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CAFs-Secreted CXCL1 Mediated Radioresistance by Regulating DNA Damage Response in an ROS-Dependent Manner in Esophageal Squamous Cell Carcinoma
ePoster Sessions S239
Volume 96 Number 2S Supplement 2016 Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, western blot analysis, Immunohistochemistry, caspases assay, MRI, and DNA fragmentation assay. Results: As expected, high dose radiation induced activation of MAPKs, ERK, p38, and JNK, in both the in vitro and in vivo models. This activation occurred via caspase-3 independent pathway and was mediated by calpain, cathepsin B/D, and apoptosis-inducing factor. Our results also demonstrated that AIF promotes chromatinolysis and necrosis by interacting with histone H2AX in RIP3+ cells. In cells provided with RIP1/RIP3 kinase-specific inhibitors, it was discovered that radiation-induced inflammatory responses (NF-kB, STAT-3, and HIF-1) were suppressed. In addition, the programmed necrosis process was disrupted. Reduced activations of MAPKs, ERK, p38, and JNK were observed, indicating that RIP3 regulates programmed necrosis via these pathways following high-dose radiation. Conclusion: RIP3 plays an essential role in the radiation-induced necrosis process via activation of MAPKs, ERK, p38, and JNK. RIP3 inhibitors can be used to modulate radiation-induced inflammatory response and to potentially control the radiation-induced necrosis process. In the future, insight into the precise role RIP3 plays in these processes may allow us the ability to differentiate necrosis from tumor progression and even harness its power for therapeutic effect. Author Disclosure: D.G. McDonald: None. D. Jacqmin: None. W.A. Vandergrift: None. S. Lindhorst: None. D. Cachia: None. V.K. Abhay: None. K.N. Vanek: None. N.L. Banik: None. J.M. Jenrette: None. P. Giglio: None. S.J. Patel: None. A. Das: None.
1178 CAFs-Secreted CXCL1 Mediated Radioresistance by Regulating DNA Damage Response in an ROS-Dependent Manner in Esophageal Squamous Cell Carcinoma H. Zhang, J. Yue, Z. Jiang, H. Wu, X. Zhang, L. Yang, Y. Li, S. Li, Q. Hou, Y. Wang, and S. Wu; Hangzhou Cancer Hospital, Hangzhou, China Purpose/Objective(s): Esophageal squamous cell carcinoma (ESCC) is a prevalent cancer with high morbidity and mortality worldwide. Radiotherapy is one major curative treatment modality for ESCC. Herein, our study aimed to investigate whether CAFs (cancer-associated fibroblasts), a main component of tumor microenvironment, were involved in mediating the development of radioresistance and further explored the mechanisms underlying CAFs-mediated radioresistance in ESCC. Materials/Methods: By primary culture, two pairs of CAFs and matched NFs (normal fibroblasts) had been successfully isolated from tumor tissues of ESCC patients and matched normal esophageal epithelial tissues, respectively. The secretion of chemokines by CAFs and matched NFs was determined by the human chemokine antibody array. The concentrations of chemokine in culture medium of CAFs, matched NFs and ESCC cells were determined by Elisa assay. The effect of CAFs-secreted chemokines on radioresistance was investigated in vitro by clonogenic survival assay, detection of DNA doublestrand breaks (DSBs) and by flow cytometry analysis on cell apoptosis. By establishment of xenograft tumor models in BALB/c nude mice, the effect of CAFs on radioresistance was investigated in vivo. Results: CAFs conferred ESCC cells KYSE-30 and KYSE-150 significant radioresistance by promoting clonogenic survival and inhibiting DSBs and cell apoptosis following radiation in comparison with their matched NFs. Mechanism studies revealed that human chemokine CXCL1 (The chemokine [C-X-C motif] ligand 1) was secreted by CAFs at a significantly higher level compared with matched NFs and ESCC cells. Inhibition of CXCL1 expression in CAFs by the human specific antibody against CXCL1 significantly reversed CAFs-mediated radioresistance by enhancing radiationinduced DSBs, and inhibiting DNA damage repair response and the activation of anti-apoptotic Mek/Erk signaling pathway. The use of CXCL1 antibody also significantly reversed CAFs-mediated radioresistance in xenograft tumor models. Further studies demonstrated that CAFs-secreted CXCL1 suppressed the expression of antioxidant enzyme SOD1 (superoxide dismutases), leading to accelerated ROS (Reactive Oxygen Species) accumulation which activated DNA damage repair response and mediated the
radioresistance. Moreover, the crosstalk of CAFs and ESCC cells induced CXCL1 expression in an autocrine/paracrine signaling loop by which the radioresistance was further enhanced. Conclusion: Our study for the first time discovered CAFs-secreted CXCL1 as an attractive target to reverse the radioresistance of ESCC. Author Disclosure: H. Zhang: None. J. Yue: None. Z. Jiang: None. H. Wu: None. X. Zhang: None. L. Yang: None. Y. Li: None. S. Li: None. Q. Hou: None. Y. Wang: None. S. Wu: None.
1179 Pharmacological Ascorbate Enhances the Response of Non-Small Cell Lung Cancer (NSCLC) to Radiation and Chemotherapy Via Disruptions of Redox Active Iron Leading to Increased H2O2 B.G. Allen,1 J.D. Schoenfeld,2 Z.A. Sibenaller,3 K.A. Mapuskar,3 M. Furqan,1 T. Abu Hejleh,4 B. Wagner,5 J.J. Cullen,3 G.R. Buettner,3 J. Buatti,1 and D. Spitz1; 1University of Iowa Hospitals & Clinics, Iowa City, IA, 2University of Iowa Hospitals and Clinics, Iowa City, IA, 3 University of Iowa Hospitals and Clinics, Iowa City, IA, 4University of Iowa Hospitals and Clinics, Iowa City, IA, 5University of Iowa Carver College of Medicine, Iowa City, IA Purpose/Objective(s): Intravenous ascorbate can result in pharmacologically active plasma ascorbate levels [ 20 mM] that have been shown to be well-tolerated and are currently being evaluated as an adjuvant to cancer therapy. We hypothesized that pharmacological ascorbate is selectively cytotoxic to NSCLC cells (H292 and H1299), relative to human bronchial epithelial cells (HBEpC) via disruptions in redox active iron metabolism leading to formation of H2O2. Materials/Methods: NCI-H292 and H1299 and HBEpC cells were assessed for clonogenic survival following treatment with ascorbate as well as ionizing radiation (IR) and carboplatin. Deferoxamine (Des) and diethylenetriaminepenta-acetic acid (DTPA) were used to inhibit the redox cycling of iron. NSCLC xenografts were treated with carboplatin (5 mg kg-1), IR (12 Gy / 2 fx), daily ascorbate (4 g kg-1), or vehicle control. Differences in intracellular H2O2 flux were assessed using, Peroxy-Orange-1. Cellular labile iron pools were assessed using Calcein-AM. Transferrin receptor levels were assessed using Western Blots and siRNA knockdown of the transferrin receptor was accomplished to test causality. Stage IV NSCLC patients were enrolled in a phase II clinical trial (NCT02420314) assessing the efficacy of pharmacological ascorbate (75 g infused twice a week) in combination with carboplatin (AUC Z 6) and paclitaxel (200 mg/m2) for 4 cycles. Responses were assessed per RECIST criteria. Results: Clonogenic and xenograft studies demonstrated that pharmacological ascorbate sensitized NSCLC cells to IR and carboplatin (P < 0.05) that was reversed by Des/DTPA or catalase treatment. Intracellular labile iron pools were increased in NCI-H292 and H1299 cells, relative to HBEpC, upon ascorbate exposure. NSCLC cell lines and patient tumor samples demonstrated increased transferrin receptor and labile iron, relative to normal lung cells, and tissues. Inhibiting transferrin receptor via siRNA, inhibited the toxicity of ascorbate in NSCLCs. Six subjects have enrolled in the phase II trial with five being evaluable for response. Four of five subjects demonstrated at least partial response with one progressing on therapy. No ascorbate treated subjects demonstrated grade III or IV toxicities related to ascorbate administration. Conclusion: These data show pharmacological ascorbate selectively sensitizes NSCLCs to IR and chemotherapy via a mechanism involving redox active iron causing increased levels of H2O2. Additionally, results from the phase II clinical trial suggest that pharmacological ascorbate is welltolerated and can be used as an adjuvant to NSCLC standard of care therapy. (Supported by ASTRO JF2014-1.) Author Disclosure: B.G. Allen: Research Grant; ASTRO. Travel Expenses; Galera Therapeutics. J.D. Schoenfeld: None. Z.A. Sibenaller: None. K.A. Mapuskar: None. M. Furqan: None. T. Abu Hejleh: None. B. Wagner: None. J.J. Cullen: None. G.R. Buettner: None. J. Buatti: None. D. Spitz: Advisory Board; Galera Therapeutics.
Volume 96 Number 2S Supplement 2016 Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, western blot analysis, Immunohistochemistry, caspases assay, MRI, and DNA fragmentation assay. Results: As expected, high dose radiation induced activation of MAPKs, ERK, p38, and JNK, in both the in vitro and in vivo models. This activation occurred via caspase-3 independent pathway and was mediated by calpain, cathepsin B/D, and apoptosis-inducing factor. Our results also demonstrated that AIF promotes chromatinolysis and necrosis by interacting with histone H2AX in RIP3+ cells. In cells provided with RIP1/RIP3 kinase-specific inhibitors, it was discovered that radiation-induced inflammatory responses (NF-kB, STAT-3, and HIF-1) were suppressed. In addition, the programmed necrosis process was disrupted. Reduced activations of MAPKs, ERK, p38, and JNK were observed, indicating that RIP3 regulates programmed necrosis via these pathways following high-dose radiation. Conclusion: RIP3 plays an essential role in the radiation-induced necrosis process via activation of MAPKs, ERK, p38, and JNK. RIP3 inhibitors can be used to modulate radiation-induced inflammatory response and to potentially control the radiation-induced necrosis process. In the future, insight into the precise role RIP3 plays in these processes may allow us the ability to differentiate necrosis from tumor progression and even harness its power for therapeutic effect. Author Disclosure: D.G. McDonald: None. D. Jacqmin: None. W.A. Vandergrift: None. S. Lindhorst: None. D. Cachia: None. V.K. Abhay: None. K.N. Vanek: None. N.L. Banik: None. J.M. Jenrette: None. P. Giglio: None. S.J. Patel: None. A. Das: None.
1178 CAFs-Secreted CXCL1 Mediated Radioresistance by Regulating DNA Damage Response in an ROS-Dependent Manner in Esophageal Squamous Cell Carcinoma H. Zhang, J. Yue, Z. Jiang, H. Wu, X. Zhang, L. Yang, Y. Li, S. Li, Q. Hou, Y. Wang, and S. Wu; Hangzhou Cancer Hospital, Hangzhou, China Purpose/Objective(s): Esophageal squamous cell carcinoma (ESCC) is a prevalent cancer with high morbidity and mortality worldwide. Radiotherapy is one major curative treatment modality for ESCC. Herein, our study aimed to investigate whether CAFs (cancer-associated fibroblasts), a main component of tumor microenvironment, were involved in mediating the development of radioresistance and further explored the mechanisms underlying CAFs-mediated radioresistance in ESCC. Materials/Methods: By primary culture, two pairs of CAFs and matched NFs (normal fibroblasts) had been successfully isolated from tumor tissues of ESCC patients and matched normal esophageal epithelial tissues, respectively. The secretion of chemokines by CAFs and matched NFs was determined by the human chemokine antibody array. The concentrations of chemokine in culture medium of CAFs, matched NFs and ESCC cells were determined by Elisa assay. The effect of CAFs-secreted chemokines on radioresistance was investigated in vitro by clonogenic survival assay, detection of DNA doublestrand breaks (DSBs) and by flow cytometry analysis on cell apoptosis. By establishment of xenograft tumor models in BALB/c nude mice, the effect of CAFs on radioresistance was investigated in vivo. Results: CAFs conferred ESCC cells KYSE-30 and KYSE-150 significant radioresistance by promoting clonogenic survival and inhibiting DSBs and cell apoptosis following radiation in comparison with their matched NFs. Mechanism studies revealed that human chemokine CXCL1 (The chemokine [C-X-C motif] ligand 1) was secreted by CAFs at a significantly higher level compared with matched NFs and ESCC cells. Inhibition of CXCL1 expression in CAFs by the human specific antibody against CXCL1 significantly reversed CAFs-mediated radioresistance by enhancing radiationinduced DSBs, and inhibiting DNA damage repair response and the activation of anti-apoptotic Mek/Erk signaling pathway. The use of CXCL1 antibody also significantly reversed CAFs-mediated radioresistance in xenograft tumor models. Further studies demonstrated that CAFs-secreted CXCL1 suppressed the expression of antioxidant enzyme SOD1 (superoxide dismutases), leading to accelerated ROS (Reactive Oxygen Species) accumulation which activated DNA damage repair response and mediated the
radioresistance. Moreover, the crosstalk of CAFs and ESCC cells induced CXCL1 expression in an autocrine/paracrine signaling loop by which the radioresistance was further enhanced. Conclusion: Our study for the first time discovered CAFs-secreted CXCL1 as an attractive target to reverse the radioresistance of ESCC. Author Disclosure: H. Zhang: None. J. Yue: None. Z. Jiang: None. H. Wu: None. X. Zhang: None. L. Yang: None. Y. Li: None. S. Li: None. Q. Hou: None. Y. Wang: None. S. Wu: None.
1179 Pharmacological Ascorbate Enhances the Response of Non-Small Cell Lung Cancer (NSCLC) to Radiation and Chemotherapy Via Disruptions of Redox Active Iron Leading to Increased H2O2 B.G. Allen,1 J.D. Schoenfeld,2 Z.A. Sibenaller,3 K.A. Mapuskar,3 M. Furqan,1 T. Abu Hejleh,4 B. Wagner,5 J.J. Cullen,3 G.R. Buettner,3 J. Buatti,1 and D. Spitz1; 1University of Iowa Hospitals & Clinics, Iowa City, IA, 2University of Iowa Hospitals and Clinics, Iowa City, IA, 3 University of Iowa Hospitals and Clinics, Iowa City, IA, 4University of Iowa Hospitals and Clinics, Iowa City, IA, 5University of Iowa Carver College of Medicine, Iowa City, IA Purpose/Objective(s): Intravenous ascorbate can result in pharmacologically active plasma ascorbate levels [ 20 mM] that have been shown to be well-tolerated and are currently being evaluated as an adjuvant to cancer therapy. We hypothesized that pharmacological ascorbate is selectively cytotoxic to NSCLC cells (H292 and H1299), relative to human bronchial epithelial cells (HBEpC) via disruptions in redox active iron metabolism leading to formation of H2O2. Materials/Methods: NCI-H292 and H1299 and HBEpC cells were assessed for clonogenic survival following treatment with ascorbate as well as ionizing radiation (IR) and carboplatin. Deferoxamine (Des) and diethylenetriaminepenta-acetic acid (DTPA) were used to inhibit the redox cycling of iron. NSCLC xenografts were treated with carboplatin (5 mg kg-1), IR (12 Gy / 2 fx), daily ascorbate (4 g kg-1), or vehicle control. Differences in intracellular H2O2 flux were assessed using, Peroxy-Orange-1. Cellular labile iron pools were assessed using Calcein-AM. Transferrin receptor levels were assessed using Western Blots and siRNA knockdown of the transferrin receptor was accomplished to test causality. Stage IV NSCLC patients were enrolled in a phase II clinical trial (NCT02420314) assessing the efficacy of pharmacological ascorbate (75 g infused twice a week) in combination with carboplatin (AUC Z 6) and paclitaxel (200 mg/m2) for 4 cycles. Responses were assessed per RECIST criteria. Results: Clonogenic and xenograft studies demonstrated that pharmacological ascorbate sensitized NSCLC cells to IR and carboplatin (P < 0.05) that was reversed by Des/DTPA or catalase treatment. Intracellular labile iron pools were increased in NCI-H292 and H1299 cells, relative to HBEpC, upon ascorbate exposure. NSCLC cell lines and patient tumor samples demonstrated increased transferrin receptor and labile iron, relative to normal lung cells, and tissues. Inhibiting transferrin receptor via siRNA, inhibited the toxicity of ascorbate in NSCLCs. Six subjects have enrolled in the phase II trial with five being evaluable for response. Four of five subjects demonstrated at least partial response with one progressing on therapy. No ascorbate treated subjects demonstrated grade III or IV toxicities related to ascorbate administration. Conclusion: These data show pharmacological ascorbate selectively sensitizes NSCLCs to IR and chemotherapy via a mechanism involving redox active iron causing increased levels of H2O2. Additionally, results from the phase II clinical trial suggest that pharmacological ascorbate is welltolerated and can be used as an adjuvant to NSCLC standard of care therapy. (Supported by ASTRO JF2014-1.) Author Disclosure: B.G. Allen: Research Grant; ASTRO. Travel Expenses; Galera Therapeutics. J.D. Schoenfeld: None. Z.A. Sibenaller: None. K.A. Mapuskar: None. M. Furqan: None. T. Abu Hejleh: None. B. Wagner: None. J.J. Cullen: None. G.R. Buettner: None. J. Buatti: None. D. Spitz: Advisory Board; Galera Therapeutics.