Biomarkers for Biodosimetry and Their Role in Predicting Radiation Injury.

Radiation-related normal tissue injury sustained during cancer radiotherapy or in a radiological or mass casualty nuclear incident is a major health concern. Reducing the risk and mitigating consequences of radiation injury could have a broad impact on cancer patients and citizens. Efforts to discover biomarkers that can determine radiation dose, predict tissue damage, and aid medical triage are underway. Exposure to ionizing radiation causes changes in gene, protein, and metabolite expression that needs to be understood to provide a holistic picture for treating acute and chronic radiation-induced toxicities. We present evidence that both RNA (mRNA, microRNA, long noncoding RNA) and metabolomic assays may provide useful biomarkers of radiation injury. RNA markers may provide information on early pathway alterations after radiation injury that can predict damage and implicate downstream targets for mitigation. In contrast, metabolomics is impacted by changes in epigenetics, genetics, and proteomics and can be considered a downstream marker that incorporates all these changes to provide an assessment of what is currently happening within an organ. We highlight research from the past 10 years to understand how biomarkers may be used to improve personalized medicine in cancer therapy and medical decision-making in mass casualty scenarios.

Long-term expression changes of immune-related genes in prostate cancer after radiotherapy.

The expression of immune-related genes in cancer cells can alter the anti-tumor immune response and thereby impact patient outcomes. Radiotherapy has been shown to modulate immune-related genes dependent on the fractionation regimen. To identify long-term changes in gene expression after irradiation, PC3 (p53 deleted) and LNCaP (p53 wildtype) prostate cancer cells were irradiated with either a single dose (SD, 10 Gy) or a fractionated regimen (MF) of 10 fractions (1 Gy per fraction). Whole human genome arrays were used to determine gene expression at 24 h and 2 months after irradiation. Immune pathway activation was analyzed with Ingenuity Pathway Analysis software. Additionally, 3D colony formation assays and T-cell cytotoxicity assays were performed. LNCaP had a higher basal expression of immunogenic genes and was more efficiently killed by cytotoxic T-cells compared to PC3. In both cell lines, MF irradiation resulted in an increase in multiple immune-related genes immediately after irradiation, while at 2 months, SD irradiation had a more pronounced effect on radiation-induced gene expression. Both immunogenic and immunosuppressive genes were upregulated in the long term in PC3 cells by a 10 Gy SD irradiation but not in LNCaP. T-cell-mediated cytotoxicity was significantly increased in 10 Gy SD PC3 cells compared to the unirradiated control and could be further enhanced by treatment with immune checkpoint inhibitors. Irradiation impacts the expression of immune-related genes in cancer cells in a fractionation-dependent manner. Understanding and targeting these changes may be a promising strategy for primary prostate cancer and recurrent tumors.

53BP1/RIF1 signaling promotes cell survival after multifractionated radiotherapy.

Multifractionated irradiation is the mainstay of radiation treatment in cancer therapy. Yet, little is known about the cellular DNA repair processes that take place between radiation fractions, even though understanding the molecular mechanisms promoting cancer cell recovery and survival could improve patient outcome and identify new avenues for targeted intervention. To address this knowledge gap, we systematically characterized how cells respond differentially to multifractionated and single-dose radiotherapy, using a combination of genetics-based and functional approaches. We found that both cancer cells and normal fibroblasts exhibited enhanced survival after multifractionated irradiation compared with an equivalent single dose of irradiation, and this effect was entirely dependent on 53BP1-mediated NHEJ. Furthermore, we identified RIF1 as the critical effector of 53BP1. Inhibiting 53BP1 recruitment to damaged chromatin completely abolished the survival advantage after multifractionated irradiation and could not be reversed by suppressing excessive end resection. Analysis of the TCGA database revealed lower expression of 53BP1 pathway genes in prostate cancer, suggesting that multifractionated radiotherapy might be a favorable option for radio-oncologic treatment in this tumor type. We propose that elucidation of DNA repair mechanisms elicited by different irradiation dosing regimens could improve radiotherapy selection for the individual patient and maximize the efficacy of radiotherapy.

Analysis of lncRNA-miRNA-mRNA expression pattern in heart tissue after total body radiation in a mouse model.

BACKGROUND: Radiation therapy is integral to effective thoracic cancer treatments, but its application is limited by sensitivity of critical organs such as the heart. The impacts of acute radiation-induced damage and its chronic effects on normal heart cells are highly relevant in radiotherapy with increasing lifespans of patients. Biomarkers for normal tissue damage after radiation exposure, whether accidental or therapeutic, are being studied as indicators of both acute and delayed effects. Recent research has highlighted the potential importance of RNAs, including messenger RNAs (mRNAs), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) as biomarkers to assess radiation damage. Understanding changes in mRNA and non-coding RNA expression will elucidate biological pathway changes after radiation. METHODS: To identify significant expression changes in mRNAs, lncRNAs, and miRNAs, we performed whole transcriptome microarray analysis of mouse heart tissue at 48 h after whole-body irradiation with 1, 2, 4, 8, and 12 Gray (Gy). We also validated changes in specific lncRNAs through RT-qPCR. Ingenuity Pathway Analysis (IPA) was used to identify pathways associated with gene expression changes. RESULTS: We observed sustained increases in lncRNAs and mRNAs, across all doses of radiation. Alas2, Aplnr, and Cxc3r1 were the most significantly downregulated mRNAs across all doses. Among the significantly upregulated mRNAs were cell-cycle arrest biomarkers Gdf15, Cdkn1a, and Ckap2. Additionally, IPA identified significant changes in gene expression relevant to senescence, apoptosis, hemoglobin synthesis, inflammation, and metabolism. LncRNAs Abhd11os, Pvt1, Trp53cor1, and Dino showed increased expression with increasing doses of radiation. We did not observe any miRNAs with sustained up- or downregulation across all doses, but miR-149-3p, miR-6538, miR-8101, miR-7118-5p, miR-211-3p, and miR-3960 were significantly upregulated after 12 Gy. CONCLUSIONS: Radiation-induced RNA expression changes may be predictive of normal tissue toxicities and may indicate targetable pathways for radiation countermeasure development and improved radiotherapy treatment plans.

Microarray analysis of miRNA expression profiles following whole body irradiation in a mouse model.

CONTEXT: Accidental exposure to life-threatening radiation in a nuclear event is a major concern; there is an enormous need for identifying biomarkers for radiation biodosimetry to triage populations and treat critically exposed individuals. OBJECTIVE: To identify dose-differentiating miRNA signatures from whole blood samples of whole body irradiated mice. METHODS: Mice were whole body irradiated with X-rays (2 Gy-15 Gy); blood was collected at various time-points post-exposure; total RNA was isolated; miRNA microarrays were performed; miRNAs differentially expressed in irradiated vs. unirradiated controls were identified; feature extraction and classification models were applied to predict dose-differentiating miRNA signature. RESULTS: We observed a time and dose responsive alteration in the expression levels of miRNAs. Maximum number of miRNAs were altered at 24-h and 48-h time-points post-irradiation. A 23-miRNA signature was identified using feature selection algorithms and classifier models. An inverse correlation in the expression level changes of miR-17 members, and their targets were observed in whole body irradiated mice and non-human primates. CONCLUSION: Whole blood-based miRNA expression signatures might be used for predicting radiation exposures in a mass casualty nuclear incident.

Developing an RNA Signature for Radiation Injury Using a Human Liver-on-a-Chip Model.

Radiation exposure in a therapeutic setting or during a mass casualty event requires improved medical triaging, where the time to delivery and quantity of medical countermeasures are critical to survival. Radiation-induced liver injury (RILI) and fibrosis can lead to death, but clinical symptoms manifest late in disease pathogenesis and there is no simple diagnostic test to determine RILI. Because animal models do not completely recapitulate clinical symptoms, we used a human liver-on-a-chip model to identify biomarkers of RILI. The goals of this study were: 1. to establish a microfluidic liver-on-a-chip device as a physiologically relevant model for studying radiation-induced tissue damage; and 2. to determine acute changes in RNA expression and biological pathway regulation that identify potential biomarkers and mechanisms of RILI. To model functional human liver tissue, we used the Emulate organ-on-a-chip system to establish a co-culture of human liver sinusoidal endothelial cells (LSECs) and hepatocytes. The chips were subject to 0 Gy (sham), 1 Gy, 4 Gy, or 10 Gy irradiation and cells were collected at 6 h, 24 h, or 7 days postirradiation for RNA isolation. To identify significant expression changes in messenger RNA (mRNA) and long non-coding RNA (lncRNA), we performed RNA sequencing (RNASeq) to conduct whole transcriptome analysis. We found distinct differences in expression patterns by time, dose, and cell type, with higher doses of radiation resulting in the most pronounced expression changes, as anticipated. Ingenuity Pathway Analysis indicated significant inhibition of the cell viability pathway 24 h after 10 Gy exposure in LSECs but activation of this pathway in hepatocytes, highlighting differences between cell types despite receiving the same radiation dose. Overall, hepatocytes showed fewer gene expression changes in response to radiation, with only 3 statistically significant differentially expressed genes at 7 days: APOBEC3H, PTCHD4, and GDNF. We further highlight lncRNA of interest including DINO and PURPL in hepatocytes and TMPO-AS1 and PRC-AS1 in LSECs, identifying potential biomarkers of RILI. We demonstrated the potential utility of a human liver-on-a-chip model with primary cells to model organ-specific radiation injury, establishing a model for radiation medical countermeasure development and further biomarker validation. Furthermore, we identified biomarkers that differentiate radiation dose and defined cell-specific targets for potential radiation mitigation therapies.

Multiomic-Based Molecular Landscape of FaDu Xenograft Tumors in Mice after a Combinatorial Treatment with Radiation and an HSP90 Inhibitor Identifies Adaptation-Induced Targets of Resistance and Therapeutic Intervention.

Treatments involving radiation and chemotherapy alone or in combination have improved patient survival and quality of life. However, cancers frequently evade these therapies due to adaptation and tumor evolution. Given the complexity of predicting response based solely on the initial genetic profile of a patient, a predetermined treatment course may miss critical adaptation that can cause resistance or induce new targets for drug and immunotherapy. To address the timescale for these evasive mechanisms, using a mouse xenograft tumor model, we investigated the rapidity of gene expression (mRNA), molecular pathway, and phosphoproteome changes after radiation, an HSP90 inhibitor, or combination. Animals received radiation, drug, or combination treatment for 1 or 2 weeks and were then euthanized along with a time-matched untreated group for comparison. Changes in gene expression occur as early as 1 week after treatment initiation. Apoptosis and cell death pathways were activated in irradiated tumor samples. For the HSP90 inhibitor and combination treatment at weeks 1 and 2 compared with Control Day 1, gene-expression changes induced inhibition of pathways including invasion of cells, vasculogenesis, and viral infection among others. The combination group included both drug-alone and radiation-alone changes. Our data demonstrate the rapidity of gene expression and functional pathway changes in the evolving tumor as it responds to treatment. Discovering these phenotypic adaptations may help elucidate the challenges in using sustained treatment regimens and could also define evolving targets for therapeutic efficacy.

Emerging evidence for adapting radiotherapy to immunotherapy.

Immunotherapy has revolutionized the clinical management of many malignancies but is infrequently associated with durable objective responses when used as a standalone treatment approach, calling for the development of combinatorial regimens with superior efficacy and acceptable toxicity. Radiotherapy, the most commonly used oncological treatment, has attracted considerable attention as a combination partner for immunotherapy owing to its well-known and predictable safety profile, widespread clinical availability, and potential for immunostimulatory effects. However, numerous randomized clinical trials investigating radiotherapy-immunotherapy combinations have failed to demonstrate a therapeutic benefit compared with either modality alone. Such a lack of interaction might reflect suboptimal study design, choice of end points and/or administration of radiotherapy according to standard schedules and target volumes. Indeed, radiotherapy has empirically evolved towards radiation doses and fields that enable maximal cancer cell killing with manageable toxicity to healthy tissues, without much consideration of potential radiation-induced immunostimulatory effects. Herein, we propose the concept that successful radiotherapy-immunotherapy combinations might require modifications of standard radiotherapy regimens and target volumes to optimally sustain immune fitness and enhance the antitumour immune response in support of meaningful clinical benefits.

Radiation-induced pulmonary fibrosis: roles of therapy-induced senescence and microRNAs.

PURPOSE: Progressive, irreversible radiation-induced pulmonary fibrosis (RIPF) is a clinically significant intermediate- to a late-occurring side effect of radiotherapy. Known mechanisms of RIPF include oxidative stress-induced activation of TGF-ß with activation of SMAD signaling, TNF-α elaboration, and activation of the Angiotensin Converting Enzyme (ACE) mediated production of angiotensin II with resulting activation of profibrotic cytokine signaling and vasoconstriction. The pioneering work of John Moulder, to whom this paper is dedicated, and several of his colleagues demonstrated that inhibiting the conversion of ACE with drugs such as Captopril, Enalapril, and Losartan can ameliorate radiation fibrosis in various tissues. While this work led several groups to probe mechanism-based pharmacological mitigation of RIPF, in this article, we explore and discuss the roles of microRNAs (miRNA) and therapy-induced senescence (TIS) in the pathogenesis of and potential biomarkers for RIPF. CONCLUSION: Our analysis of the published literature in the last decade on RIPF, miRNA, and TIS identifies TIS as a mechanism in the onset and progression of RIPF, which is regulated through several miRNAs. This work may lead to the discovery and development of the next generation of miRNA therapeutics and/or the repurposing of approved pharmaceutical agents and the development of early biomarker panels to predict RIPF.

Comparative Analysis of miRNA Expression after Whole-Body Irradiation Across Three Strains of Mice.

Whole- or partial-body exposure to ionizing radiation damages major organ systems, leading to dysfunction on both acute and chronic timescales. Radiation medical countermeasures can mitigate acute damages and may delay chronic effects when delivered within days after exposure. However, in the event of widespread radiation exposure, there will inevitably be scarce resources with limited countermeasures to distribute among the affected population. Radiation biodosimetry is necessary to separate exposed from unexposed victims and determine those who requires the most urgent care. Blood-based, microRNA signatures have great potential for biodosimetry, but the affected population in such a situation will be genetically heterogeneous and have varying miRNA responses to radiation. Thus, there is a need to understand differences in radiation-induced miRNA expression across different genetic backgrounds to develop a robust signature. We used inbred mouse strains C3H/HeJ and BALB/c mice to determine how accurate miRNA in blood would be in developing markers for radiation vs. no radiation, low dose (1 Gy, 2 Gy) vs. high dose (4 Gy, 8 Gy), and high risk (8 Gy) vs. low risk (1 Gy, 2 Gy, 4 Gy). Mice were exposed to whole-body doses of 0 Gy, 1 Gy, 2 Gy, 4 Gy, or 8 Gy of X rays. MiRNA expression changes were identified using NanoString nCounter panels on blood RNA collected 1, 2, 3 or 7 days postirradiation. Overall, C3H/HeJ mice had more differentially expressed miRNAs across all doses and timepoints than BALB/c mice. The highest amount of differential expression occurred at days 2 and 3 postirradiation for both strains. Comparison of C3H/HeJ and BALB/c expression profiles to those previously identified in C57BL/6 mice revealed 12 miRNAs that were commonly expressed across all three strains, only one of which, miR-340-5p, displayed a consistent regulation pattern in all three miRNA data. Notably multiple Let-7 family members predicted high-dose and high-risk radiation exposure (Let-7a, Let-7f, Let-7e, Let-7g, and Let-7d). KEGG pathway analysis demonstrated involvement of these predicted miRNAs in pathways related to: Fatty acid metabolism, Lysine degradation and FoxO signaling. These findings indicate differences in the miRNA response to radiation across various genetic backgrounds, and highlights key similarities, which we exploited to discover miRNAs that predict radiation exposure. Our study demonstrates the need and the utility of including multiple animal strains in developing and validating biodosimetry diagnostic signatures. From this data, we developed highly accurate miRNA signatures capable of predicting exposed and unexposed subjects within a genetically heterogeneous population as quickly as 24 h of exposure to radiation.

Microarray analysis identifies coding and non-coding RNA markers of liver injury in whole body irradiated mice.

Radiation injury from medical, accidental, or intentional sources can induce acute and long-term hepatic dysregulation, fibrosis, and cancer. This long-term hepatic dysregulation decreases quality of life and may lead to death. Our goal in this study is to determine acute changes in biological pathways and discover potential RNA biomarkers predictive of radiation injury. We performed whole transcriptome microarray analysis of mouse liver tissue (C57BL/6 J) 48 h after whole-body irradiation with 1, 2, 4, 8, and 12 Gray to identify significant expression changes in mRNAs, lncRNAs, and miRNAs, We also validated changes in specific RNAs through qRT-PCR. We used Ingenuity Pathway Analysis (IPA) to identify pathways associated with gene expression changes. We observed significant dysregulation of multiple mRNAs across all doses. In contrast, miRNA dysregulation was observed upwards of 2 Gray. The most significantly upregulated mRNAs function as tumor suppressors: Cdkn1a, Phlda3, and Eda2r. The most significantly downregulated mRNAs were involved in hemoglobin synthesis, inflammation, and mitochondrial function including multiple members of Hbb and Hba. The most significantly upregulated miRNA included: miR-34a-5p, miR-3102-5p, and miR-3960, while miR-342-3p, miR-142a-3p, and miR-223-3p were most significantly downregulated. IPA predicted activation of cell cycle checkpoint control pathways and inhibition of pathways relevant to inflammation and erythropoietin. Clarifying expression of mRNA, miRNA and lncRNA at a short time point (48 h) offers insight into potential biomarkers, including radiation markers shared across organs and animal models. This information, once validated in human models, can aid in development of bio-dosimetry biomarkers, and furthers our understanding of acute pathway dysregulation.

Microarray analysis of hub genes, non-coding RNAs and pathways in lung after whole body irradiation in a mouse model.

PURPOSE: Previous research has highlighted the impact of radiation damage, with cancer patients developing acute disorders including radiation induced pneumonitis or chronic disorders including pulmonary fibrosis months after radiation therapy ends. We sought to discover biomarkers that predict these injuries and develop treatments that mitigate this damage and improve quality of life. MATERIALS AND METHODS: Six- to eight-week-old female C57BL/6 mice received 1, 2, 4, 8, 12 Gy or sham whole body irradiation. Animals were euthanized 48 h post exposure and lungs removed, snap frozen and underwent RNA isolation. Microarray analysis was performed to determine dysregulation of messenger RNA (mRNA), microRNA (miRNA), and long non-coding RNA (lncRNA) after radiation injury. RESULTS: We observed sustained dysregulation of specific RNA markers including: mRNAs, lncRNAs, and miRNAs across all doses. We also identified significantly upregulated genes that can indicate high dose exposure, including Cpt1c, Pdk4, Gdf15, and Eda2r, which are markers of senescence and fibrosis. Only three miRNAs were significantly dysregulated across all radiation doses: miRNA-142-3p and miRNA-142-5p were downregulated and miRNA-34a-5p was upregulated. IPA analysis predicted inhibition of several molecular pathways with increasing doses of radiation, including: T cell development, Quantity of leukocytes, Quantity of lymphocytes, and Cell viability. CONCLUSIONS: These RNA biomarkers might be highly relevant in the development of treatments and in predicting normal tissue injury in patients undergoing radiation treatment. We are conducting further experiments in our laboratory, which includes a human lung-on-a-chip model, to develop a decision tree model using RNA biomarkers.

Gene expression profile of coronary artery cells treated with nonsteroidal anti-inflammatory drugs reveals off-target effects.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have come under scrutiny because of the gastrointestinal, renal, and cardiovascular toxicity associated with prolonged use of these drugs. The purpose of this study was to identify molecular targets for NSAIDs related to cellular toxicity with a view to optimize drug efficacy in the clinic. Coronary artery smooth muscle cells and endothelial cells were treated with low (clinically achievable) and high (typically used in preclinical studies) concentrations of celecoxib, NS398, and ibuprofen for 24 hours. NSAIDs-induced gene expression changes were evaluated by microarray analysis and validated by real-time reverse-transcription polymerase chain reaction and western blotting. The functional significance of differentially expressed genes was evaluated by Ingenuity Pathway Analysis. At high concentrations, NSAIDs altered the expression of genes regulating cell proliferation and cell death. NSAIDs also altered genes associated with cardiovascular functions including inflammation, thrombosis, fibrinolysis, coronary artery disease, and hypertension. The gene expression was most impacted by ibuprofen, celecoxib, and NS398, in that order. This study revealed that NSAIDs altered expression of an array of genes associated with cardiovascular events and emphasizes the potential for fingerprinting drugs in preclinical studies to assess the potential drug toxicity and to optimize the drug efficacy in clinical settings.

Profiling mRNA, miRNA and lncRNA expression changes in endothelial cells in response to increasing doses of ionizing radiation.

Recent and past research have highlighted the importance of the endothelium in the manifestation of radiation injury. Our primary focus is on medical triage and management following whole body or partial-body irradiation. Here we investigated the usability of endothelial cells' radiation response for biodosimetry applications. We profiled the transcriptome in cultured human endothelial cells treated with increasing doses of X-rays. mRNA expression changes were useful 24 h and 72 h post-radiation, microRNA and lncRNA expression changes were useful 72 h after radiation. More mRNA expressions were repressed than induced while more miRNA and lncRNA expressions were induced than repressed. These novel observations imply distinct radiation responsive regulatory mechanisms for coding and non-coding transcripts. It also follows how different RNA species should be explored as biomarkers for different time-points. Radiation-responsive markers which could classify no radiation (i.e., '0 Gy') and dose-differentiating markers were also predicted. IPA analysis showed growth arrest-related processes at 24 h but immune response coordination at the 72 h post-radiation. Collectively, these observations suggest that endothelial cells have a precise dose and time-dependent response to radiation. Further studies in the laboratory are examining if these differences could be captured in the extracellular vesicles released by irradiated endothelial cells.

Serum RNA biomarkers for predicting survival in non-human primates following thoracic radiation.

In a mass radiation exposure, the healthcare system may rely on differential expression of miRNA to determine exposure and effectively allocate resources. To this end, miRNome analysis was performed on non-human primate serum after whole thorax photon beam irradiation of 9.8 or 10.7 Gy with dose rate 600 cGy/min. Serum was collected up to 270 days after irradiation and sequenced to determine immediate and delayed effects on miRNA expression. Elastic net based GLM methods were used to develop models that predicted the dose vs. controls at 81% accuracy at Day 15. A three-group model at Day 9 achieved 71% accuracy in determining if an animal would die in less than 90 days, between 90 and 269 days, or survive the length of the study. At Day 21, we achieved 100% accuracy in determining whether an animal would later develop pleural effusion. These results demonstrate the potential ability of miRNAs to determine thorax partial-body irradiation dose and forecast survival or complications early following whole thorax irradiation in large animal models. Future experiments incorporating additional doses and independent animal cohorts are warranted to validate these results. Development of a serum miRNA assay will facilitate the administration of medical countermeasures to increase survival and limit normal tissue damage following a mass exposure.

Radiotherapy alters expression of molecular targets in prostate cancer in a fractionation- and time-dependent manner.

The efficacy of molecular targeted therapy depends on expression and enzymatic activity of the target molecules. As radiotherapy modulates gene expression and protein phosphorylation dependent on dose and fractionation, we analyzed the long-term effects of irradiation on the post-radiation efficacy of molecular targeted drugs. We irradiated prostate cancer cells either with a single dose (SD) of 10 Gy x-ray or a multifractionated (MF) regimen with 10 fractions of 1 Gy. Whole genome arrays and reverse phase protein microarrays were used to determine gene expression and protein phosphorylation. Additionally, we evaluated radiation-induced pathway activation with the Ingenuity Pathway Analysis software. To measure cell survival and sensitivity to clinically used molecular targeted drugs, we performed colony formation assays. We found increased activation of several pathways regulating important cell functions such as cell migration and cell survival at 24 h after MF irradiation or at 2 months after SD irradiation. Further, cells which survived a SD of 10 Gy showed a long-term upregulation and increased activity of multiple molecular targets including AKT, IGF-1R, VEGFR2, or MET, while HDAC expression was decreased. In line with this, 10 Gy SD cells were more sensitive to target inhibition with Capivasertib or Ipatasertib (AKTi), BMS-754807 (IGF-1Ri), or Foretinib (VEGFR2/METi), but less sensitive to Panobinostat or Vorinostat (HDACi). In summary, understanding the molecular short- and long-term changes after irradiation can aid in optimizing the efficacy of multimodal radiation oncology in combination with post-irradiation molecularly-targeted drug treatment and improving the outcome of prostate cancer patients.

Long and short non-coding RNA and radiation response: a review.

Once thought of as arising from "junk DNA," noncoding RNAs (ncRNAs) have emerged as key molecules in cellular processes and response to stress. From diseases such as cancer, coronary artery disease, and diabetes to the effects of ionizing radiation (IR), ncRNAs play important roles in disease progression and as biomarkers of damage. Noncoding RNAs regulate cellular processes by competitively binding DNA, mRNA, proteins, and other ncRNAs. Through these interactions, specific ncRNAs can modulate the radiosensitivity of cells and serve as diagnostic and prognostic biomarkers of radiation damage, whether from incidental exposure in radiotherapy or in accidental exposure scenarios. Analysis of RNA expression after radiation exposure has shown alterations not only in mRNAs, but also in ncRNAs (primarily miRNA, circRNA, and lncRNA), implying an important role in cellular stress response. Due to their abundance and stability in serum and other biofluids, ncRNAs also have great potential as minimally invasive biomarkers with advantages over current biodosimetry methods. Several studies have examined changes in ncRNA expression profiles in response to IR and other forms of oxidative stress. Furthermore, some studies have reported modulation of radiosensitivity by altering expression levels of these ncRNAs. This review discusses the roles of ncRNAs in the radiation response and evaluates prior research on ncRNAs as biomarkers of radiation damage. Future directions and applications of ncRNAs in radiation research are introduced, including the potential for a clinical ncRNA assay for assessing radiation damage and for the therapeutic use of RNA interference (RNAi).

The lncRNAs LINC00261 and LINC00665 are upregulated in long-term prostate cancer adaptation after radiotherapy.

Long non-coding RNAs (lncRNAs) have been shown to impact important biological functions such as proliferation, survival, and genomic stability. To analyze radiation-induced lncRNA expression in human tumors, we irradiated prostate cancer cells with a single dose of 10 Gy or a multifractionated radiotherapeutic regimen of 10 fractions with a dose of 1 Gy (10 × 1 Gy) during 5 days. We found a stable upregulation of the lncRNA LINC00261 and LINC00665 at 2 months after radiotherapy that was more pronounced after single-dose irradiation. Analysis of the The Cancer Genome Atlas (TCGA) and The Atlas of Non-coding RNAs in Cancer (TANRIC) databases showed that high expression of these two lncRNAs may be a potential negative prognostic marker for overall survival of prostate cancer patients. Knockdown of LINC00261 and LINC00665 in long-term survivors decreased survival after re-irradiation and affected DNA double-strand break repair. Mechanistically, both lncRNAs showed an interdependent expression and regulated expression of the DNA repair proteins CtIP (RBBP8) and XPC as well as the microRNA miR-329. Identifying long-term tumor adaptation mechanisms can lead to the discovery of new molecular targets, in effect opening up new research directions and improving multimodal radiation oncologic treatment.

Whole blood gene expression within days after total-body irradiation predicts long term survival in Gottingen minipigs.

Gottingen minipigs mirror the physiological radiation response observed in humans and hence make an ideal candidate model for studying radiation biodosimetry for both limited-sized and mass casualty incidents. We examined the whole blood gene expression profiles starting one day after total-body irradiation with increasing doses of gamma-rays. The minipigs were monitored for up to 45 days or time to euthanasia necessitated by radiation effects. We successfully identified dose- and time-agnostic (over a 1-7 day period after radiation), survival-predictive gene expression signatures derived using machine-learning algorithms with high sensitivity and specificity. These survival-predictive signatures fare better than an optimally performing dose-differentiating signature or blood cellular profiles. These findings suggest that prediction of survival is a much more useful parameter for making triage, resource-utilization and treatment decisions in a resource-constrained environment compared to predictions of total dose received. It should hopefully be possible to build such classifiers for humans in the future.

NS-398, ibuprofen, and cyclooxygenase-2 RNA interference produce significantly different gene expression profiles in prostate cancer cells.

Cyclooxygenase-2 (COX-2) plays a significant role in tumor development and progression. Nonsteroidal anti-inflammatory drugs (NSAID) exhibit potent anticancer effects in vitro and in vivo by COX-2-dependent and COX-2-independent mechanisms. In this study, we used microarray analysis to identify the change of expression profile regulated by a COX-2-specific NSAID NS-398 (0.01 and 0.1 mmol/L), a nonspecific NSAID ibuprofen (0.1 and 1.5 mmol/L) and RNA interference (RNAi)-mediated COX-2 inhibition in PC3 prostate cancer cells. A total of 3,362 differentially expressed genes with 2-fold change and P<0.05 were identified. Low concentrations of NSAIDs and COX-2 RNAi altered very few genes (1-3%) compared with the higher concentration of NS-398 (17%) and ibuprofen (80%). Ingenuity Pathway Analysis was used for distributing the differentially expressed genes into biological networks and for evaluation of functional significance. The top 3 networks for both NSAIDs included functional categories of DNA replication, recombination and repair, and gastrointestinal disease. Immunoresponse function was specific to NS-398, and cell cycle and cellular movement were among the top functions for ibuprofen. Ingenuity Pathway Analysis also identified renal and urologic disease as a function specific for ibuprofen. This comprehensive study identified several COX-2-independent targets of NSAIDs, which may help explain the antitumor and radiosensitizing effects of NSAIDs. However, none of these categories were reflected in the identified networks in PC3 cells treated with clinically relevant low concentrations of NS-398 and ibuprofen or with COX-2 RNAi, suggesting the benefit to fingerprinting preclinical drug concentrations to improve their relevance to the clinical setting.