Targeting K-Ras-mediated DNA damage response in radiation oncology: Current status, challenges and future perspectives

Approximately 60% of cancer patients receive curative or palliative radiation. Despite the significant role of radiotherapy (RT) as a curative approach for many solid tumors, tumor recurrence occurs, partially because of intrinsic radioresistance. Accumulating evidence indicates that the success of RT is hampered by activation of the DNA damage response (DDR). The intensity of DDR signaling is affected by multiple parameters, e.g., loss-of-function mutations in tumor suppressor genes, gain-of-function mutations in protooncogenes as well as radiation-induced alterations in signal-transduction pathways. Therefore, the response to irradiation differs in tumors of different types, which makes the individualization of RT as a rational but challenging goal. One contributor to tumor cell radiation survival is signaling through the Ras pathway. Three RAS genes encode 4 Ras isoforms: K-Ras4A, K-Ras4B, H-Ras, and N-Ras. RAS family members are found to be mutated in approximately 19% of human cancers. Mutations in RAS lead to constitutive activation of the gene product and activation of multiple Ras-dependent signal-transduction cascades. Preclinical studies have shown that the expression of mutant KRAS affects DDR and increases cell survival after irradiation. Approximately 70% of RAS mutations occur in KRAS. Thus, applying targeted therapies directly against K-Ras as well as K-Ras upstream activators and downstream effectors might be a tumor-specific approach to overcome K-Ras-mediated RT resistance. In this review, the role of K-Ras in the activation of DDR signaling will be summarized. Recent progress in targeting DDR in KRAS-mutated tumors in combination with radiochemotherapy will be discussed

Targeting DNA Double-Strand Break Repair Pathways to Improve Radiotherapy Response

More than half of cancer patients receive radiotherapy as a part of their cancer treatment. DNA double-strand breaks (DSBs) are considered as the most lethal form of DNA damage and a primary cause of cell death and are induced by ionizing radiation (IR) during radiotherapy. Many malignant cells carry multiple genetic and epigenetic aberrations that may interfere with essential DSB repair pathways. Additionally, exposure to IR induces the activation of a multicomponent signal transduction network known as DNA damage response (DDR). DDR initiates cell cycle checkpoints and induces DSB repair in the nucleus by non-homologous end joining (NHEJ) or homologous recombination (HR). The canonical DSB repair pathways function in both normal and tumor cells. Thus, normal-tissue toxicity may limit the targeting of the components of these two pathways as a therapeutic approach in combination with radiotherapy. The DSB repair pathways are also stimulated through cytoplasmic signaling pathways. These signaling cascades are often upregulated in tumor cells harboring mutations or the overexpression of certain cellular oncogenes, e.g., receptor tyrosine kinases, PIK3CA and RAS. Targeting such cytoplasmic signaling pathways seems to be a more specific approach to blocking DSB repair in tumor cells. In this review, a brief overview of cytoplasmic signaling pathways that have been reported to stimulate DSB repair is provided. The state of the art of targeting these pathways will be discussed. A greater understanding of the underlying signaling pathways involved in DSB repair may provide valuable insights that will help to design new strategies to improve treatment outcomes in combination with radiotherapy

Mechanismus und Rolle der EGFR-Tyrosinekinase-Inhibition während der Strahlenantwort von Tumor- und Normalzellen

Cancer is a public health problem worldwide and the main cause of mortality. Surgery, radiotherapy and chemotherapy are the three major cancer treatment modalities. Applying advanced technical developments in radiation oncology has improved the quality of cancer treatment by radiotherapy alone as well as in combination with chemotherapy. Nevertheless, further progress in clinical efficiency of radiotherapy can only be expected when in addition to technological advances biological parameters of the radiation response profile of tumors are taken into account for the development of treatment strategies. Therefore clarifying the underlying molecular mechanisms of radiation responses and identifying molecular targets for intervention will create the potential to develop specific therapeutic strategies in radiation oncology based on individual biological parameters of the tumors to be treated. Accelerated repopulation of tumors during fractionated radiotherapy is one of the phenomenons that limits the success and effectiveness of radiation treatment. One proposed mechanism for tumor repopulation is the potential of ionizing radiation to activate the epidermal growth factor receptor (EGFR) which is linked to several components of mitogenic and survival signaling pathways mediating resistance to ionizing radiation and failure in tumor treatment. Based on the prominent role of EGFR in accelerated repopulation as well as cellular radioresistance, molecular-targeting approaches of this receptor were proposed to enhance efficacy of radiotherapy. For this purpose, differential pharmacological and biological approaches have been developed favoring two strategies: Monoclonal antibodies against ligand binding domain of EGFR and low molecular weight receptor tyrosine kinase (RTK) inhibitors. The aim of the present study was to investigate the molecular principles of radioresistance, radiation-induced EGFR-autophosphorylation and activation of downstream signal transduction pathways in EGFR-overexpressing human tumor cells. Since it is known that in addition to EGFR-overexpression, mutations in the RAS gene are not only very frequent in human tumors but also influence cellular radiation sensitivity, the analyses were performed with human tumor cells with either K-RAS-wildtype (K-RASwt) or K-RAS-mutated (K-RASmt) status. Special emphasis was given to the radiosensitizing potential of the selective EGFR TK inhibitor BIBX1382BS and its molecular mode of action. The following major results were obtained: 1. As shown for a panel of human tumor cell lines and fibroblasts exposure to ionizing radiation mediated stimulation of EGFR autophosphorylation in a ligand independent manner. 2. Blockage of EGFR-tyrosine kinase activity by BIBX1382BS led to a differential antiproliferative effect for all cells tested. 3. Inhibition of EGFR-TK-activity by BIBX1382BS resulted in an enhanced radiation toxicity only in tumor cells presenting a point mutation in the K-RAS gene. 4. Analyses of the three major EGFR downstream pathways (PI3K-AKT, MAPK, and JAK-STATs) revealed that blockage of EGFR-TK by BIBX1382BS primarily results in an inhibition of the phosphatidylinositol 3- kinase (PI3K)-AKT pathway leading to enhanced radiation sensitivity. 5. Radioresistance of K-RASmt tumor cells was found to be dependent on autocrine/paracrine secretion of EGFR ligand, i.e. amphiregulin (AREG). Due to constitutively active K-RAS these cells produce significantly elevated levels of secreted AREG which in turn leads to an autocrine activation of the EGFR-PI3K-AKT survival pathway. 6. Expression of K-RAS-siRNA in K-RASmt cells blocked autocrine activation of EGFR-PI3K-AKT pathway and enhanced radiation sensitivity. 7. Blockage of EGFR-tyrosine kinase activity by BIBX1382BS affected DNA repair mainly by significantly reducing nuclear activation of DNA-PKcs (an important enzyme in NEHJ-repair) resulting in increased micronuclei formation. The data presented provided for the first time direct evidence that radiosensitization of human tumor cells by EGFR-targeting approaches applying the EGFR-specific TK-inhibitor BIBX1382BS requires the presence of a K-RAS mutation. These findings specifically point to a mechanism that promotes radioresistance in K-RASmt human tumor cells via EGFR dependent but Ras-GTP independent autocrine activation of PI3K/AKT pathway through modulation of DNA repair processes, e.g. NHEJ. In conclusion the present study provides molecular and biochemical evidence which may help to explain at least in part the heterogeneity of EGFR-targeting approaches for induction of enhanced radiation sensitivity of EGFR overexpressing tumor cells and underlines the importance of additional mutations in related pathways which may promote or abolish the expected effect.Krebs ist ein weltweites Gesundheitsproblem und die Haupttodesursache. Chirurgie, Strahlentherapie und Chemotherapie sind die drei wesentlichen Behandlungsstrategien. Durch Applikation fortgeschrittener technischer Entwicklungen in der Radioonkologie wurde die Qualität der Tumorbehandlung sowohl durch Strahlentherapie allein als auch in Kombination mit Chemotherapie verbessert. Dennoch kann ein weiterer Fortschritt in der klinischen Effizienz der Strahlentherapie nur erwartet werden, wenn zusätzlich zu den technologischen Fortschritten, biologische Parameter des Strahlenreaktionsprofils von Tumoren bei der Entwicklung von Behandlungsstrategien berücksichtigt werden. Daher wird die Aufklärung der zugrunde liegenden molekularen Mechanismen der Strahlenreaktion und die Identifikation molekularer Ziele für die Intervention, eine Entwicklung spezifischer therapeutischer Strategien in der Radioonkologie ermöglichen, die auf individuellen biologischen Parametern des zu behandelnden Tumors basieren. Die beschleunigte Repopulierung des Tumors während einer fraktionierten Strahlentherapie ist eines der Phänomene, die den Erfolg und die Effektivität der Bestrahlungsbehandlung limitieren. Eine mögliche Ursache dieser Tumorrepopulierung könnte die durch ionisierende Strahlung verursachte Aktivierung des "epidermal growth factor receptor" (EGFR) sein, welcher die Regulation verschiedener für Zellteilung und Überleben verantwortlicher Signalwege (z.B. PI3K-AKT- und MAPK-Kaskade) modulieren kann. Dadurch kann Resistenz gegenüber ionisierender Strahlung vermittelt werden, die den Erfolg der Radiotherapie Tumorbehandlung deutlich beeinträchtigen kann. Basierend auf der herausragenden Rolle des EGFR in der beschleunigten Repopulation sowie zellulären Strahlenresistenz, wurden Ansatze vorgeschlagen, diesen Rezeptor auf molekularer Ebene anzugreifen und zu blockieren, um die Effizienz der Strahlentherapie zu erhöhen. Zu diesem Zweck werden zwei verschiedene pharmakologische und biologische Ansätze favorisiert: monoklonale Antikörper gegen die Ligandenbindungsstelle des EGFR und Rezeptor-Tyrosinkinase (RTK)-Inhibitoren. Ziel der vorliegenden Arbeit war es, das molekulare Prinzip der Strahlenresistenz, der strahleninduzierten EGFR-Autophosphorylierung und die Aktivierung von nachfolgenden Signaltransduktionswegen in EGFR-überexprimierenden menschlichen Tumorzellen zu ermitteln. Es ist bekannt, dass neben der EGFR-Überexpression auch Mutationen des RAS Gens sehr häufig in menschlichen Tumoren vorkommen und darüber hinaus die zelluläre Strahlensensitivität beeinflussen. Daher wurden die Analysen mit menschlichen Tumorzellen durchgeführt, die entweder einen K-RAS-Wildtyp-Status aufwiesen oder K-RAS- mutiert waren. Besonderes Gewicht wurde auf das radiosensitivierende Potential des selektiven EGFR-Tyrosinkinaseinhibitors BIBX1382BS und seinen molekularen Wirkmechanismus gelegt. Folgende wesentliche Ergebnisse wurden erzielt: 1. Es wurde für mehrere Tumorzelllinien und Fibroblasten gezeigt, dass Bestrahlung mit ionisierender Strahlung die EGFR-Autophosphorylierung ligandenunabhängig stimuliert. 2. Die Blockade der EGFR-Tyrosinkinaseaktivität durch BIBX1382BS führte zu einem unterschiedlich ausgeprägten antiproliferativen Effekt in allen untersuchten Zelllinien. 3. Die Inhibition der EGFR-Tyrosinkinaseaktivität durch BIBX1382BS führte nur in Tumorzellen, die eine Punktmutation im K-RAS Gen aufweisen, zu einer erhöhten Strahlentoxizität. 4. Die Analyse der drei wesentlichen EGFR abhängigen intrazellulären Signal-kaskaden(PI3K-AKT, MAPK und JAK-STATs) zeigte, dass die Blockade der EGFR-Tyrosinkinase durch BIBX1382BS hauptsächlich zu einer Inhibition des Phosphatidylinositol 3-Kinase (PI3K)-AKT Weges führt, aus der eine erhöhte Strahlensensitivität resultiert. 5. Es wurde festgestellt, dass die Strahlenresistenz von K-RAS mutierten Tumorzellen abhängig von der autokrinen/parakrinen Sekretion des EGFR-Liganden Amphiregulin (AREG) ist. Aufgrund des konstitutiv aktiven K-RAS weisen diese Zellen signifikant erhöhte AREG-Werte auf, die wiederum zur autokrinen Aktivierung des EGFR-PI3K-AKT Überlebenssignalweg führen. 6. Die Expression von K-RAS-siRNA in K-RAS-mutierten Zellen blockierte die autokrine Aktivierung des EGFR-PI3K-AKT Signalwegs und erhöhte die Radiosensitivität. 7. Die Blockade der EGFR-Tyrosinkinaseaktivität durch BIBX1382BS beeinflusste die DNA-Reparatur im wesentlichen durch eine signifikante Reduktion der nukleären Aktivierung von DNA-PKC (einem wichtigen Enzym der NEHJ-Reparatur) und führte so zu einer vermehrten Bildung von Mikronuklei. Mit diesen Daten konnte zum ersten Mal gezeigt werden, dass Radiosensitivierung von menschlichen Tumorzellen durch gezieltes Angreifen des EGF-Rezeptors mittels BIBX1382BS, einem EGFR-spezifischen Tyrosinkinaseinhibitor, das Vorhandensein einer K-RAS-Mutation voraussetzt. Diese Ergebnisse weisen spezifisch auf einen Mechanismus hin, der die Strahlenresistenz von K-RAS-mutierten menschlichen Tumorzellen über EGFR-abhängige, aber Ras-GTP-unabhängige autokrine Aktivierung des PI3K/AKT Signalwegs durch Modulation von DNA-Reparaturprozessen, z.B. NHEJ, fördert. Zusammenfassend lässt sich sagen, dass diese Untersuchungen molekulare und biochemische Beweise lieferten, die hilfreich sein können, zumindest teilweise die Heterogenität klinischer Studian zu erklären, die versuchten durch gezielte Blockade des EGF-Rezeptors eine erhöhte Strahlensensitivität in EGFR-überexprimierenden Tumoren zu induzieren. Dadurch wird die Bedeutung von weiteren Mutationen die den erwarteten Effekt fördern oder aufheben könnten in mit EGFR-vernetzten Signaltransduktionswegen unterstrichen

Close Links between Cold Shock Proteins and Cancer

Nine of the ten papers published in this Special Issue explore various aspects of the multifunctional protein Y-box binding protein-1 (YB-1) and its role in cancer [...

Cell-line dependent effects of hypoxia prior to irradiation in squamous cell carcinoma lines

Purpose: To assess the impact of hypoxia exposure on cellular radiation sensitivity and survival of tumor cells with diverse intrinsic radiation sensitivity under normoxic conditions. Materials and methods: Three squamous cell carcinoma (SCC) cell lines, with pronounced differences in radiation sensitivity, were exposed to hypoxia prior, during or post irradiation. Cells were seeded in parallel for colony formation assay (CFA) and stained for γH2AX foci or processed for western blot analysis. Results: Hypoxia during irradiation led to increased cellular survival and reduced amount of residual γH2AX foci in all the cell lines with similar oxygen enhancement ratios (OER SKX: 2.31, FaDu: 2.44, UT-SCC5: 2.32), while post-irradiation hypoxia did not alter CFA nor residual γH2AX foci. Interestingly, prolonged exposure to hypoxia prior to irradiation resulted in differential outcome, assessed as Hypoxia modifying factor (HMF) namely radiosensitization (SKX HMF: 0.76), radioresistance (FaDu HMF: 1.54) and no effect (UT SCC-5 HMF: 1.1). Notably, radiosensitization was observed in the ATM-deficient SKX cell line while UT SCC-5 and to a lesser extent also FaDu cells showed radiation- and hypoxia-induced upregulation of ATM phosphorylation. Across all the cell lines Rad51 was downregulated whereas phosphor-DNA-PKcs was enhanced under hypoxia for FaDu and UTSCC-5 and was delayed in the SKX cell line. Conclusion: We herein report a key role of ATM in the cellular fitness of cells exposed to prolonged moderate hypoxia prior to irradiation. While DNA damage response post-irradiation seem to be mainly driven by non-homologous end joining repair pathway in these conditions, our data suggest an important role for ATM kinase in hypoxia-driven modification of radiation response

Nanog Signaling Mediates Radioresistance in ALDH-Positive Breast Cancer Cells

Recently, cancer stem cells (CSCs) have been identified as the major cause of both chemotherapy and radiotherapy resistance. Evidence from experimental studies applying both in vitro and in vivo preclinical models suggests that CSCs survive after conventional therapy protocols. Several mechanisms are proposed to be involved in CSC resistance to radiotherapy. Among them, stimulated DNA double-strand break (DSB) repair capacity in association with aldehyde dehydrogenase (ALDH) activity seems to be the most prominent mechanism. However, thus far, the pathway through which ALDH activity stimulates DSB repair is not known. Therefore, in the present study, we investigated the underlying signaling pathway by which ALDH activity stimulates DSB repair and can lead to radioresistance of breast cancer cell lines in vitro. When compared with ALDH-negative cells, ALDH-positive cells presented significantly enhanced cell survival after radiation exposure. This enhanced cell survival was associated with stimulated Nanog, BMI1 and Notch1 protein expression, as well as stimulated Akt activity. By applying overexpression and knockdown approaches, we clearly demonstrated that Nanog expression is associated with enhanced ALDH activity and cellular radioresistance, as well as stimulated DSB repair. Akt and Notch1 targeting abrogated the Nanog-mediated radioresistance and stimulated ALDH activity. Overall, we demonstrate that Nanog signaling induces tumor cell radioresistance and stimulates ALDH activity, most likely through activation of the Notch1 and Akt pathways