Continued expansion of the nanotechnology industry has necessitated the self-assessment of manufa... more Continued expansion of the nanotechnology industry has necessitated the self-assessment of manufacturing processes, specifically in regards to understanding the health related aspects following exposure to nanomaterials. There exists a growing concern over potential occupational exposure in the semiconductor industry where Al2O3, CeO2 and SiO2 nanoparticles are commonly featured as part of the chemical mechanical planarization (CMP) process. Chronic exposure to toxicants can result not only in acute cytotoxicity but also initiation of a chronic inflammatory state associated with diverse pathologies. In the current investigation, pristine nanoparticles and CMP slurry formulations of Al2O3, SiO2 and CeO2 were employed to assess their ability to induce cytotoxicity, inflammatory responses and reactive oxygen species in a mouse alveolar macrophage cell model. The pristine nanoparticles and slurries were not intrinsically cytotoxic and did not generate free radicals but were found to act as scavengers in the presence of an oxidant stimulant. Al2O3 and SiO2 nanoparticles increased levels of pro-inflammatory cytokines while pristine SiO2 nanoparticles induced generation of F2-Isoprostanes. In co-treatment studies, the pristine nanomaterials modulated the response to the inflammatory stimulant lipopolysaccharide. The studies have established that pristine nanoparticles and slurries do not impact the cells in a similar way indicating that they should not be used as slurry substitutes in toxicity evaluations. Further, we have defined how an alveolar cell line, which would likely be the first challenged upon nanomaterial aerosolization, responds to diverse mixtures of nanomaterials. Moreover, our findings reinforce the importance of using multiple analytic methods to define the redox state of the cell following exposure to commonly used industrial nanomaterials and toxicants.
Experimental and Molecular Therapeutics, Jul 1, 2021
Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer ... more Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer subtype. Targeted therapy for HGSOC is challenging as the disease is characterized by copy number variation and not somatic mutations. HGSOC displays well-conserved features such as expression of transcription factor PAX8. We propose employing therapies that capitalize on interactions between PAX8 and specific DNA-binding sites to develop therapeutic approaches to target HGSOC. Our hypothesis is to use a biomimetic approach wherein PAX8 is induced to transcribe an ectopically transfected CRISPR-base editor (BE)-guide RNA (gRNA) plasmid containing a PAX8 binding site in its promoter. The gRNA will impart a dominant-negative allele of ataxia telangiectasia and Rad3-related protein (ATR), which has been shown to be synthetically lethal with mutant p53 in cancer cells. In the presence of PAX8, the plasmid will be transcribed, while in the absence of PAX8, the plasmid is degraded, with minimal toxicity observed in healthy cells. Methods: PAX8-expressing ovarian cancer cell lines including COV362, Kuramochi, OVCAR8 and OVSAHO or PAX8-null cell lines including MCF-7 and HeLa were transfected with polyplexes bearing the plasmid of interest. Polyplex diameter, polydispersity indices and surface zeta potentials were measured on a Malvern Zetasizer. Polyplexes were imaged for uptake and functionality using fluorescence microscopy and Amnis ImageStream. Polyplex toxicity was assessed in vitro using cell viability assays. Preliminary in vivo experiments to demonstrate luciferase plasmid-bearing polyplexes have been performed in NSG mice. To monitor in vivo transfection efficiencies, we will deliver fluorescently labeled plasmids or luciferase-encoding plasmids to test bio-distribution and pharmacokinetics. Results: We have synthesized sub-200nm (diameter) polyplexes using the polyamine PPLG-g-azidopropylamine (PPLG) and BE or gRNA plasmids. Using -/- ATR gRNA, we observed that the BE and the gRNA can be efficiently packaged into polymeric nanoplexes and delivered to ovarian cancer cells or control cells to impart a dominant-negative mutant of ATR. Ongoing work includes cell cycle analysis using flow cytometry to monitor checkpoint activation, quantifying percent of total sequencing reads with target base pair conversions and testing gRNAs against other targets for efficacy in ovarian cancer targeting. Conclusion: Combining nanotechnology and gene editing can improve specificity toward ovarian cancer cells as well as incorporate precision therapy aimed at the underlying genetic architecture of this disease. Due to the synthetic lethality nature of ATR and p53, addition of a combinatorial PARP inhibitor to the proposed therapy will reduce tumor burden. While resistance can develop to ATR inhibitors, ovarian cancer cells are unable to downregulate PAX8, thus ensuring continued response to the PAX8-directed therapy. Citation Format: Akshaya Chandrasekaran, Mariam Ahmed, Nicole Holub, Wade Wang, Paula T. Hammond, Kevin M. Elias. PAX8-directed nanotherapeutics for high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1316.
Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogeni... more Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogenic potential. While senescent cells have lost the ability to divide, they remain metabolically active and adapt a deleterious senescence associated secretory phenotype (SASP) central to the progression of several age-associated disease pathologies. The SASP is mechanistically regulated by the pro-inflammatory cytokine interleukin-1 alpha (IL-1α) whose expression and activity is responsive to the senescence associated (SA) oxidant production and the accompanying disruption of calcium (Ca2+) homeostasis. Using primary IMR-90 human fetal lung fibroblasts as a model of replicative senescence, we explored the molecular underpinnings facilitating increased Ca2+ entry in senescent cells. We establish that the redox-responsive Transient Receptor Potential TRPC6 channel is compromised due to desensitization owing to SA increases in steady state hydrogen peroxide (H2O2) production. SA dysregulation of Ca2+ is also accompanied by loss of response to H2O2-induced Ca2+ influx that can be rescued with catalase pre-treatments. Senescent cells are also insensitive to Ca2+ entry induced by hyperforin, a specific activator of TRPC6 that can be restored by catalase pre-treatments, further suggesting redox regulation of TRPC6 in senescence. Inhibition of TRPC6 channel activity restores ability of senescent cells to respond to peroxide-induced Ca2+ in addition to suppressing SASP gene expression. Furthermore, mammalian target of rapamycin (mTOR) signaling regulates SASP by means of modulating TRPC6 channel expression. Together, our findings provide compelling evidence that H2O2 and mTOR-mediated regulation of TRPC6 channel modulates SASP gene expression. As TRP channels emerge as targets of pharmacologic intervention for numerous disease pathologies, it is exciting to speculate that effect of TRP interventions may be attributed in part to inhibition of senescence and the SASP.
The lethality of most cancers can be attributed to metastatic progression. Metastatic tumor cells... more The lethality of most cancers can be attributed to metastatic progression. Metastatic tumor cells adapt and cope with various stressful environments to enable survival during their metastatic journey which starts with the escape from the primary tumor and ends in colonization of secondary sites. Stressors of metastatic tumor microenvironments include deprivation of oxygen (hypoxia), inflammation, and chemo- and radiotherapeutic exposure, which have the propensity to expose cells to reactive oxygen species (ROS). Further, many tumor-associated cells, including cancer-associated fibroblasts, macrophages, and senescent cells within the tumor microenvironment, contribute to this ROS production. In addition to these exogenous sources, tumor cells themselves produce and are able to cope with elevated intracellular ROS. Increased ROS production outside and within metastatic tumor cells has been associated with a number of pro-metastatic events including angiogenesis, invasion, migration, survival, and anchorage-independent cell survival (anoikis resistance). Large surges of ROS can lead to oxidation of macromolecules and largely irreversible damage that may cause mitochondrial damage, leading to alterations in cancer metabolism, and genomic instability and carcinogenesis. More subtle changes in ROS lead to redox-signaling, primarily by reversible oxidation of thiols, which has been shown to contribute to pro-metastatic behavior. To cope with these changes in both intracellular and extracellular redox environments, tumor cells have uniquely evolved to alter their antioxidant enzyme expression. The present review focuses on antioxidant enzymes important in the regulation of H2O2 balance within metastatic tumor cells. It aims to highlight some of the dichotomous roles demonstrated for these enzymes in cancer etiology, and how their enzymatic activity may further influence the tumor redox environment and consequently regulate carcinogenesis and metastasis. We focus on the role of superoxide dismutases, catalase, and glutathione peroxidases and give examples on their demonstrated roles as both tumor suppressors and promoters. Specifically, we discuss how metastatic cancer cells uniquely adapt to alter expression of these enzymes and how this may contribute to changes in the intracellular redox environment to drive certain metastatic phenotypes. To therapeutically target these ROS-mediated pathways in metastatic disease will require further insights into the specificity of the ROS involved and their spatiotemporal regulation in the context of the antioxidant enzymes present.
Experimental and Molecular Therapeutics, Aug 13, 2020
Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer ... more Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer subtype. Targeted therapy for HGSOC is challenging because the disease is characterized by copy number variation rather than recurrent somatic mutations. HGSOC displays well-conserved epigenetic features such as expression of transcription factor PAX8. We propose employing epigenetic therapies that capitalize on interactions between PAX8 and specific DNA-binding sites to develop potential therapeutic approaches to target HGSOC. Our hypothesis is to use a biomimetic approach wherein PAX8 is induced to transcribe an ectopically transfected CRISPR-base editor (BE)-guide RNA (gRNA) plasmid containing a PAX8 binding site in its promoter. The gRNA will impart a dominant-negative allele of Polo Like Kinase 1 (PLK1) which has been shown to be synergistically lethal with deficient tp53 in cancer cells. In the presence of PAX8, the plasmid will be transcribed, while in the absence of PAX8, the plasmid is degraded, with minimal toxicity observed in healthy cells. Methods: PAX8-expressing ovarian cancer cell lines including COV362, Kuramochi, OVCAR8 and OVSAHO or PAX8-negative control cell lines including JEG3, MCF-7, HeLa and HEK293T were transfected with polyplexes bearing the plasmid of interest. Polyplex diameter, polydispersity indices, and surface zeta potentials were measured on a Malvern Zetasizer. Polyplexes were imaged for uptake and functionality using fluorescence microscopy and Amnis ImageStream. Polyplex toxicity was assessed in vitro using cell viability assays. Preliminary in vivo experiments to demonstrate luciferase plasmid-bearing polyplexes have been performed in NOD-SCID mice. To monitor in vivo transfection efficiencies, we will deliver fluorescently labeled plasmids or luciferase-encoding plasmids to test bio-distribution and pharmacokinetics. Results: We have synthesized sub-200nm (diameter) polyplexes using the polyamine PPLG-g-azidopropylamine (PPLG) and BE or gRNA plasmids. Using -/-PLK1 gRNA, we observed that the BE and the gRNA can be efficiently packaged into two distinct polymeric nanoplexes and co-delivered to ovarian cancer cells or control cells to impart a dominant-negative mutant of PLK1, which is also synthetically lethal with mutated tp53, as observed from cancer cell death compared to controls. Ongoing work includes repetitive polyplex injections in vitro and in vivo, quantifying percentage of total sequencing reads with target base pair conversions after treatment, determination of plasmid loading efficiency, and testing gRNAs against other targets for efficacy in ovarian cancer targeting. Conclusion: Well-defined lineage transcription factors have the potential to serve as novel targets for gene-editing therapies in ovarian cancer and other malignancies. Combining nanotechnology and gene editing can improve specificity toward ovarian cancer cells as well as incorporate precision therapy aimed at the underlying genetic architecture of this disease. Citation Format: Akshaya Chandrasekaran, Mariam Ahmed, Wade Wang, Paula T. Hammond, Kevin M. Elias. PAX8-directed nanotherapeutics for high-grade serous ovarian carcinoma [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 6241.
As an innate defense mechanism, macrophages produce reactive oxygen species (ROS) which weaken pa... more As an innate defense mechanism, macrophages produce reactive oxygen species (ROS) which weaken pathogens and serve as secondary messengers involved in immune function. The gram-negative bacterium F. tularensis utilizes its antioxidant armature to limit the host immune response but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca2+ entry in macrophages thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild-type (LVS) or catalase deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 pM/sec and 0.015 pM/sec, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca2+ concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca2+ influx in response to ionomycin, as a result of the ionophores H2O2-sensitivity. Exogenously added H2O2 or H2O2 generated...
Aberrant matrix metalloproteinase-1 (MMP-1) expression contributes to the pathogenesis of many de... more Aberrant matrix metalloproteinase-1 (MMP-1) expression contributes to the pathogenesis of many degenerative disease processes that are associated with increased oxidative damage or stress. We and others have established that shifts in steady-state H2O2 production resulting from enforced antioxidant gene expression, senescence, or UV irradiation control MMP-1 expression. Here we establish that histone deacetylase-2 (HDAC2) protein levels and its occupancy of the MMP-1 promoter are decreased in response to enforced manganese superoxide dismutase (Sod2) expression. Inhibition of HDAC activity further accentuates the redox-dependent expression of MMP-1. Sod2-dependent decreases in HDAC2 are associated with increases in a proteasome-sensitive pool of ubiquitinylated HDAC2 and MMP-1-specific histone H3 acetylation. Sod2 overexpression also enhanced recruitment of Ets-1, c-Jun, c-Fos, and the histone acetyltransferase PCAF to the distal and proximal regions of the MMP-1 promoter. Furthermo...
Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer ... more Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer subtype. Targeted therapy for HGSOC is challenging as the disease is characterized by copy number variation and not somatic mutations. HGSOC displays well-conserved features such as expression of transcription factor PAX8. We propose employing therapies that capitalize on interactions between PAX8 and specific DNA-binding sites to develop therapeutic approaches to target HGSOC. Our hypothesis is to use a biomimetic approach wherein PAX8 is induced to transcribe an ectopically transfected CRISPR-base editor (BE)-guide RNA (gRNA) plasmid containing a PAX8 binding site in its promoter. The gRNA will impart a dominant-negative allele of ataxia telangiectasia and Rad3-related protein (ATR), which has been shown to be synthetically lethal with mutant p53 in cancer cells. In the presence of PAX8, the plasmid will be transcribed, while in the absence of PAX8, the plasmid is degraded, with minima...
Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogeni... more Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogenic potential but is accompanied by the often pathologically deleterious senescence-associated secretory phenotype (SASP). Here we demonstrate an H2O2-dependent functional disruption controlling senescence-associated Ca2+ homeostasis and the SASP. Senescent cells fail to respond to H2O2-dependent plasma lamellar Ca2+ entry when compared to pre-senescent cells. Limiting exposure to senescence-associated H2O2 restores H2O2-dependent Ca2+ entry as well as transient receptor potential cation channel subfamily C member 6 (TRPC6) function. SA-TRPC6 and SASP expression is blocked by restoring Ca2+ entry with the TRP channel antagonist SKF-96365 or by the mTOR inhibitors rapamycin and Ku0063794. Together, our findings provide compelling evidence that redox and mTOR-mediated regulation of Ca2+ entry through TRPC6 modulates SASP gene expression and approaches which preserve normal Ca2+ homeostasis m...
Continued expansion of the nanotechnology industry has necessitated the self-assessment of manufa... more Continued expansion of the nanotechnology industry has necessitated the self-assessment of manufacturing processes, specifically in regards to understanding the health related aspects following exposure to nanomaterials. There exists a growing concern over potential occupational exposure in the semiconductor industry where Al2O3, CeO2 and SiO2 nanoparticles are commonly featured as part of the chemical mechanical planarization (CMP) process. Chronic exposure to toxicants can result not only in acute cytotoxicity but also initiation of a chronic inflammatory state associated with diverse pathologies. In the current investigation, pristine nanoparticles and CMP slurry formulations of Al2O3, SiO2 and CeO2 were employed to assess their ability to induce cytotoxicity, inflammatory responses and reactive oxygen species in a mouse alveolar macrophage cell model. The pristine nanoparticles and slurries were not intrinsically cytotoxic and did not generate free radicals but were found to act as scavengers in the presence of an oxidant stimulant. Al2O3 and SiO2 nanoparticles increased levels of pro-inflammatory cytokines while pristine SiO2 nanoparticles induced generation of F2-Isoprostanes. In co-treatment studies, the pristine nanomaterials modulated the response to the inflammatory stimulant lipopolysaccharide. The studies have established that pristine nanoparticles and slurries do not impact the cells in a similar way indicating that they should not be used as slurry substitutes in toxicity evaluations. Further, we have defined how an alveolar cell line, which would likely be the first challenged upon nanomaterial aerosolization, responds to diverse mixtures of nanomaterials. Moreover, our findings reinforce the importance of using multiple analytic methods to define the redox state of the cell following exposure to commonly used industrial nanomaterials and toxicants.
Experimental and Molecular Therapeutics, Jul 1, 2021
Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer ... more Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer subtype. Targeted therapy for HGSOC is challenging as the disease is characterized by copy number variation and not somatic mutations. HGSOC displays well-conserved features such as expression of transcription factor PAX8. We propose employing therapies that capitalize on interactions between PAX8 and specific DNA-binding sites to develop therapeutic approaches to target HGSOC. Our hypothesis is to use a biomimetic approach wherein PAX8 is induced to transcribe an ectopically transfected CRISPR-base editor (BE)-guide RNA (gRNA) plasmid containing a PAX8 binding site in its promoter. The gRNA will impart a dominant-negative allele of ataxia telangiectasia and Rad3-related protein (ATR), which has been shown to be synthetically lethal with mutant p53 in cancer cells. In the presence of PAX8, the plasmid will be transcribed, while in the absence of PAX8, the plasmid is degraded, with minimal toxicity observed in healthy cells. Methods: PAX8-expressing ovarian cancer cell lines including COV362, Kuramochi, OVCAR8 and OVSAHO or PAX8-null cell lines including MCF-7 and HeLa were transfected with polyplexes bearing the plasmid of interest. Polyplex diameter, polydispersity indices and surface zeta potentials were measured on a Malvern Zetasizer. Polyplexes were imaged for uptake and functionality using fluorescence microscopy and Amnis ImageStream. Polyplex toxicity was assessed in vitro using cell viability assays. Preliminary in vivo experiments to demonstrate luciferase plasmid-bearing polyplexes have been performed in NSG mice. To monitor in vivo transfection efficiencies, we will deliver fluorescently labeled plasmids or luciferase-encoding plasmids to test bio-distribution and pharmacokinetics. Results: We have synthesized sub-200nm (diameter) polyplexes using the polyamine PPLG-g-azidopropylamine (PPLG) and BE or gRNA plasmids. Using -/- ATR gRNA, we observed that the BE and the gRNA can be efficiently packaged into polymeric nanoplexes and delivered to ovarian cancer cells or control cells to impart a dominant-negative mutant of ATR. Ongoing work includes cell cycle analysis using flow cytometry to monitor checkpoint activation, quantifying percent of total sequencing reads with target base pair conversions and testing gRNAs against other targets for efficacy in ovarian cancer targeting. Conclusion: Combining nanotechnology and gene editing can improve specificity toward ovarian cancer cells as well as incorporate precision therapy aimed at the underlying genetic architecture of this disease. Due to the synthetic lethality nature of ATR and p53, addition of a combinatorial PARP inhibitor to the proposed therapy will reduce tumor burden. While resistance can develop to ATR inhibitors, ovarian cancer cells are unable to downregulate PAX8, thus ensuring continued response to the PAX8-directed therapy. Citation Format: Akshaya Chandrasekaran, Mariam Ahmed, Nicole Holub, Wade Wang, Paula T. Hammond, Kevin M. Elias. PAX8-directed nanotherapeutics for high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1316.
Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogeni... more Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogenic potential. While senescent cells have lost the ability to divide, they remain metabolically active and adapt a deleterious senescence associated secretory phenotype (SASP) central to the progression of several age-associated disease pathologies. The SASP is mechanistically regulated by the pro-inflammatory cytokine interleukin-1 alpha (IL-1α) whose expression and activity is responsive to the senescence associated (SA) oxidant production and the accompanying disruption of calcium (Ca2+) homeostasis. Using primary IMR-90 human fetal lung fibroblasts as a model of replicative senescence, we explored the molecular underpinnings facilitating increased Ca2+ entry in senescent cells. We establish that the redox-responsive Transient Receptor Potential TRPC6 channel is compromised due to desensitization owing to SA increases in steady state hydrogen peroxide (H2O2) production. SA dysregulation of Ca2+ is also accompanied by loss of response to H2O2-induced Ca2+ influx that can be rescued with catalase pre-treatments. Senescent cells are also insensitive to Ca2+ entry induced by hyperforin, a specific activator of TRPC6 that can be restored by catalase pre-treatments, further suggesting redox regulation of TRPC6 in senescence. Inhibition of TRPC6 channel activity restores ability of senescent cells to respond to peroxide-induced Ca2+ in addition to suppressing SASP gene expression. Furthermore, mammalian target of rapamycin (mTOR) signaling regulates SASP by means of modulating TRPC6 channel expression. Together, our findings provide compelling evidence that H2O2 and mTOR-mediated regulation of TRPC6 channel modulates SASP gene expression. As TRP channels emerge as targets of pharmacologic intervention for numerous disease pathologies, it is exciting to speculate that effect of TRP interventions may be attributed in part to inhibition of senescence and the SASP.
The lethality of most cancers can be attributed to metastatic progression. Metastatic tumor cells... more The lethality of most cancers can be attributed to metastatic progression. Metastatic tumor cells adapt and cope with various stressful environments to enable survival during their metastatic journey which starts with the escape from the primary tumor and ends in colonization of secondary sites. Stressors of metastatic tumor microenvironments include deprivation of oxygen (hypoxia), inflammation, and chemo- and radiotherapeutic exposure, which have the propensity to expose cells to reactive oxygen species (ROS). Further, many tumor-associated cells, including cancer-associated fibroblasts, macrophages, and senescent cells within the tumor microenvironment, contribute to this ROS production. In addition to these exogenous sources, tumor cells themselves produce and are able to cope with elevated intracellular ROS. Increased ROS production outside and within metastatic tumor cells has been associated with a number of pro-metastatic events including angiogenesis, invasion, migration, survival, and anchorage-independent cell survival (anoikis resistance). Large surges of ROS can lead to oxidation of macromolecules and largely irreversible damage that may cause mitochondrial damage, leading to alterations in cancer metabolism, and genomic instability and carcinogenesis. More subtle changes in ROS lead to redox-signaling, primarily by reversible oxidation of thiols, which has been shown to contribute to pro-metastatic behavior. To cope with these changes in both intracellular and extracellular redox environments, tumor cells have uniquely evolved to alter their antioxidant enzyme expression. The present review focuses on antioxidant enzymes important in the regulation of H2O2 balance within metastatic tumor cells. It aims to highlight some of the dichotomous roles demonstrated for these enzymes in cancer etiology, and how their enzymatic activity may further influence the tumor redox environment and consequently regulate carcinogenesis and metastasis. We focus on the role of superoxide dismutases, catalase, and glutathione peroxidases and give examples on their demonstrated roles as both tumor suppressors and promoters. Specifically, we discuss how metastatic cancer cells uniquely adapt to alter expression of these enzymes and how this may contribute to changes in the intracellular redox environment to drive certain metastatic phenotypes. To therapeutically target these ROS-mediated pathways in metastatic disease will require further insights into the specificity of the ROS involved and their spatiotemporal regulation in the context of the antioxidant enzymes present.
Experimental and Molecular Therapeutics, Aug 13, 2020
Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer ... more Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer subtype. Targeted therapy for HGSOC is challenging because the disease is characterized by copy number variation rather than recurrent somatic mutations. HGSOC displays well-conserved epigenetic features such as expression of transcription factor PAX8. We propose employing epigenetic therapies that capitalize on interactions between PAX8 and specific DNA-binding sites to develop potential therapeutic approaches to target HGSOC. Our hypothesis is to use a biomimetic approach wherein PAX8 is induced to transcribe an ectopically transfected CRISPR-base editor (BE)-guide RNA (gRNA) plasmid containing a PAX8 binding site in its promoter. The gRNA will impart a dominant-negative allele of Polo Like Kinase 1 (PLK1) which has been shown to be synergistically lethal with deficient tp53 in cancer cells. In the presence of PAX8, the plasmid will be transcribed, while in the absence of PAX8, the plasmid is degraded, with minimal toxicity observed in healthy cells. Methods: PAX8-expressing ovarian cancer cell lines including COV362, Kuramochi, OVCAR8 and OVSAHO or PAX8-negative control cell lines including JEG3, MCF-7, HeLa and HEK293T were transfected with polyplexes bearing the plasmid of interest. Polyplex diameter, polydispersity indices, and surface zeta potentials were measured on a Malvern Zetasizer. Polyplexes were imaged for uptake and functionality using fluorescence microscopy and Amnis ImageStream. Polyplex toxicity was assessed in vitro using cell viability assays. Preliminary in vivo experiments to demonstrate luciferase plasmid-bearing polyplexes have been performed in NOD-SCID mice. To monitor in vivo transfection efficiencies, we will deliver fluorescently labeled plasmids or luciferase-encoding plasmids to test bio-distribution and pharmacokinetics. Results: We have synthesized sub-200nm (diameter) polyplexes using the polyamine PPLG-g-azidopropylamine (PPLG) and BE or gRNA plasmids. Using -/-PLK1 gRNA, we observed that the BE and the gRNA can be efficiently packaged into two distinct polymeric nanoplexes and co-delivered to ovarian cancer cells or control cells to impart a dominant-negative mutant of PLK1, which is also synthetically lethal with mutated tp53, as observed from cancer cell death compared to controls. Ongoing work includes repetitive polyplex injections in vitro and in vivo, quantifying percentage of total sequencing reads with target base pair conversions after treatment, determination of plasmid loading efficiency, and testing gRNAs against other targets for efficacy in ovarian cancer targeting. Conclusion: Well-defined lineage transcription factors have the potential to serve as novel targets for gene-editing therapies in ovarian cancer and other malignancies. Combining nanotechnology and gene editing can improve specificity toward ovarian cancer cells as well as incorporate precision therapy aimed at the underlying genetic architecture of this disease. Citation Format: Akshaya Chandrasekaran, Mariam Ahmed, Wade Wang, Paula T. Hammond, Kevin M. Elias. PAX8-directed nanotherapeutics for high-grade serous ovarian carcinoma [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 6241.
As an innate defense mechanism, macrophages produce reactive oxygen species (ROS) which weaken pa... more As an innate defense mechanism, macrophages produce reactive oxygen species (ROS) which weaken pathogens and serve as secondary messengers involved in immune function. The gram-negative bacterium F. tularensis utilizes its antioxidant armature to limit the host immune response but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca2+ entry in macrophages thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild-type (LVS) or catalase deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 pM/sec and 0.015 pM/sec, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca2+ concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca2+ influx in response to ionomycin, as a result of the ionophores H2O2-sensitivity. Exogenously added H2O2 or H2O2 generated...
Aberrant matrix metalloproteinase-1 (MMP-1) expression contributes to the pathogenesis of many de... more Aberrant matrix metalloproteinase-1 (MMP-1) expression contributes to the pathogenesis of many degenerative disease processes that are associated with increased oxidative damage or stress. We and others have established that shifts in steady-state H2O2 production resulting from enforced antioxidant gene expression, senescence, or UV irradiation control MMP-1 expression. Here we establish that histone deacetylase-2 (HDAC2) protein levels and its occupancy of the MMP-1 promoter are decreased in response to enforced manganese superoxide dismutase (Sod2) expression. Inhibition of HDAC activity further accentuates the redox-dependent expression of MMP-1. Sod2-dependent decreases in HDAC2 are associated with increases in a proteasome-sensitive pool of ubiquitinylated HDAC2 and MMP-1-specific histone H3 acetylation. Sod2 overexpression also enhanced recruitment of Ets-1, c-Jun, c-Fos, and the histone acetyltransferase PCAF to the distal and proximal regions of the MMP-1 promoter. Furthermo...
Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer ... more Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer subtype. Targeted therapy for HGSOC is challenging as the disease is characterized by copy number variation and not somatic mutations. HGSOC displays well-conserved features such as expression of transcription factor PAX8. We propose employing therapies that capitalize on interactions between PAX8 and specific DNA-binding sites to develop therapeutic approaches to target HGSOC. Our hypothesis is to use a biomimetic approach wherein PAX8 is induced to transcribe an ectopically transfected CRISPR-base editor (BE)-guide RNA (gRNA) plasmid containing a PAX8 binding site in its promoter. The gRNA will impart a dominant-negative allele of ataxia telangiectasia and Rad3-related protein (ATR), which has been shown to be synthetically lethal with mutant p53 in cancer cells. In the presence of PAX8, the plasmid will be transcribed, while in the absence of PAX8, the plasmid is degraded, with minima...
Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogeni... more Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogenic potential but is accompanied by the often pathologically deleterious senescence-associated secretory phenotype (SASP). Here we demonstrate an H2O2-dependent functional disruption controlling senescence-associated Ca2+ homeostasis and the SASP. Senescent cells fail to respond to H2O2-dependent plasma lamellar Ca2+ entry when compared to pre-senescent cells. Limiting exposure to senescence-associated H2O2 restores H2O2-dependent Ca2+ entry as well as transient receptor potential cation channel subfamily C member 6 (TRPC6) function. SA-TRPC6 and SASP expression is blocked by restoring Ca2+ entry with the TRP channel antagonist SKF-96365 or by the mTOR inhibitors rapamycin and Ku0063794. Together, our findings provide compelling evidence that redox and mTOR-mediated regulation of Ca2+ entry through TRPC6 modulates SASP gene expression and approaches which preserve normal Ca2+ homeostasis m...
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Papers by Akshaya Chandrasekaran