherapy response in head and neck cancer

The immune infiltration in the tumor microenvironment has been demonstrated to be relevant to radiotherapy response. Here, we sought to understand the immune infiltration in head and neck cancer (HNC) and evaluate its significance in predicting prognosis and radiotherapy response. Using RNA sequencing data of 522 retrospective head and neck squamous cell carcinomas (HNSCCs), we constructed an immune content score based on genes related to 6 prognostic infiltrating cell types. Unsupervised hallmark pathway clustering demonstrated an immune-related tumor cluster containing the immune content score. Patients with high immune content scores exhibited favorable overall survival and disease-free survival (DFS). Moreover, the immune content score was an independent prognostic factor for DFS in HNSCC. Interestingly, the immune content score was strongly associated with radiation response pathways. These results were also extended to nasopharyngeal carcinoma. Furthermore, patients in the low immune content score group significantly gained overall survival benefits from postoperative radiotherapy (PORT), whereas patients in the high immune content score group did not. Therefore, this study identifies the immune content score as a prognostic tool, which might have a potential association with PORT response, thereby facilitating outcome prediction and treatment decision in HNC.

Racial Disparity in the Genomic Basis of Radiosensitivity – An Exploration of Whole-Transcriptome Sequencing Data via a Machine-Learning Approach

There is emerging evidence for the existence of intrinsic biological differences in cancer responsiveness to therapeutic radiation between African-American (AA) men compared to non-Hispanic White (NHW) men (Spratt et al. [abstract ASTRO 2018]). The basis of this racial difference in radiosensitivity is likely based on variations in the expression of genes encoding for radiation response pathways. For example, decreased double strand break repair gene expression is associated increased radiosensitivity in somatic and cancer cell lines. We hypothesized that a racial difference in the expression levels of genes participating in radiation response pathways could be identified via a machine learning approach. We extracted the gene expression level data of 7,470 patients from the Genomic Data Commons Pan-Cancer database who had race identified as AA (n=802) or NHW (n=6,668). For each patient, the expression levels of 741 genes that are known to be involved in radiation response pathways were selected for subsequent analysis. An ensemble of five machine learning methods (support vector machine (SVM), linear discriminant analysis (LDA), gradient boosted machine (GBM), Bayesian generalized linear model (BGLM), and sample mean (SM)) was trained on 80% of the data to predict for race based on this 741-gene expression panel. Out-of-sample error was estimated using 5-fold cross validation. The trained ensemble model was used to predict on the remaining 20% of the data. Performance of the ensemble model was evaluated via area under the curve (AUC) of the receiver operating characteristic curve. The mean squared error for the SVM, LDA, GBM, BGLM, and SM methods were 0.071, 0.075, 0.081, 0.076, and 0.096 respectively. The ensemble model achieved a mean square error of 0.068. Prediction by the ensemble model yielded an AUC of 0.861 (95% CI 0.844-0.878). Expression levels of radiation response pathway genes can be used to accurately identify race via an ensemble of machine learning models. This supports the emerging evidence that race may be associated with radiosensitivity via intrinsic biologic differences in gene expression levels. Further studies are warranted to investigate whether these gene expression differences translate to clinically detectable variation in radiosensitivity and tumor control among different patient populations.

Distinct vascular genomic response of proton and gamma radiation-A pilot investigation.

The cardiovascular biology of proton radiotherapy is not well understood. We aimed to compare the genomic dose-response to proton and gamma radiation of the mouse aorta to assess whether their vascular effects may diverge. We performed comparative RNA sequencing of the aorta following (4 hrs) total-body proton and gamma irradiation (0.5–200 cGy whole body dose, 10 dose levels) of conscious mice. A trend analysis identified genes that showed a dose response. While fewer genes were dose-responsive to proton than gamma radiation (29 vs. 194 genes; q-value ≤ 0.1), the magnitude of the effect was greater. Highly responsive genes were enriched for radiation response pathways (DNA damage, apoptosis, cellular stress and inflammation; p-value ≤ 0.01). Gamma, but not proton radiation induced additionally genes in vasculature specific pathways. Genes responsive to both radiation types showed almost perfectly superimposable dose-response relationships. Despite the activation of canonical radiation response pathways by both radiation types, we detected marked differences in the genomic response of the murine aorta. Models of cardiovascular risk based on photon radiation may not accurately predict the risk associated with proton radiation.

Dotting the eyes: mouse strain dependency of the lens epithelium to low dose radiation-induced DNA damage

Purpose: Epidemiological evidence regarding the radiosensitivity of the lens of the eye and radiation cataract development has led to changes in the EU Basic Safety Standards for protection of the lens against ionizing radiation. However, mechanistic details of lens radiation response pathways and their significance for cataractogenesis remain unclear. Radiation-induced DNA damage and the potential impairment of repair pathways within the lens epithelium, a cell monolayer that covers the anterior hemisphere of the lens, are likely to be involved.Materials and Methods: In this work, the lens epithelium has been analyzed for its DNA double-strand break (DSB) repair response to ionizing radiation. The responses of epithelial cells located at the anterior pole (central region) have been compared to at the very periphery of the monolayer (germinative and transitional zones). Described here are the different responses in the two regions and across four strains (C57BL/6, 129S2, BALB/c and CBA/Ca) over a low dose (0–25 mGy) in-vivo whole body X-irradiation range up to 24 hours post exposure.Results: DNA damage and repair as visualized through 53BP1 staining was present across the lens epithelium, although repair kinetics appeared non-uniform. Epithelial cells in the central region have significantly more 53BP1 foci. The sensitivities of different mouse strains have also been compared.Conclusions: 129S2 and BALB/c showed higher levels of DNA damage, with BALB/c showing significantly less inter-individual variability and appearing to be a more robust model for future DNA damage and repair studies. As a result of this study, BALB/c was identified as a suitable radiosensitive lens strain to detect and quantify early low dose ionizing radiation DNA damage effects in the mouse eye lens specifically, as an indicator of cataract formation.

The Immune Landscape of Prostate Cancer and Nomination of PD-L2 as a Potential Therapeutic Target.

Immunotherapy has been less successful in treating prostate cancer than other solid tumors. We sought to better understand the immune landscape in prostate cancer and identify immune-related biomarkers and potential therapeutic targets. We analyzed gene expression data from 7826 prospectively collected prostatectomy samples (2013-2016), and 1567 retrospective samples with long-term clinical outcomes, for a total of 9393 samples, all profiled on the same commercial clinical platform in a CLIA-certified lab. The primary outcome was distant metastasis-free survival (DMFS). Secondary outcomes included biochemical recurrence-free survival (bRFS), prostate cancer-specific survival (PCSS), and overall survival (OS). All statistical tests were two-sided. Unsupervised hierarchical clustering of hallmark pathways demonstrated an immune-related tumor cluster. Increased estimated immune content scores based on immune-specific genes from the literature were associated with worse bRFS (hazard ratio [HR] = 1.26 [95% confidence interval [CI] = 1.12 to 1.42]; P < .001), DMFS (HR = 1.34 [95% CI = 1.13 to 1.58]; P < .001), PCSS (HR = 1.53 [95% CI = 1.21 to 1.92]; P < .001), and OS (HR = 1.27 [95% CI = 1.07 to 1.50]; P = .006). Deconvolution using Cibersort revealed that mast cells, natural killer cells, and dendritic cells conferred improved DMFS, whereas macrophages and T-cells conferred worse DMFS. Interestingly, while PD-L1 was not prognostic, consistent with its low expression in prostate cancer, PD-L2 was expressed at statistically significantly higher levels (P < .001) and was associated with worse bRFS (HR = 1.17 [95% CI = 1.03 to 1.33]; P = .01), DMFS (HR = 1.25 [95% CI = 1.05 to 1.49]; P = .01), and PCSS (HR = 1.45 [95% CI = 1.13 to 1.86]; P = .003). PD-L2 was strongly associated with immune-related pathways on gene set enrichment analysis suggesting that it is playing an important role in immune modulation in clinical prostate cancer samples. Furthermore, PD-L2 was correlated with radiation response pathways, and also predicted response to postoperative radiation therapy (PORT) on multivariable interaction analysis (P = .03). In the largest study of its kind to date, these results illustrate the complex relationship between the tumor-immune interaction, prognosis, and response to radiotherapy, and nominate PD-L2 as a potential novel therapeutic target in prostate cancer, potentially in combination with radiotherapy.

Gene Expression in Parp1 Deficient Mice Exposed to a Median Lethal Dose of Gamma Rays.

There is a current interest in the development of biodosimetric methods for rapidly assessing radiation exposure in the wake of a large-scale radiological event. This work was initially focused on determining the exposure dose to an individual using biological indicators. Gene expression signatures show promise for biodosimetric application, but little is known about how these signatures might translate for the assessment of radiological injury in radiosensitive individuals, who comprise a significant proportion of the general population, and who would likely require treatment after exposure to lower doses. Using Parp1-/- mice as a model radiation-sensitive genotype, we have investigated the effect of this DNA repair deficiency on the gene expression response to radiation. Although Parp1 is known to play general roles in regulating transcription, the pattern of gene expression changes observed in Parp1-/- mice 24 h postirradiation to a LD50/30 was remarkably similar to that in wild-type mice after exposure to LD50/30. Similar levels of activation of both the p53 and NFκB radiation response pathways were indicated in both strains. In contrast, exposure of wild-type mice to a sublethal dose that was equal to the Parp1-/- LD50/30 resulted in a lower magnitude gene expression response. Thus, Parp1-/- mice displayed a heightened gene expression response to radiation, which was more similar to the wild-type response to an equitoxic dose than to an equal absorbed dose. Gene expression classifiers trained on the wild-type data correctly identified all wild-type samples as unexposed, exposed to a sublethal dose or exposed to an LD50/30. All unexposed samples from Parp1-/- mice were also correctly classified with the same gene set, and 80% of irradiated Parp1-/- samples were identified as exposed to an LD50/30. The results of this study suggest that, at least for some pathways that may influence radiosensitivity in humans, specific gene expression signatures have the potential to accurately detect the extent of radiological injury, rather than serving only as a surrogate of physical radiation dose.

Radiation‐induced cataracts

SummaryThe radiosensitive nature of the lens has been increasingly reported, although the exact mechanistic details of the radiation response pathways for cataractogenesis are unclear. Radiation‐induced DNA damage and the subsequent impairment of repair pathways within the lens epithelium are involved. Here, two distinct regions of the murine lens epithelium have been analysed for their differences in double strand break (DSB) repair responses to ionising radiation. The responses of epithelial cells located at the anterior pole (central region) have been compared to those in other locations, including the proliferative compartment, and including the very periphery of the monolayer (peripheral region). Described here are the responses between the two regions, across four strains, over a low dose (0–25 mGy) X‐irradiation range up to 24 hours. Damage visualised through biomarker 53BP1 staining was present across the epithelium, repair kinetics appear non‐uniform. Epithelial cells in the central region have significantly more 53BP1 positive foci. In this study, BALB/c were identified as the most suitable strain for low dose ionising radiation exposure investigation.

Space-type radiation induces multimodal responses in the mouse gut microbiome and metabolome

BackgroundSpace travel is associated with continuous low dose rate exposure to high linear energy transfer (LET) radiation. Pathophysiological manifestations after low dose radiation exposure are strongly influenced by non-cytocidal radiation effects, including changes in the microbiome and host gene expression. Although the importance of the gut microbiome in the maintenance of human health is well established, little is known about the role of radiation in altering the microbiome during deep-space travel.ResultsUsing a mouse model for exposure to high LET radiation, we observed substantial changes in the composition and functional potential of the gut microbiome. These were accompanied by changes in the abundance of multiple metabolites, which were related to the enzymatic activity of the predicted metagenome by means of metabolic network modeling. There was a complex dynamic in microbial and metabolic composition at different radiation doses, suggestive of transient, dose-dependent interactions between microbial ecology and signals from the host’s cellular damage repair processes. The observed radiation-induced changes in microbiota diversity and composition were analyzed at the functional level. A constitutive change in activity was found for several pathways dominated by microbiome-specific enzymatic reactions like carbohydrate digestion and absorption and lipopolysaccharide biosynthesis, while the activity in other radiation-responsive pathways like phosphatidylinositol signaling could be linked to dose-dependent changes in the abundance of specific taxa.ConclusionsThe implication of microbiome-mediated pathophysiology after low dose ionizing radiation may be an unappreciated biologic hazard of space travel and deserves experimental validation. This study provides a conceptual and analytical basis of further investigations to increase our understanding of the chronic effects of space radiation on human health, and points to potential new targets for intervention in adverse radiation effects.

Abstract 1647: Radiation response genome-wide analysis using paired pre and post-radiation FFPE human breast tumor samples

Abstract Background: Breast cancer is the most common female malignancy in the US, and radiotherapy is a routine part of multi-disciplinary breast cancer care. Radiation treatment (RT) tailored to target radiation response pathways could maximize treatment benefit in radiation resistant tumors, while minimizing unnecessary toxicity and cost in more radiation responsive breast tumor subtypes. Here we report on genome-wide analysis, gene expression profiles and biological pathways associated with RT response in breast cancer patients. Methods: Between 2010-2013, a total of 32 breast cancer patients were enrolled at Duke University Medical Center on an IRB approved Phase I clinical trial testing preoperative radiotherapy. Paired pre and post-RT FFPE biopsy samples were available for N = 26. Genome wide-analysis was performed using two distinct techniques: i) Microarray analysis using the Affymetrix GeneChip ® Human Transcriptome Array 2.0 (HTA 2.0); ii) RNA-Seq using TruSeq ®RNA Access Library kit (Illumina). All libraries were sequenced on the Illumina HiSeq 2000 generating 50 bp single reads at a sequencing depth of 40 million reads. The RNA-Seq data was processed using the TrimGalore toolkit. For the paired pre and post-RT differential gene expression analysis we used the linear model. Genes that had an FDR ≤ 5% in the RNA-Seq experiment were compared with all microarray probesets that had an FDR ≤ 5%. If at least one probeset that belonged to a gene in the RNA-Seq list was significant and showed the same direction of change between pre and post-RT samples, they were identified as consistent. Gene Set Enrichment Analysis (GSEA) and Database for Annotation, Visualization and Integrated Discovery (DAVID) were used to identify significant pathways and gene ontologies that were differentially regulated between the conditions. Results: 2648 genes showed the same significant results in response to RT in microarray and RNA-Seq including: i) a total of 1401 genes that had significantly higher expression levels, and ii) a total of 1247 genes that had significantly lower expression levels following RT. Principal Components Analysis (PCA) demonstrated distinct separation of pre and post-RT gene expression signatures. Differentially expressed genes were involved in major biological pathways such as cell cycle checkpoint, DNA replication and DNA repair pathways. A subset of these genes also demonstrated differential expression in radiation responsive vs. non-responsive breast cancer cell lines. Conclusion: New laboratory techniques have become available which provide the opportunity to use FFPE samples for microarray and RNA-seq analysis. Using these new techniques and our rare pre and post-RT FFPE breast tumor samples, we have been able to demonstrate distinct differences in gene expression profiles and modulation of biological pathways in response to RT in human breast tumors. Citation Format: Sharareh Siamakpour-Reihani, Wei Chen, David Corcoran, Chen-Ting Lee, Ying Zhou, Mark W. Dewhirst, Jen-Tsan A. Chi, Janet K. Horton. Radiation response genome-wide analysis using paired pre and post-radiation FFPE human breast tumor samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1647.

Integrated Bioinformatics for Radiation-Induced Pathway Analysis from Proteomics and Microarray Data

Functional analysis and interpretation of large-scale proteomics and gene expression data require effective use of bioinformatics tools and public knowledge resources coupled with expert-guided examination. An integrated bioinformatics approach was used to analyze cellular pathways in response to ionizing radiation. ATM, or ataxia-telangiectasia mutated , a serine-threonine protein kinase, plays critical roles in radiation responses, including cell cycle arrest and DNA repair. We analyzed radiation responsive pathways based on 2D-gel/MS proteomics and microarray gene expression data from fibroblasts expressing wild type or mutant ATM gene. The analysis showed that metabolism was significantly affected by radiation in an ATM dependent manner. In particular, purine metabolic pathways were differentially changed in the two cell lines. The expression of ribonucleoside-diphosphate reductase subunit M2 (RRM2) was increased in ATM-wild type cells at both mRNA and protein levels, but no changes were detected in ATM-mutated cells. Increased expression of p53 was observed 30min after irradiation of the ATM-wild type cells. These results suggest that RRM2 is a downstream target of the ATM-p53 pathway that mediates radiation-induced DNA repair. We demonstrated that the integrated bioinformatics approach facilitated pathway analysis, hypothesis generation and target gene/protein identification.

Human fibroblast growth factor 20 (FGF-20; CG53135-05): A novel cytoprotectant with radioprotective potential

The aim was to evaluate the radioprotective properties of recombinant human fibroblast growth factor 20 (FGF-20; CG53135-05) in vitro and in vivo and to examine its effects on known cellular pathways of radioprotection. Relative transcript levels of the cyclooxygenase 2 (COX2), Mn-super oxide dismutase (SOD), CuZn-SOD, extracellular (EC)-SOD, nuclear respiratory factor 2 (Nrf2), glutathione peroxidase 1 (GPX1) and intestinal trefoil factor 3 (ITF3) genes, which are involved in radiation response pathways, were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) in NIH/3T3, IEC18, CCD-18Co, CCD-1070sk and human umbilical vein endothelial cells (HUVEC) cells exposed to FGF-20. Activation of the radioprotective signal transduction pathways initiating with the serine/threonine Akt kinase and the extracellular regulated kinase (ERK) were analysed. Levels of intracellular hydrogen peroxide and cytosolic redox potential were also measured in irradiated and unirradiated cells in the presence or absence of FGF-20. The effects of FGF-20 on cell survival in vitro following ionizing radiation were evaluated using clonogenic assays. To test the potential activity of FGF-20 as a radioprotectant in vivo, mice were administered a single dose of FGF-20 (4 mg kg−1, intraperitoneally (i.p.) 1 day before lethal total-body irradiation and evaluated for survival. In vitro exposure to FGF-20 increased expression of the Nrf2 transcription factor and oxygen radical scavenging enzymes such as MnSOD, activated signal transduction pathways (ERK and Akt) and resulted in increased survival of irradiated cells in vitro. FGF-20 treatment also resulted in a concomitant reduction in intracellular levels of injurious reactive oxygen species (ROS) following acute ionizing irradiation. Finally, prophylactic administration of FGF-20 to mice before potentially lethal, whole-body X-irradiation led to significant increases in overall survival. FGF-20 reduced the lethal effects of acute ionizing radiation exposure in cells by up-regulating important signalling and free radical scavenging pathways. Survival-sparing effects of FGF-20 prophylaxis in acutely irradiated mice presumably are elicited by comparable mechanisms. These results indicate that FGF-20, has significant radioprotective attributes with potential applications in clinical and non-clinical exposure settings.