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.

Focal adhesion signaling and therapy resistance in cancer.

Interlocking gene mutations, epigenetic alterations and microenvironmental features perpetuate tumor development, growth, infiltration and spread. Consequently, intrinsic and acquired therapy resistance arises and presents one of the major goals to solve in oncologic research today. Among the myriad of microenvironmental factors impacting on cancer cell resistance, cell adhesion to the extracellular matrix (ECM) has recently been identified as key determinant. Despite the differentiation between cell adhesion-mediated drug resistance (CAMDR) and cell adhesion-mediated radioresistance (CAMRR), the underlying mechanisms share great overlap in integrin and focal adhesion hub signaling and differ further downstream in the complexity of signaling networks between tumor entities. Intriguingly, cell adhesion to ECM is per se also essential for cancer cells similar to their normal counterparts. However, based on the overexpression of focal adhesion hub signaling receptors and proteins and a distinct addiction to particular integrin receptors, targeting of focal adhesion proteins has been shown to potently sensitize cancer cells to different treatment regimes including radiotherapy, chemotherapy and novel molecular therapeutics. In this review, we will give insight into the role of integrins in carcinogenesis, tumor progression and metastasis. Additionally, literature and data about the function of focal adhesion molecules including integrins, integrin-associated proteins and growth factor receptors in tumor cell resistance to radio- and chemotherapy will be elucidated and discussed.

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.

Unilateral Diaphragmatic Paralysis After Stereotactic Ablative Radiation Therapy to a Lung Tumor Abutting the Course of the Phrenic Nerve.

We present the case of a woman with metastatic adenoid cystic carcinoma who received stereotactic ablative radiation therapy with a total dose of 50 Gy in 4 fractions to 2 lung metastases and developed symptomatic left phrenic nerve injury 2 years after radiation. The maximum dose to the approximate location of the phrenic nerve was 57.7 Gy, which corresponds to a biologically effective dose for late effects (using α/ß ratio = 3) of 335.14 Gy. Here, we discuss the case, planning considerations by radiation oncologists and medical physicists, and the multidisciplinary medical management of this patient.

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.

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.

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.

Radiation-induced Adaptive Response: New Potential for Cancer Treatment.

Radiotherapy is highly effective due to its ability to physically focus the treatment to target the tumor while sparing normal tissue and its ability to be combined with systemic therapy. This systemic therapy can be utilized before radiotherapy as an adjuvant or induction treatment, during radiotherapy as a radiation "sensitizer," or following radiotherapy as a part of combined modality therapy. As part of a unique concept of using radiation as "focused biology," we investigated how tumors and normal tissues adapt to clinically relevant multifraction (MF) and single-dose (SD) radiation to observe whether the adaptations can induce susceptibility to cell killing by available drugs or by immune enhancement. We identified an adaptation occurring after MF (3 × 2 Gy) that induced cell killing when AKT-mTOR inhibitors were delivered following cessation of radiotherapy. In addition, we identified inducible changes in integrin expression 2 months following cessation of radiotherapy that differ between MF (1 Gy × 10) and SD (10 Gy) that remain targetable compared with preradiotherapy. Adaptation is reflected across different "omics" studies, and thus the range of possible molecular targets is not only broad but also time, dose, and schedule dependent. While much remains to be studied about the radiation adaptive response, radiation should be characterized by its molecular perturbations in addition to physical dose. Consideration of the adaptive effects should result in the design of a tailored radiotherapy treatment plan that accounts for specific molecular changes to be targeted as part of precision multimodality cancer treatment.

Expression of integrin-linked kinase is increased in differentiated cells.

Integrin-linked kinase (ILK), a mediator of beta integrin signals, has emerged as a therapeutic target in malignant tumors. Because malignant transformation is accompanied by dedifferentiation, ILK expression was evaluated in diverse normal and tumor tissue samples with regard to tissue differentiation. In single sections and in a tissue microarray (323 tumor tissues, 181 normal tissues), immunohistochemistry was performed [ILK, Akt, phospho-Akt-S473, loricrin, transforming growth factor beta2 (TGFbeta2)], and staining intensities were semiquantitatively scored. Increased ILK expression was clearly associated with increased differentiation in normal gastrointestinal, neural, bone marrow, renal tissue, and in more differentiated areas of malignant tumors. ILK colocalized with its putative downstream target Akt and with loricrin or TGFbeta2. Our findings clearly show that elevated levels of ILK are associated with cellular differentiation in high turnover tissues but not generally with a malignant phenotype. Our study indicates that ILK is not a general molecular target for cancer therapy but rather an indicator of differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Integrin-linked kinase: dispensable for radiation survival of three-dimensionally cultured fibroblasts.

PURPOSE: Cancer treatment by conventional radiotherapy is limited by normal tissue side-effects. Fibroblasts as "non-target" stromal cell type are considered as strong promoter of tumor growth and for developing a therapy resistant phenotype. Regarding application of novel molecular therapeutics combined with radiotherapy, evaluation of a specific targeted molecule in both tumor and normal cells is mandatory for efficacy and tolerability assessment. Previous work showed integrin-linked kinase (ILK), a mediator of beta-integrin signals and putative phosphorylator of AKT, as potent anti-survival regulator in human cancer cell lines. MATERIALS AND METHODS: To evaluate the role of ILK in normal fibroblast survival, ILK-wild-type (ILK(fl/fl)), ILK(-/-) and ILK(N-terminal) and ILK(C-terminal) domain expressing fibroblasts were irradiated with X-rays on different substrata or in three-dimensional laminin-rich extracellular matrix (lrECM). RESULTS: On control substrata, ILK-deficient and ILK-mutant fibroblasts showed significant increase in radiation survival relative to ILK-wild-type cells. This effect was compensated by growth on ECM proteins and in 3D lrECM. ILK regulated AKT activity in a phosphatidylinositol-3 kinase (PI3K)-dependent manner. Upon PI3K inhibition, only ILK-wild-type fibroblasts showed significant radiosensitization. CONCLUSIONS: These findings obtained in 3D cell cultures suggest ILK to be dispensable for the radiation survival response of normal fibroblasts. However, targeting the PI3K/AKT signaling axis pharmacologically might be critical for survival of normal fibroblasts exposed to ionizing radiation.

Radiation-Induced Long Noncoding RNAs in a Mouse Model after Whole-Body Irradiation.

Long noncoding RNAs (lncRNAs) are emerging as key molecules in regulating many biological processes and have been implicated in development and disease pathogenesis. Biomarkers of cancer and normal tissue response to treatment are of great interest in precision medicine, as well as in public health and medical management, such as for assessment of radiation injury after an accidental or intentional exposure. Circulating and functional RNAs, including microRNAs (miRNAs) and lncRNAs, in whole blood and other body fluids are potential valuable candidates as biomarkers. Early prediction of possible acute, intermediate and delayed effects of radiation exposure enables timely therapeutic interventions. To address whether long noncoding RNAs (lncRNAs) could serve as biomarkers for radiation biodosimetry we performed whole genome transcriptome analysis in a mouse model after whole-body irradiation. Differential lncRNA expression patterns were evaluated at 16, 24 and 48 h postirradiation in total RNA isolated from whole blood of mice exposed to 1, 2, 4, 8 and 12 Gy of X rays. Sham-irradiated animals served as controls. Significant alterations in the expression patterns of lncRNAs were observed after different radiation doses at the various time points. We identified several radiation-induced lncRNAs known for DNA damage response as well as immune response. Long noncoding RNA targets of tumor protein 53 (P53), Trp53cor1, Dino, Pvt1 and Tug1 and an upstream regulator of p53, Meg3, were altered in response to radiation. Gm14005 ( Morrbid) and Tmevpg1 were regulated by radiation across all time points and doses. These two lncRNAs have important potential as blood-based radiation biomarkers; Gm14005 ( Morrbid) has recently been shown to play a key role in inflammatory response, while Tmevpg1 has been implicated in the regulation of interferon gamma. Precise molecular biomarkers, likely involving a diverse group of inducible molecules, will not only enable the development and effective use of medical countermeasures but may also be used to detect and circumvent or mitigate normal tissue injury in cancer radiotherapy.

Long-term Tumor Adaptation after Radiotherapy: Therapeutic Implications for Targeting Integrins in Prostate Cancer.

Adaptation of tumor cells to radiotherapy induces changes that are actionable by molecular targeted agents and immunotherapy. This report demonstrates that radiation-induced changes in integrin expression can be targeted 2 months later. Integrins are transmembrane cell adhesion molecules that are essential for cancer cell survival and proliferation. To analyze the short- and long-term effects of radiation on the integrin expression, prostate cancer cells (DU145, PC3, and LNCaP) were cultured in a 3D extracellular matrix and irradiated with either a single dose of radiation (2-10 Gy) or a multifractionated regimen (2-10 fractions of 1 Gy). Whole human genome microarrays, immunoblotting, immunoprecipitation assays, and immunofluorescence staining of integrins were performed. The results were confirmed in a prostate cancer xenograft model system. Interestingly, ß1 and ß4 integrins (ITGB1 and ITGB4) were upregulated after radiation in vitro and in vivo. This overexpression lasted for more than 2 months and was dose dependent. Moreover, radiation-induced upregulation of ß1 and ß4 integrin resulted in significantly increased tumor cell death after treatment with inhibitory antibodies. Combined, these findings indicate that long-term tumor adaptation to radiation can result in an increased susceptibility of surviving cancer cells to molecular targeted therapy due to a radiation-induced overexpression of the target. IMPLICATIONS: Radiation induces dose- and schedule-dependent adaptive changes that are targetable for an extended time; thus suggesting radiotherapy as a unique strategy to orchestrate molecular processes, thereby providing new radiation-drug treatment options within precision cancer medicine.

Exploiting Radiation-Induced Signaling to Increase the Susceptibility of Resistant Cancer Cells to Targeted Drugs: AKT and mTOR Inhibitors as an Example.

Implementing targeted drug therapy in radio-oncologic treatment regimens has greatly improved the outcome of cancer patients. However, the efficacy of molecular targeted drugs such as inhibitory antibodies or small molecule inhibitors essentially depends on target expression and activity, which both can change during the course of treatment. Radiotherapy has previously been shown to activate prosurvival pathways, which can help tumor cells to adapt and thereby survive treatment. Therefore, we aimed to identify changes in signaling induced by radiation and evaluate the potential of targeting these changes with small molecules to increase the therapeutic efficacy on cancer cell survival. Analysis of "The Cancer Genome Atlas" database disclosed a significant overexpression of AKT1, AKT2, and MTOR genes in human prostate cancer samples compared with normal prostate gland tissue. Multifractionated radiation of three-dimensional-cultured prostate cancer cell lines with a dose of 2 Gy/day as a clinically relevant schedule resulted in an increased protein phosphorylation and enhanced protein-protein interaction between AKT and mTOR, whereas gene expression of AKT, MTOR, and related kinases was not altered by radiation. Similar results were found in a xenograft model of prostate cancer. Pharmacologic inhibition of mTOR/AKT signaling after activation by multifractionated radiation was more effective than treatment prior to radiotherapy. Taken together, our findings provide a proof-of-concept that targeting signaling molecules after activation by radiotherapy may be a novel and promising treatment strategy for cancers treated with multifractionated radiation regimens such as prostate cancer to increase the sensitivity of tumor cells to molecular targeted drugs. Mol Cancer Ther; 17(2); 355-67. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

The Future of Radiobiology.

Innovation and progress in radiation oncology depend on discovery and insights realized through research in radiation biology. Radiobiology research has led to fundamental scientific insights, from the discovery of stem/progenitor cells to the definition of signal transduction pathways activated by ionizing radiation that are now recognized as integral to the DNA damage response (DDR). Radiobiological discoveries are guiding clinical trials that test radiation therapy combined with inhibitors of the DDR kinases DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), ataxia telangiectasia related (ATR), and immune or cell cycle checkpoint inhibitors. To maintain scientific and clinical relevance, the field of radiation biology must overcome challenges in research workforce, training, and funding. The National Cancer Institute convened a workshop to discuss the role of radiobiology research and radiation biologists in the future scientific enterprise. Here, we review the discussions of current radiation oncology research approaches and areas of scientific focus considered important for rapid progress in radiation sciences and the continued contribution of radiobiology to radiation oncology and the broader biomedical research community.

An essential role of integrin-linked kinase in the cellular radiosensitivity of normal fibroblasts during the process of cell adhesion and spreading.

PURPOSE: In addition to focal adhesion kinase (FAK), Paxillin and p130 Crk-associated substrate (p130Cas), integrin-linked kinase (ILK) mediates signals from beta integrins for controlling, e.g., survival, adhesion and spreading. To evaluate the role of ILK in the cellular radiosensitivity at different stages of cell adhesion and spreading, ILK(floxed/floxed (fl/fl)) and ILK(-/-) mouse fibroblasts were examined. MATERIALS AND METHODS: Cells were irradiated (0 - 4 Gy, X-rays) in suspension, after varying time periods on fibronectin (FN) or after 24 h on different matrix proteins. Irradiation was combined with phosphatidylinositol-3 kinase (PI3K) inhibition using Ly294002. Clonogenic radiation survival, cell adhesion, and kinetics of protein expression and phosphorylation during FN adhesion (ILK, v-akt murine thymoma viral oncogene homolog 1 (AKT), FAK, Paxillin, p130Cas) were examined. RESULTS: In suspension and during the first hour on FN, irradiated ILK(fl/fl) cells survived significantly better than ILK(-/-) cells in a PI3K- and serum-dependent manner. 24-h cell cultures on different matrix proteins showed no difference in radiosensitivity. During FN adhesion, which was slightly impaired in ILK(-/-) cells, protein kinetics uncovered differences in AKT, FAK, Paxillin and p130Cas phosphorylation in the two cell lines. Phosphorylation of FAK, Paxillin and p130Cas was downregulated upon exposure to ionizing radiation in an ILK-independent manner. CONCLUSIONS: These findings indicate a critical function of ILK in the cellular radiosensitivity during the early stages of adhesion to and spreading on FN. On the basis of the presented data, a precise correlation of adhesion-, serum- and PI3K-mediated changes in PI3K/AKT and FAK/Paxillin/p130Cas signaling cascades was not found. However, identifying the underlying mechanisms of adhesion- and spreading-related changes in the cellular radiosensitivity might be relevant for an optimization of radiotherapeutic strategies specifically targeting cells located at the invasive edge of a malignant tumor.

Pharmacological inhibition of EGFR tyrosine kinase affects ILK-mediated cellular radiosensitization in vitro.

PURPOSE: Integrin-linked kinase (ILK) mediates signals from beta integrins and links integrins to epidermal growth factor receptor (EGFR). Previous studies have identified an antisurvival effect of ILK in irradiated cells. The aim of this study was to evaluate the role of EGFR tyrosine kinase (tk) activity for ILK-mediated radiosensitization. MATERIALS AND METHODS: Human FaDu squamous cell carcinoma (SCC) cells stably transfected with hyperactive ILK (ILK-hk) and ILK(fl/fl) and ILK(-/-) mouse fibroblasts were treated with the pharmacological EGFR-tk inhibitor BIBX1382BS without or in combination with single doses of X-rays. Clonogenic radiation survival, protein expression and phosphorylation (EGFR, v-akt murine thymoma viral oncogene homolog 1 (Akt), p42/44 mitogen-activated protein kinase), DNA-double strand break (DSB) repair measured by gammaH2AX foci, cell morphology and cell cycle distribution were examined. RESULTS: Expression of ILK-hk or ILK(fl/fl) status resulted in significant radiosensitization relative to vector controls or ILK(-/-). Following BIBX1382BS, clonogenic survival of normal fibroblasts and vector controls remained unaffected while ILK-hk-related radiosensitization was significantly diminished. In contrast to BIBX1382BS, which did not affect DNA-DSB repair, ILK-hk-mediated radiosensitization was associated with reduced DNA-DSB repair. At 10 days after BIBX1382BS treatment, FaDu transfectants, in contrast to fibroblasts, showed reduced cell size, accumulation of G1 phase cells and reduced Akt-serine(S)473 phosphorylation. CONCLUSIONS: Our findings confirm ILK as a cell type-independent antisurvival factor in irradiated cells, which actions in terms of radiosensitization critically depend on proper EGFR-tk activity.

Radiation-Induced Fibrosis: Mechanisms and Opportunities to Mitigate. Report of an NCI Workshop, September 19, 2016.

A workshop entitled "Radiation-Induced Fibrosis: Mechanisms and Opportunities to Mitigate" (held in Rockville, MD, September 19, 2016) was organized by the Radiation Research Program and Radiation Oncology Branch of the Center for Cancer Research (CCR) of the National Cancer Institute (NCI), to identify critical research areas and directions that will advance the understanding of radiation-induced fibrosis (RIF) and accelerate the development of strategies to mitigate or treat it. Experts in radiation biology, radiation oncology and related fields met to identify and prioritize the key areas for future research and clinical translation. The consensus was that several known and newly identified targets can prevent or mitigate RIF in pre-clinical models. Further, basic and translational research and focused clinical trials are needed to identify optimal agents and strategies for therapeutic use. It was felt that optimally designed preclinical models are needed to better study biomarkers that predict for development of RIF, as well as to understand when effective therapies need to be initiated in relationship to manifestation of injury. Integrating appropriate endpoints and defining efficacy in clinical trials testing treatment of RIF were felt to be critical to demonstrating efficacy. The objective of this meeting report is to (a) highlight the significance of RIF in a global context, (b) summarize recent advances in our understanding of mechanisms of RIF,