- Email: [email protected]
218. Monitoring ER Stress Activation of the ATF6 Pathway Using Nanoluciferase
Cancer-Targeted Gene and Cell Therapy I HEK293-miR122 and miR122T sequence together increase yields of AAV vectors carrying suicide genes. HEK293 Batch 1 Batch 2 Batch 3 Ave SD 2.68E + 9.96E 2.78E + 2.15E + 1.00E + ssAAV8-FLuc 12 + 11 12 12 12 ssAAV8-FLuc8.72E 2.28E + 1.34E + 1.50E + 7.17E 122T + 11 12 12 12 + 11 6.23E + 1.34E + 3.75E + 7.79E + 5.01E + ssAAV8-TCS 08 09 08 08 08 ssAAV8-TCS2.34E + 1.68E + 4.27E + 1.48E + 9.72E + 122T 09 09 08 09 08 HEK293-miR122 ssAAV8-FLuc ssAAV8-FLuc122T ssAAV8-TCS ssAAV8-TCS122T
Batch 1 4.04E + 12 5.61E + 12 3.45E + 09 1.66E + 11
Batch 2 6.60E + 12 2.74E + 12 1.39E + 09 8.50E + 10
Batch 3 1.05E + 12 7.80E + 11 8.50E + 08 9.30E + 10
Ave 3.90E + 12 7.80E + 11 1.90E + 09 1.15E + 11
SD 2.78E + 12 2.43E + 12 1.37E + 09 4.46E + 10
216. Exploiting Redox-Based Vulnerabilities in Field Cancerization by Targeting Cancer Associated Fibroblasts
Jeremy Soon Kiat Chan, Ming Jie Tan, Nguan Soon Tan School of Biological Sciences, Nanyang Technological University, Singapore, Singapore Tumor development and progression to malignancy is increasingly viewed as a consequence of disrupted homeostatic interactions between epithelial cells and the underlying stromal elements. In squamous cell carcinoma of the skin, genomic and histological abnormalities have been observed in keratinocytes and stroma bordering multifocal neoplastic lesions, suggesting a regional carcinogenic signal that promotes aberrant cellular behavior within the tumor microenvironment. This has been termed field cancerization. Cancer associated fibroblasts (CAFs) are the major cell type in the tumor stroma and are known producers of extracellular protumor signaling molecules. While growth factors and cytokines are candidate molecules for targeted therapy, reactive oxygen species (ROS) are gaining attention for their roles in heterotypic paracrine signaling in cancer. The importance of ROS in field cancerization remains poorly understood. Histological analysis of on sections of patient tumor biopsies revealed that ROS was elevated in the tumor stroma. CAFs explanted from patient tumors also released high levels of hydrogen peroxide into the extracellular space. Interestingly, normal fibroblasts exposed to CAF-conditioned medium displayed increased ROS production, an observation that could be reversed by the antioxidant N-acetyl cysteine. Exposure to hydrogen peroxide also resulted in elevated ROS production in normal fibroblasts. Taken together, these findings suggest that CAFs are a potent source of ROS in the tumor microenvironment and can induce a field effect that transforms adjacent normal fibroblasts into a CAF-like state. Transformed fibroblasts in turn produce ROS to spread oxidative stress in the microenvironment, potentially exacerbating field cancerization. Therefore, antioxidant-based therapies targeting the tumor microenvironment may be important adjuvant treatments to current anticancer chemotherapy or targeted therapy.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
217. Combinatorial Anti-Angiogenic Gene Therapy in a Human Malignant Mesothelioma Model
Shuji Kubo1, Yuki Mawatari-Furukawa1, Noriyuki Kasahara2 Genetics, Hyogo College of Medicine, Nishinomiya, Japan, 2Cell Biology and Pathology, University of Miami, Miami, FL
1
Anti-angiogenic gene therapy represents a promising strategy for cancer; however, it has rarely been tested in malignant mesothelioma, a highly aggressive tumor associated with asbestos that has poor prognosis. In this study, we investigated whether anti-angiogenic factors such as angiostatin, endostatin, and the soluble form of vascular endothelial growth factor receptor 2 (sFlk1) were able to inhibit endothelial cell proliferation via lentivirus-mediated gene transfer into malignant mesothelioma cells in culture. We also assessed whether a dual-agent strategy had greater therapeutic benefit. Human malignant pleural mesothelioma MSTO-211H cells were transduced using lentiviral vectors that individually expressed angiostatin, endostatin, and sFlk1 and linked to enhanced green fluorescent protein (EGFP) marker gene expression via an internal ribosome entry site. The lentivirus expressing EGFP alone was used as a control. The resultant cells designated as MSTO-A, MSTO-E, MSTO-F, and MSTO-C were confirmed by Western blot and fluorescence microscopy to stably express the corresponding proteins. No differences were observed in the in vitro growth rates between any of these cells. However, coculture of MSTO-A, MSTO-E, and MSTO-F showed significant suppression of human umbilical endothelial cell growth in vitro compared with that of MSTO-C. Furthermore, a combination of any two among MSTO-A, MSTO-E, and MSTO-F significantly enhanced efficacy. These results suggest that combinational anti-angiogenic gene therapy targeting different pathways of endothelial growth factor signaling has the potential for greater therapeutic efficacy than that of a single-agent regimen.
218. Monitoring ER Stress Activation of the ATF6 Pathway Using Nanoluciferase
Emily S. Wires, Doug Howard, Mark J. Henderson, Xiaokang Yan, Kathleen A. Trychta, Emily J. Heathward, Yajun Zhang, Molly Lutrey, Christopher Richie, Brandon K. Harvey OTTC, NIDA/NIH, Baltimore, MD The endoplasmic reticulum (ER) is an invaluable component of the secretory pathway; serving as the main site for protein synthesis and modification, lipid metabolism, lipoprotein secretion, and calcium homeostasis. In response to “stress”, the ER maintains homeostasis through the unfolded protein response (UPR). The UPR is an adaptive response employed by the ER to alleviate stress caused by misfolded proteins. UPR activation is mediated by three transmembrane proteins; IRE1 (inositolrequiring protein-1), ATF6 (activating transcription factor- 6), and PERK (protein kinase RNA-like ER kinase). Here we describe the construction and characterization of a Nanoluciferase-based assay to look at the activation of the ATF6 prong of the UPR. We demonstrate the ability to simultaneously monitor ER calcium homeostasis using a novel Gaussia luciferasebased sensor. Using in vitro and in vivo assays, we demonstrate the ability to monitor ATF6 activation in human neuroblastoma cells, rat hepatoma cell line, and rat liver. With this approach we hope to gain better understanding of the contributions of ATF6 activation and ER calcium depletion in disease pathogenesis. Ongoing studies will examinegenetically or pharmacologically augmenting or inhibiting these pathways as a therapeutic approach.
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Batch 1 4.04E + 12 5.61E + 12 3.45E + 09 1.66E + 11
Batch 2 6.60E + 12 2.74E + 12 1.39E + 09 8.50E + 10
Batch 3 1.05E + 12 7.80E + 11 8.50E + 08 9.30E + 10
Ave 3.90E + 12 7.80E + 11 1.90E + 09 1.15E + 11
SD 2.78E + 12 2.43E + 12 1.37E + 09 4.46E + 10
216. Exploiting Redox-Based Vulnerabilities in Field Cancerization by Targeting Cancer Associated Fibroblasts
Jeremy Soon Kiat Chan, Ming Jie Tan, Nguan Soon Tan School of Biological Sciences, Nanyang Technological University, Singapore, Singapore Tumor development and progression to malignancy is increasingly viewed as a consequence of disrupted homeostatic interactions between epithelial cells and the underlying stromal elements. In squamous cell carcinoma of the skin, genomic and histological abnormalities have been observed in keratinocytes and stroma bordering multifocal neoplastic lesions, suggesting a regional carcinogenic signal that promotes aberrant cellular behavior within the tumor microenvironment. This has been termed field cancerization. Cancer associated fibroblasts (CAFs) are the major cell type in the tumor stroma and are known producers of extracellular protumor signaling molecules. While growth factors and cytokines are candidate molecules for targeted therapy, reactive oxygen species (ROS) are gaining attention for their roles in heterotypic paracrine signaling in cancer. The importance of ROS in field cancerization remains poorly understood. Histological analysis of on sections of patient tumor biopsies revealed that ROS was elevated in the tumor stroma. CAFs explanted from patient tumors also released high levels of hydrogen peroxide into the extracellular space. Interestingly, normal fibroblasts exposed to CAF-conditioned medium displayed increased ROS production, an observation that could be reversed by the antioxidant N-acetyl cysteine. Exposure to hydrogen peroxide also resulted in elevated ROS production in normal fibroblasts. Taken together, these findings suggest that CAFs are a potent source of ROS in the tumor microenvironment and can induce a field effect that transforms adjacent normal fibroblasts into a CAF-like state. Transformed fibroblasts in turn produce ROS to spread oxidative stress in the microenvironment, potentially exacerbating field cancerization. Therefore, antioxidant-based therapies targeting the tumor microenvironment may be important adjuvant treatments to current anticancer chemotherapy or targeted therapy.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
217. Combinatorial Anti-Angiogenic Gene Therapy in a Human Malignant Mesothelioma Model
Shuji Kubo1, Yuki Mawatari-Furukawa1, Noriyuki Kasahara2 Genetics, Hyogo College of Medicine, Nishinomiya, Japan, 2Cell Biology and Pathology, University of Miami, Miami, FL
1
Anti-angiogenic gene therapy represents a promising strategy for cancer; however, it has rarely been tested in malignant mesothelioma, a highly aggressive tumor associated with asbestos that has poor prognosis. In this study, we investigated whether anti-angiogenic factors such as angiostatin, endostatin, and the soluble form of vascular endothelial growth factor receptor 2 (sFlk1) were able to inhibit endothelial cell proliferation via lentivirus-mediated gene transfer into malignant mesothelioma cells in culture. We also assessed whether a dual-agent strategy had greater therapeutic benefit. Human malignant pleural mesothelioma MSTO-211H cells were transduced using lentiviral vectors that individually expressed angiostatin, endostatin, and sFlk1 and linked to enhanced green fluorescent protein (EGFP) marker gene expression via an internal ribosome entry site. The lentivirus expressing EGFP alone was used as a control. The resultant cells designated as MSTO-A, MSTO-E, MSTO-F, and MSTO-C were confirmed by Western blot and fluorescence microscopy to stably express the corresponding proteins. No differences were observed in the in vitro growth rates between any of these cells. However, coculture of MSTO-A, MSTO-E, and MSTO-F showed significant suppression of human umbilical endothelial cell growth in vitro compared with that of MSTO-C. Furthermore, a combination of any two among MSTO-A, MSTO-E, and MSTO-F significantly enhanced efficacy. These results suggest that combinational anti-angiogenic gene therapy targeting different pathways of endothelial growth factor signaling has the potential for greater therapeutic efficacy than that of a single-agent regimen.
218. Monitoring ER Stress Activation of the ATF6 Pathway Using Nanoluciferase
Emily S. Wires, Doug Howard, Mark J. Henderson, Xiaokang Yan, Kathleen A. Trychta, Emily J. Heathward, Yajun Zhang, Molly Lutrey, Christopher Richie, Brandon K. Harvey OTTC, NIDA/NIH, Baltimore, MD The endoplasmic reticulum (ER) is an invaluable component of the secretory pathway; serving as the main site for protein synthesis and modification, lipid metabolism, lipoprotein secretion, and calcium homeostasis. In response to “stress”, the ER maintains homeostasis through the unfolded protein response (UPR). The UPR is an adaptive response employed by the ER to alleviate stress caused by misfolded proteins. UPR activation is mediated by three transmembrane proteins; IRE1 (inositolrequiring protein-1), ATF6 (activating transcription factor- 6), and PERK (protein kinase RNA-like ER kinase). Here we describe the construction and characterization of a Nanoluciferase-based assay to look at the activation of the ATF6 prong of the UPR. We demonstrate the ability to simultaneously monitor ER calcium homeostasis using a novel Gaussia luciferasebased sensor. Using in vitro and in vivo assays, we demonstrate the ability to monitor ATF6 activation in human neuroblastoma cells, rat hepatoma cell line, and rat liver. With this approach we hope to gain better understanding of the contributions of ATF6 activation and ER calcium depletion in disease pathogenesis. Ongoing studies will examinegenetically or pharmacologically augmenting or inhibiting these pathways as a therapeutic approach.
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