Role In Radiation Resistance Research Articles

FtsZ phosphorylation brings about growth arrest upon DNA damage in Deinococcus radiodurans.

The polymerization/depolymerization dynamics of FtsZ play a pivotal role in cell division in the majority of the bacteria. Deinococcus radiodurans, a radiation-resistant bacterium, shows an arrest of growth in response to DNA damage with no change in the level of FtsZ. This bacterium does not deploy LexA/RecA type of DNA damage response and cell cycle regulation, and its genome does not encode SulA homologues of Escherichia coli, which attenuate FtsZ functions in response to DNA damage in other bacteria. A radiation-responsive Ser/Thr quinoprotein kinase (RqkA), characterized for its role in radiation resistance in this bacterium, could phosphorylate several cognate proteins, including FtsZ (drFtsZ) at Serine 235 (S235) and Serine 335 (S335) residues. Here, we reported the detailed characterization of S235 and S335 phosphorylation effects in the regulation of drFtsZ functions and demonstrated that the phospho-mimetic replacements of these residues in drFtsZ had grossly affected its functions that could result in cell cycle arrest in response to DNA damage in D. radiodurans. Interestingly, the phospho-ablative replacements were found to be nearly similar to drFtsZ, whereas the phospho-mimetic mutant lost the wild-type protein's signature characteristics, including its dynamics under normal conditions. The kinetics of post-bleaching recovery for drFtsZ and phospho-mimetic mutant were nearly similar at 2h post-irradiation recovery but were found to be different under normal conditions. These results highlighted the role of S/T phosphorylation in the regulation of drFtsZ functions and cell cycle arrest in response to DNA damage, which is demonstrated for the first time, in any bacteria.

SPP1 promotes radiation resistance through JAK2/STAT3 pathway in esophageal carcinoma.

Therapeutic resistance to radiotherapy is one of the major obstacles in clinical practice that significantly affect the therapeutic efficiency and prognosis of human esophageal carcinoma (ESCA). Thus, it is critical to understand the molecular mechanisms of radiation resistance in ESCA. Secreted phosphoprotein 1 (SPP1) plays an essential role in various human cancers, but its role in radiation resistance remains unclear. Cell culture and transfection; Cell Counting Kit-8 (CCK-8) assays; EdU incorporation assays; Patient sample collection and medical records review; Transwell assays; Colony formation assays; Wound healing assays; Western blot; Immunofluorescence; Immunohistochemistry; Irradiation; Flow cytometry; Animal studies; Human Apoptosis Array Kit; Bioinformatics. In the current study, we reported the novel phenomenon that radiation-treated human ESCA cells upregulated SPP1 expression, which in turn contributed to the ESCA resistance to radiotherapy. We also reported the tumor-promoting effect of SPP1 in ESCA systematically and comprehensively. Furthermore, subsequent studies by knocking down or overexpressing SPP1 in human ESCA cells showed that SPP1 could facilitate the repair of DNA damage and the survival of tumor cells post-radiation in ESCA, which might contribute to the development of radiation resistance during the radiotherapy process. More detailed investigations on the downstream molecular pathway suggested that radiation could increase the phosphorylation level of JAK2 and STAT3 by increasing SPP1 expression. Further in vivo validation using a mouse ESCA xenograft model showed that SPP1 overexpression significantly increased tumor volume while either SPP1 knockdown or pharmacological inhibition of the JAK2-STAT3 pathway reduced tumor volume in a synergistic manner with radiotherapy. Collectively, these findings suggested that the SPP1/JAK2/STAT3 axis is a critical player in ESCA progression and radiation resistance, which is a potential therapeutic target for combined therapy with the standard radiotherapy regimen to improve curative effect and increase patients' survival with ESCA.

Inhibition of lncRNA RET enhances radio-sensitivity of tumor cells via miR-3179/Slug/PTEN axis.

Radioresistance is one of the key obstacles that may lead to the failure of cancer treatment. The underlying mechanisms of radioresistance remain largely unknown; however, increasing evidence has shown that long noncoding RNAs (lncRNAs) are involved in radiotherapy resistance of several cancers. In the present study, we demonstrated that radiation-elevated transcript (RET), a newly identified lnRNA, was highly expressed in cancer cells. Knockdown of RET significantly inhibited the proliferation and colony formation of cancer cells and markedly inhibited apoptosis. Furthermore, downregulation of RET in cancer cells significantly inhibited cell growth, decreased colony survival fractions, and promoted apoptosis in response to radiation treatment, indicating a role in radiation resistance. Moreover, RET knockdown significantly increased the expression of γ-H2AX, an indicator of DNA double strand damage, and reversed radiation-induced EMT, both of which contributed to its radiation resistance. In addition, a negative correlation was found between the expression of RET and PTEN. Rescue assays confirmed RET knockdown enhanced radiosensitivity of cancer cells by upregulating the expression of PTEN. Mechanistically, RET positively regulated Slug, a repressor of PTEN transcription, by acting as a molecular sponge of miR-3179. Our present study showed that RET conferred radioresistance by regulating miR-3179/Slug/PTEN axis, indicating that RET may be a potential target for the clinical application in cancer patients with radioresistance.

Gallotannin from Bouea macrophylla Seed Extract Suppresses Cancer Stem-like Cells and Radiosensitizes Head and Neck Cancer

Cancer stem cells (CSCs) play a critical role in radiation resistance and recurrence. Thus, drugs targeting CSCs can be combined with radiotherapy to improve its antitumor efficacy. Here, we investigated whether a gallotannin extract from Bouea macrophylla seed (MPSE) and its main bioactive compound, pentagalloyl glucose (PGG), could suppress the stemness trait and further confer the radiosensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines. In this study, we evaluate the effect of MPSE or PGG to suppress CSC-like phenotypes and radiosensitization of HNSCC cell lines using a series of in vitro experiments, tumorsphere formation assay, colony formation assay, apoptosis assay, and Western blotting analysis. We demonstrate that MPSE or PGG is able to suppress tumorsphere formation and decrease protein expression of cancer stem cell markers. MPSE or PGG also enhanced the radiosensitivity in HNSCC cells. Pretreatment of cells with MPSE or PGG increased IR-induced DNA damage (γ-H2Ax) and enhanced radiation-induced cell death. Notably, we observed that pretreatment with MPSE or PGG attenuated the IR-induced stemness-like properties characterized by tumorsphere formation and the CD44 CSC marker. Our findings describe a novel strategy for increasing therapeutic efficacy for head and neck cancer patients using the natural products MPSE and PGG.

PLCε knockdown enhances the radiosensitivity of castration‑resistant prostate cancer via the AR/PARP1/DNA‑PKcs axis.

Radiotherapy (RT) has been used as a therapeutic option for treatment of prostate cancer (PCa) for a number of years; however, patients frequently develop RT resistance, particularly in castration-resistant PCa (CRPC), although the underlying mechanisms remain unknown. Understanding the underlying mechanism of RT resistance in CRPC may potentially highlight novel targets to improve therapeutic options for patients with PCa. In the present study, the expression levels of phospholipase Cε (PLCε), androgen receptor (AR) and DNA-dependent protein kinase catalytic subunit (PKcs) were examined in PCa tissue samples and PCa cells, and the effects of PLCε knockdown on AR and DNA damage repair (DDR)-related molecules were determined. The association between PLCε, AR and Poly (ADP-ribose) polymerase 1 (PARP1), as well as their respective roles in radiation resistance, were assessed using gene knockdown and pharmaceutical inhibitors or activators. A chromatin immunoprecipitation assay was used to determine the epigenetic regulatory effects of PLCε on PARP1. Animal experiments were performed to assess whether the mechanisms observed in vitro could be replicated in vivo. The expression levels of PLCε, AR and DNA-PKcs were significantly upregulated in PCa, particularly in CRPC. PLCε knockdown reduced the viability and increased apoptosis of cells subjected to radiation. Additionally, PLCε deficiency suppressed DDR progression by downregulating an AR and PARP1 positive feedback loop and the associated downstream molecules following radiation. PLCε depletion also increased the presence of histone H3 lysine 27 trimethylation in the PARP1 promoter region, suggesting increased methylation of the PARP1 gene and thus resulting in reduced expression of PARP1. In vivo, PLCε knockdown significantly potentiated the effects of radiation on tumor growth. Taken together, the results of the present study demonstrated that PLCε knockdown enhanced the radiosensitivity of CRPC by downregulating the AR/PARP1/DNA-PKcs axis.

The Role of the Tumor Microenvironment in Neuropilin 1-Induced Radiation Resistance in Lung Cancer Cells.

Background: Neuropilin 1 (NRP1) is a pleiotropic receptor which can interact with multiple ligands and their receptors. It plays an important role in the process of axonal growth, angiogenesis, tumor metastasis and radiation resistance in endothelial cells and some tumor cells. Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis, and has received considerable attention in recent years.Material and Methods: In this study, A549 lung cancer cell lines with different NRP1 expression levels were constructed in vitro, a two-dimensional (2D), three-dimensional (3D) co-culture system and tumor-bearing model was established in SCID mice. Western blot, qRT-PCR, immunofluorescence, cytometric bead array and flow cytometry were used to investigate the effect of the tumor microenvironment in NRP1-induced lung cancer cell radiation resistance.Results: In 2D or 3D co-culture system, NRP1 could be regulated inflammatory factors such as TNF, IL-6 IL-8 and IL-17 and the related chemokines MCP-1, IP-10 and RANTES in the tumor microenvironment, which in turn induced radiation resistance in lung cancer cells. In addition, different expression levels of NRP1 in 2D, 3D culture systems and tumor-bearing models were able to significantly regulate cell phenotype, proliferative capacity, epithelial-mesenchymal transition (EMT) and the radiation resistance of A549 cells.Conclusion: Our results verified that NRP1, inflammatory factors, chemokines and related signaling pathways, which affect the transformation of related cell components and thus lung cancer cell immune tolerance and migratory ability, all play an important role in radiation resistance.

Knockdown of long non-coding RNA PCAT1 in glioma stem cells promotes radiation sensitivity.

This study aimed to investigate the function of glioma stem cells (GSCs) and the role of PCAT1. This study dissociated the differences between GSCs and glioma cells in terms of apoptosis rate and γH2AX positive cells levels after radiation. Microarray was carried out to detect that expressed PCAT1, and it was testified by RT-qPCR. After transfection, GSCs were used to investigate the influence of PCAT1 on radiation sensitivity. Sphere-formation capability was first examined. Cell apoptosis rate after radiation of 0Gy or 6Gy was analyzed by flow cytometry, and the level of γH2AX positive cells after 6Gy radiation were compared. CCK8 assay was used to investigate the cell proliferation and RT-qPCR was used to examine miR-129-5p and HMGB1 expression. GSCs exhibited great capability in sphere formation and lower expression in apoptosis and γH2AX positive cells rates after 6Gy radiation. PCAT1 had higher expression in GSCs. PCAT1 knockdown restrained the sphere-formation ability, increased the apoptosis rate and DNA damage under the treatment of radiation. Moreover, knockdown of PCAT1 inhibited the cell proliferation. In addition, silencing PCAT1 could increase the expression of miR-129-5p and decrease the expression of HMGB1. PCAT1 was overexpressed in GSCs and played a facilitating role in radiation resistance.

Stress Induced Overexpression of Stanniocalcin 2 (STC2) Plays Key Role in Radiation Resistance and Metastasis of Nasopharyngeal Carcinomas

Stanniocalcin2 (STC2) expression is up-regulated by multiple stress conditions including hypoxia, nutrient depletion and radiation, and overexpression of STC2 correlates with tumor progression and poor prognosis. Particularly, it has been demonstrated that overexpression of STC2 in nasopharyngeal carcinomas (NPC) positively correlates with radiation resistance and metastasis, two major clinical obstacles to further improvement of NPC management. However, a contributing role of STC2 overexpression in radiation resistance and metastasis of NPC has not been established. We hypothesize that stress-induced overexpression of STC2 plays a key role in radiation resistance and metastasis of NPC. This study has two objectives: 1) to define the importance of STC2 overexpression in radiation resistance in NPC; and 2) to determine if targeting STC2 can be explored as a strategy to sensitize NPC to radiation. Radiation resistant CNE2 cell line was used as a model system. CRISPR/Cas9-based genome editing was used to create STC2 knockout lines (CNE2-STC2-KO). The genomic status of KO lines was confirmed by T7E1 assays. The STC2 mRNA and protein levels of parental and KO cell lines were examined using quantitative reverse transcription polymerase chain reaction (PCR) and Western blotting analysis, respectively. NPC cells were radiated with an ELEKTA accelerator (6MVx, Sweden) at distance of 100 cm at 285cGy/min. Cell survival was assayed by flow cytometric analysis, TrypanBlue staining and clonogenic analysis. Cell cycle distribution was analyzed by flow cytometry, and invasion and migration abilities were gauged with Transwell/MatriGel-based assays. SPSS 17.0 was used for statistical analysis, and p<0.05 was set as significant. CRISP/Cas9 construct was made and transfected into CNE2 cells to create CNE2-KO lines. The KO lines were verified at genomic, mRNA and protein levels. Compared with the parental radiation resistant CNE2 line, the STC2-KO lines show increased radiation sensitivity under either normoxic or hypoxic stress. In response to radiation, STC2-KO cells manifest decreased invasion and migration ability. Cell cycle analyses reveal that loss of STC2 significantly increases NPC cells at G2/M phase post radiation, consistent with an enhanced cytotoxicity. These data demonstrate that elevated expression of STC2 under various stress conditions promotes NPC resistance to radiation, facilitates cell survival and metastasis. Targeting STC2 function may provide a novel strategy to effectively overcome radiation resistance and to prevent metastasis of NPC.

Targeting Tumor Microenvironment and Metabolism to Overcome Radiation Resistance.

The tumor microenvironment comprises multiple different cell types and structural and functional components, and defines a unique tumor milieu on the individual tumor level. Several biological processes in the microenvironment are pivotal for tumor growth, like the formation of an intact tumor vasculature and the composition of the extracellular matrix. Furthermore, tumor heterogeneity also derives from the complex and dynamic interactions of the tumor cells with the stromal compartment during tumor growth and in response to treatment, and thereby represents a major treatment hurdle. Here we give an overview of the different entities of the tumor microenvironment and present their role in radiation resistance. Dynamic changes in response to irradiation will be outlined, and relevant approaches to target critical elements of the tumor microenvironment will be discussed. We are only now starting to understand how these different entities cooperate biologically, and thereby determine tumor aggressiveness and treatment resistance on the individual level. At the same time, these dynamic and even treatment-induced interactions within the tumor represent promising targets for novel combined treatment modalities with radiotherapy.

Conservation and diversity of the IrrE/DdrO-controlled radiation response in radiation-resistant Deinococcus bacteria.

The extreme radiation resistance of Deinococcus bacteria requires the radiation‐stimulated cleavage of protein DdrO by a specific metalloprotease called IrrE. DdrO is the repressor of a predicted radiation/desiccation response (RDR) regulon, composed of radiation‐induced genes having a conserved DNA motif (RDRM) in their promoter regions. Here, we showed that addition of zinc ions to purified apo‐IrrE, and short exposure of Deinococcus cells to zinc ions, resulted in cleavage of DdrO in vitro and in vivo, respectively. Binding of IrrE to RDRM‐containing DNA or interaction of IrrE with DNA‐bound DdrO was not observed. The data are in line with IrrE being a zinc peptidase, and indicate that increased zinc availability, caused by oxidative stress, triggers the in vivo cleavage of DdrO unbound to DNA. Transcriptomics and proteomics of Deinococcus deserti confirmed the IrrE‐dependent regulation of predicted RDR regulon genes and also revealed additional members of this regulon. Comparative analysis showed that the RDR regulon is largely well conserved in Deinococcus species, but also showed diversity in the regulon composition. Notably, several RDR genes with an important role in radiation resistance in Deinococcus radiodurans, for example pprA, are not conserved in some other radiation‐resistant Deinococcus species.

Cancer stem cells in radiation resistance of esophageal cancer: role and molecular mechanism

Objective To investigate the role of cancer stem cells in radiation resistance of esophageal cancer and its molecular mechanism, and to provide a theoretical basis for radiotherapy for esophageal cancer. Methods Esophageal cancer cell line TE1 was treated with 8 Gy of radiation. Esophageal cancer cell line with resistance to radiation, TE1-res, was established and screened. Cell counting was used to evaluate cell proliferation. Flow cytometry was used to determine the expression of CD44(high) CD24(-)CD133(+) and apoptosis in cells. The colony formation assay was used to determine the colony-forming rate and cell survival curve. Bisulfite sequencing PCR was used to determine the methylation status of cancer suppressor genes. Comparison of the data was made by group t test or analysis of variance. Results Compared with TE1 cells, TE1-res cells had significantly enhanced proliferation, a significantly higher proportion of CD44(high) CD24(-)CD133(+) cells, and significantly enhanced resistance to apoptosis (mean value 20.84×105vs. 4.46×105/day, P=0.008; (38.0±2.9)% vs. (10.1±1.3)%, P=0.001; mean value 33.23% vs. 10.50%, P=0.003). After treatment with 8 Gy of radiation, TE1-res cells had significantly higher colony-forming rate and D0 value than TE1 cells ((14.3±2.6)% vs. (0.9±0.3)%, P=0.011; 3.28 vs. 2.19 Gy, P=0.125). Moreover, the promoter methylation in cancer suppressor genes including SPINT2, CDKN1B, DKK1, TP53, and PPP2R1B was significantly enhanced in TE1-res cells than in TE1 cells ((89.7±4.9)% vs. (5.0±0.5)%, P=0.001; (92.3±4.7)% vs. (10.4±0.7)%, P=0.001; (90.7±3.7)% vs. (7.9±0.4)%, P=0.001; (83.4±5.7)% vs. (17.2±1.2)%, P=0.002; (90.2±6.7)% vs. (4.4±1.2)%, P=0.002). Conclusions Cancer stem cells play an important role in radiation resistance of esophageal cancer. The resistance to radiation is closely associated with promoter hypermethylation in cancer suppressor genes including SPINT2, CDKN1B, DKK1, TP53, and PPP2R1B. Key words: Esophageal neoplasms cell line; Irradioresistance; Cancer suppressor gene; Methylation

Abstract 2227: Effects of cathepsin S in differentiated and stem-like glioblastoma cells

Abstract Background: Glioblastoma (GBM) is the most common and aggressive brain tumour subtype with a poor prognosis of ∼15 months. A GBM stem-like population within the tumour is hypothesised to promote resistance to current treatment and invasion through normal tissue. Cathepsin S (Cat S) is a lysosomal cysteine protease that remains catalytically active at neutral pH and has been shown to play a role in radiation resistance and invasion of cancer cells. It is also an independent predictor of GBM patient survival. Materials and methods: GBM cell lines (U87MG and LN229) with lentiviral shRNA knockdown of Cat S are grown in serum-free medium with growth factors that allow de-differentiation of the cells into a stem-like state. A transwell system is used for quantifying monolayer invasion. Under stem conditions, spheroid structures are formed and their invasion in 3D is analysed with GFP expression. Protein expression determined via Western blot. Radiation response determined via clonogenic survival assay. Proliferation measured using MTT assay. Cat S-like activity measured using fluorogenic peptide substrate cleavage. Results: Radiation induces Cat S protein expression and protease activity in both GBM cell lines at 24 hours. Knockdown of Cat S induces radiation sensitivity in both cell lines when differentiated. In stem-like conditions, this is observed for U87MG but not for LN229 cells. Similarly, Cat S knockdown has no effect on proliferation of cell lines, except LN229 stem-like cells where it is increased. Finally, Cat S knockdown reduces the invasive capacity of both differentiated cell lines and U87MG stem-like cells 3D invasion through both collagen and matrigel, but has no effect on LN229 stem-like cells. Cat S-like activity is reduced in all but stem LN229, despite complete knockdown. Further investigation into differential effects in stem-like cells will examine possible role of autophagy, which is linked to radiation resistance and Cat S inhibition. Conclusion Cat S is a potential drug target in GBM as it reduces invasion and radio-resistance of glioma cell lines. The differential effect seen in stem-like cells suggests that the genetic background of tumours may be an important factor in determining utility of Cat S therapy in targeting the stem-like GBM population thought to be responsible for recurrence. Citation Format: Robyn A. Foster, Shahnaz T. Al Rashid, Roberta E. Burden, Christopher J. Scott, Kevin M. Prise, Thomas J. Flannery. Effects of cathepsin S in differentiated and stem-like glioblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2227. doi:10.1158/1538-7445.AM2015-2227

Nimotuzumab enhances radiation sensitivity of NSCLC H292 cells in vitro by blocking epidermal growth factor receptor nuclear translocation and inhibiting radiation-induced DNA damage repair.

BackgroundThe epidermal growth factor receptor (EGFR) signaling pathway plays a significant role in radiation resistance. There is evidence that EGFR nuclear translocation is associated with DNA double-strand breaks (DSB) repair. Nimotuzumab has shown the effect of radiosensitization in various cancer cells, but little is known about the relationship between nimotuzumab and EGFR nuclear translocation in non-small cell lung cancer (NSCLC) cell lines. In this study, we selected two NSCLC cell lines, namely, H292 (with high EGFR expression) and H1975 (with low EGFR expression) and explored the mechanisms underlying radiation sensitivity.MethodsMTT assay, clonogenic survival assay, and flow cytometry were performed separately to test cell viability, radiation sensitivity, cell cycle distribution, and apoptosis. Protein γ-H2AX, DNA-PK/p-DNA-PK, and EGFR/p-EGFR expression were further compared both in the cytoplasm and the nucleus with the western blot.ResultsNimotuzumab reduced the viability of H292 cells and sensitized H292 cells to ionizing radiation. The radiation sensitivity enhancement ratio (SER) was 1.304 and 1.092 for H292 and H1975 cells, respectively. H292 cells after nimotuzumab administration were arrested at the G0/G1 phase in response to radiation. Apoptosis was without statistical significance in both cell lines. γ-H2AX formation in the combination group (nimotuzumab and radiation) increased both in the cytoplasm and the nucleus along with the decreased expression of nuclear EGFR/p-EGFR and p-DNA-PK in H292 cells (P<0.05) that was more significant than that in H1975 cells.ConclusionOur research revealed a possible mechanism to explain the radiosensitivity in H292 cells. Nimotuzumab decreased the radiation-induced activation of DNA-PK by blocking EGFR nuclear translocation and impairing DNA DSB repair, thus enhancing radiosensitivity in H292 cells. Because these results represent early research, the matters of how γ-H2AX and DNA-PK dynamically change simultaneously with nuclear EGFR and the best time to administer nimotuzumab will require further exploration.

Integrated Omic Analysis of Oropharyngeal Carcinomas Reveals Human Papillomavirus (HPV)–dependent Regulation of the Activator Protein 1 (AP-1) Pathway

HPV-positive oropharyngeal carcinoma (OPC) patients have superior outcomes relative to HPV-negative patients, but the underlying mechanisms remain poorly understood. We conducted a proteomic investigation of HPV-positive (n = 27) and HPV-negative (n = 26) formalin-fixed paraffin-embedded OPC biopsies to acquire insights into the biological pathways that correlate with clinical behavior. Among the 2,633 proteins identified, 174 were differentially abundant. These were enriched for proteins related to cell cycle, DNA replication, apoptosis, and immune response. The differential abundances of cortactin and methylthioadenosine phosphorylase were validated by immunohistochemistry in an independent cohort of 29 OPC samples (p = 0.023 and p = 0.009, respectively). An additional 1,124 proteins were independently corroborated through comparison to a published proteomic dataset of OPC. Furthermore, utilizing the Cancer Genome Atlas, we conducted an integrated investigation of OPC, attributing mechanisms underlying differential protein abundances to alterations in mutation, copy number, methylation, and mRNA profiles. A key finding of this integration was the identification of elevated cortactin oncoprotein levels in HPV-negative OPCs. These proteins might contribute to reduced survival in these patients via their established role in radiation resistance. Through interrogation of Cancer Genome Atlas data, we demonstrated that activation of the β1-integrin/FAK/cortactin/JNK1 signaling axis and associated differential regulation of activator protein 1 transcription factor target genes are plausible consequences of elevated cortactin protein levels.

Genome-wide association analysis of radiation resistance in Drosophila melanogaster.

BackgroundIonizing radiation is genotoxic to cells. Healthy tissue toxicity in patients and radiation resistance in tumors present common clinical challenges in delivering effective radiation therapies. Radiation response is a complex, polygenic trait with unknown genetic determinants. The Drosophila Genetic Reference Panel (DGRP) provides a model to investigate the genetics of natural variation for sensitivity to radiation.Methods and FindingsRadiation response was quantified in 154 inbred DGRP lines, among which 92 radiosensitive lines and 62 radioresistant lines were classified as controls and cases, respectively. A case-control genome-wide association screen for radioresistance was performed. There are 32 single nucleotide polymorphisms (SNPs) associated with radio resistance at a nominal p<10−5; all had modest effect sizes and were common variants with the minor allele frequency >5%. All the genes implicated by those SNP hits were novel, many without a known role in radiation resistance and some with unknown function. Variants in known DNA damage and repair genes associated with radiation response were below the significance threshold of p<10−5 and were not present among the significant hits. No SNP met the genome-wide significance threshold (p = 1.49×10−7), indicating a necessity for a larger sample size.ConclusionsSeveral genes not previously associated with variation in radiation resistance were identified. These genes, especially the ones with human homologs, form the basis for exploring new pathways involved in radiation resistance in novel functional studies. An improved DGRP model with a sample size of at least 265 lines and ideally up to 793 lines is recommended for future studies of complex traits.

Hypothetical Proteins Present During Recovery Phase of Radiation Resistant Bacterium Deinococcus radiodurans are Under Purifying Selection

Deinococcus radiodurans has an unusual capacity to recover from intense doses of ionizing radiation. The DNA repair proteins of this organism play an important role in repairing the heavily damaged DNA by employing a novel mechanism of DNA double-strand break repair. An earlier report stated that genes of many of these repair proteins are under positive selection implying that these genes have a tendency to mutate, which in turn provides selective advantage to this bacterium. Several "hypothetical proteins" are also present during the recovery phase and some of them have also been shown for their roles in radiation resistance. Therefore, we tested the selection pressure on the genes encoding these poorly characterized proteins. Our results show that a number of "hypothetical proteins" present during the repair phase have structural adaptations compared to their orthologs and the genes encoding them as well as those for the DNA repair proteins present during this phase are under purifying selection. Evidence of purifying selection in these hypothetical proteins suggests that certain novel characteristics among these proteins are conserved and seem to be under functional constraints to perform important functions during recovery process after gamma radiation damage.

Abstract 290: EMT and cancer stem cells: role in radiation resistance of breast cancer.

Abstract Epithelial-Mesenchymal Transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. During EMT, epithelial cells lose epithelial characteristics, such as E-cadherin expression and gain mesenchymal properties, such as vimentin expression. EMT cells have stem cells properties and possess the characteristics of cell motility, invasiveness and chemotherapy resistance and these cancer stem cells may be responsible for mediating tumor metastasis and resistance to cancer treatments. However, very little is known about the role of EMT and cancer stem cells in modulating radiation response of human breast cancer cells. We compared expression levels of E-cadherin and other EMT related markers in ER-a negative (MDA-MB-231 and Hs578t) and ER-a positive (MCF-7) human breast cancer cells. Clonogenic cell survival assays showed that the cell lines expressing estrogen receptor (MCF-7) were more sensitive to increasing doses of radiation and had high expression of E-cadherin. In contrast, ER negative cells (MDA-MB-231 and Hs578t) had no detectable expression of E-cadherin and were more radioresistant. We transfected MDA-MB-231 and Hs578t cells with a CDH1-expression vector and isolated stable clones. These clones were selected and tested for radiosensitivity. MDA-MB-231 and Hs578t cells transfected with a control vector (pCMV) served as controls. Restoring E-cadherin expression radiosensitized the cells compared to the control vector cell line, suggesting that restoration of E-cadherin expression in mesenchymal-like cells produces a radiosensitizing effect. Our preliminary data demonstrates that EMT, detected as the loss of E-cadherin expression, may regulate tumor cell radiosensitivity, i.e. cells that have undergone EMT are relatively radioresistant compared to the lines that have retained the epithelial phenotype, which are relatively radiosensitive. Thus, there was a general correlation between EMT, based on loss of E-cadherin expression, and radioresistance. Overall, our results suggest that E-cadherin interacts with radiation and enhances the radioresponse of human breast cancer cells. Since it has been demonstrated that the process of EMT contributes to drug resistance and results in cells with CSC-like characteristics, we compared ALDH1 expression in ER positive (MCF-7) and ER negative (MDA-MB-231 and Hs578t) cell lines. Our data shows that ALDH1 expression correlates with the ER status of the breast cancer cells, with a higher number of ALDH1+ cells in ER negative compared to ER positive cell lines. Our preliminary investigation leads us to believe that potentially resistant breast cancer stem cell populations appear to be overrepresented in ER negative breast cancer cell lines. Our observation concurs with clinical data that ER negative cancers are resistant to radiation therapy compared to ER positive breast cancers. Citation Format: Anupama Munshi, Meghna Mehta, Yuhui Yuan, Huifeng Liu, Raymond E. Meyn, Rajagopal Ramesh. EMT and cancer stem cells: role in radiation resistance of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 290. doi:10.1158/1538-7445.AM2013-290

Abstract 3370: DNA repair in glioma stem cell-mediated radiation resistance

Abstract Glioblastoma multiforme is the most common type of primary brain cancer in adults and has a 14-month median survival after diagnosis. This low survival is due to resistance to the currently available therapies, which include surgery, radiation, chemotherapy, and anti-angiogenic drugs. Resistance to chemotherapy and radiation in these tumors has been linked to a subgroup of highly tumorigenic cells, called glioblastoma stem cells (GSCs), that resemble normal neural progenitor cells. GSCs have the ability to self-renew, differentiate into various lineages, and re-propagate the tumor, making GSCs an attractive target for treatment. Radiation-treated GSCs retain their tumorigenic capacity and have decreased apoptosis compared to non-GSCs. In addition, GSCs have enhanced DNA damage response activation and faster DNA double-stranded break (DSB) resolution relative to non-GSCs after exposure to ionizing radiation (IR). Exposure to low dose radiation induces DSBs, which are repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). Targeting key proteins involved in HR, such as RAD51, has been shown to reduce glioma cell viability. We are investigating the molecular mechanisms by which DNA repair mediates radiation resistance in GSCs, with some lead candidates identified. Based on a DNA repair gene array, several candidates were identified to be preferentially expressed in GSCs. Current work is focusing on targeting these genes and elucidating their role in radiation resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3370. doi:1538-7445.AM2012-3370

Abstract 3455: p53 status determines the radio-sensitizing effect of an Akt kinase inhibitor in malignant gliomas

Abstract Purpose: It has been reported that PI3K-Akt pathway, which plays a role in cell survival and apoptosis, is deregulated in many cancer types. In gliomas, amplification and constitutive activation of Akt, which can result from amplifications in TKIs including EGFR, IGFR1α, PDGFRα, etc. and mutations in PI3K, PTEN, etc. are thought to play a role in radiation resistance. Some have tried to modulate the PI3K/Akt pathway by targeting molecules upstream of Akt such as Ras and PI3K, while others have used inhibitors targeting molecules downstream of Akt, such as mTOR. These inhibitors have been shown to reduce cancer cell growth in vitro and in vivo, but have had limited success in clinical settings as single agents. In our study, we have investigated the use of a phosphatidylinositol ether lipid analogue (PIA) that directly inhibits Akt activation as a radiosensitizer for GBM. Materials and Methods: We utilized multiple established (U87, LN18, LN229) and primary (VC3, MGH8, OSU61) cell lines with varying molecular and genetic signatures to determine radiosensitizing effects of PIA. We performed Annexin V, clonogenic survival, and COMET assays on glioma cells treated with PIA, radiation (RT), and a combination of PIA and RT. Western blot was performed using these cell lines to examine the changes in expression of PI3K-Akt pathway, apoptotic, and pro-survival proteins following treatment. Furthermore, we sequenced tumor suppressor genes p53 and PTEN in all cell lines used in this study. Gene silencing of p53 was done in U87 cells via a lentiviral transfection of shRNA. Results and Conclusions: PIA decreases the activation of Akt to a considerable extent (∼80%), induces apoptosis, and reduces clonogenicity of all cell lines tested. A subset of cell lines did show enhanced radiosensitization when PIA is combined with RT. Further detailed investigation of differential radiosensitizing effects led us to discover the link between p53 status, Akt kinase inhibition, and radiosensitization. GBMs with intact p53 had no radiosensitizing effect after Akt kinase inhibition, whereas radiosensitization was observed if either p53 is null or mutated. This observation was further validated using isogenic U87 p53 knockdown cells. Surprisingly, MGMT status, a predictive marker of outcome in GBM, did not determine radiosensitizing effects of PIA in the GBM cells tested. Through this preclinical study, we have identified which subset of GBM patients will derive benefit from Akt kinase inhibition and the investigations conducted justify future clinical trials and lays ground work for phase I study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3455. doi:1538-7445.AM2012-3455

DR2417, a hypothetical protein characterized as a novel β-CASP family nuclease in radiation resistant bacterium, Deinococcus radiodurans

BackgroundDeinococcus radiodurans survives extreme doses of radiations contributed by efficient DNA repair pathways. DR2417 (DncA) was detected separately both in a pool of nucleotide binding proteins and multiprotein complex isolated from cells undergoing DNA repair. Scope of reviewDR_2417m ORF was sequenced and amino acid sequence of DncA was search for structural similarities with other proteins and functional motifs. Recombinant DncA was characterized for its DNA metabolic functions in vitro and its role in radiation resistance. Major conclusionsSequencing of DR_2417m did not show the reported frame shift at 996th nucleotide position of this gene. DncA showed similarities with β-CASP family nucleases. Recombinant protein acted efficiently on dsDNA and showed an Mn2+ dependent 3′→5′ exonuclease and ssDNA/dsDNA junction endonuclease activities while a very low level activity on RNA. The DNase activity of this protein was inhibited in presence of ATP. Its transcription was induced upon γ radiation exposure and a reduction in its copy number resulted in reduced growth rate and loss of γ radiation resistance in Deinococcus. Conclusion — our results suggest that DncA was a novel nuclease of β CASP family having a strong dsDNA end processing activity and it seems to be an essential gene required for both growth and γ radiation resistance of this bacterium. General significanceTraditionally DncA should have shown both DNase and RNase functions as other members of β CASP family nucleases. A strong DNase and poor RNase activity possibly made it functionally significant in the radioresistance of D. radiodurans, which would be worth investigating independently.