Acquired Radiation Resistance Research Articles

Acquired Radiation Resistance Induces Thiol-dependent Cisplatin Cross-resistance.

Resistance to radiation remains a significant clinical challenge in non-small cell lung carcinoma (NSCLC). It is therefore important to identify the underlying molecular and cellular features that drive acquired resistance. We generated genetically matched NSCLC cell lines to investigate characteristics of acquired resistance. Murine Lewis lung carcinoma (LLC) and human A549 cells acquired an approximate 1.5-2.5-fold increase in radiation resistance as compared to their parental match, which each had unique intrinsic radio-sensitivities. The radiation resistance (RR) was reflected in higher levels of DNA damage and repair marker γH2AX and reduced apoptosis induction after radiation. Morphologically, we found that radiation resistance A549 (A549-RR) cells exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its parental counterpart. Since the N/C ratio is linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 cells had a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double-positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR cells, as compared to A549 cells. Subsequently, we confirmed invivo that the LLC-RR and A549-RR cells are also more resistance to radiation than their isogenic-matched counterpart. Moreover, we found that the acquired radiation resistance also induced resistance to cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevation of thiol levels. Gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) sensitized the resistant cells to cisplatin by decreasing the amount of thiols to levels prior to obtaining acquired radiation resistance. By generating radiation-resistance genetically matched NSCLC we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for combinatorial treatment regimens including glutathione pathway disruptors in patients with the potential of improving clinical outcomes in the future.

Abstract 1088: Acquired radiation resistance induces thiol-dependent cisplatin cross-resistance

Abstract Purpose. Resistance to radiation, both intrinsic and acquired, remains a significant clinical challenge. It is therefore import to identify the underlying molecular and cellular features involved in this resistance. Materials and Methods. We generated genetically-matched counterparts of various degrees of radiation sensitivity in non-small cell lung carcinoma (NSCLC) tumor models to define characteristics of acquired resistance through repeated exposures to conventional X-ray radiation. Results. As assessed by cell viability and clonogenic assays murine Lewis lung carcinoma (LLC) and human A549 cell lines acquired an approximate 1.5 - 2-fold increase in radiation resistance (RR) as compared to its parental counterpart. Morphologically, we found that A549-RR exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its respective counterpart. Since the N/C ratio has been linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 have a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR, as compared to A549. Subsequently, we confirmed that the LLC-RR and A549-RR are also more resistant to radiotherapy than their isogenic-matched counterpart in vivo. Remarkably, we found that the acquired radiation resistance also induced resistance to first-line chemotherapeutic cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevated thiol levels. Decreasing the amount of thiols with buthionine sulfoximine (BSO), a gamma-glutamylcysteine synthetase inhibitor, sensitized the resistant cells to cisplatin, to levels similar as parental cells. Conclusions. By generating genetically-matched radiation resistant NSCLC tumor models we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for multi-modality treatment regimens with the potential of improving clinical outcomes in the future. Citation Format: Samir V. Jenkins, Shruti Shah, Azemat Jamshidi-Parsian, Amir Mortazavi, Gunnar Boysen, Kieng B. Vang, Robert J. Griffin, Narasimhan Rajaram, Ruud P. Dings. Acquired radiation resistance induces thiol-dependent cisplatin cross-resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1088.

An in vivo model of glioblastoma radiation resistance identifies long noncoding RNAs and targetable kinases.

Key molecular regulators of acquired radiation resistance in recurrent glioblastoma (GBM) are largely unknown, with a dearth of accurate preclinical models. To address this, we generated 8 GBM patient-derived xenograft (PDX) models of acquired radiation therapy–selected (RTS) resistance compared with same-patient, treatment-naive (radiation-sensitive, unselected; RTU) PDXs. These likely unique models mimic the longitudinal evolution of patient recurrent tumors following serial radiation therapy. Indeed, while whole-exome sequencing showed retention of major genomic alterations in the RTS lines, we did detect a chromosome 12q14 amplification that was associated with clinical GBM recurrence in 2 RTS models. A potentially novel bioinformatics pipeline was applied to analyze phenotypic, transcriptomic, and kinomic alterations, which identified long noncoding RNAs (lncRNAs) and targetable, PDX-specific kinases. We observed differential transcriptional enrichment of DNA damage repair pathways in our RTS models, which correlated with several lncRNAs. Global kinomic profiling separated RTU and RTS models, but pairwise analyses indicated that there are multiple molecular routes to acquired radiation resistance. RTS model–specific kinases were identified and targeted with clinically relevant small molecule inhibitors. This cohort of in vivo RTS patient-derived models will enable future preclinical therapeutic testing to help overcome the treatment resistance seen in patients with GBM.

CD146 interaction with integrin β1 activates LATS1-YAP signaling and induces radiation-resistance in breast cancer cells

Radiotherapy is an indispensable modality in comprehensive treatment of breast cancer. However, inherent or acquired radiation resistance of tumors compromises the efficacy of radiotherapy. Herein, we found that CD146, a unique epithelial-to-mesenchymal transition (EMT) inducer particularly highly expressed in triple-negative breast cancer (TNBC), is dramatically induced by ionizing irradiation. Further study demonstrates that CD146 promotes tumor cell radioresistance in vitro and in vivo. Specifically, we report the underlying mechanism that CD146 activates YAP protein, and drives its relocation from plasma to nucleus by regulating LATS1, and promoting abnormal DNA damage repair, as well as inducing EMT and stemness. Moreover, CD146 can form a novel co-receptor complex with integrin β1 and induces radiation-resistance in breast cancer. Dual inhibition of CD146 and integrin β1 activity had a stronger inhibitory effect on breast cancer tumor growth and synergistically increased their sensitivity to radiotherapy. This study identifies a unique function of CD146 implicates with integrin β1 and YAP signaling, contributing to radiation resistance. Targeted therapy against CD146 or inhibition of integrin β1 is a potential strategy to overcome radiotherapeutic resistance of breast cancer.

Abstract 5429: Therapeutic targeting of oncogenic long non-coding RNAs utilizing antisense oligonucleotides

Abstract Non-coding RNAs (ncRNAs) are emerging as potent regulators of biological process including human diseases such as cancer. Long non-coding RNAs (lncRNAs) are a dynamic and versatile class of ncRNAs for which there is extensive evidence of these transcripts modulating epigenetic programs to affect cellular phenotypes. Our initial studies using a model of glioblastoma (GBM) tumor recurrence has revealed a number of oncogenic lncRNAs associated with therapy resistance and/or tumor recurrence. We have validated the expression of these oncogenic lncRNAs in another cancer, pediatric rhabdomyosarcoma (RMS). There have been few effective therapeutic advances made in past decades for either RMS or GBM. Radiation therapy is commonly employed alongside surgery and standard chemotherapy, especially in the case of unresectable disease, advanced disease, or upon tumor recurrence. Tumors may inherently possess or acquire resistance to radiation therapy and other treatment modalities over the course of disease. Tumor therapy resistance and recurrence are mediated by complex epigenetic programs which lead to increased capacity for DNA damage repair or tolerance of double stranded breaks. LncRNAs may regulate mechanisms of tumor recurrence and therapy resistance across multiple cancer types. The functional roles of ZFAS1 modulating epithelial to mesenchymal transition in colon cancers and SAMMSON which drives aberrant mitochondrial function in melanoma have been described. CASC19/PCAT2, ZFAS1, DUXAP9, and SOX2-OT are overexpressed in multiple cancers with CASC19 having a significant negative association with GBM patient survival. In silico analysis identifies DNA-binding capacities harbored by these lncRNA transcripts, and their predicted binding sites are proximal to several cancer hallmark genes. We have verified the expression of isoforms of these lncRNA transcripts that contain DNA-binding domains through sequencing and qPCR. We hypothesize that these lncRNAs regulate transcriptional networks via recruitment of transcriptional or epigenetic machinery to genomic loci to modulate oncogenic programs and/or tumor suppressor pathways. As potent epigenetic regulators, lncRNAs present an attractive class of therapeutic targets that could complement standard treatments to reduce disease burden and improve patient survival. It is possible that acquired radiation resistance mediated by lncRNAs is conserved across multiple malignancies. We are developing chemically modified, novel antisense oligonucleotides to target these transcripts in GBM and RMS patient-derived xenografts. Using these systems, we hope to reveal novel associations with disease progression and oncogenic mechanisms that could be exploited through the direct targeting of lncRNAs. We hope our work will lead to the development of novel clinical agents to treat patients with RMS, GBM, and potentially other intractable cancers. Citation Format: Christian Tyler Stackhouse, Corinne Linardic, Malaika Kimmons, Samantha Weitzel. Therapeutic targeting of oncogenic long non-coding RNAs utilizing antisense oligonucleotides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5429.

Multi-omic Exploration of Inherent and Acquired Radiation Resistance of Glioblastoma Patient-derived Xenografts

Glioblastoma (GBM), the most common primary brain malignancy, has a very poor prognosis despite decades of research. Current treatment with maximal safe resection, concurrent and adjuvant temozolomide (TMZ), and radiation treatment (XRT) with tumor-treating fields have provided marginal improvement in outcomes, yet median survival is only 15 months. Our group believes that inadequate preclinical models have hampered translational efforts for GBM. We posit that patient-derived xenografts (PDX) are superior to traditional immortalized glioma lines and can be an important tool for exploring mechanisms of therapy resistance. We screened a large GBM PDX panel to identify those with inherent XRT sensitivity, then produced the largest cohort of acquired XRT resistant PDX lines through serial in vivo XRT selection. PDX were profiled at the transcriptome and kinome levels. GBM PDX were propagated in athymic nude mice in vivo and screened for XRT sensitivity. Acquired XRT resistance was produced through serial flank XRT of sensitive tumors with serial passaging. GBM PDX were transcriptionally profiled by RNA-Seq and global kinase activity (kinomic) profiling using a microarray processor. Informatics tools include Differential Gene Expression; Differential Gene Correlation Analysis; BEDtools; Weighted Correlation Network Analysis; Fast Exponential Monte Carlo; Pathway, Annotated-list, and Gene-signature Electronic Repository; Biomedical Entity Expansion, Ranking, and Exploration; GeneOntology and GeneTerrain visualization; and using the long non-coding RNA (lncRNA) tools Triplexator and ASSA. 20 PDX were screened for baseline XRT sensitivity by measuring tumor doubling time (TDT) following flank XRT (2 Gy x 6 over 2 weeks) and PDX were deemed XRT sensitive if TDT>20 days. Of the 10 inherently XRT sensitive PDX, 8 lines were rendered XRT resistant through serial flank irradiation (2 Gy x 6 over 2 weeks) repeated during up to 6 in vivo passages. Median parental line TDT was 35 days versus 5 days for the XRT resistant lines (p<0.0001, Gehan-Breslow-Wilcoxon test). Differential RNA-Seq revealed that acquired XRT resistance was associated with a number of altered lncRNA transcripts that were mapped to corresponding gene expression targets with high correlation. Interestingly, many of the lncRNA transcripts were decreased in the XRT resistant setting. Similarly, acquired XRT resistance promoted a global reduction in kinomic activity as compared to parental and inherently resistant tumors, though JAK, FGFR, and Ephrin family kinases were increased. Interactive bioinformatic visualization tools allow for exploration of these data sets. This robust cohort of inherent and acquired XRT resistant GBM PDX provide a suitable preclinical model for novel therapeutic testing.

Abstract 279: Long non-coding RNAs in glioblastoma tumor recurrence and therapy resistance

Abstract Glioblastoma multiforme (GBM) is a devastating disease which invariably recurs and is often resistant to standard therapies. GBM patient-derived xenoline (PDX) models of tumor recurrence and radiation resistance were created using serial in vivo selection against radiation therapy (6 × 2Gy fractions over 2 weeks for 6+ rounds). This produced 8 isogenic pairs of patient matched radiation-sensitive, primary GBM PDX to radiation-resistant, recurrent GBM PDX. Long non-coding RNAs (lncRNA) represent novel regulatory mechanisms for therapy resistance and tumor recurrence in GBM. An in silico informatics pipeline employing differential expression, differential gene correlation, machine learning, and semantic networking was devised to identify coding and non-coding transcripts related to radiation resistance from deep total RNA sequencing. This analysis revealed 269 lncRNA and 947 coding transcripts that are associated with adaptive radiation resistance. LncRNA:RNA and lncRNA:DNA interaction prediction software was employed to further uncover mechanisms of epigenetic regulation by lncRNAs. A subset of the lncRNA transcripts were predicted to interact directly with other coding transcripts while other lncRNAs have predicted interactions with DNA in regulatory regions of the human genome. We are further validating lncRNA:DNA interactions through in silico methods to determine if genes proximal to these interaction sites are related to tumorigenesis or therapy response. Semantic linkages have been predicted from references in the current literature for lncRNAs and key molecular processes such as DNA damage response, extracellular matrix, and chromatin remodeling. Modules of highly correlated genes, including lncRNAs, have been identified that are differentially regulated between radiation sensitive and resistant tumors. Cross-validation between our in silico approaches have confirmed high-confidence associations of a subset of lncRNAs with acquired radiation resistance. Subsequently, we will validate the expression and phenotypic relevance of high-confidence transcripts in our cohort of PDX models derived from recurrent GBM tumors. In conclusion, recurrence of therapy resistant GBM is responsible for patient mortality. Not all patients qualify for surgical resection or for chemotherapy, but radiation is almost a universally tolerated therapy. We have generated a novel model of tumor recurrence and radiation resistance that recapitulates recurrent tumor physiology. We have discovered 269 lncRNAs associated with radiation resistance that suggests potential regulatory mechanisms through nucleic acid binding. Evidence suggests that lncRNAs most likely contribute to acquired resistance through epigenetic regulation of transcription and chromatin state. These transcripts may provide novel, druggable targets for treating therapy resistant, recurrent GBM. Citation Format: Christian Tyler Stackhouse, James R. Rowland, Jelai Wang, Thanh Nguyen, Zongliang Yue, Jake Y. Chen, Lara Ianov, G. Yancey Gillespie, Christopher D. Willey. Long non-coding RNAs in glioblastoma tumor recurrence and therapy resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 279.

Novel role of PAF1 in attenuating radiosensitivity in cervical cancer by inhibiting IER5 transcription

BackgroundRadiosensitivity is limited in cervical cancer (CC) patients due to acquired radiation resistance. In our previous studies, we found that immediate-early response 5 (IER5) is upregulated in CC cells upon radiation exposure and decreases cell survival by promoting apoptosis. The details on the transcriptional regulation of radiation-induced IER5 expression are unknown. Studies in recent years have suggested that Pol II-associated factor 1 (PAF1) is a pivotal transcription factor for certain genes “induced” during tumor progression. In this study, we investigated the role of PAF1 in regulating IER5 expression during CC radiotherapy.MethodsPAF1 expression in CC cells was measured by western blotting, immunohistochemistry, and qRT-PCR, and the localization of PAF1 and IER5 was determined by immunofluorescence. The effect of PAF1 and IER5 knockdown by siRNA in Siha and Hela cells was studied by western blotting, qRT-PCR, CCK-8 assay, and flow cytometry. The physical interaction of PAF1 with the IER5 promoter and enhancers was confirmed using chromatin immunoprecipitation and qPCR with or without enhancers knockout by CRISPR/Cas9.ResultsWe confirmed that PAF1 was highly expressed in CC cells and that relatively low expression of IER5 was observed in cells with highly expressed PAF1 in the nucleus. PAF1 knockdown in Siha and Hela cells was associated with increased expression of IER5, reduced cell viability and higher apoptosis rate in response to radiation exposure, while simultaneous PAF1 and IER5 knockdown had little effect on the proportion of apoptotic cells. We also found that PAF1 hindered the transcription of IER5 by promoting Pol II pausing at the promoter-proximal region, which was primarily due to the binding of PAF1 at the enhancers.ConclusionsPAF1 reduces CC radiosensitivity by inhibiting IER5 transcription, at least in part by regulating its enhancers. PAF1 might be a potential therapeutic target for overcoming radiation resistance in CC patients.

Radiation-induced extracellular vesicle (EV) release of miR-603 promotes IGF1-mediated stem cell state in glioblastomas

BackgroundRecurrence after radiation therapy is nearly universal for glioblastomas, the most common form of adult brain cancer. The study aims to define clinically pertinent mechanisms underlying this recurrence. MethodsmicroRNA (miRNA) profiling was performed using matched pre- and post-radiation treatment glioblastoma specimens from the same patients. All specimens harbored unmethylated O6-methylguanine-DNA methyltransferase promoters (umMGMT) and wild-type isocitrate dehydrogenase (wtIDH). The most altered miRNA, miR-603, was characterized. FindingsWhile nearly all miRNAs remained unchanged after treatment, decreased levels of few, select miRNAs in the post-treatment specimens were observed, the most notable of which involved miR-603. Unbiased profiling of miR-603 targets revealed insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R). Ionizing radiation (IR) induced cellular export of miR-603 through extracellular vesicle (EV) release, thereby de-repressing IGF1 and IGF1R. This de-repression, in turn, promoted cancer stem-cell (CSC) state and acquired radiation resistance in glioblastomas. Export of miR-603 additionally de-repressed MGMT, a DNA repair protein responsible for detoxifying DNA alkylating agents, to promote cross-resistance to these agents. Ectopic miR-603 expression overwhelmed cellular capacity for miR-603 export and synergized with the tumoricidal effects of IR and DNA alkylating agents. InterpretationProfiling of matched pre- and post-treatment glioblastoma specimens revealed altered homeostasis of select miRNAs in response to radiation. Radiation-induced EV export of miR-603 simultaneously promoted the CSC state and up-regulated DNA repair to promote acquired resistance. These effects were abolished by exogenous miR-603 expression, suggesting potential for clinical translation. FundingNIH 1R01NS097649-01, 9R44GM128223-02, 1R01CA240953-01, the Doris Duke Charitable Foundation Clinical Scientist Development Award, The Sontag Foundation Distinguished Scientist Award, the Kimmel Scholar Award, and BWF 1006774.01 (C.C.C).

DRES-04. LONG NON-CODING RNAS IN GLIOBLASTOMA TUMOR RECURRENCE AND THERAPY RESISTANCE

Abstract Recurrence of therapy resistant Glioblastoma multiforme (GBM) is responsible for patient mortality. Not all patients qualify for surgical resection or for chemotherapy, but radiation is almost a universally tolerated therapy. We are modeling acquired radiation resistance using 8 radiation-sensitive GBM patient-derived xenolines (PDX) made resistant by six irradiation series (6x2Gy each) in vivo. In 4 resistance-induced PDX, MGMT (O6-methylguanine–DNA methyltransferase) protein expression increased over original isogenic PDX. Paradoxically, temozolomide screening of orthotopic radiation-resistant PDX with increased MGMT expression revealed increased, not decreased chemo-sensitivity. This suggests an unanticipated mechanism associating acquired radiation resistance and alkylating chemotherapy sensitivity. RNA-seq data of long non-coding RNAs (lncRNAs) has revealed associations with patient overall survival and age at diagnosis. Out of 24,076 lncRNAs, 5 are significantly differentially expressed with regard to patient overall survival and age at diagnosis. The functions of lnc-ZNF117-1, lnc-DCUN1D4-1, and LINC01397 are unknown, but they are enriched in brain over other tissues. Tissue specific expression and function are unknown for lnc-TBL1XR1-5, but it is highly expressed in HepG2 (hepatocellular carcinoma) as well as in non-functioning pituitary adenomas (NFPAs). The lnc-CDH17-1 transcript is also highly expressed in NFPAs. All 5 of these lncRNAs have complex secondary and tertiary structures, but their physiologic or pathologic functions are unknown. LncRNAs most likely contribute to acquired resistance through epigenetic regulation of transcription and chromatin state. Deep sequencing of total RNA isolated from intracranial radiation–sensitive/–resistant PDX to relate transcriptional programs and therapy resistance is underway. These data will be paired with kinomic profiling of matched tumors to elucidate basal signaling modality changes between radiation–sensitive/–resistant tumors. Analysis of differentially expressed lncRNA will be used to predict lncRNA structure/function, elucidating druggable mechanisms mediated by lncRNAs.

PO-115 Radiation resistance mechanisms of breast cancer cells

IntroductionDespite the high efficacy of Radiation therapy (RT) to treat breast carcinoma (BC), the problem of radioresistance and tumour recurrence is still not solved. It is believed that radioresistance can be associated with the presence of a subpopulation of cells, called ‘cancer stem cells’ (CSCs). However, it is still unclear which molecular profiling of BC cells should be used as a predictor of poor clinical outcome after RT. Therefore, the main aim of this study was to determine the molecular phenotype of BC cells with acquired radiation resistance and stemness properties.Material and methodsTwo carcinoma cell lines, MDA-MB-231 and T47D, representing the most common molecular subtypes of breast cancer (triple negative and luminal A) were repetitively irradiated at a total dose of 100 Gy, and finally, radioresistant (RR) BC cell lines were obtained. To confirm the radioresistant phenotype of the newly received RR cells, their radiation sensitivities were compared with those in their parental counterparts using a clonogenic survival assay. FACS analysis was performed to detect the content of BC stem cell markers CD44+/CD24-. Additionally, a sphere forming assay was performed to detect the stemness abilities of the investigated breast carcinoma cells. Parental and RR cells were examined for their protein patterns using nano-LC mass spectrometry followed by PathwayStudio-based computational analysis. Western Blot, FACS, and metabolic patterns of parental and RR cells were performed to prove the proteomic results.Results and discussionsNewly obtained RR breast carcinoma cell lines possess a prominent radiation resistance accompanied by enhanced sphere forming abilities and increased number of CD44+/CD24- cells in comparison with parental cells. Proteome analysis has shown an up-regulation of proteins involved in different molecular pathways protecting carcinoma cells from ionising radiation. Molecular pathways regulating DNA repair processes, cell cycle distribution and cell death development, metabolic activities, cell differentiation/dedifferentiation, have demonstrated the most pronounced connectivities with radiation resistance.ConclusionRadioresistant BC cells demonstrate their aggressive behaviour through changed metabolic properties and activation of pro-survival mechanisms associated with affected therapy response.

Abstract B05: Ecto-5’-nucleotidase (CD73) confers radioresistance in pancreatic cancer

Abstract Pancreatic cancer is an exceptional aggressive cancer, and acquired radiation resistance in this type of cancer is common. The objective of this study was to use a comparative proteomic method to identify the proteins that are responsible for acquired radioresistance in pancreatic cancer cells. In order to eliminate the complications caused by genetic background variations, we first generated a pancreatic cancer isogenic stable cell line that differs in radioresistance. The isogenic stable cell line was generated by exposing the pancreatic cancer MIA PaCa-2 cells (an undifferentiated pancreatic cancer cell line) to a total dose of 60 Gy of ionizing radiation (IR; gamma radiation) in 30 fractions over a period of 6 weeks, a protocol that is commonly used in clinical radiotherapy. The clones that survived from the fractionated exposures showed strong radioresistance, which could be demonstrated by both cell apoptotic assays and the long-term clonogenic survival assays. We then used a mass spectrometry-based quantitative proteomic method to systematically compare the expression of proteins between the radiosensitive parental cells and the selected radioresistant cells. One protein identified to be substantially upregulated in the radioresistant cells was ecto-5’-nucleotidase (CD73), a cell surface enzyme that converts adenosine monophosphate (AMP) into adenosine and is known to play important roles in cell-cell/cell-matrix interactions and immune suppression. To examine whether the upregulated CD73 was functionally related to the acquired radioresistance of pancreatic cancer cells, we silenced or ectopically overexpressed CD73 gene in the cells and examined the effect of the expression manipulations on long-term survival and apoptosis of the cells in response to IR exposure. The results demonstrated that knockdown of CD73 in the radioresistant cells significantly decreased the long-term survival rates, and resensitized the cells to apoptosis after the cells were exposed to IR. Concomitantly, stable overexpression of CD73 in the radiosensitive parental cells significantly increased the long-term survival rates, and enhanced resistance of the cells to apoptosis upon exposure of the cells to IR. To determine if the enzyme activity of CD73 is important for its effect on radioresistance, we generated an enzyme-dead mutant CD73 and overexpressed the mutant CD73 in the parental cells. Functional assays demonstrated that the mutant CD73 affected radioresistance of pancreatic cancer cells in a similar way as wild-type CD73, suggesting that the enzyme activity is not related to its effect on radioresistance. In summary, our results suggest that upregulation of CD73 expression is an important mechanism by which pancreatic cancer cells acquire resistance to IR, and the effect of CD73 on radioresistance in pancreatic cancer cells is enzyme-independent and at least partially through affecting the sensitivity of the cells to IR-induced apoptosis. Citation Format: Anna M. Nguyen, Jianhong Zhou, Yuchun Du.{Authors}. Ecto-5’-nucleotidase (CD73) confers radioresistance in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B05.

Transcriptional regulation of IER5 in response to radiation in HepG2.

Radiotherapy is one of the important treatments for patients with hepatocellular carcinoma. The treatment response (or efficacy), however, is limited in many patients due to acquired radiation resistance of cancer cells. Immediate-early response 5 (IER5) is one of the genes upregulated on radiation. The gene could modulate cell cycle checkpoint, leading to a decrease of cancer cell survival in response to radiation. To better understand how IRE5 expression is regulated on radiation, this study aims to identify transcription factors that interact with IER5 promoter region in liver cancer cell line. Using bioinformatic tool, we identified promoter region of IER5 gene. Subsequent luciferase reporter assay revealed two putative GC binding factor (GCF) binding sites. We found mutations of these binding sites increased the luciferase activity, suggesting a negative regulation of GCF on IER5 transcriptional activity. The physical interaction of GCF with the gene promoter was confirmed using chromatin immunoprecipitation and electrophoretic mobility shift assay assays. Different doses of radiation were also applied in these experiments, and we found the formation of protein-DNA complex reduced with the increasing dose of radiation. Together, we propose the GCF regulated transcriptional activity, at least in part, contributed to the upregulation of IER5 on radiation. The present findings provide insights into understanding the regulatory mechanisms of IER5.

Abstract 3243: Notch inhibitors and chemoradiation in an orthotopic glioblastoma model

Abstract Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults. Aggressive multimodal treatment using surgery followed by radiotherapy and chemotherapy extends the median survival of GBM patients to approximately one year after diagnosis. Treatment is not curative because of the intrinsic and acquired radiation resistance of a subpopulation of tumor cells. Notch inhibition has been shown to impair the tumorigenic capacity of these cells as well as enhance their sensitivity towards radiation. Therefore, we investigated if a highly potent and clinically approved Notch pathway inhibitor (g-secretase inhibitor) improves tumor control when combined with radiotherapy and chemotherapy in an orthotopic GBM mouse model. To investigate the treatment efficacy of combinations treatments between standard of care treatment (radiotherapy and temozolomide) and Notch inhibitors, we used a three dimensional spheroid growth assay using both established and primary human glioma cell lines in which spheroid volume growth delay was quantitatively monitored. In addition, we assessed the expression of the putative glioma stem cell marker CD133 using flow cytometry. Furthermore, therapeutic efficacy of these combination treatments was als assessed in vivo in an orthotopic glioma tumor model (U87-luc) wherein tumor progression was evaluated using bioluminescence (BLI) and contrast-enhanced micro-computed tomography (CT) imaging. A small animal precision irradiation platform (PXI, XRAD 225Cx, CT, USA) was used for micro-CT imaging and irradiation delivery of conformal doses between 2- 10 Gy to intracranial tumors in mice receiving single or combination treatments with Notch inhibitors and chemotherapy. Results: Notch blockade alone did not affect the sphere volume compared to control, whereas combination treatment with radiation and / or temozolomide resulted in a substantial spheroid growth delay (p = 0.004). Irradiation enhanced the expression of the stem cell marker CD133, while Notch blockade reduced CD133 expression. In intracranial tumours we found a strong correlation between CT and BLI imaging of tumor growth (Pearson coefficient (r) = 0.85, p = 0.001). The potential of Notch inhibition combined with precision radiotherapy and chemotherapy in our orthotopic GBM model is currently being determined and the outcome of this study will be presented. Citation Format: Sanaz Yahyanejad, Patrick Granton, Stefan van Hoof, Lydie Barbeau, Jan Theys, Frank Verhaegen, Marc Vooijs. Notch inhibitors and chemoradiation in an orthotopic glioblastoma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3243. doi:10.1158/1538-7445.AM2015-3243

Abstract B72: Serine/threonine kinase MLK4 is a master regulator for proneural-mesenchymal transformation of glioma stem cells

Abstract High-grade glioma is a highly aggressive form of brain cancer associated with a poor prognosis. Recently, we identified that mesenchymal glioma stem cells (Mes GSCs) of HGG are linked to radiation resistance and proneural (PN) GSCs acquire Mes identity via radiation treatment. Here we performed a combined kinome-wide RNA interference screen with genome wide expression analysis to identify kinase-encoding genes that are essential for mesenchymal identity of GSCs. Through this stepwise screening with patient-derived GSC samples, we identified the serine/threonine kinase MLK4 as a key regulator for Mes GSC survival and cell cycle progression in vitro and tumor initiation and propagation in vivo. In addition, overexpression of MLK4 promotes, while both its gene-specific shRNA and pharmacological inhibition attenuate, proneural-mesenchymal transformation (PMT) of GSCs. In patient-derived GSCs, expression of MLK4 protein positively correlated with endogenous EGFRvIII expression and the in vivo growth of mouse xenografts derived from glioma cells with EGFRvIII expression was prominently inhibited by MLK4 knockdown. MLK4-overexpressing PN GSCs acquired radiation resistance, while MLK4 elimination by shRNA radiosensitized Mes GSCs. Patients with a higher MLK4 expression showed reduced survival in CD44-high populations. Collectively, these data indicate that MLK4 as a plausible target for therapy against Mes GBM as well as post-irradiation PN GBM. Citation Format: Kim Sunghak, Rosenstiel-Goidts Violaine, Shi-Yuan Cheng, Nakano Ichiro. Serine/threonine kinase MLK4 is a master regulator for proneural-mesenchymal transformation of glioma stem cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B72. doi:10.1158/1538-7445.CHTME14-B72

Nanoparticles in radiation therapy: a summary of various approaches to enhance radiosensitization in cancer

Acquired radiation resistance is one of the major causes of radio therapy failure and subsequent tumor relapse. Multiple approaches have been utilized to limit the radiation resistance while simultaneously enhancing the efficacy and safety of radiation therapy. The three major approaches for the improvement of radiation therapy have involved (I) ehancing radiosensitization of tumor tissue; (II) rversing of radiation resistance in tumor tissue; and (III) ehancing radioresistance of the healthy tissue. Nanoparticles have played a key role in the enhancement of the radiation therapy by acting both as a therapeutic as well as a carrier for other therapeutics. In this review we summarize the research being carried out using different species of nanoparticles for enhanced radiosensitization in cancer.

DNA-dependent protein kinase catalytic subunit inhibitor reverses acquired radioresistance in lung adenocarcinoma by suppressing DNA repair.

The mechanisms underlying lung cancer radioresistance remain to be fully elucidated. The DNA repair pathway is a predominant target of radiotherapy, which is considered to be involved in the acquired radioresistance of cancer cells. The present study aimed to establish a radioresistant cell model using the A549 human lung cancer cell line, and to further investigate the potential mechanisms underlying the radioresistance. The A549R radioresistant lung cancer cell variant was established by exposing the parental A549 cells to repeated γ-ray irradiation at a total dose of 60 Gy. Colony formation assays were then used to determine cell survival following γ-ray exposure. The established radioresistant cells were subsequently treated with or without the NU7026 DNA-PKcs inhibitor. The levels of DNA damage were determined by counting the number of fluorescent γ-H2AX foci in the cells. The cellular capacity for DNA repair was assessed using antibodies for the detection of various DNA repair pathway proteins. The radioresistant sub-clones exhibited significantly decreased survival following NU7026 treatment, compared with the parental cells, as determined by colony formation assays (P<0.05), and this finding was found to be dose-dependent. Treatment with the DNA-dependent protein kinase (DNA-PK) inhibitor significantly reduced γ-H2AX foci formation (P<0.05) following acute radiation exposure in the radioresistant sub-clones, compared with the parental control cells. The decreased levels of γ-H2AX were accompanied by an increase in the percentage of apoptotic cells in the radioresistant cell line following post-radiation treatment with the DNA-PKcs inhibitor. The expression levels of proteins associated with the DNA repair pathway were altered markedly in the cells treated with NU7026. The results of the present study suggested that radioresistance may be associated with enhanced DNA repair following exposure to radiation, resulting in reduced apoptosis. Therefore, the quantity of γ-H2AX determines the radioresistance of cells. The DNA repair pathway is important in mediating radioresistance, and treatment with the DNA-PKcs inhibitor, NU7026 restored the acquired radiation resistance.

S1P lyase regulates DNA damage responses in vitro and in vivo

The injurious consequences of radiation to normal human cells and the acquired radiation resistance of cancer cells represent limitations to cancer radiotherapy. Radiation induces DNA damage response pathways that orchestrate cell cycle arrest, DNA repair and apoptosis such that irradiated cells are either repaired or eliminated. Concomitantly, radiation activates acid sphingomyelinase (ASMase), which generates ceramide, thereby promoting apoptosis. However, ceramide can also be metabolized to sphingosine-1-phosphate (S1P), which acts paradoxically as a radioprotectant. Thus, sphingolipid metabolism represents a radiosensitivity pivot point, a notion supported by genetic evidence in radiation resistant cancer cells. S1P lyase (SPL) catalyzes degradation of S1P in the final step of sphingolipid metabolism. We show that by reducing S1P and enhancing ASMase-mediated ceramide generation, SPL modulates the kinetics of DNA repair, the speed of recovery from G2 cell cycle arrest, and the extent of apoptosis after irradiation. In contrast, inhibiting SPL in vitro enhances DNA repair, and oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body irradiation. Our findings reveal SPL to be a regulator of ASMase, the G2 checkpoint and DNA repair and a novel target for radioprotection.

Blockade of NF-κB activation by IκBα gene therapy enhances radiation sensitivity and abolishes acquired resistance to radiation

Radiation is one of the main treatment modalities in lung cancer, but low sensitivity and acquired resistance of lung cancer cells to radiation frequently result in treatment failure. The activation of nuclear factor (NF)-κB is reportedly the main mechanism by which cancer cells exhibit resistance to external stresses, such as chemotherapy or radiation therapy. In this study, we blocked the activation of NF-κB by adenovirus-expressing IκBα-SR and investigated the effect this had on radiation sensitivity. Transduction with ad-IκBα effectively blocked the activation of NF-κB by radiation in all the cancer cell lines tested, except for NCI H460. Clonogenic assay after radiation demonstrated that ad-IκBα transduction enhanced the sensitivity to radiation of the lung cancer cell lines and HeLa cells, except for NCI H460. The radiosensitizing effect of IκBα was more potent in lung cancer cell lines with radioresistance (SKMESres) (sensitizer enhancement ratio 1:61). From these findings, NF-κB blockade by ad-IκBα enhanced the radiation sensitivity and also abolished the acquired radiation resistance of lung cancer cell lines. This study provides a therapeutic rationale for combining an NF-κB-blocking strategy with radiation to both increase sensitivity and overcome acquired resistance to radiation.

In vivo radiosensitizing effects of recombinant interleukin 6 on radiation resistant BCL-1 B-lineage leukemia cells in a murine syngeneic bone marrow transplant model system.

The ability of total body irradiation (TBI) to eradicate clonogenic leukemia cells from B-lineage acute lymphoblastic leukemia patients prior to bone marrow transplantation (BMT) is greatly hampered by their inherent or acquired radiation resistance. The radiorefractory nature of these cells is believed to contribute to the high relapse rate subsequent to TBI and BMT in patients with B-lineage acute lymphoblastic leukemia (ALL). A method by which clonogenic leukemia cells could be radiosensitized in vivo could be clinically beneficial. In the present study, we used a highly radiation resistant subclone of the murine B-lineage leukemia cell line BCL-1 in a syngeneic BMT model to investigate if any of the B-cell stimulatory cytokines interleukin 2, interleukin 4, interleukin 5, or interleukin 6 could have radiosensitizing effects. All untreated BALB/c mice (N = 33) inoculated with 1 x 10(6) BCL-1 cells died of disseminated leukemia within 24 days with a median survival of 13.3 days. TBI (700 cGy = LD100/30 for BALB/c mice) followed by syngeneic BMT (N = 70) extended the median survival to 23.6 days (P < 0.001 by log-rank test). A single intraperitoneal bolus injection of 100 ng, 500 ng, or 2500 ng recombinant murine interleukin 6(rmIL-6) 2-4 hours before TBI extended the median survival to 32.5 days, 31.0 days, and 30.5 days, respectively (P < 0.01 by log-rank test for all dose groups). The improved survival was not due to any direct anti-leukemic activity of rmIL-6 and all control BALB/c mice (N = 15) that received the same doses of rmIL-6 but did not undergo TBI and BMT died of BCL-1 leukemia within 28 days with a median survival of 13.6 days. In contrast to rmIL-6, recombinant murine interleukin 5 (rmIL-5) had minimal radiosensitizing effects.(ABSTRACT TRUNCATED AT 250 WORDS)