Fisetin overcomes non-targetability of mutated KRAS induced YB-1 signaling in colorectal cancer cells and improves radiosensitivity by blocking repair of radiation-induced DNA double-strand breaks.

BACKGROUND AND PURPOSE: KRAS is frequently mutated, and the Y-box binding protein 1 (YB-1) is overexpressed in colorectal cancer (CRC). Mutant KRAS (KRASmut) stimulates YB-1 through MAPK/RSK and PI3K/AKT, independent of epidermal growth factor receptor (EGFR). The p21-activated kinase (PAK) family is a switch-site upstream of AKT and RSK. The flavonoid compound fisetin inhibits RSK-mediated YB-1 signaling. We sought the most effective molecular targeting approach that interferes with DNA double strand break (DSB) repair and induces radiosensitivity of CRC cells, independent of KRAS mutation status. MATERIALS AND METHODS: KRAS activity and KRAS mutation were analyzed by Ras-GTP assay and NGS. Effect of dual targeting of RSK and AKT (DT), the effect of fisetin as well as targeting PAK by FRAX486 and EGFR by erlotinib on YB-1 activity was tested by Western blotting after irradiation in vitro and ex vivo. Additionally, the effect of DT and FRAX486 on DSB repair pathways was tested in cells expressing reporter constructs for the DSB repair pathways by flow cytometry analysis. Residual DSBs and clonogenicity were examined by γH2AX- and clonogenic assays, respectively. RESULTS: Erlotinib neither blocked DSB repair nor inhibited YB-1 phosphorylation under KRAS mutation condition in vitro and ex vivo. DT and FRAX486 effectively inhibited YB-1 phosphorylation independent of KRAS mutation status and diminished homologous recombination (HR) and alternative non-homologous end joining (NHEJ) repair. DT and FRAX486 inhibited DSB repair in CaCo2 but not in isogenic KRASG12V cells. Fisetin inhibited YB-1 phosphorylation, blocked DSB repair and increased radiosensitivity, independent of KRAS mutation status. CONCLUSION: Combination of fisetin with radiotherapy may improve CRC radiation response, regardless of KRASmut status.

YB-1 activating cascades as potential targets in KRAS-mutated tumors.

Y­box binding protein­1 (YB-1) is a multifunctional protein that is highly expressed in human solid tumors of various entities. Several cellular processes, e.g. cell cycle progression, cancer stemness and DNA damage signaling that are involved in the response to chemoradiotherapy (CRT) are tightly governed by YB­1. KRAS gene with about 30% mutations in all cancers, is considered the most commonly mutated oncogene in human cancers. Accumulating evidence indicates that oncogenic KRAS mediates CRT resistance. AKT and p90 ribosomal S6 kinase are downstream of KRAS and are the major kinases that stimulate YB­1 phosphorylation. Thus, there is a close link between the KRAS mutation status and YB­1 activity. In this review paper, we highlight the importance of the KRAS/YB­1 cascade in the response of KRAS-mutated solid tumors to CRT. Likewise, the opportunities to interfere with this pathway to improve CRT outcome are discussed in light of the current literature.

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 [...].

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.

Fisetin induces DNA double-strand break and interferes with the repair of radiation-induced damage to radiosensitize triple negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is associated with aggressiveness and a poor prognosis. Besides surgery, radiotherapy serves as the major treatment modality for TNBC. However, response to radiotherapy is limited in many patients, most likely because of DNA damage response (DDR) signaling mediated radioresistance. Y-box binding protein-1 (YB-1) is a multifunctional protein that regulates the cancer hallmarks among them resisting to radiotherapy-induced cell death. Fisetin, is a plant flavonol of the flavonoid family of plant polyphenols that has anticancer properties, partially through inhibition of p90 ribosomal S6 kinase (RSK)-mediated YB-1 phosphorylation. The combination of fisetin with radiotherapy has not yet been investigated. METHODS: Activation status of the RSK signaling pathway in total cell lysate and in the subcellular fractions was analyzed by Western blotting. Standard clonogenic assay was applied to test post-irradiation cell survival. γH2AX foci assay and 3 color fluorescence in situ hybridization analyses were performed to study frequency of double-strand breaks (DSB) and chromosomal aberrations, respectively. The underlying repair pathways targeted by fisetin were studied in cells expressing genomically integrated reporter constructs for the DSB repair pathways via quantifying the expression of green fluorescence protein by flow cytometry. Flow cytometric quantification of sub-G1 cells and the protein expression of LC3-II were employed to measure apoptosis and autophagy, respectively. Kinase array and phosphoproteomics were performed to study the effect of fisetin on DDR response signaling. RESULTS: We showed that the effect of fisetin on YB-1 phosphorylation in TNBC cells is comparable to the effect of the RSK pharmacological inhibitors. Similar to ionizing radiation (IR), fisetin induces DSB. Additionally, fisetin impairs repair of IR-induced DSB through suppressing the classical non-homologous end-joining and homologous recombination repair pathways, leading to chromosomal aberration as tested by metaphase analysis. Effect of fisetin on DSB repair was partially dependent on YB-1 expression. Phosphoproteomic analysis revealed that fisetin inhibits DDR signaling, which leads to radiosensitization in TNBC cells, as shown in combination with single dose or fractionated doses irradiation. CONCLUSION: Fisetin acts as a DSB-inducing agent and simultaneously inhibits repair of IR-induced DSB. Thus, fisetin may serve as an effective therapeutic strategy to improve TNBC radiotherapy outcome.

Targeting HER3-dependent activation of nuclear AKT improves radiotherapy of non-small cell lung cancer.

BACKGROUND: AKT1 must be present and activated in the nucleus immediately after irradiation to stimulate AKT1-dependent double-strand breaks (DSB) repair through the fast non-homologous end-joining (NHEJ) repair process. We investigated the subcellular distribution of AKT1 and the role of HER family receptor members on the phosphorylation of nuclear AKT and radiation response. MATERIALS AND METHODS: Using genetic approaches and pharmacological inhibitors, we investigated the subcellular distribution of AKT1 and the role of HER family receptor members on the activation of nuclear AKT in non-small cell lung cancer (NSCLC) cells in vitro. ɤH2AX foci assay was applied to investigate the role of AKT activating signaling pathway on DSB repair. A mouse tumor xenograft model was used to study the impact of discovered signaling pathway activating nuclear AKT on the radiation response of tumors in vivo. RESULTS: Our data suggests that neither ionizing radiation (IR) nor stimulation with HER family receptor ligands induced rapid nuclear translocation of endogenous AKT1. GFP-tagged exogenous AKT1 translocated to the nucleus under un-irradiated conditions and IR did not stimulate this translocation. Nuclear translocation of GFP-AKT1 was impaired by the AKT inhibitor MK2206 as shown by its accumulation in the cytoplasmic fraction. IR-induced phosphorylation of nuclear AKT was primarily dependent on HER3 expression and tyrosine kinase activation of epidermal growth factor receptor. In line with the role of AKT1 in DSB repair, the HER3 neutralizing antibody patritumab as well as HER3-siRNA diminished DSB repair in vitro. Combination of patritumab with radiotherapy improved the effect of radiotherapy on tumor growth delay in a xenograft model. CONCLUSION: IR-induced activation of nuclear AKT occurs inside the nucleus that is mainly dependent on HER3 expression in NSCLC. These findings suggest that targeting HER3 in combination with radiotherapy may provide a logical treatment option for investigation in selected NSCLC patients.

Targeting the Y-box Binding Protein-1 Axis to Overcome Radiochemotherapy Resistance in Solid Tumors.

Multifunctional Y-box binding protein-1 (YB-1) is highly expressed in different human solid tumors and is involved in various cellular processes. DNA damage is the major mechanism by which radiochemotherapy (RCT) induces cell death. On induction of DNA damage, a multicomponent signal transduction network, known as the DNA damage response, is activated to induce cell cycle arrest and initiate DNA repair, which protects cells against damage. YB-1 regulates nearly all cancer hallmarks described to date by participating in DNA damage response, gene transcription, mRNA splicing, translation, and tumor stemness. YB-1 lacks kinase activity, and p90 ribosomal S6 kinase and AKT are the key kinases within the RAS/mitogen-activated protein kinase and phosphoinositide 3-kinase pathways that directly activate YB-1. Thus, the molecular targeting of ribosomal S6 kinase and AKT is thought to be the most effective strategy for blocking the cellular function of YB-1 in human solid tumors. In this review, after describing the prosurvival effect of YB-1 with a focus on DNA damage repair and cancer cell stemness, clinical evidence will be provided indicating an inverse correlation between YB-1 expression and the treatment outcome of solid tumors after RCT. In the interest of being concise, YB-1 signaling cascades will be briefly discussed and the current literature on YB-1 posttranslational modifications will be summarized. Finally, the current status of targeting the YB-1 axis, especially in combination with RCT, will be highlighted.

Simultaneous Targeting of RSK and AKT Efficiently Inhibits YB-1-Mediated Repair of Ionizing Radiation-Induced DNA Double-Strand Breaks in Breast Cancer Cells.

PURPOSE: Y-box binding protein 1 (YB-1) overexpression is associated with chemotherapy- and radiation therapy resistance. Ionizing radiation (IR), receptor tyrosine kinase ligands, and mutation in KRAS gene stimulate activation of YB-1. YB-1 accelerates the repair of IR-induced DNA double-strand breaks (DSBs). Ribosomal S6 kinase (RSK) is the main kinase inducing YB-1 phosphorylation. We investigated the impact of RSK targeting on DSB repair and radiosensitivity. MATERIALS AND METHODS: The triple negative breast cancer (TNBC) cell lines MDA-MB-231, MDA-MB-468, and Hs 578T, in addition to non-TNBC cell lines MCF7, HBL-100, and SKBR3, were used. MCF-10A cells were included as normal breast epithelial cells. The RSK inhibitor LJI308 was used to investigate the role of RSK activity in S102 phosphorylation of YB-1 and YB-1-associated signaling pathways. The activation status of the underlying pathways was investigated by Western blotting after treatment with pharmacologic inhibitors or transfection with siRNA. The impact of LJI308 on DSB repair and postirradiation cell survival was tested by the γH2AX foci and the standard clonogenic assays, respectively. RESULTS: LJI308 inhibited the phosphorylation of RSK (T359/S363) and YB-1 (S102) after irradiation, treatment with EGF, and in cells expressing a KRAS mutation. LJI308 treatment slightly inhibited DSB repair only in some of the cell lines tested. This was shown to be due to PI3K-dependent stimulation of AKT or constitutive AKT activity mainly in cancer cells but not in normal breast epithelial MCF-10A cells. Simultaneous targeting of AKT and RSK strongly blocked DSB repair in all cancer cell lines, independent of TNBC status or KRAS mutation, with a minor effect in MCF-10A cells. Cotargeting of RSK- and AKT-induced radiation sensitivity in TNBC MDA-MB-231 and non-TNBC MCF7 cells but not in MCF-10A cells. CONCLUSIONS: Simultaneous targeting of RSK and AKT might be an efficient approach to block the repair of DSBs after irradiation and to induce radiosensitization of breast cancer cells.

Blocking Y-Box Binding Protein-1 through Simultaneous Targeting of PI3K and MAPK in Triple Negative Breast Cancers.

The multifunctional protein Y-box binding protein-1 (YB-1) regulates all the so far described cancer hallmarks including cell proliferation and survival. The MAPK/ERK and PI3K/Akt pathways are also the major pathways involved in cell growth, proliferation, and survival, and are the frequently hyperactivated pathways in human cancers. A gain of function mutation in KRAS mainly leads to the constitutive activation of the MAPK pathway, while the activation of the PI3K/Akt pathway occurs either through the loss of PTEN or a gain of function mutation of the catalytic subunit alpha of PI3K (PIK3CA). In this study, we investigated the underlying signaling pathway involved in YB-1 phosphorylation at serine 102 (S102) in KRAS(G13D)-mutated triple-negative breast cancer (TNBC) MDA-MB-231 cells versus PIK3CA(H1047R)/PTEN(E307K) mutated TNBC MDA-MB-453 cells. Our data demonstrate that S102 phosphorylation of YB-1 in KRAS-mutated cells is mainly dependent on the MAPK/ERK pathway, while in PIK3CA/PTEN-mutated cells, YB-1 S102 phosphorylation is entirely dependent on the PI3K/Akt pathway. Independent of the individual dominant pathway regulating YB-1 phosphorylation, dual targeting of MEK and PI3K efficiently inhibited YB-1 phosphorylation and blocked cell proliferation. This represents functional crosstalk between the two pathways. Our data obtained from the experiments, applying pharmacological inhibitors and genetic approaches, shows that YB-1 is a key player in cell proliferation, clonogenic activity, and tumor growth of TNBC cells through the MAPK and PI3K pathways. Therefore, dual inhibition of these two pathways or single targeting of YB-1 may be an effective strategy to treat TNBC.

Dual Targeting of Y-Box Binding Protein-1 and Akt Inhibits Proliferation and Enhances the Chemosensitivity of Colorectal Cancer Cells.

KRAS-mutated colorectal cancers (CRCs) are resistant to cetuximab treatment. The multifunctional Y-box binding protein 1 (YB-1) is overexpressed in CRC and is associated with chemoresistance. In this study, the effects of oncogenic mutated KRAS(G12V) and KRAS(G13D) on YB-1 phosphorylation were investigated in CRC cells. The effects of the inhibition of p90 ribosomal S6 kinase (RSK) on YB-1 phosphorylation, cell proliferation and survival were tested with and without treatment with 5-fluorouracil using pharmacological inhibitors and siRNA. YB-1 phosphorylation status and subcellular distribution in CRC patient tissues were determined by immunofluorescence staining and confocal microscopy. Endogenous expression of mutated KRAS(G13D) and conditional expression of KRAS(G12V) significantly stimulated YB-1 phosphorylation via RSK and were associated with cetuximab resistance. Inhibition of YB-1 by targeting RSK stimulated the Akt signaling pathway, and this stimulation occurred independently of KRAS mutational status. Akt activation interfered with the antiproliferative effect of the RSK inhibitor. Consequently, dual targeting of RSK and Akt efficiently inhibited cell proliferation in KRAS(G13D)-mutated HCT116 and KRAS wild-type SW48 cells. Treatment with 5-fluorouracil (5-FU) significantly enhanced YB-1 phosphorylation in KRAS(G13D)-mutated HCT116 cells but not in KRAS wild-type SW48 cells. Dual targeting of Akt and RSK sensitized HCT116 cells to 5-FU by stimulating 5-FU-induced apoptosis and inhibiting repair of 5-FU-induced DNA damage. YB-1 was highly phosphorylated in CRC patient tumor tissues and was mainly localized in the nucleus. Together, dual targeting of RSK and Akt may be an alternative molecular targeting approach to cetuximab for treating CRC in which YB-1 is highly phosphorylated.

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.

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.

Stress-Induced Phosphorylation of Nuclear YB-1 Depends on Nuclear Trafficking of p90 Ribosomal S6 Kinase.

Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced DNA double-strand breaks (DSBs) in the nucleus. Thus far, the YB-1 nuclear translocation pattern after cell exposure to various cellular stressors is not clear. In the present study, we investigated the pattern of YB-1 phosphorylation and its possible translocation to the nucleus in KRASwt cells after exposure to IR, EGF treatment, and conditional expression of mutated KRAS(G12V). IR, EGF, and conditional KRAS(G12V) expression induced YB-1 phosphorylation in both the cytoplasmic and nuclear fractions of KRASwt cells. None of the stimuli induced YB-1 nuclear translocation, while p90 ribosomal s6 kinase (RSK) translocation was enhanced in KRASwt cells after any of the stimuli. EGF-induced RSK translocation to the nucleus and nuclear YB-1 phosphorylation were completely blocked by the EGF receptor kinase inhibitor erlotinib. Likewise, RSK inhibition blocked RSK nuclear translocation and nuclear YB-1 phosphorylation after irradiation and KRAS(G12V) overexpression. In summary, acute stimulation of YB-1 phosphorylation does not lead to YB-1 translocation from the cytoplasm to the nucleus. Rather, irradiation, EGF treatment, or KRAS(G12V) overexpression induces RSK activation, leading to its translocation to the nucleus, where it activates already-existing nuclear YB-1. Our novel finding illuminates the signaling pathways involved in nuclear YB-1 phosphorylation and provides a rationale for designing appropriate targeting strategies to block YB-1 in oncology as well as in radiation oncology.

Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells.

Akt1 through the C-terminal domain interacts with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and stimulates the repair of DNA double-strand breaks (DSBs) in K-RAS-mutated (K-RASmut) cells. We investigated the interactions of distinct domain(s) of DNA-PKcs in binding to full-length Akt1. Similarly, we analyzed potential interactions of DNA-PKcs with Akt2 and Akt3. Finally the effect of Akt isoforms in cell proliferation and tumor growth was tested. We demonstrated that Akt1 preferentially binds to the N-terminal domain of DNA-PKcs using pull-down studies with distinct eGFP-tagged DNA-PKcs fragments that were expressed by plasmids in combination with mCherry-tagged full-length Akt isoforms. These binding studies also indicated an interaction with the intermediate and C-terminal domains of DNA-PKcs. In contrast, Akt3 interacted with all four DNA-PKcs fragments without a marked preference for any specific domain. Notably, we could not see binding of Akt2 to any of the tested DNA-PKcs fragments. In subsequent studies, we demonstrated that Akt inhibition interferes with binding of Akt1 to the N-terminal domain of DNA-PKcs. This indicated a correlation between Akt1 activity and the Akt1/DNA-PKcs complex formation. Finally, knockdown studies revealed that the depletion of endogenous Akt1 and Akt3, but not Akt2, inhibit clonogenic activity and repair of ionizing radiation (IR)-induced DNA DSBs, leading to radiosensitization. Furthermore, in a xenograft study the expression of shAkt1 or shAkt3, but not shAkt2 in K-RASmut breast cancer cell line MDA-MB-231 showed major tumor growth delay. Together, these data indicate that Akt1 and Akt3, but not Akt2, physically interact with DNA-PKcs, thus stimulating the repair of DSBs and therefore protecting K-RASmut cells against IR. Likewise, interaction of Akt isoforms with DNA-PKcs could be crucial for their role in regulating tumor growth.

Akt1 Stimulates Homologous Recombination Repair of DNA Double-Strand Breaks in a Rad51-Dependent Manner.

Akt1 is known to promote non-homologous end-joining (NHEJ)-mediated DNA double-strand break (DSB) repair by stimulation of DNA-PKcs. In the present study, we investigated the effect of Akt1 on homologous recombination (HR)-dependent repair of radiation-induced DSBs in non-small cell lung cancer (NSCLC) cells A549 and H460. Akt1-knockdown (Akt1-KD) significantly reduced Rad51 protein level, Rad51 foci formation and its colocalization with γH2AX foci after irradiation. Moreover, Akt1-KD decreased clonogenicity after treatment with Mitomycin C and HR repair, as tested by an HR-reporter assay. Double knockdown of Akt1 and Rad51 did not lead to a further decrease in HR compared to the single knockdown of Rad51. Consequently, Akt1-KD significantly increased the number of residual DSBs after irradiation partially independent of the kinase activity of DNA-PKcs. Likewise, the number of residual BRCA1 foci, indicating unsuccessful HR events, also significantly increased in the irradiated cells after Akt1-KD. Together, the results of the study indicate that Akt1 seems to be a regulatory component in the HR repair of DSBs in a Rad51-dependent manner. Thus, based on this novel role of Akt1 in HR and the previously described role of Akt1 in NHEJ, we propose that targeting Akt1 could be an effective approach to selectively improve the killing of tumor cells by DSB-inducing cytotoxic agents, such as ionizing radiation.

AKT2 suppresses pro-survival autophagy triggered by DNA double-strand breaks in colorectal cancer cells.

DNA double-strand breaks (DSBs) are critical DNA lesions, which threaten genome stability and cell survival. DSBs are directly induced by ionizing radiation (IR) and radiomimetic agents, including the cytolethal distending toxin (CDT). This bacterial genotoxin harbors a unique DNase-I-like endonuclease activity. Here we studied the role of DSBs induced by CDT and IR as a trigger of autophagy, which is a cellular degradation process involved in cell homeostasis, genome protection and cancer. The regulatory mechanisms of DSB-induced autophagy were analyzed, focusing on the ATM-p53-mediated DNA damage response and AKT signaling in colorectal cancer cells. We show that treatment of cells with CDT or IR increased the levels of the autophagy marker LC3B-II. Consistently, an enhanced formation of autophagosomes and a decrease of the autophagy substrate p62 were observed. Both CDT and IR concomitantly suppressed mTOR signaling and stimulated the autophagic flux. DSBs were demonstrated as the primary trigger of autophagy using a DNase I-defective CDT mutant, which neither induced DSBs nor autophagy. Genetic abrogation of p53 and inhibition of ATM signaling impaired the autophagic flux as revealed by LC3B-II accumulation and reduced formation of autophagic vesicles. Blocking of DSB-induced apoptotic cell death by the pan-caspase inhibitor Z-VAD stimulated autophagy. In line with this, pharmacological inhibition of autophagy increased cell death, while ATG5 knockdown did not affect cell death after DSB induction. Interestingly, both IR and CDT caused AKT activation, which repressed DSB-triggered autophagy independent of the cellular DNA-PK status. Further knockdown and pharmacological inhibitor experiments provided evidence that the negative autophagy regulation was largely attributable to AKT2. Finally, we show that upregulation of CDT-induced autophagy upon AKT inhibition resulted in lower apoptosis and increased cell viability. Collectively, the findings demonstrate that DSBs trigger pro-survival autophagy in an ATM- and p53-dependent manner, which is curtailed by AKT2 signaling.

Role of Na+/Ca2+ Exchangers in Therapy Resistance of Medulloblastoma Cells.

BACKGROUND/AIMS: Alterations of cytosolic Ca2+-activity ([Ca2+]i) are decisive in the regulation of tumor cell proliferation, migration and survival. Transport processes participating in the regulation of [Ca2+]i include Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether medulloblastoma cells express Na+/Ca2+-exchangers, whether expression differs between therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells, and whether Na+/Ca2+-exchangers participate in the regulation of cell survival. METHODS: In therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells transcript levels were estimated by RT-PCR, protein abundance by Western blotting, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/ Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free by Na+ free and Ca2+ containing (2 mM) extracellular perfusate as well as cell death from PI -staining and annexin-V binding in flow cytometry. RESULTS: The transcript levels of NCX3, NCKX2, and NCKX5, protein abundance of NCX3, slope and peak of Δ[Ca2+]i as well as Ica were significantly lower in therapy sensitive D283 than in therapy resistant UW228-3 medulloblastoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i, and augmented the ionizing radiation-induced apoptosis but did not significantly modify clonogenicity of medulloblastoma cells. Apoptosis was further enhanced by NCX3 silencing. CONCLUSIONS: Na+/Ca2+-exchanger activity significantly counteracts apoptosis but does not significantly affect clonogenicity after radiation of medulloblastoma cells.

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.

Pharmacological targeting of glucose-6-phosphate dehydrogenase in human erythrocytes by Bay 11-7082, parthenolide and dimethyl fumarate.

In mature erythrocytes, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) yield NADPH, a crucial cofactor of the enzyme glutathione reductase (GR) converting glutathione disulfide (GSSG) into its reduced state (GSH). GSH is essential for detoxification processes in and survival of erythrocytes. We explored whether the anti-inflammatory compounds Bay 11-7082, parthenolide and dimethyl fumarate (DMF) were able to completely deplete a common target (GSH), and to impair the function of upstream enzymes of GSH recycling and replenishment. Treatment of erythrocytes with Bay 11-7082, parthenolide or DMF led to concentration-dependent eryptosis resulting from complete depletion of GSH. GSH depletion was due to strong inhibition of G6PDH activity. Bay 11-7082 and DMF, but not parthenolide, were able to inhibit the GR activity. This approach "Inhibitors, Detection of their common target that is completely depleted or inactivated when pharmacologically relevant concentrations of each single inhibitor are applied, Subsequent functional analysis of upstream enzymes for this target" (IDS), can be applied to a broad range of inhibitors and cell types according to the selected target. The specific G6PDH inhibitory effect of these compounds may be exploited for the treatment of human diseases with high NADPH and GSH consumption rates, including malaria, trypanosomiasis, cancer or obesity.

Dual targeting of PI3K and MEK enhances the radiation response of K-RAS mutated non-small cell lung cancer.

Despite the significant contribution of radiotherapy to non-small lung cancer (NSCLC), radioresistance still occurs. One of the major radioresistance mechanisms is the hyperactivation of the PI3K/Akt pathway in which Akt facilitates the repair of DNA double-strand breaks (DSBs) through the stimulation of DNA-PKcs. We investigated if targeting PI3K would be a potential approach for enhancing the radiosensitivity of K-RAS mutated (K-RASmut) NSCLC cell lines A549 and H460. Short-term (1-2 h) pre-treatment of cells with the PI3K inhibitor PI-103 (1 µM) inhibited Akt/DNA-PKcs activity, blocked DSBs repair and induced radiosensitivity, while long-term (24 h) pre-treatment did not. Lack of an effect after 24 h of PI-103 pre-treatment was due to reactivation of K-Ras/MEK/ERK-dependent Akt. However, long-term treatment with the combination of PI-103 and MEK inhibitor PD98059 completely blocked reactivation of Akt and impaired DSBs repair through non-homologous end joining (NHEJ) leading to radiosensitization. The effect of PI3K inhibition on Akt signaling was also tested in A549 mouse xenografts. P-Akt and P-DNA-PKcs were inhibited 30 min post-irradiation in xenografts, which were pretreated by PI-103 30 min before irradiation. However, Akt was reactivated 30 min post-irradiation in tumors, which were pre-treated for 3 h with PI-103 before irradiation. After a 24 h pretreatment with PI-103, a significant reactivation of Akt was achieved 24 h after irradiation. Thus, due to MEK/ERK-dependent reactivation of Akt, targeting PI3K alone is not a suitable approach for radiosensitizing K-RASmut NSCLC cells, indicating that dual targeting of PI3K and MEK is an efficient approach to improve radiotherapy outcome.