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Venous thromboembolism (VTE) is one of the leading causes of direct maternal deaths. It has been estimated that approximately 50% of these deaths are potentially preventable. The UK Confidential Enquiries into Maternal Deaths and the National Partnership for Maternal Safety have proposed strategies for the prevention of maternal deaths from VTE based on current guideline recommendations. The main strategies include: – Early recognition and comprehensive assessment of risk factors for VTE at different times from the beginning of pregnancy until patient’s discharge from hospital. – Appropriate risk stratification using standardized VTE risk assessment tools (e.g. the Royal College Scoring System). – Risk–based antenatal and postnatal heparin thromboprophylaxis adjusting the heparin dosage and the duration of prophylaxis to the individual patient’s risk. – Adequate management of heparin prophylaxis before and after delivery. – Preference of universal rather than selective post-cesarean heparin thromboprophylaxis and application of perioperative mechanical prophylaxis. – Avoidance of gaps in the postpartum prescription of heparin. – Good communication and cooperation between primary and secondary care including community midwifery staff in the postpartum period. – Immediate intravenous administration of (unfractionated) heparin, if pulmonary embolism is suspected. – Critical analysis of all thromboembolic events, particularly in association with maternal death to learn from failures and to realize, if and where improvement is needed. Adequate pharmacological thromboprophylaxis has the potential to reduce the risk of VTE by 60–70% in pregnant women at increased risk.

A)Effect of radiation on H3K27me3 levels in U251 cells. B)Effects of GSKJ4 on cell cycle distribution of U251 or MDA-MB-231 cells.

Gene expression of mTOR/AKT related genes after single dose and multifractionated radiation. DU145 human prostate carcinoma cells were irradiated with 10 Gy single dose (SD) or 5 fractions of 2 Gy or 10 fractions of 1 Gy (2 Gy a day) as multifractionated (MF) radiotherapy. At indicated time points after radiation, cells were lysed and RNA was isolated.

Average weights of surviving mice in each treatment group.

Radiation schedule for the cell culture and xenograft experiments. Human prostate cancer cells were either plated or injected subcutaneously into the flanks of the right hind legs of athymic nude mice. After 24 h (in vitro) or tumors reached a tumor size of 5 mm x 5 mm (in vivo), tumors were irradiated with different fractionation regimens including a single dose of 10 Gy at day 5, 10 fractions of 1 Gy (2 fractions per day), or 5 fractions of 2 Gy (1 fraction per day). Unirradiated cells or tumors were used as control.

Densitometric analysis of basal protein expression and phosphorylation in DU145 and PC3 cells. Western blots of 3D-grown prostate cancer cell lines were evaluated with ImageJ. Phospho-protein expression was normalized to total protein expression. Results show mean {plus minus} SD (n = 3) (See also Figure 3A).

Analysis of the radiation-induced translatome of glioblastoma stem-like cells (GSC) identified an interacting network in which XPO1 serves as a major hub protein. To determine whether this nuclear export protein provides a target for radiosensitization, we defined the effects of clinically relevant XPO1 inhibitor selinexor on the radiosensitivity of glioblastoma cells. As determined by clonogenic survival analysis, selinexor enhanced the radiosensitivity of GSCs but not normal fibroblast cell lines. On the basis of γH2AX foci and neutral comet analyses, selinexor inhibited the repair of radiation-induced DNA double-strand breaks in GSCs, suggesting that the selinexor-induced radiosensitization is mediated by an inhibition of DNA repair. Consistent with a role for XPO1 in the nuclear to cytoplasm export of rRNA, selinexor reduced 5S and 18S rRNA nuclear export in GSCs, which was accompanied by a decrease in gene translation efficiency, as determined from polysome profiles, as well as in protein synthesis. In contrast, rRNA nuclear export and protein synthesis were not reduced in normal cells treated with selinexor. Orthotopic xenografts initiated from a GSC line were then used to define the in vivo response to selinexor and radiation. Treatment of mice bearing orthotopic xenografts with selinexor decreased tumor translational efficiency as determined from polysome profiles. Although selinexor treatment alone had no effect on the survival of mice with brain tumors, it significantly enhanced the radiation-induced prolongation of survival. These results indicate that selinexor enhances the radiosensitivity of glioblastoma cells and suggest that this effect involves the global inhibition of gene translation. Mol Cancer Ther; 17(8); 1717–26. ©2018 AACR.

The processes mediating the repair of DNA double-strand breaks (DSB) are critical determinants of radiosensitivity and provide a source of potential targets for tumor radiosensitization. Among the events required for efficient DSB repair are a variety of post-translational histone modifications, including methylation. Because trimethylation of histone H3 on lysine 27 (H3K27me3) has been associated with chromatin condensation, which can influence DSB repair, we determined the effects of radiation on H3K27me3 levels in tumor and normal cell lines. Irradiation of tumor cells resulted in a rapid loss of H3K27me3, which was prevented by the siRNA-mediated knockdown of the H3K27 demethylase UTX. Knockdown of UTX also enhanced the radiosensitivity of each tumor cell line. Treatment of tumor cells with the H3K27 demethylase inhibitor GSKJ4 immediately before irradiation prevented the radiation-induced decrease in H3K27me3 and enhanced radiosensitivity. As determined by neutral comet analysis and γH2AX expression, this GSKJ4 treatment protocol inhibited the repair of radiation-induced DSBs. Consistent with in vitro results, treatment of mice bearing leg tumor xenografts with GSKJ4 significantly enhance radiation-induce tumor growth delay. In contrast with results generated from tumor cell lines, radiation had no effect on H3K27me3 levels in normal fibroblast cell lines and GSKJ4 did not enhance their radiosensitivity. These data suggest that H3K27me3 demethylation contributes to DSB repair in tumor cells and that UTX, the demethylase responsible, provides a target for selective tumor cell radiosensitization. Mol Cancer Ther; 17(5); 1070–8. ©2018 AACR.

Oligonucleotides used for CRISPR/Cas9 gene editing

Subcellular localization of AKT and mTOR after radiation. Immunofluorescence staining of AKT and mTOR in irradiated and unirradiated DU145 and PC3 cells. Nuclear staining was performed with DAPI.

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

PDF - 89K, Functions associated with the top networks for the genes no longer decreased by radiation after INK128 treatment.

PDF - 93K, Genes comprising the DNA replication, recombination and repair biological function whose radiation-induced up-regulation was inhibited by INK128.

Supplementary Tables S1-S2.

Influence of INK128 on GSC radiosensitivity

Changes in polysome-bound mRNA (translatome) are correlated closely with changes in the proteome in cells. Therefore, to better understand the processes mediating the response of glioblastoma to ionizing radiation (IR), we used polysome profiling to define the IR-induced translatomes of a set of human glioblastoma stem-like cell (GSC) lines. Although cell line specificity accounted for the largest proportion of genes within each translatome, there were also genes that were common to the GSC lines. In particular, analyses of the IR-induced common translatome identified components of the DNA damage response, consistent with a role for the translational control of gene expression in cellular radioresponse. Moreover, translatome analyses suggested that IR enhanced cap-dependent translation processes, an effect corroborated by the finding of increased eIF4F–cap complex formation detected after irradiation in all GSC lines. Translatome analyses also predicted that Golgi function was affected by IR. Accordingly, Golgi dispersal was detected after irradiation of each of the GSC lines. In addition to the common responses seen, translatome analyses predicted cell line–specific changes in mitochondria, as substantiated by changes in mitochondrial mass and DNA content. Together, these results suggest that analysis of radiation-induced translatomes can provide new molecular insights concerning the radiation response of cancer cells. More specifically, they suggest that the translational control of gene expression may provide a source of molecular targets for glioblastoma radiosensitization. Cancer Res; 76(10); 3078–87. ©2016 AACR.

Purpose: Radiotherapy remains a primary treatment modality for pancreatic carcinoma, a tumor characterized by aberrant mTOR activity. Given the regulatory role of mTOR in gene translation, in this study, we defined the effects of the clinically relevant, ATP-competitive mTOR inhibitor, INK128 on the radiosensitivity of pancreatic carcinoma cell lines.Experimental Design: Clonogenic survival was used to determine the effects of INK128 on in vitro radiosensitivity of three pancreatic carcinoma cell lines and a normal fibroblast cell line with mTOR activity defined using immunoblots. DNA double-strand breaks were evaluated according to γH2AX foci. The influence of INK128 on radiation-induced gene translation was determined by microarray analysis of polysome-bound mRNA. Leg tumor xenografts grown from pancreatic carcinoma cells were evaluated for mTOR activity, eIF4F cap complex formation, and tumor growth delay.Results: INK128, while inhibiting mTOR activity in each of the cell lines, enhanced the in vitro radiosensitivity of the pancreatic carcinoma cells but had no effect on normal fibroblasts. The dispersal of radiation-induced γH2AX foci was inhibited in pancreatic carcinoma cells by INK128 as were radiation-induced changes in gene translation. Treatment of mice with INK128 resulted in an inhibition of mTOR activity as well as cap complex formation in tumor xenografts. Whereas INK128 alone had no effect of tumor growth rate, it enhanced the tumor growth delay induced by single and fractionated doses of radiation.Conclusion: These results indicate that mTOR inhibition induced by INK128 enhances the radiosensitivity of pancreatic carcinoma cells and suggest that this effect involves the inhibition of DNA repair. Clin Cancer Res; 20(1); 110–9. ©2013 AACR.

A consequence of the intratumor heterogeneity (ITH) of glioblastoma (GBM) is the susceptibility to treatment-driven evolution. To determine the potential of radiotherapy to influence GBM evolution, we used orthotopic xenografts initiated from CD133+ GBM stem–like cells (GSC). Toward this end, orthotopic xenografts grown in nude mice were exposed to a fractionated radiation protocol, which resulted in a significant increase in animal survival. Brain tumors from control and irradiated mice were then collected at morbidity and compared in terms of growth pattern, clonal diversity, and genomic architecture. In mice that received fractionated radiation, tumors were less invasive, with more clearly demarcated borders and tumor core hypercellularity as compared with controls, suggesting a fundamental change in tumor biology. Viral integration site analysis indicated a reduction in clonal diversity in the irradiated tumors, implying a decrease in ITH. Changes in clonal diversity were not detected after irradiation of GSCs in vitro, suggesting that the radiation-induced reduction in ITH was dependent on the brain microenvironment. Whole-exome sequencing revealed differences in mutation patterns between control and irradiated tumors, which included modifications in the presence and clonality of driver mutations associated with GBM. Moreover, changes in the distribution of mutations as a function of subpopulation size between control and irradiated tumors were consistent with subclone expansion and contraction, that is, subpopulation evolution. Taken together, these results indicate that radiation drives the evolution of the GSC-initiated orthotopic xenografts and suggest that radiation-driven evolution may have therapeutic implications for recurrent GBM.Significance:Radiation drives the evolution of glioblastoma orthotopic xenografts; when translated to the clinic, this may have therapeutic implications for recurrent tumors.

PDF - 99K, Functions associated with the top networks corresponding to the genes whose radiation-induced upregulation was inhibited by INK128

higher magnification of xenograft histology

Purpose: Brain tumor xenografts initiated from human glioblastoma (GBM) stem-like cells (TSC) simulate the biological characteristics of GBMs in situ. Therefore, to determine whether the brain microenvironment affects the intrinsic radiosensitivity of GBM cells, we compared the radioresponse of GBM TSCs grown in vitro and as brain tumor xenografts.Experimental Design: As indicators of DNA double-strand breaks (DSB), γH2AX, and 53BP1 foci were defined after irradiation of 2 GBM TSC lines grown in vitro and as orthotopic xenografts in nude mice. Microarray analysis was conducted to compare gene expression patterns under each growth condition.Results: Dispersal of radiation-induced γH2AX and 53BP1 foci was faster in the tumor cells grown as orthotopic xenografts compared with cells irradiated in vitro. In addition, cells irradiated in vivo were approximately 3-fold less susceptible to foci induction as compared with cells grown in vitro. Microarray analysis revealed a significant number of genes whose expression was commonly affected in the 2 GBM models by orthotopic growth conditions. Consistent with the decrease in sensitivity to foci induction, genes related to reactive oxygen species (ROS) metabolism were expressed at higher levels in the brain tumor xenografts.Conclusion: γH2AX and 53BP1 foci analyses indicate that GBM cells irradiated within orthotopic xenografts have a greater capacity to repair DSBs and are less susceptible to their induction than tumor cells irradiated under in vitro growth conditions. Because DSB induction and repair are critical determinants of radiosensitivity, these results imply that the brain microenvironment contributes to GBM radioresistance.Clin Cancer Res; 16(24); 6049–59. ©2010 AACR.

Genes upregulated in IR-induced translatome;Genes down-regulated in the radiation-induced translatome; Top IPA networks of genes down-regulated in the common IR-induced translatome; GSEA enrichment of Mitochondrial GO terms in the radiation-induced translatome

A consequence of the intratumor heterogeneity (ITH) of glioblastoma (GBM) is the susceptibility to treatment-driven evolution. To determine the potential of radiotherapy to influence GBM evolution, we used orthotopic xenografts initiated from CD133+ GBM stem–like cells (GSC). Toward this end, orthotopic xenografts grown in nude mice were exposed to a fractionated radiation protocol, which resulted in a significant increase in animal survival. Brain tumors from control and irradiated mice were then collected at morbidity and compared in terms of growth pattern, clonal diversity, and genomic architecture. In mice that received fractionated radiation, tumors were less invasive, with more clearly demarcated borders and tumor core hypercellularity as compared with controls, suggesting a fundamental change in tumor biology. Viral integration site analysis indicated a reduction in clonal diversity in the irradiated tumors, implying a decrease in ITH. Changes in clonal diversity were not detected after irradiation of GSCs in vitro, suggesting that the radiation-induced reduction in ITH was dependent on the brain microenvironment. Whole-exome sequencing revealed differences in mutation patterns between control and irradiated tumors, which included modifications in the presence and clonality of driver mutations associated with GBM. Moreover, changes in the distribution of mutations as a function of subpopulation size between control and irradiated tumors were consistent with subclone expansion and contraction, that is, subpopulation evolution. Taken together, these results indicate that radiation drives the evolution of the GSC-initiated orthotopic xenografts and suggest that radiation-driven evolution may have therapeutic implications for recurrent GBM. Significance: Radiation drives the evolution of glioblastoma orthotopic xenografts; when translated to the clinic, this may have therapeutic implications for recurrent tumors.

Evolution of the structure and surface morphology of thin films composed of two selenium based chalcogenide phases, AgInSe2 and Ag2Se on 140 MeV ion irradiation is reported. At this high energy, the projectile Ni ions sputtered the surface of the films through energy deposition to the target electrons. The sputtering led to decrease of silver and selenium concentration, but did not affect indium concentration. Surface analysis by field emission scanning electron microscopy study revealed nanoparticles of widely varying shapes and sizes on the surface of the films. Irradiation did not affect the particle size distribution which peaked at ~100 nm. The two phases, AgInSe2 and Ag2Se exhibited different sensitivity to 140 MeV Ni ion irradiations. While Ag2Se phase was insensitive, the AgInSe2 phase showed complex structural modifications at intermediate fluences, finally getting amorphized at high ion fluences. Analysis of the fluence dependence of Grazing Incidence X-ray Diffraction (GIXRD) peak area of AgInSe2 phase indicated that each ion that penetrated the film, created an amorphous column of 1.6 nm radius. Surrounding the amorphous column, a cylindrical crystalline AgInSe2 region of radius 8.2 nm, but with reduced lattice parameters formed. High resolution transmission electron microscopy image also confirmed registration of amorphized tracks and the modified crystalline region of similar radii around the path of 140 MeV Ni ions in a grain of AgInSe2 as obtained from GIXRD study.

Metformin is used to treat patients with diabetes mellitus and that was found to lower the incidence of cancer. The present study investigated the effects of metformin on human bone-derived mesenchymal stromal cells (BM-MSC) and their breast cancer cell line interactions. BM-MSCs were tested for growth stimulation and migration controlling activity on four breast cancer cell lines employing MTT tests, migration scratch tests and assays of the expression of adipokines in Western Blot arrays. Compared to breast cancer cell lines, metformin significantly inhibited the proliferation of BM-MSC lines. Pretreatment of BM-MSCs with metformin showed variable effects on breast cancer cell lines depending on the specific BM-MSC cancer line combination. Metformin significantly impaired the migration of MDA-MB-231 and MDA-MB-436 in response to conditioned media (CM) of drug pretreated BM-MSCs. Metformin-induced alterations of adipokines by BM-MSC CM indicated increased osteogenic signaling and possibly impairment of metastasis. The anticancer activities of metformin seem to be the result of direct and indirect mechanisms. A lower metformin-induced protumor activity of BM-MSCs in the bone microenvironment seem to contribute to the anticancer effects of this drug in breast cancer patients.

In the present study, the AgInSe2 (AIS) thin films 500 nm in thickness were prepared by selenization of the Ag/In/Ag/In multilayer deposited by DC magnetron sputtering. The selenization was carried out at 250 °C and was followed by annealing at 450 °C, 475 °C and 500 °C. The selenized and annealed films were characterized by grazing incidence X-ray diffraction (GIXRD), UV-Visible-NIR spectroscopy, Field-Emission Scanning Electron Microscopy (FESEM) and Raman spectroscopy. The XRD analysis revealed the formation of the desired AgInSe2 phase along with Ag2Se (AS) impurity phase. The impurity Ag2Se phase is being diminished at higher annealing temperature (500 °C) resulting the formation of AgInSe2 phase. The super ionic nature of α-Ag2Se phase facilities the diffusion of Ag ion with its neighbouring tetrahedral sites. The low band gap Ag2Se impurity phase suppression by annealing at 500 °C lead to optical band gap increase. The Raman spectrum shows individual AgInSe2 and Ag2Se peaks and also the overlapping of these two phases. The Ag2Se peaks are vanished after annealing. The EDXRF study revealed that the film is off-stoichiometric and exhibit n-type conductivity. The RBS analysis indicates the diffusion of the film material to the glass substrate. From Hall measurement it is confirmed that the film shows n-type conductivity and the resistivity decreases with increasing the annealing temperature.

Analysis of the radiation-induced translatome of glioblastoma stem-like cells (GSC) identified an interacting network in which XPO1 serves as a major hub protein. To determine whether this nuclear export protein provides a target for radiosensitization, we defined the effects of clinically relevant XPO1 inhibitor selinexor on the radiosensitivity of glioblastoma cells. As determined by clonogenic survival analysis, selinexor enhanced the radiosensitivity of GSCs but not normal fibroblast cell lines. On the basis of γH2AX foci and neutral comet analyses, selinexor inhibited the repair of radiation-induced DNA double-strand breaks in GSCs, suggesting that the selinexor-induced radiosensitization is mediated by an inhibition of DNA repair. Consistent with a role for XPO1 in the nuclear to cytoplasm export of rRNA, selinexor reduced 5S and 18S rRNA nuclear export in GSCs, which was accompanied by a decrease in gene translation efficiency, as determined from polysome profiles, as well as in protein synthesis. In contrast, rRNA nuclear export and protein synthesis were not reduced in normal cells treated with selinexor. Orthotopic xenografts initiated from a GSC line were then used to define the in vivo response to selinexor and radiation. Treatment of mice bearing orthotopic xenografts with selinexor decreased tumor translational efficiency as determined from polysome profiles. Although selinexor treatment alone had no effect on the survival of mice with brain tumors, it significantly enhanced the radiation-induced prolongation of survival. These results indicate that selinexor enhances the radiosensitivity of glioblastoma cells and suggest that this effect involves the global inhibition of gene translation. Mol Cancer Ther; 17(8); 1717–26. ©2018 AACR.

Radiotherapy is a primary treatment modality for glioblastomas (GBM). Because DNA-PKcs is a critical factor in the repair of radiation-induced double strand breaks (DSB), this study evaluated the potential of VX-984, a new DNA-PKcs inhibitor, to enhance the radiosensitivity of GBM cells. Treatment of the established GBM cell line U251 and the GBM stem-like cell (GSC) line NSC11 with VX-984 under in vitro conditions resulted in a concentration-dependent inhibition of radiation-induced DNA-PKcs phosphorylation. In a similar concentration-dependent manner, VX-984 treatment enhanced the radiosensitivity of each GBM cell line as defined by clonogenic analysis. As determined by γH2AX expression and neutral comet analyses, VX-984 inhibited the repair of radiation-induced DNA double-strand break in U251 and NSC11 GBM cells, suggesting that the VX-984-induced radiosensitization is mediated by an inhibition of DNA repair. Extending these results to an in vivo model, treatment of mice with VX-984 inhibited radiation-induced DNA-PKcs phosphorylation in orthotopic brain tumor xenografts, indicating that this compound crosses the blood–brain tumor barrier at sufficient concentrations. For mice bearing U251 or NSC11 brain tumors, VX-984 treatment alone had no significant effect on overall survival; radiation alone increased survival. The survival of mice receiving the combination protocol was significantly increased as compared with control and as compared with radiation alone. These results indicate that VX-984 enhances the radiosensitivity of brain tumor xenografts and suggest that it may be of benefit in the therapeutic management of GBM. Mol Cancer Ther; 17(6); 1207–16. ©2018 AACR.

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

Mutated or rearranged driver kinases in non-small cell lung cancer (NSCLC) cells are clinically amenable to treatment with tyrosine kinase inhibitors (TKIs) resulting in prolonged survival and significant benefit compared to cytotoxic chemotherapy. The most frequent genomic alterations are observed for epidermal growth factor receptor and anaplastic lymphoma kinase, which can be blocked by a range of specific TKIs in sequence. In clinics, resistance to TKIs emerges after approximately one year and comprises secondary mutations of the kinases (on-target) or alternative pathways circumventing the original kinase (off-target) alterations. A special feature of NSCLC is the occurrence of histological transformation to small cell lung cancer (SCLC) in up to 14% of cases, which, in general, is accompanied by resistance to the original TKIs. SCLC transformed tumors may be treated with the classical platinum/etoposide regimen but thus far there are no definitive guidelines. Four transformed pleural SCLC lines in our lab indicate the presence of a gradual NSCLC-SCLC shift with overlapping drug sensitivities. In conclusion, the treatment of NSCLC-SCLC transformed cancer cells would need a better chemosensitivity assessment using functional genomics to guide further therapy.

Multifractionated irradiation is the mainstay of radiation treatment in cancer therapy. Yet, little is known about the cellular DNA repair processes that take place between radiation fractions, even though understanding the molecular mechanisms promoting cancer cell recovery and survival could improve patient outcome and identify new avenues for targeted intervention. To address this knowledge gap, we systematically characterized how cells respond differentially to multifractionated and single-dose radiotherapy, using a combination of genetics-based and functional approaches. We found that both cancer cells and normal fibroblasts exhibited enhanced survival after multifractionated irradiation compared with an equivalent single dose of irradiation, and this effect was entirely dependent on 53BP1-mediated NHEJ. Furthermore, we identified RIF1 as the critical effector of 53BP1. Inhibiting 53BP1 recruitment to damaged chromatin completely abolished the survival advantage after multifractionated irradiation and could not be reversed by suppressing excessive end resection. Analysis of the TCGA database revealed lower expression of 53BP1 pathway genes in prostate cancer, suggesting that multifractionated radiotherapy might be a favorable option for radio-oncologic treatment in this tumor type. We propose that elucidation of DNA repair mechanisms elicited by different irradiation dosing regimens could improve radiotherapy selection for the individual patient and maximize the efficacy of radiotherapy.

Glioblastoma (GBM) is the most common form of malignant brain tumor corresponding to approximately 20,000 new cases in the US per year (1). While the best available treatment involves surgery followed by the combination of radiotherapy and temozolomide (2), the survival of the vast majority of patients with GBM remains less than 2 years after diagnosis. Accordingly, considerable research has continued into the development of more effective GBM therapy. At the fundamental and preclinical levels such research has focused on developing experimental models that accurately reflect GBM biology, defining the molecules mediating GBM cell survival and treatment resistance and identifying drugs suitable for targeting those molecules within the constraints of CNS physiology. In the recent article by Lee et al . (3), each of these topics has been addressed in an attempt to suggest a novel, more effective GBM treatment strategy.

Changes in polysome-bound mRNA (translatome) are correlated closely with changes in the proteome in cells. Therefore, to better understand the processes mediating the response of glioblastoma to ionizing radiation (IR), we used polysome profiling to define the IR-induced translatomes of a set of human glioblastoma stem-like cell (GSC) lines. Although cell line specificity accounted for the largest proportion of genes within each translatome, there were also genes that were common to the GSC lines. In particular, analyses of the IR-induced common translatome identified components of the DNA damage response, consistent with a role for the translational control of gene expression in cellular radioresponse. Moreover, translatome analyses suggested that IR enhanced cap-dependent translation processes, an effect corroborated by the finding of increased eIF4F–cap complex formation detected after irradiation in all GSC lines. Translatome analyses also predicted that Golgi function was affected by IR. Accordingly, Golgi dispersal was detected after irradiation of each of the GSC lines. In addition to the common responses seen, translatome analyses predicted cell line–specific changes in mitochondria, as substantiated by changes in mitochondrial mass and DNA content. Together, these results suggest that analysis of radiation-induced translatomes can provide new molecular insights concerning the radiation response of cancer cells. More specifically, they suggest that the translational control of gene expression may provide a source of molecular targets for glioblastoma radiosensitization. Cancer Res; 76(10); 3078–87. ©2016 AACR.

Small cell lung cancer (SCLC) is distinguished by aggressive growth, early dissemination and a poor prognosis at advanced stage. The remarkably high count of circulating tumor cells (CTCs) of SCLC allowed for the establishment of permanent CTC cultures at our institution for the first time. CTCs are assumed to have characteristics of cancer stem cells (CSCs) and an epithelial-mesenchymal transition (EMT) phenotype, but extravasation of tumors at distal sites is marked by epithelial features. Two SCLC CTC cell lines, namely BHGc7 and BHGc10, as well as SCLC cell lines derived from primary tumors and metastases were analyzed for the expression of pluripotent stem cell markers and growth factors. Expression of E-cadherin and β-Catenin were determined by flow cytometry. Stem cell-associated markers SOX17, α-fetoprotein, OCT-3/4, KDR, Otx2, GATA-4, Nanog, HCG, TP63 and Goosecoid were not expressed in the 2 CTC lines. In contrast, high expression was found for HNF-3β/FOXA2, SOX2, PDX-1/IPF1 and E-cadherin. E-cadherin expression was restricted to the 2 CTCs and 2 cell lines derived from pleural effusion (SCLC26A) and bone metastases (NCI-H526), respectively. Thus, these SCLC CTCs established from extended disease SCLC patients lack expression of stem cell markers which suppress the epithelial phenotype. Instead they express high levels of E-cadherin consistent with a mesenchymal-epithelial transition (MET or EMrT) and form large tumorospheres possibly in response to the selection pressure of first-line chemotherapy. HNF-3β/FOXA2 and PDX-1/IPF1 expression seem to be related to growth factor dependence on insulin/IGF-1 receptors and IGF-binding proteins.

Cancer cell spheroids are used for drug screening as these three-dimensional (3D) assemblies recapitulate tumors more realistic than the widely employed 2D

Sensitivity of the anatase and rutile phases of titanium dioxide to Swift Heavy Ion (SHI) irradiation was experimentally probed and compared with the predictions of the Coulomb explosion, analytical and inelastic thermal spike models of ion-matter interaction. Conforming to the predictions of all these models, our study indicated higher sensitivity of anatase to these ions than the rutile phase. A detailed examination however revealed that Coulomb explosion model cannot explain either the nature of variation of the interaction cross section of SHI with the energy deposited by these ions, Se to the target electrons, or the relative values of the threshold electronic energy loss, Seth of anatase and rutile. The analytical thermal spike (a-TS) model, using the available physicochemical data for this oxide, predicted that tracks cannot form either in anatase or in rutile by 297 MeV and 511 MeV Ni ions, while inelastic thermal spike (i-TS) model predicted formation of ion tracks by 297 MeV Ni ions and their absence with 511 MeV Ni ions in both anatase and rutile. Our observation agreed with the predictions of i-TS model albeit with a difference in the radius of the tracks. In addition, we observed halo of defect ridden crystalline region of much larger radius around the ion track. Interestingly, the radius of the halo scales with the velocity of the ions, which is opposite to the conventionally observed velocity effect.

Modifications induced by 79 MeV Br ions in rutile titanium dioxide thin films, synthesized by dc magnetron sputtering are presented. Irradiations did not induce any new XRD peak corresponding to any other phase. The area and the width of the XRD peaks were considerably affected by irradiation, and peaks shifted to lower angles. But the samples retained their crystallinity at the highest fluence (1 × 10 13 ions cm −2 ) of irradiation even though the electronic energy loss of 79 MeV Br ions far exceeds the reported threshold value for amorphization of rutile TiO 2 . Fitting of the fluence dependence of the XRD peak area to Poisson equation yielded the radius of ion tracks as 2.4 nm. Ion track radius obtained from the simulation based on the thermal spike model matches closely with that obtained from the fluence dependence of the area under XRD peaks. Williamson–Hall analysis of the XRD spectra indicated broadening and shifting of the peaks are a consequence of irradiation induced defect accumulation leading to microstrains, as was also indicated by Raman and UV–Visible absorption study.

Toward developing a model system for investigating the role of the microenvironment in the radioresistance of glioblastoma (GBM), human glioblastoma stem-like cells (GSCs) were grown in coculture with human astrocytes. Using a trans-well assay, survival analyses showed that astrocytes significantly decreased the radiosensitivity of GSCs compared to standard culture conditions. In addition, when irradiated in coculture, the initial level of radiation-induced γH2AX foci in GSCs was reduced and foci dispersal was enhanced suggesting that the presence of astrocytes influenced the induction and repair of DNA double-strand breaks. These data indicate that astrocytes can decrease the radiosensitivity of GSCs in vitro via a paracrine-based mechanism and further support a role for the microenvironment as a determinant of GBM radioresponse. Chemokine profiling of coculture media identified a number of bioactive molecules not present under standard culture conditions. The gene expression profiles of GSCs grown in coculture were significantly different as compared to GSCs grown alone. These analyses were consistent with an astrocyte-mediated modification in GSC phenotype and, moreover, suggested a number of potential targets for GSC radiosensitization that were unique to coculture conditions. Along these lines, STAT3 was activated in GSCs grown with astrocytes; the JAK/STAT3 inhibitor WP1066 enhanced the radiosensitivity of GSCs under coculture conditions and when grown as orthotopic xenografts. Further, this coculture system may also provide an approach for identifying additional targets for GBM radiosensitization.

Small cell lung cancer (SCLC) is a highly malignant disease with dismal prognosis. Although great progress has been made in investigating genetic aberrations and putative drivers of this tumor entity, the mechanisms of rapid dissemination and acquisition of drug resistance are not clear. The majority of SCLC cases are characterized by inactivation of the tumor suppressors p53 and retinoblastoma (Rb) and, therefore, interchangeable drivers will be difficult to target successfully. Access to pure cultures of SCLC circulating tumor cells (CTCs) and study of their tumor biology has revealed a number of new potential targets. Most important, expression of chitinase-3-like-1/YKL-40 (CHI3L1) which controls expression of vascular epithelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP9) was newly described in these cells. The process switching CHI3L1-negative SCLC cells to CHI3L1-positive CTCs seems to be associated with cytokines released by inflammatory immune cells. Furthermore, these CTCs were found to promote monocyte-macrophage differentiation, most likely of the M2 tumor-promoting type, recently described to express PD-1 immune checkpoint antigen in SCLC. In conclusion, dissemination of SCLC seems to be linked to conversion of regular tumor cells to highly invasive CHI3L1-positive CTCs, which are protected by immune system suppression. Besides the classical targets VEGF, MMP-9 and PD-1, CHI3L1 constitutes a new possibly drugable molecule to retard down dissemination of SCLC cells, which may be similarly relevant for glioblastoma and other tumor entities.

Small cell lung cancer (SCLC) has a poor prognosis and is found disseminated at first presentation in the majority of cases. The cell biological mechanisms underlying metastasis and drug resistance are not clear. SCLC is characterized by high numbers of circulating tumor cells (CTCs) and we were able to expand several CTC lines ex vivo and to relate chitinase-3-like-1/YKL-40 (CHI3L1) as marker. Availability of expanded SCLC CTC cells allowed for a screening of receptor tyrosine kinases (RTKs) expressed. The metastatic CHI3L1-negative SCLC cell line SCLC26A, established from a pleural effusion was used for comparison. The CTC cell line BHGc10 was found to exhibit increased expression of RYK, AXL, Tie-1, Dtk, ROR1/2, several ephrins (Eph) and FGF/EGF receptors compared to SCLC26A. All of these RTKs have been associated with cell motility, invasion and poor prognosis in diverse cancer entities without knowledge of their association with CTCs. The identification of RYK, AXL and ROR1/2 as pseudokinases, lacking activity, seems to be related to the observed failure of RTK inhibitors in SCLC. These kinases are involved in the noncanonical WNT pathway and their expression in SCLC CTCs represents a cancer stem cell-like phenotype.

Two sets of gold thin films of thickness of about 20 and 50 nm, grown by thermal evaporation method on (100) silicon wafers were irradiated by 197 MeV Au ions. Grazing incidence X-Ray diffraction (GIXRD) study has been revealed lattice expansion on decreasing the film thickness. 197 MeV Au ion irradiation was not affect either the cubic crystal structure of gold or its lattice parameter. Atomic force microscopy (AFM) study indicated that the evolution of the surface morphology with ion fluence crucially depended on the film thickness, the thinner film being more sensitive than the thicker one. Irradiation led to nanoparticles formation on the surface of the films. This observation is in contrast to the generally perceived damaging role of swift heavy ion (SHI) irradiation. Power spectral density analysis of the roughness along both the lateral and vertical directions demonstrated dominance of surface diffusion over volume diffusion induced by SHI irradiation. A comparison of the sputtering yield obtained from Rutherford back scattering (RBS) spectra of the irradiated films and transmission electron microscopy (TEM) of the particles sputtered from the films and collected by a catcher grid during irradiation indicated that more than the surface and volume diffusion processes, it is the irradiation induced sputtering that controls the overall surface morphology of the films. The surface roughness increase with ion fluence and the irradiation induced sputtering yield was found to be larger in thinner films. Film thickness dependence of the evolution of surface morphology and sputtering yield with 197 MeV Au ion irradiation clearly indicates the dominance of the electronic energy loss over the nuclear energy loss of the projectiles ions in the target medium and opens up the ways for examining the applicability of different models of ion-matter interaction in systems with reduced dimensions. Copyright © 2015 VBRI press.

Analysis of the radiation-induced translatome of glioblastoma stem-like cells (GSCs) identified an interacting network in which XPO1 serves as a major hub protein. To determine whether this nuclear export protein provides a target for radiosensitization, we defined the effects of the clinically relevant XPO1 inhibitor Selinexor on the radiosensitivity of glioblastoma cells. As determined by clonogenic survival analysis, Selinexor enhanced the radiosensitivity of GSCs but not normal fibroblast cell lines. Based on γH2AX foci and neutral comet analyses, Selinexor inhibited the repair of radiation-induced DNA double strand breaks in GSCs suggesting that the Selinexor-induced radiosensitization is mediated by an inhibition of DNA repair. Consistent with a role for XPO1 in the nuclear to cytoplasm export of rRNA, Selinexor reduced 5S and 18S rRNA nuclear export in GSCs, which was accompanied by a decrease in gene translation efficiency, as determined from polysome profiles, as well as in protein synthesis. In contrast, rRNA nuclear export and protein synthesis were not reduced in normal cells treated with Selinexor. Orthotopic xenografts initiated from a GSC line were then used to define the in vivo response to Selinexor and radiation. Treatment of mice bearing orthotopic xenografts with Selinexor decreased tumor translational efficiency as determined from polysome profiles. Although Selinexor treatment alone had no effect on the survival of mice with brain tumors, it significantly enhanced the radiation-induced prolongation of survival. These results indicate that Selinexor enhances the radiosensitivity of glioblastoma cells and suggest that this effect involves a global inhibition of gene translation.

Radiotherapy is a primary treatment modality for glioblastomas (GBM). Because DNA-PKcs is a critical factor in the repair of radiation-induced double strand breaks (DSB), this study evaluated the potential of VX-984, a new DNA-PKcs inhibitor, to enhance the radiosensitivity of GBM cells. Treatment of the established GBM cell line U251 and the GBM stem-like cell (GSC) line NSC11 with VX-984 under in vitro conditions resulted in a concentration-dependent inhibition of radiation-induced DNAPKcs phosphorylation. In a similar concentration-dependent manner, VX-984 treatment enhanced the radiosensitivity of each GBM cell line as defined by clonogenic analysis. As determined by γH2AX expression and neutral comet analyses, VX-984 inhibited the repair of radiation-induced DNA double-strand break in U251 and NSC11 GBM cells, suggesting that the VX-984-induced radio-sensitization is mediated by an inhibition of DNA repair. Extending these results to an in vivo model, treatment of mice with VX-984 inhibited radiation-induced DNA-PKcs phosphorylation in orthotopic brain tumor xenografts, indicating that this compound crosses the blood–brain tumor barrier at sufficient concentrations. For mice bearing U251 or NSC11 brain tumors, VX-984 treatment alone had no significant effect on overall survival; radiation alone increased survival. The survival of mice receiving the combination protocol was significantly increased as compared with control and as compared with radiation alone. These results indicate that VX-984 enhances the radiosensitivity of brain tumor xenografts and suggest that it may be of benefit in the therapeutic management of GBM.