The Chaperone Protein GRP78 Promotes Survival and Migration of Head and Neck Cancer After Direct Radiation Exposure and Extracellular Vesicle-Transfer.

Background and Purpose: Increased levels of the chaperone protein GRP78 have been implicated in poorer outcomes of cancer therapy. We have therefore explored the functional connection between the expression of GRP78 and the development of radioresistance and metastatic behavior in HNSCC. Material and Methods: The association between gene expression of GRP78 and survival in HNSCC patients was examined using the TCGA database. The influence of ionizing radiation on the GRP78 levels in HNSCC cell lines, their secreted extracellular vesicles (EV) and non-irradiated EV-recipient cells was investigated by Western Blot and FACS. The consequences of chemical inhibition or experimental overexpression of GRP78 on radioresistance and migration of HNSCC cells were analyzed by clonogenic survival and gap closure assays. Results: Elevated levels of GRP78 RNA in HNSCC correlated with poorer overall survival. Radiation increased GRP78 protein expression on the surface of HNSCC cell lines. Experimental overexpression of GRP78 increased both radioresistance and migratory potential. Chemical inhibition of GRP78 impaired cell migration. EVs were identified as a potential source of increased GRP78 content as elevated levels of surface GRP78 were found in EVs released by irradiated cells. These vesicles transferred GRP78 to non-irradiated recipient cells during co-cultivation. Conclusions: We have identified the chaperone protein GRP78 as a potential driver of increased radioresistance and motility in HNSCC. The uptake of GRP78-rich EVs originating from irradiated cells may contribute to a poorer prognosis through bystander effects mediated by the transfer of GRP78 to non-irradiated cells. Therefore, we consider the chaperone protein GRP78 to be an attractive target for improving radiotherapy strategies.

Radiation Exposure of Peripheral Mononuclear Blood Cells Alters the Composition and Function of Secreted Extracellular Vesicles.

Normal tissue toxicity is a dose-limiting factor in radiation therapy. Therefore, a detailed understanding of the normal tissue response to radiation is necessary to predict the risk of normal tissue toxicity and to development strategies for tissue protection. One component of normal tissue that is continuously exposed during therapeutic irradiation is the circulating population of peripheral blood mononuclear cells (PBMC). PBMCs are highly sensitive to ionizing radiation (IR); however, little is known about how IR affects the PBMC response on a systemic level. It was the aim of this study to investigate whether IR was capable to induce changes in the composition and function of extracellular vesicles (EVs) secreted from PBMCs after radiation exposure to different doses. Therefore, whole blood samples from healthy donors were exposed to X-ray radiation in the clinically relevant doses of 0, 0.1, 2 or 6 Gy and PBMC-secreted EVs were isolated 72 h later. Proteome and miRNome analysis of EVs as well as functional studies were performed. Secreted EVs showed a dose-dependent increase in the number of significantly deregulated proteins and microRNAs. For both, proteome and microRNA data, principal component analysis showed a dose-dependent separation of control and exposed groups. Integrated pathway analysis of the radiation-regulated EV proteins and microRNAs consistently predicted an association of deregulated molecules with apoptosis, cell death and survival. Functional studies identified endothelial cells as an efficient EV recipient system, in which irradiation of recipient cells further increased the uptake. Furthermore an apoptosis suppressive effect of EVs from irradiated PBMCs in endothelial recipient cells was detected. In summary, this study demonstrates that IR modifies the communication between PBMCs and endothelial cells. EVs from irradiated PBMC donors were identified as transmitters of protective signals to irradiated endothelial cells. Thus, these data may lead to the discovery of biomarker candidates for radiation dosimetry and even more importantly, they suggest EVs as a novel systemic communication pathway between irradiated normal, non-cancer tissues.

Comparison of Radiosensitization by HDAC Inhibitors CUDC-101 and SAHA in Pancreatic Cancer Cells.

Pancreatic cancer has a poor prognosis. New treatment options are urgently required to improve patient outcomes. One promising new class of anticancer drugs are synthetic histone deacetylase inhibitors (HDACi) which modulate chromatin structure and gene expression by blocking histone deacetylation. In this study, we aimed at comparing the in vitro capacities of the HDACi SAHA and CUDC-101 to increase radiosensitivity of human pancreatic tumor cell lines. Therefore, three pancreatic cancer cell lines (Su.86.86, MIA Paca-2, T3M-4) were treated with SAHA (1.5-5 µM) or CUDC-101 (0.25-3 µM) and after 24 h irradiated. Cell proliferation, clonogenic survival and apoptosis was determined. Additionally, cell lysates were investigated for the expression of apoptosis-related proteins. CUDC-101 and SAHA increased the radiation sensitivity of pancreatic tumor cell lines in a dose-dependent manner. This was evidenced by cell proliferation and clonogenic survival. Furthermore, enhanced radiation sensitivity after CUDC-101 or SAHA treatment was confirmed for Su.86.86 and T3M-4 cells in a 3-D microtissue approach. Increased amounts of subG1 cells and diminished full length PARP-1 suggest increased radiation-induced apoptosis after SAHA or CUDC-101 treatment. The comparison of both inhibitors in these assays manifested CUDC-101 as more potent radiosensitizer than SAHA. In line, western blot quantification of the apoptosis-inhibitory proteins XIAP and survivin showed a stronger down-regulation in response to CUDC-101 treatment than after SAHA application. These proteins may contribute to the synergy between HDAC inhibition and radiation response. In conclusion, these preclinical results suggest that treatment with the HDAC inhibitors CUDC-101 or SAHA can enhance radiation-induced cytotoxicity in human pancreatic cells. However, comparison of both inhibitors identified the multi target inhibitor CUDC-101 as more potent radiosensitizer than the HDAC inhibitor SAHA.

The circRNA interactome-innovative hallmarks of the intra- and extracellular radiation response.

Generated by Quaking (QKI), circular RNAs (circRNAs) are newly recognised non-coding RNA (ncRNA) members characterised by tissue specificity, increased stability and enrichment within exosomes. Studies have shown that ionizing radiation (IR) can influence ncRNA transcription. However, it is unknown whether circRNAs or indeed QKI are regulated by IR. Microarray circRNA profiling and next generation sequencing revealed that circRNA expression was altered by low and medium dose exposure sourced predominantly from genes influencing the p53 pathway. CircRNAs KIRKOS-71 and KIRKOS-73 transcribed from the WWOX (WW Domain Containing Oxidoreductase) tumor suppressor (a p53 regulator) responded within hours to IR. KIRKOS-71 and KIRKOS-73 were present in exosomes yet exhibited differential transcript clearance between irradiated cell lines. Dual-quasar labelled probes and in-situ hybridization demonstrated the intercellular distribution of KIRKOS-71 and KIRKOS-73 predominantly within the perinucleus. QKI knockdown removed nuclear expression of these circRNAs with no significant effect on cytosolic KIRKOS-71 and KIRKOS-73. Distinct QKI transcription between cell lines and its augmented interaction with KIRKOS-71 and KIRKOS-73 was noted post IR. This foremost study provides evidence that QKI and circRNAs partake in the cellular irradiation response. KIRKOS-71 and KIRKOS-73 as stable secreted circRNAs may afford vital characteristics worth syphoning as promising diagnostic radiotherapy biomarkers.

Radiation alters the cargo of exosomes released from squamous head and neck cancer cells to promote migration of recipient cells.

Radiation is a highly efficient therapy in squamous head and neck carcinoma (HNSCC) treatment. However, local recurrence and metastasis are common complications. Recent evidence shows that cancer-cell-derived exosomes modify tumour cell movement and metastasis. In this study, we link radiation-induced changes of exosomes to their ability to promote migration of recipient HNSCC cells. We demonstrate that exosomes isolated from irradiated donor cells boost the motility of the HNSCC cells BHY and FaDu. Molecular data identified enhanced AKT-signalling, manifested through increased phospho-mTOR, phospho-rpS6 and MMP2/9 protease activity, as underlying mechanism. AKT-inhibition blocked the pro-migratory action, suggesting AKT-signalling as key player in exosome-mediated migration. Proteomic analysis of exosomes isolated from irradiated and non-irradiated BHY donor cells identified 39 up- and 36 downregulated proteins. In line with the observed pro-migratory effect of exosomes isolated from irradiated cells protein function analysis assigned the deregulated exosomal proteins to cell motility and AKT-signalling. Together, our findings demonstrate that exosomes derived from irradiated HNSCC cells confer a migratory phenotype to recipient cancer cells. This is possibly due to radiation-regulated exosomal proteins that increase AKT-signalling. We conclude that exosomes may act as driver of HNSCC progression during radiotherapy and are therefore attractive targets to improve radiation therapy strategies.

Radiation induced transcriptional and post-transcriptional regulation of the hsa-miR-23a~27a~24-2 cluster suppresses apoptosis by stabilizing XIAP.

The non-coding transcriptome, in particular microRNAs (miRNA), influences cellular survival after irradiation. However, the underlying mechanisms of radiation-induced miRNA expression changes and consequently target expression changes are poorly understood. In this study we show that a single dose of 5Gy ɣ-radiation decreases expression of the miR-23a~27a~24-2 cluster in the human endothelial cell-line EA.hy926 and the mammary epithelial cell-line MCF10A. In the endothelial cells this was facilitated through transcriptional regulation by promoter methylation and also at the post-transcriptional level by reduced miRNA processing through phosphorylation of Argonaute (AGO). Furthermore, we demonstrate that all three mature cluster miRNAs reduce apoptosis by increasing expression of the common target protein XIAP. These findings link a temporal succession of transcriptional and post-transcriptional regulatory mechanisms of the miR~23a~24-2~27a cluster, enabling a dynamic stress response and assuring cellular survival after radiation exposure.

Exosomes Derived from Squamous Head and Neck Cancer Promote Cell Survival after Ionizing Radiation.

Exosomes are nanometer-sized extracellular vesicles that are believed to function as intercellular communicators. Here, we report that exosomes are able to modify the radiation response of the head and neck cancer cell lines BHY and FaDu. Exosomes were isolated from the conditioned medium of irradiated as well as non-irradiated head and neck cancer cells by serial centrifugation. Quantification using NanoSight technology indicated an increased exosome release from irradiated compared to non-irradiated cells 24 hours after treatment. To test whether the released exosomes influence the radiation response of other cells the exosomes were transferred to non-irradiated and irradiated recipient cells. We found an enhanced uptake of exosomes isolated from both irradiated and non-irradiated cells by irradiated recipient cells compared to non-irradiated recipient cells. Functional analyses by exosome transfer indicated that all exosomes (from non-irradiated and irradiated donor cells) increase the proliferation of non-irradiated recipient cells and the survival of irradiated recipient cells. The survival-promoting effects are more pronounced when exosomes isolated from irradiated compared to non-irradiated donor cells are transferred. A possible mechanism for the increased survival after irradiation could be the increase in DNA double-strand break repair monitored at 6, 8 and 10 h after the transfer of exosomes isolated from irradiated cells. This is abrogated by the destabilization of the exosomes. Our results demonstrate that radiation influences both the abundance and action of exosomes on recipient cells. Exosomes transmit prosurvival effects by promoting the proliferation and radioresistance of head and neck cancer cells. Taken together, this study indicates a functional role of exosomes in the response of tumor cells to radiation exposure within a therapeutic dose range and encourages that exosomes are useful objects of study for a better understanding of tumor radiation response.

Cell survival following radiation exposure requires miR-525-3p mediated suppression of ARRB1 and TXN1.

BACKGROUND: microRNAs (miRNAs) are non-coding RNAs that alter the stability and translation efficiency of messenger RNAs. Ionizing radiation (IR) induces rapid and selective changes in miRNA expression. Depletion of the miRNA processing enzymes Dicer or Ago2 reduces the capacity of cells to survive radiation exposure. Elucidation of critical radiation-regulated miRNAs and their target proteins offers a promising approach to identify new targets to increase the therapeutic effectiveness of the radiation treatment of cancer. PRINCIPAL FINDINGS: Expression of miR-525-3p is rapidly up-regulated in response to radiation. Manipulation of miR-525-3p expression in irradiated cells confirmed that this miRNA mediates the radiosensitivity of a variety of non-transformed (RPE, HUVEC) and tumor-derived cell lines (HeLa, U2-Os, EA.hy926) cell lines. Thus, anti-miR-525-3p mediated inhibition of the increase in miR-525-3p elevated radiosensitivity, while overexpression of precursor miR-525-3p conferred radioresistance. Using a proteomic approach we identified 21 radiation-regulated proteins, of which 14 were found to be candidate targets for miR-525-3p-mediated repression. Luciferase reporter assays confirmed that nine of these were indeed direct targets of miR-525-3p repression. Individual analysis of these direct targets by RNAi-mediated knockdown established that ARRB1, TXN1 and HSPA9 are essential miR-525-3p-dependent regulators of radiation sensitivity. CONCLUSION: The transient up-regulation of miR-525-3p, and the resultant repression of its direct targets ARRB1, TXN1 and HSPA9, is required for cell survival following irradiation. The conserved function of miR-525-3p across several cell types makes this microRNA pathway a promising target for modifying the efficacy of radiotherapy.

MicroRNA-mediated processes are essential for the cellular radiation response.

A detailed understanding of the mechanisms that determine the variable cellular sensitivity to radiation is needed for improved radiation therapy as well as for the identification of individuals with innate radiation hypersensitivity. MicroRNAs (miRNAs) are a class of small non-coding RNAs that post-transcriptionally regulate protein expression. Alterations in miRNA expression patterns in response to ionizing radiation have been shown, but there are almost no data describing the functional impact of these miRNA changes. We report here the results of studies on the functional roles of miRNAs in the radiation response in immortalized and primary endothelial cells. Global suppression of miRNA expression was achieved through downregulation of Argonaut e-2 (AGO2) or DICER proteins using RNAi. The reductions in either DICER or AGO2 led to increased cell death after irradiation, indicating a prosurvival function of miRNAs. Furthermore, while cell cycle checkpoint activation and apoptosis were compromised, DNA double-strand break repair was not affected by the lack of miRNAs. The differential sensitivity of these pathways implies the independent activation of the two response pathways rather than a concerted DNA damage response. The miRNAs that were changed after 2.5 Gy irradiation were identified by TaqMan-based low-density array technology. Of the miRNAs showing an upregulation 4 h or 24 h after radiation exposure, we were able to establish prosurvival and antiapoptotic functions for three miRNAs. Taken together, our data indicate a general prosurvival role for miRNA-mediated gene regulation during the radiation response. We show a functional association between miRNAs, apoptosis and cell cycle checkpoint activation in irradiated cells.

A novel function for the Mre11-Rad50-Xrs2 complex in base excision repair.

The Mre11/Rad50/Xrs2 (MRX) complex in Saccharomyces cerevisiae has well-characterized functions in DNA double-strand break processing, checkpoint activation, telomere length maintenance and meiosis. In this study, we demonstrate an involvement of the complex in the base excision repair (BER) pathway. We studied the repair of methyl-methanesulfonate-induced heat-labile sites in chromosomal DNA in vivo and the in vitro BER capacity for the repair of uracil- and 8-oxoG-containing oligonucleotides in MRX-deficient cells. Both approaches show a clear BER deficiency for the xrs2 mutant as compared to wildtype cells. The in vitro analyses revealed that both subpathways, long-patch and short-patch BER, are affected and that all components of the MRX complex are similarly important for the new function in BER. The investigation of the epistatic relationship of XRS2 to other BER genes suggests a role of the MRX complex downstream of the AP-lyases Ntg1 and Ntg2. Analysis of individual steps in BER showed that base recognition and strand incision are not affected by the MRX complex. Reduced gap-filling activity and the missing effect of aphidicoline treatment, an inhibitor for polymerases, on the BER efficiency indicate an involvement of the MRX complex in providing efficient polymerase activity.