Clonogenic Death Research Articles

Combined EGFR1 and PARP1 Inhibition Enhances the Effect of Radiation in Head and Neck Squamous Cell Carcinoma Models

Head and neck squamous cell carcinoma (HNSCC) is a challenging cancer with little change in five-year overall survival rate of 50-60% over the last two decades. Radiation with or without platinum-based drugs remains the standard of care despite limited benefit and high toxicity. HNSCCs often overexpress epidermal growth factor receptor (EGFR) and inhibition of EGFR signaling enhances radiation sensitivity by interfering with repair of radiation-induced DNA breaks. Poly (adenosine diphosphate-ribose) polymerase-1 (PARP1) also participates in DNA damage repair, but its inhibition provides benefit in cancers that lack DNA repair by homologous recombination (HR) such as BRCA-mutant breast cancer. HNSCCs in contrast are typically BRCA wild-type and proficient in HR repair, making it challenging to apply anti-PARP1 therapy in this model. A recently published study showed that a combination of EGFR and PARP1 inhibition induced more DNA damage and greater growth control than each single agent in HNSCC cells. This led us to hypothesize that a combination of EGFR and PARP1 inhibition would enhance the efficacy of radiation to a greater extent than each single agent, providing a rationale for paradigm-shifting combinatorial approaches to improve the standard of care in HNSCC. Here, we report a proof-of-concept study using Detroit562 HNSCC cells, which are proficient for DNA repair by both HR and non-homologous end joining (NHEJ) mechanisms. We tested the effect of adding cetuximab and/or olaparib (inhibitors of EGFR and PARP1, respectively) to radiation and compared it to that of cisplatin and radiation combination, which is the standard of care. Our results demonstrate that the combination of cetuximab and olaparib with radiation was superior to the combination of any single drug with radiation in terms of induction of unrepaired DNA damage, induction of senescence, apoptosis and clonogenic death, and tumor growth control in mouse xenografts. Combined with our recently published phase I safety data on cetuximab/olaparib/radiation triple combination, the data reported here demonstrate a potential for combining biologically-based therapies that might optimize radiosensitization in HNSCC.

Abstract LB-070: Design and modular development of I-PARTS: An integrated platform for anti-cancer radiation therapeutics screening

Abstract Tumor irradiation during cancer treatment induces cell death by apoptosis, mitotic death, and clonogenic death. With expanded global access to cancer radiotherapy, there is a need to discover agents with anti-cancer and radiation sensitizing properties. in vitro high-throughput screening (HTS) systems routinely used to screen agents for cytotoxicity assays unfortunately do not directly measure clonogenic potential and fail to accurately predict the efficacy of an agent either in subsequent preclinical animal model testing or in clinical trials. Clonogenic assays unlike colorimetric and metabolic assays for determining cell viability and apoptosis, are less susceptible to artifacts. We are addressing these requirements through an integrated I-PARTS platform development and optimization. Here, the design and the prototype of a fully enclosed imaging/X-ray irradiation system will be presented. The proof of principle experimentation to obtain high content information on the cellular response to specific primary human cells (normal or tumors) to a variety of radiation doses and fraction modality will be described. The initial approaches taken towards the establishment of an integrated platform to screen anti-cancer drugs will be discussed. Citation Format: Rao V. Papineni, Sylvain Costes, Shayoni Ray, William McLaughlin, Brendan Lukaniec, Kurt Kruger, Keith Kanugi. Design and modular development of I-PARTS: An integrated platform for anti-cancer radiation therapeutics screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-070.

Abstract 5858: Development of a translation elongation inhibitor for the treatment of cancer

Abstract Translation is essential for cellular maintenance and growth, as well as survival in response to a variety of stresses. Translation factors are often overexpressed in cancers, and their experimental inhibition has been shown to retard cancer cell growth in a variety of pre-clinical models. Further, clinically relevant forms of cellular stress (e.g. ionizing radiation (IR), hypoxia) result in altered translational programs, and inhibition of translation following such stress results in impaired cellular recovery. Cumulatively these observations suggest that modulation of translation, alone or in combination with genotoxic stress, is of potential therapeutic relevance in oncology. Bouvardin (BVD) is a plant-derived protein synthesis inhibitor that we identified in a screen for enhancers of ionizing radiation (IR). We previously demonstrated that BVD functions as a translation elongation inhibitor by blocking dissociation of elongation factor 2 (eEF2) from the ribosome. In preclinical models of head and neck cancer (HNC) and glioblastoma two cancer indications for which IR is a standard of care, we demonstrated that BVD enhanced clonogenic death induced by IR. Further, BVD enhanced the growth inhibitory effects of IR in HNC tumor xenografts. We show here that a fully synthetic derivative of BVD, SVC112, exhibits strong therapeutic properties alone, and in combination with standards of care, in a variety of solid and hematological cancer models. As an example for solid tumors: in HNC cell lines SVC112 exhibits antiproliferative activity with an average IC50 of ≈150 nM, with no IC50 exceeding 500 nM. In clonogenic assays, physiologically relevant levels of SVC112 robustly synergistically enhance clonogenic death induced by HNC standards of care (e.g. IR and cisplatin). Finally, in cell line based HNC xenografts, SVC112 exhibits antitumorigenic activity as a single agent and enhances the growth inhibitory effects of IR-cisplatin treatment. As an example for hematological cancers: in acute myeloid leukemia (AML) cell lines SVC112 exhibits antiproliferative activity in sensitive cells with an average IC50 of ≈25 nM. Brief exposure to physiologically relevant levels of SVC112 induces rapid and robust apoptosis in sensitive AML cell lines concomitant with the loss of known drivers of AML growth (e.g. MCL-1). Finally, in cell line based AML xenograft models, SVC112 as a single agent induces robust and sustained antiproliferative activity. Cumulatively these data demonstrate that SVC112 mediated inhibition of translation elongation alone, and in combination with existing standards of care, is a potentially promising treatment option for a variety of solid and hematological cancers. This project has been funded in part with Federal funds from the NCI/NIH, Department of Health and Human Services, under Contract No. HHSN261201300021C Citation Format: Nathan Gomes, Barb Frederick, Daniel Gustafson, Bert Pronk, Tin Tin Su. Development of a translation elongation inhibitor for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5858.

MutT homolog 1 counteracts the effect of anti-neoplastic treatments in adult and pediatric glioblastoma cells

Glioblastoma, a fatal disease in both adult and pediatric patients, currently has limited treatment options that offer no more than temporary relief. Our experiments with adult and pediatric glioblastoma cell lines showed that radiation induces a dose-dependent increase in the level of MutT homolog 1 (MTH1) - an enzyme that hydrolyzes oxidized purine nucleoside triphosphates. Similarly, the combination of vorinostat, which is a histone deacetylase inhibitor, and ABT-888, which is a PARP-1 inhibitor, enhanced clonogenic death and increased the MTH1 level, relative to each treatment alone. This result suggests that the MTH1 level is directly related to the damage that is inflicted upon the cells, and its activity protects them against anti-neoplastic therapy. Indeed, the MTH1 inhibitor TH588 and MTH1 siRNA increased glioblastoma's response to both radiation and the combination of vorinostat and ABT-888. TH588 also inhibited glioblastoma's capacity for migration and invasion. In normal fibroblasts, low radiation doses and the combination of vorinostat and ABT-888 decreased the level of the enzyme. TH588 did not alter the fibroblasts’ response to radiation and only mildly affected their response to the combination of vorinostat and ABT-888.In summary, the inhibition of MTH1 is required to better realize the therapeutic potential of anti-neoplastic treatments in glioblastoma.

Induction of reproductive cell death in Caenorhabditis elegans across entire linear-energy-transfer range of carbon-ion irradiation

Heavy-ion radiation has attracted extensive attention as an effective cancer therapy because of the varying energy deposition along its track and its high cell-killing effect. Reproductive cell death (RCD), also known as clonogenic death, is an important mode of death of the cancer cells after radiotherapy. Although RCD induced by heavy-ion irradiation with various linear energy transfers has been demonstrated using clonogenic assay in vitro, little is known about the distribution of RCD across the range of heavy-ion irradiation at the level of whole organisms. In this study, a vulval tissue model of Caenorhabditis elegans was for the first time used to assess RCD in vivo induced by carbon-ion irradiation. A polymethyl methacrylate wedge was designed to provide a gradually varying thickness of shielding, so worms could be exposed to the entire range of carbon-ion irradiation. The carbon-ion irradiation led to a significant induction of RCD over the entire range in a dose-dependent manner. The biological peak did not correspond to the physical Bragg peak and moved forward, rather than spread forward, as radiation dose increased. The degree and shape of the range-distribution of RCD were also affected by the developmental stages of the worms. The gene mutations in DNA-damage checkpoints did not affect the responses of mutant worms positioned in biological peaks, compared to wild-type worms, but decreased radio-sensitivity in the entrance region. An increased induction of RCD was observed in the worms impaired in homologous recombination (HR), but not in non-homologous end jointing pathway, suggesting a crucial role of HR repair in vulval cells of C. elegans in dealing with the carbon-ion-induced DNA damage. These unique manifestations of RCD in vivo in response to carbon-ion irradiation might provide new clues for further investigating the biological effects of heavy-ion irradiation.

Feasibility study on the use of gold nanoparticles in fractionated kilovoltage X-ray treatment of melanoma

Purpose: Despite the high radioresistance of melanoma, unresectable lesions can be subjected to radiation treatment with the use of gold nanoparticles (AuNPs) as a dose-enhancing agent preferentially loaded on these lesions. The modality of single high-dose treatment has been investigated to confirm its therapeutic efficiency for AuNP-treated melanoma cells. This study explores the feasibility of utilizing AuNPs in fractionated radiation therapy of melanoma for further therapeutic gain.Materials and methods: The responses of human skin melanoma cells to 150-kVp X-ray exposure at 2 and 4 Gy were assessed by quantify gamma-H2AX expression and clonogenic survival, with or without 320 μM of 50 nm AuNP treatment in a culture medium. The influence of AuNPs on cell cycle distribution was observed before irradiation and during 3 d period after irradiation.Results: The AuNP treatment of melanoma cells influenced the cellular response to kilovoltage X-rays to similar extents in terms of the percentage of gamma-H2AX-positive cells and the fractional loss of clonogenicity. Without radiation exposure, AuNPs reduced the portion of melanoma cells at the G2/M phase from 11 to 7%. After irradiation, the progression of the melanoma cells treated with AuNPs toward the G2/M phase was more rapid than that of the AuNP-free cells, and the release of the former from the G2/M phase was slower than that of the latter. At 24 h after irradiation with AuNPs, the cell cycle was rearranged in a pattern that increased the vulnerability of the cells to radiation damage.Conclusions: In addition to the benefit of AuNP treatment to the control of melanoma in single high-dose treatment, further therapeutic gain is expected through fractionated X-ray treatment that involves daily exposure. The AuNP-treated melanoma cells of an increased portion in the radiosensitive G2/M phase following a fractionated dose delivery would respond to the next treatment with an enhanced chance of clonogenic death.

Abstract 831: TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM

Abstract Transforming growth factor β (TGFβ) is a well-documented tumor suppressor; a poorly studied aspect of TGFβ biology is its control of genomic stability. Our prior work showed that TGFβ compromises ATM (ataxia telangiectasia mutated) kinase activity, which mediates recognition and repair of DNA damage. Consequently, inhibiting TGFβ following ionizing radiation increases clonogenic death in vitro and tumor control in vivo in breast, brain, and lung cancer preclinical models. As yet unknown is how TGFβ controls ATM kinase. We recently determined that TGFβ regulates mammary lineage commitment by post-transcriptional control of BRCA1 mRNA and protein by its suppression of miR-182 (Martinez-Ruiz et al., Science Signaling, in press). miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors in cancer cell lines (10.1016/j.molcel.2010.12.005) Here we used two cell culture models to investigate the effect of TGFβ regulation of BRCA1 and miR-182 on DNA damage response. Spontaneously immortalized Tgfb1 heterozygote and wild type fibroblasts from primary mammary epithelial preparations were synchronized in S-phase and exposed to UV radiation before fixation to detect unrepaired DNA damage by quantitative image analysis of 53BP1 foci. Consistent with compromised BRCA1 function, more than twice as many cells with 53BP1 foci were evident in Tgfb1 heterozygote fibroblasts compared to wild type cells (p<0.01). The second model consisted of non-malignant human breast epithelial cell line, MCF10A, stably transduced with a scrambled or miR-182 antagomir, grown under serum free conditions and treated with a small molecule inhibitor of TGFβ type I receptor kinase, LY364937. FOXO3 is a target of miR-182 and thus also regulated by TGFβ. and FOXO3 association with ATM is required for its kinase activity (10.1038/ncb1709). As reported, FOXO3 and ATM co-immunoprecipitated, but was eliminated by TGFβ inhibition and this effect was lost in MCF10A cells expressing a miR-182 antagomir. The impaired DNA repair phenotype induced by TGFβ inhibition can be fully rescued by antagonizing miR-182. These studies support TGFβ as stringent regulator of the DNA damage response via suppression miR-182, which directly targets BRCA1 and indirectly affects ATM activity via FOXO3. We predict that cancers that have lost TGFβ signaling capacity will be genomically unstable due to defective DNA damage control, which is consistent with our studies showing that TGFβ inhibition sensitize cancers to radiation therapy. Citation Format: Qi Liu, Haydeliz Martinez-Ruiz, John Murnane, Simon N. Powell, Mary Helen Barcellos-Hoff. TGFβ controls the DNA damage response via miR-182 regulation of BRCA1 and ATM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 831. doi:10.1158/1538-7445.AM2017-831

Abstract B24: Development of a radiation modulator that functions via translation inhibition

Abstract Translation is essential for cellular maintenance and growth, as well as survival in response to a variety of stresses. Translation factors are often overexpressed in cancers, and their experimental inhibition has been shown to retard cancer cell growth in a variety of pre-clinical models. Further, clinically relevant forms of cellular stress (e.g. ionizing radiation, hypoxia) result in altered translational programs. Inhibition of translation following such stress results in impaired cellular recovery. Taken together these observations suggest that modulation of translation, alone or in combination with genotoxic stress, is of potential therapeutic relevance in oncology. Bouvardin (BVD) is a plant-derived protein synthesis inhibitor that we identified in a screen for enhancers of ionizing radiation (IR) in D. melanogaster. We previously demonstrated that BVD functions as a translation elongation inhibitor by blocking dissociation of elongation factor 2 (eEF2) from the ribosome. In preclinical models of head and neck cancer (HNC) and glioma, two cancer indications for which IR is a standard of care, we demonstrated that BVD enhanced clonogenic death induced by IR. Further, BVD enhanced the growth inhibitory effects of IR in HNC tumor xenografts in mice. We show here that a fully synthetic derivative of BVD, SVC112, exhibits promising therapeutic properties in tissue culture and xenograft models of HNC. Like BVD, SVC112 inhibits general de novo protein synthesis in cell culture. In a panel of 19 HNC cell lines SVC112 exhibits antiproliferative activity with an average IC50 of ~160 nM, with no IC50 exceeding 500 nM. Combinations of SVC112 and IR dose-response curves in HNC cells yield average combination-index (CI) values generally lower than 1, especially in maximally tolerated dose ranges. CI values of 1 are indicative of additive interactions, while CI values <1 are indicative of synergy between two agents. Further, in clonogenic assays physiologically achievable levels of SVC112 consistently exhibit dose modifying factors (DMF) of >1 in HNC cells, when combined with a range of clinically relevant IR doses. DMF values of 1 are indicative of additive interactions, while DMF values >1 indicate synergistic enhancement of IR mediated clonogenic death. Additionally in clonogenic assays, SVC112 enhances cisplatin-IR mediated clonogenic death. Finally, SVC112 enhances the growth inhibitory effects of IR-cisplatin treatment (alone and in combination) in HNC tumor xenografts in mice. Taken together these data demonstrate that inhibition of translation elongation in combination with existing standards of care is a potentially promising treatment option for cancers of the head and neck. *This project has been funded in whole or in part with Federal funds from the NCI/NIH, Department of Health and Human Services, under Contract No. HHSN261201300021C Citation Format: Nathan Gomes, Barb Frederick, Ryan Hansen, Gan Zhang, Daniel Gustafson, Gijsbertus Pronk, Tin Tin Su. Development of a radiation modulator that functions via translation inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B24.

PDTB-10. INHIBITION OF RADIATION-INDUCED DNA DAMAGE REPAIR MEDIATED CHROMATIN MODIFICATION BY HISTONE H3 DEMETHYLASE INHIBITOR IN PEDIATRIC BRAINSTEM GLIOMA

Diffuse intrinsic pontine glioma (DIPG) is among the most devastating childhood cancers, and virtually all patients die within two years after diagnosis. Since these tumors occur in the brainstem, there are no surgical options for providing relief to patients, the only therapeutic modality routinely used in the treatment of DIPG is radiation. Radiation often provides transient relief from DIPG symptoms, but cancer recurrence and patient demise is inevitable. Identifying efficacious therapeutics to enhance the radiation effect is desperately needed. We have recently shown that inhibition of the histone H3K27 demethylase JMJD3 acts to restore H3K27 methylation in DIPG harboring H3K27M mutation, while demonstrating potent anti-tumor activity, both in vitro and in vivo. Our expression profiling of K27M DIPG treated with GSKJ4 JMJD3 demethylase inhibitor revealed several decreases in the genes encoded proteins involved with DNA double-strand break (DSB) repair. We investigate the hypothesis that GSKJ4 may inhibits radiation-induced DNA repair by chromatin modification, in association with analysis of therapeutic combination of GSKJ4 and radiation. DNA damage repair is evaluated by western blotting, DNA repair assay, and immunocytochemistry of DSB markers gH2AX and 53BP1. MNase digestion and FAIRE-seq will analyze nucleosome assembly and transcriptional activity in cellular chromatin. Clonogenic survival assays assess the response to radiation in combination with GSKJ4. In vivo response to radiation therapy (RT) and combination of RT + GSKJ4 were measured by bioluminescence imaging and animal survival. GSKJ4 sustained high levels of gH2AX and 53BP1 in irradiated cells treated with GSKJ4 and increase clonogenic death compared to cells exposed to radiation alone. GSKJ4 + RT combination therapy shows further reduction of tumor growth rate and increase of animal survival relative to either monotherapy. We are currently examining the effect of GSKJ4 on chromatin modification in DIPG, which will be reported at the meeting.

Abstract 1668: Bouvardinis a radiation modulator with a novel mechanism of action

Abstract Protein synthesis is essential for growth, proliferation and survival of cells. Translation factors are overexpressed in many cancers and their experimental inhibition curbs cancer growth in preclinical models. Differential regulation of translation also occurs upon exposure to cancer-relevant stresses such as hypoxia and ionizing radiation (IR). The failure to regulate translation has been shown to interfere with recovery after genotoxic stress. These findings suggest that modulation of translation, alone or in conjunction with genotoxins, may be therapeutic in oncology. Yet, only two drugs that directly inhibit translation are FDA-approved for oncology use today. We described before the identification of protein synthesis inhibitor bouvardin in a screen for small molecule enhancers of IR in Drosophila melanogaster. Bouvardin was independently identified in a screen for selective inhibitors of engineered human breast cancer stem cells. Here we report the effect of bouvardin in preclinical models of head and neck cancer (HNC) and glioma, two cancer types for which IR is a key therapy choice. Our data show for the first time that bouvardin blocked translation elongation on human ribosomes and suggest that it did so by blocking the dissociation of elongation factor 2 from the ribosome. Bouvardin enhanced the induction of clonogenic death by IR in HNC and glioma cells, although by different mechanisms. Bouvardin enhanced the anti-tumor effect of IR in HNC tumor xenografts in mice. These data suggest that inhibition of translation elongation, particularly in combination with IR, may be a promising treatment option for cancer. Citation Format: Tin tin SU. Bouvardinis a radiation modulator with a novel mechanism of action. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1668.

Effects of chronic exposure to acidic environment on the response of tumor cells to radiation

Purpose: The influence of short-term exposure to an acidic environment on the radiosensitivity of tumor cells has been extensively explored, but the implication of chronic exposure to an acidic environment for the response of tumor cells to radiation has not been fully elucidated. This study aimed to investigate the effects of chronic pre- and post-irradiation exposure of tumor cells to an acidic environment on the radiation-induced clonogenic death.Materials and methods: Rat gliosarcoma cells were used throughout the in vitro study. Cells were exposed to pH 6.6 medium for varying durations of up to 4 days before and after X-irradiation. Cell viability, apoptosis, clonogenic cell death and cell cycle distribution were observed.Results: Incubation of tumor cells in pH 6.6 medium for 2 or 4 days extended cell cycle, decreased cell viability, and induced apoptotic and clonogenic cell death. The radiation-induced clonogenic death was increased by 2- or 4-day pre-irradiation exposure of tumor cells to pH 6.6 medium, whereas it was reduced by 4-day post-irradiation exposure to an acidic medium.Conclusion: Prolonged exposure to an acidic environment enhanced the sensitivity of tumor cells to subsequent X-irradiation. However, the radiosensitization by pre-irradiation exposure was almost completely nullified by prolonged post-irradiation exposure to an acidic environment.

Carbon ion irradiation withstands cancer stem cells' migration/invasion process in Head and Neck Squamous Cell Carcinoma (HNSCC).

Cancer Stem Cells (CSCs) in Head and Neck Squamous Cell Carcinoma (HNSCC) have extremely aggressive profile (high migratory and invasive potential). These characteristics can explain their resistance to conventional treatment. Efficacy of photon and carbon ion irradiation with addition of cetuximab (5 nM) is studied on clonogenic death, migration and invasion of two HNSCC populations: SQ20B and SQ20B/CSCs. SQ20B express E-cadherin and overexpress EGFR while SQ20B/CSCs express N-cadherin and low EGFR. Cetuximab strongly inhibits SQ20B proliferation but has no effect on SQ20B/CSCs. 2 Gy photon irradiation enhances migration and invasiveness in both populations (p < 0.05), while cetuximab only stops SQ20B migration (p < 0.005). Carbon irradiation significantly inhibits invasion in both populations (p < 0.05), and the association with cetuximab significantly inhibits invasion in both populations (p < 0.005). These results highlight CSCs characteristics: EGFRLow, cetuximab-resistant, and highly migratory. Carbon ion irradiation appears to be a very promising therapeutic modality counteracting migration/invasion process in both parental cells and CSCs in contrast to photon irradiation.

Deubiquitylating enzyme USP9x regulates radiosensitivity in glioblastoma cells by Mcl-1-dependent and -independent mechanisms.

Glioblastoma is a very aggressive form of brain tumor with limited therapeutic options. Usually, glioblastoma is treated with ionizing radiation (IR) and chemotherapy after surgical removal. However, radiotherapy is frequently unsuccessful, among others owing to resistance mechanisms the tumor cells have developed. Antiapoptotic B-cell leukemia (Bcl)-2 family members can contribute to radioresistance by interfering with apoptosis induction in response to IR. Bcl-2 and the closely related Bcl-xL and Mcl-1 are often overexpressed in glioblastoma cells. In contrast to Bcl-2 and Bcl-xL, Mcl-1 is a short-lived protein whose stability is closely regulated by ubiquitylation-dependent proteasomal degradation. Although ubiquitin ligases facilitate degradation, the deubiquitylating enzyme ubiquitin-specific protease 9x (USP9x) interferes with degradation by removing polyubiquitin chains from Mcl-1, thereby stabilizing this protein. Thus, an inability to downregulate Mcl-1 by enhanced USP9x activity might contribute to radioresistance. Here we analyzed the impact of USP9x on Mcl-1 levels and radiosensitivity in glioblastoma cells. Correlating Mcl-1 and USP9x expressions were significantly higher in human glioblastoma than in astrocytoma. Downregulation of Mcl-1 correlated with apoptosis induction in established glioblastoma cell lines. Although Mcl-1 knockdown by siRNA increased apoptosis induction after irradiation in all glioblastoma cell lines, USP9x knockdown significantly improved radiation-induced apoptosis in one of four cell lines and slightly increased apoptosis in another cell line. In the latter two cell lines, USP9x knockdown also increased radiation-induced clonogenic death. The massive downregulation of Mcl-1 and apoptosis induction in A172 cells transfected with USP9x siRNA shows that the deubiquitinase regulates cell survival by regulating Mcl-1 levels. In contrast, USP9x regulated radiosensitivity in Ln229 cells without affecting Mcl-1 levels. We conclude that USP9x can control survival and radiosensitivity in glioblastoma cells by Mcl-1-dependent and Mcl-1-independent mechanisms.

Bouvardin is a Radiation Modulator with a Novel Mechanism of Action.

Protein synthesis is essential for growth, proliferation and survival of cells. Translation factors are overexpressed in many cancers and in preclinical models, their experimental inhibition has been shown to inhibit cancer growth. Differential regulation of translation also occurs upon exposure to cancer-relevant stressors such as hypoxia and ionizing radiation. The failure to regulate translation has been shown to interfere with recovery after genotoxic stress. These findings suggest that modulation of translation, alone or in conjunction with genotoxins, may be therapeutic in oncology. Yet, only two drugs that directly inhibit translation are FDA-approved for oncology therapies used today. We have previously identified the protein synthesis inhibitor, bouvardin in a screen for small molecule enhancers of ionizing radiation in Drosophila melanogaster . Bouvardin was independently identified in a screen for selective inhibitors of engineered human breast cancer stem cells. Here we report the effect of bouvardin treatment in preclinical models of head and neck cancer (HNC) and glioma, two cancer types for which radiation therapy is the most common treatment. Our data show that bouvardin treatment blocked translation elongation on human ribosomes and suggest that it did so by blocking the dissociation of elongation factor 2 from the ribosome. Bouvardin and radiation enhanced the induction of clonogenic death in HNC and glioma cells, although by different mechanisms. Bouvardin treatment enhanced the radiation-induced antitumor effects in HNC tumor xenografts in mice. These data suggest that inhibition of translation elongation, particularly in combination with radiation treatment, may be a promising treatment option for cancer.

γH2AX is a biomarker of modulated cytostatic drug resistance.

ONE reason for reduced therapeutic efficiency of cancer chemotherapies is the appearance of multidrug resistance. Plasma membrane glycoprotein (P-gp) is recognized as one of the best characterized drug efflux pumps playing a central role in the absorption and disposition of many drugs, including chemotherapeutic agents. Increased P-gp activity has been suggested and reported to be an indicator of chemotherapeutic failure. Therefore, P-gp activity has been used as a potential target in being able to reverse multidrug resistance. Cytostatic drugs such as etoposide in combination with the immunosuppressive cyclosporine A may inhibit P-gp activity, ultimately resulting in intracellular enrichment of the targeted drug (1). One issue in cancer therapy involves the highly varying drug response among patients. To assess the multidrug resistance status for personalized cancer therapy, an assay that can provide results to monitor the cytostatic effect of antineoplastic agents in combination with different drugs is highly desirable. Many of these drugs, like etoposide, cause DNA double strand breaks (DSBs) that may eventuate in clonogenic death. DSBs are one of the most biologically significant DNA damage lesions that leads to chromosome breakage and/or rearrangement, mutagenesis and loss or gain of genetic information (2). The phosphorylation of H2AX histone proteins which are located in the vicinity of the DSBs is known as one of the earliest responses to DNA DSBs in cells. Induction of DNA DSBs in live mammalian cells triggers the phosphorylation of Ser139 in the SQ motif near the C-terminal of H2AX protein, which results in the phosphorylated form of H2AX, termed cH2AX. cH2AX signals appear as discrete nuclear “foci” which can be visualized and quantified by a number of methods including fluorescence microscopy and flow cytometry, following immunostaining procedures with fluorescence-coupled antibodies. The cH2AX foci counting approach has been used in numerous studies to assess the relationship between cH2AX foci removal and the rate of DSBs repair (3). Each DSB is represented by an individual cH2AX focus with a ratio of 1:1 and after successful repair of DSBs, the level of cH2AX foci decline over time in the presence of normal physiological repair mechanisms. The presence of persistent cH2AX indicates impaired DNA repair. Therefore, cH2AX quantification may prove to be a sensitive biomarker of DNA DSBs in human cells and has been suggested for biomonitoring tumor progression and treatment response (4). DSBs are directly generated by exogenous agents such as ionizing radiation and antitumor drugs (bleomycin, mitoxantrone, etoposide). Because mammalian cells respond to DSBs by activating a multitude of proteins involved in signalling and DNA repair pathways, the very nature of DSBs poses such a threat to cell survival that DNA damage checkpoint proteins may be activated to initiate cellular division arrest. This provides time for DNA repair to proceed before mitosis is completed or in the case of overwhelming damage, apoptosis ensues (5). Therefore, detection of accumulated cH2AX foci may be an efficient method for assessing multidrug resistance in cancer therapy as presented by Reddig et al. in this issue of Cytometry A (page XXX).

Autocrine CSF-1R signaling drives mesothelioma chemoresistance via AKT activation.

Clinical management of malignant pleural mesothelioma (MPM) is very challenging because of the uncommon resistance of this tumor to chemotherapy. We report here increased expression of macrophage colony-stimulating-factor-1-receptor (M-CSF/CSF-1R) mRNA in mesothelioma versus normal tissue specimens and demonstrate that CSF-1R expression identifies chemoresistant cells of mesothelial nature in both primary cultures and mesothelioma cell lines. By using RNAi or ligand trapping, we demonstrate that the chemoresistance properties of those cells depend on autocrine CSF-1R signaling. At the single-cell level, the isolated CSF-1Rpos cells exhibit a complex repertoire of pluripotency, epithelial–mesenchymal transition and detoxifying factors, which define a clonogenic, chemoresistant, precursor-like cell sub-population. The simple activation of CSF-1R in untransformed mesothelial cells is sufficient to confer clonogenicity and resistance to pemetrexed, hallmarks of mesothelioma. In addition, this induced a gene expression profile highly mimicking that observed in the MPM cells endogenously expressing the receptor and the ligands, suggesting that CSF-1R expression is mainly responsible for the phenotype of the identified cell sub-populations. The survival of CSF1Rpos cells requires active AKT (v-akt murine thymoma viral oncogene homolog 1) signaling, which contributed to increased levels of nuclear, transcriptionally competent β-catenin. Inhibition of AKT reduced the transcriptional activity of β-catenin-dependent reporters and sensitized the cells to senescence-induced clonogenic death after pemetrexed treatment. This work expands what is known on the non-macrophage functions of CSF-1R and its role in solid tumors, and suggests that CSF-1R signaling may have a critical pathogenic role in a prototypical, inflammation-related cancer such as MPM and therefore may represent a promising target for therapeutic intervention.

Effect of Brazilian propolis (AF-08) on genotoxicity, cytotoxicity and clonogenic death of Chinese hamster ovary (CHO-K1) cells irradiated with 60Co gamma-radiation

The present study was conducted in order to evaluate the effect of Brazilian propolis (AF-08; 5, 10, 15, 30, 50, 100, and 200μg/mL) in protecting CHO-K1 cells against genotoxic and cytotoxic damage and clonogenic death induced by 60Co gamma-radiation (1.0, 2.0, 4.0, and 6.0Gy). For this purpose, three interlinked endpoints were analyzed: induction of DNA damage by use of the micronucleus (MN) test (genotoxic damage), cell viability by means of the MTS assay, and differential staining (cytotoxic damage) and clonogenic death via the colony-formation test (cytotoxic damage). The MN test revealed that propolis alone (5–100μg/mL) was not genotoxic up to 100μg/mL and that 30μg/mL of propolis reduced the radiation-induced DNA damage (∼56% reduction, p<0.05), exhibiting a radio-protective effect on irradiated CHO-K1 cells. On the other hand, analysis of cytotoxicity showed that a concentration of 50μg/mL presented a significant proliferative effect (p<0.001) when associated with radiation, decreasing the percentage of necrotic cells (p<0.01). No mediated cytotoxic effect was found, but the concentration of 200μg/mL was toxic when analyzed at 24 and 48h via the differential staining technique, but not at 72h after irradiation, analyzed with the MTS assay. Differential staining also showed that necrosis was the main death modality in irradiated cells and that apoptosis was induced only at the toxic concentration of propolis (200μg/mL). Concerning the clonogenic capacity, a concentration of 50μg/mL also exhibited a significant stimulating effect on cell proliferation (p<0.001), in agreement with the data from differential staining. Taken together, these data suggest that the use of propolis AF-08 for the prevention of the adverse effects of ionizing radiation is promising. Nevertheless, additional investigations are necessary for a better understanding of potential applications of propolis to improve human health.

Protumorigenic effects of mir-145 loss in malignant pleural mesothelioma

We identified a discrete number of microRNAs differentially expressed in benign or malignant mesothelial tissues. We focused on mir-145 whose levels were significantly downregulated in malignant mesothelial tissues and malignant pleural mesothelioma (MPM) cell lines as compared to benign tissues (pleura, peritoneum or cysts). We show that promoter hyper-methylation caused very low levels in MPM cell lines and specimens. Treatment of MPM cell lines with mir-145 agonists negatively modulated some protumorigenic properties of MPM cells, such as clonogenicity, cell migration and resistance to pemetrexed treatment. The main effector mechanism of the clonogenic death induced by mir-145 was that of accelerated senescence. We found that mir-145 targeted OCT4 via specific binding to its 3'-UTR. Increased intracellular levels of mir-145 decreased the levels of OCT4 and its target gene ZEB1, thereby counteracting the increase of OCT4 induced by pemetrexed treatment which is known to favor the development of chemoresistant cells. In line with this, reintroduction of OCT4 into mimic-145 treated cells counteracted the effects on clonogenicity and replicative senescence. This further supports the relevance of the mir-145-OCT4 interaction for the survival of MPM cells. The potential use of mir-145 expression levels to classify benign vs malignant mesothelial tissues and the differences between pemetrexed-induced senescence and that induced by the re-expression of mir-145 are discussed.

Eukaryotic Initiation Factor 2α - a Downstream Effector of Mammalian Target of Rapamycin - Modulates DNA Repair and Cancer Response to Treatment

In an effort to circumvent resistance to rapamycin – an mTOR inhibitor - we searched for novel rapamycin-downstream-targets that may be key players in the response of cancer cells to therapy. We found that rapamycin, at nM concentrations, increased phosphorylation of eukaryotic initiation factor (eIF) 2α in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2α phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal – an inhibitor of eIF2α dephosphorylation – decreased expression of a surface marker associated with capacity for self renewal, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2α is detrimental to the cells' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2α phosphorylation than non-irradiated ones. Similar to salubrinal - the phosphomimetic eIF2α variant - S51D - increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2α in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks that were induced by I-SceI in green fluorescent protein reporter plasmids. In addition to its effect on radiation, salubrinal enhanced eIF2α phosphorylation and clonogenic death in response to the histone deacetylase inhibitor – vorinostat. Finally, the catalytic competitive inhibitor of mTOR - Ku-0063794 - increased phosphorylation of eIF2α demonstrating further the involvement of mTOR activity in modulating eIF2α phosphorylation. These experiments suggest that excessive phosphorylation of eIF2α decreases survival of cancer cells; making eIF2α a worthy target for drug development, with the potential to enhance the cytotoxic effects of established anti-neoplastic therapies and circumvent resistance to rapalogues and possibly to other drugs that inhibit upstream components of the mTOR pathway.

Radioprotection and Cell Cycle Arrest of Intestinal Epithelial Cells by Darinaparsin, a Tumor Radiosensitizer

It was recently reported that the organic arsenic compound darinaparsin (DPS) is a cytotoxin and radiosensitizer of tumor cells in vitro and in subcutaneous xenograft tumors. Surprisingly, it was also found that DPS protects normal intestinal crypt epithelial cells (CECs) from clonogenic death after ionizing radiation (IR). Here we tested the DPS radiosensitizing effect in a clinically relevant model of prostate cancer and explored the radioprotective effect and mechanism of DPS on CECs. The radiation modification effect of DPS was tested in a mouse model of orthotopic xenograft prostate cancer and of IR-induced acute gastrointestinal syndrome. The effect of DPS on CEC DNA damage and DNA damage responses was determined by immunohistochemistry. In the mouse model of IR-induced gastrointestinal syndrome, DPS treatment before IR accelerated recovery from body weight loss and increased animal survival. DPS decreased post-IR DNA damage and cell death, suggesting that the radioprotective effect was mediated by enhanced DNA damage repair. Shortly after DPS injection, significant cell cycle arrest was observed in CECs at both G1/S and G2/M checkpoints, which was accompanied by the activation of cell cycle inhibitors p21 and growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A). Further investigation revealed that DPS activated ataxia telangiectasia mutated (ATM), an important inducer of DNA damage repair and cell cycle arrest. DPS selectively radioprotected normal intestinal CECs and sensitized prostate cancer cells in a clinically relevant model. This effect may be, at least in part, mediated by DNA damage response activation and has the potential to significantly increase the therapeutic index of radiation therapy.