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Modulation of mitochondrial fission activity maintains ovarian tumor initiating cells dependent on mitochondrial energetics
S74
A. Newman / Free Radical Biology and Medicine 128 (2018) S61–S78
PRDX1 with doses up to 10 Gy found Prdx1-/- MEFs were more sensitive to IR and exhibited reduced homologous recombination in DRGFP reporter assays. Prdx1 -/- MEFs also displayed enhanced DNA damage as measured by nuclear ɣH2AX phosphorylation and reduced RAD51 foci formation following IR. As PRDX1 has been previously identified to bind proteins under heightened oxidative insult, immunoprecipitation assays were conducted, which found PRDX1 directly bound RAD51 with increasing IR. Oxidation of cysteine residues to the sulfenic acid form was specifically probed with DAZ2 and loss of PRDX1 enhanced sulfenylation of RAD51. Site-directed mutagenesis revealed C319 of RAD51 as a primary target of oxidation. Cells harboring overexpression of mutant RAD51 C319 revealed suppression of HR, IR-induced RAD51 foci formation, RAD51 enhanced malignancy in an anchorage-independent growth soft agar colony formation and proliferation. MDA-MB-231 breast cancer cells deficient of PRDX1 had decreased IR-induced RAD51 foci formation and were more sensitive to doxorubicin. In conclusion, PRDX1 binds RAD51 during heighted oxidative conditions and protects the functionally important RAD51 residue C319 from oxidation.
https://doi.org/10.1016/j.freeradbiomed.2018.10.159
156
Modulation of mitochondrial fission activity maintains ovarian tumor initiating cells dependent on mitochondrial energetics Brian Spurlock*, Danitra Parker, Malay Basu, Anita Hjelmeland, Kasturi Mitra University of Alabama at Birmingham, USA
157
Overcoming cell adhesion-mediated drug resistance (CAM-DR) in multiple myeloma by targeting possible crosstalk between the thioredoxin system and mucins Jun Hui (Samantha) Sze* Griffith University, Australia
Multiple myeloma (MM) is a clonal plasma B-cell neoplasm that resides in the bone marrow (BM) cavity. While in the BM, the cells interact with BM stromal cells resulting in the secretion of chemokines and growth factors as well as expression of cell adhesion molecules that allow the myeloma cells to migrate to secondary BM sites where they eventually invade and proliferate. These events allow the cells to not only disseminate but also to become chemo-resistant to MM drugs through cell-adhesion mediated drug resistance (CAM-DR). The thioredoxin (Trx) system is one of the major cellular antioxidant systems involved in maintaining redox homeostasis in the cell. Studies have shown that the Trx system system redox-regulates a large number of transcription factors including NF-κβ, p53 and PTEN and is responsible for increased cancer cell proliferation, survival and chemo-resistance. Although inhibition of the Trx system has been shown to re-sensitise bortezomib-resistant MM cells, the effects of its redox activity to overcome CAM-DR remains to be fully elucidated. Mucins are a group of glycoproteins and their functions range from establishing molecular barriers for epithelial surfaces to signal transduction. In addition the mucins are able to mediate cell adhesion properties in cancer cells. Mucins are aberrantly expressed in MM and are also involved in the regulation of ROS. These findings suggest a possibility of crosstalk between the redox systems and mucins that may be involved in the pathogenesis of MM. This project aims to discover the possible crosstalk between the Trx system and mucins with respect to CAM-DR in MM.
https://doi.org/10.1016/j.freeradbiomed.2018.10.161 Self-renewing, therapy resistant ovarian tumor initiating cells (ovTICs) clearly contribute to development and recurrence of epithelial ovarian cancer (EOC), and recurrent EOC is incurable. Depending on the genetic and physiological context of a patient ovTICs can be dependent on mitochondrial energetics, specifically ATP production and redox homeostasis. Mitochondrial energetics can be repressed through fragmentation of mitochondria through elevated activity of mitochondrial fission protein Dynamin-related protein 1 (Drp1). Conversely, repression of Drp1 activity can augment mitochondrial energetics. Drp1 has also been implicated in maintenance of some stem cell lineages. However, Drp1 regulation of the stem-like ovTICs and the impact of fission activity on the mitochondrial energetics of these cells have not been well characterized. Hypothesis: Modulation of mitochondrial energetics by fission, likely mediated by cyclic changes in Drp1 activity, maintains undifferentiated ovTICs. We studied mitochondrial properties in a derived chemoresistant EOC cell line previously shown to be enriched for ovTICs compared to its parental line. We found that the chemoresistant line has boosted mitochondrial energetics and repressed Drp1 activity compared to its parental line. We then FACS-sorted the chemoresistant line based on a TIC marker (Aldh) and mitochondrial membrane potential (ΔΨ) into 4 populations (Aldh-ΔΨlo, Aldh-ΔΨhi, Aldh þ ΔΨlo, and Aldh þ ΔΨhi). The sorted Aldh þ populations are dependent on mitochondrial energetics and cycle between ΔΨhi and ΔΨlo energetic states as they form in vitro tumorspheres, unlike the Aldh- populations. These energetic states have characteristic mitochondrial fission activity. Importantly, regulation of ATP production or redox state by fission is mutually exclusive for each energetic state. Understanding how fission can modulate the mitochondrial energetics of ovTICs that depend on mitochondria for ATP production and/or redox homeostasis will allow us to better identify and directly target TICs.
https://doi.org/10.1016/j.freeradbiomed.2018.10.160
158
Mn porphyrin/ascorbate sensitizes serous epithelial ovarian cancer to chemotherapy Ines Batinic-Haberle, Artak Tovmasyan*, Ashley Veade, Ivan Spasojevic, Shara Reihani, Angeles Secord Duke University School of Medicine, USA
Ovarian cancer is the most lethal gynecologic malignancy. Even with aggressive frontline treatment, the majority of patients with advanced disease will develop a recurrence within the first 3 years and ultimately die of disease. Serous epithelial ovarian cancers [high-grade serous ovarian cancers(HGSC) and low-grade serous ovarian cancers(LGSC)] are especially challenging to treat due to primary and/or secondary chemoresistance that ultimately result in death. Oxidative stress plays important role in the carcinogenesis, cancer progression and metastases. Mn porphyrin(MnP)based SOD mimics, when combined with ascorbate (Asc) and/or radiation (RT), largely suppress 4T1 mouse breast tumor growth through MnP-catalyzed H2O2/GSH-mediated oxidative modification of signaling proteins, with NF-kB/TAB3/p38MAPK/p38α(MAPK14)/HSP60 as a key S-glutathionylated network. Here we extended our studies of MnP/Asc tumor therapy to ovarian tumor. Genomic RNA microarray data from HGSC patients point to the key role of Bcl-2, which is transcriptionally regulated by NF-kB. Bcl-2
A. Newman / Free Radical Biology and Medicine 128 (2018) S61–S78
PRDX1 with doses up to 10 Gy found Prdx1-/- MEFs were more sensitive to IR and exhibited reduced homologous recombination in DRGFP reporter assays. Prdx1 -/- MEFs also displayed enhanced DNA damage as measured by nuclear ɣH2AX phosphorylation and reduced RAD51 foci formation following IR. As PRDX1 has been previously identified to bind proteins under heightened oxidative insult, immunoprecipitation assays were conducted, which found PRDX1 directly bound RAD51 with increasing IR. Oxidation of cysteine residues to the sulfenic acid form was specifically probed with DAZ2 and loss of PRDX1 enhanced sulfenylation of RAD51. Site-directed mutagenesis revealed C319 of RAD51 as a primary target of oxidation. Cells harboring overexpression of mutant RAD51 C319 revealed suppression of HR, IR-induced RAD51 foci formation, RAD51 enhanced malignancy in an anchorage-independent growth soft agar colony formation and proliferation. MDA-MB-231 breast cancer cells deficient of PRDX1 had decreased IR-induced RAD51 foci formation and were more sensitive to doxorubicin. In conclusion, PRDX1 binds RAD51 during heighted oxidative conditions and protects the functionally important RAD51 residue C319 from oxidation.
https://doi.org/10.1016/j.freeradbiomed.2018.10.159
156
Modulation of mitochondrial fission activity maintains ovarian tumor initiating cells dependent on mitochondrial energetics Brian Spurlock*, Danitra Parker, Malay Basu, Anita Hjelmeland, Kasturi Mitra University of Alabama at Birmingham, USA
157
Overcoming cell adhesion-mediated drug resistance (CAM-DR) in multiple myeloma by targeting possible crosstalk between the thioredoxin system and mucins Jun Hui (Samantha) Sze* Griffith University, Australia
Multiple myeloma (MM) is a clonal plasma B-cell neoplasm that resides in the bone marrow (BM) cavity. While in the BM, the cells interact with BM stromal cells resulting in the secretion of chemokines and growth factors as well as expression of cell adhesion molecules that allow the myeloma cells to migrate to secondary BM sites where they eventually invade and proliferate. These events allow the cells to not only disseminate but also to become chemo-resistant to MM drugs through cell-adhesion mediated drug resistance (CAM-DR). The thioredoxin (Trx) system is one of the major cellular antioxidant systems involved in maintaining redox homeostasis in the cell. Studies have shown that the Trx system system redox-regulates a large number of transcription factors including NF-κβ, p53 and PTEN and is responsible for increased cancer cell proliferation, survival and chemo-resistance. Although inhibition of the Trx system has been shown to re-sensitise bortezomib-resistant MM cells, the effects of its redox activity to overcome CAM-DR remains to be fully elucidated. Mucins are a group of glycoproteins and their functions range from establishing molecular barriers for epithelial surfaces to signal transduction. In addition the mucins are able to mediate cell adhesion properties in cancer cells. Mucins are aberrantly expressed in MM and are also involved in the regulation of ROS. These findings suggest a possibility of crosstalk between the redox systems and mucins that may be involved in the pathogenesis of MM. This project aims to discover the possible crosstalk between the Trx system and mucins with respect to CAM-DR in MM.
https://doi.org/10.1016/j.freeradbiomed.2018.10.161 Self-renewing, therapy resistant ovarian tumor initiating cells (ovTICs) clearly contribute to development and recurrence of epithelial ovarian cancer (EOC), and recurrent EOC is incurable. Depending on the genetic and physiological context of a patient ovTICs can be dependent on mitochondrial energetics, specifically ATP production and redox homeostasis. Mitochondrial energetics can be repressed through fragmentation of mitochondria through elevated activity of mitochondrial fission protein Dynamin-related protein 1 (Drp1). Conversely, repression of Drp1 activity can augment mitochondrial energetics. Drp1 has also been implicated in maintenance of some stem cell lineages. However, Drp1 regulation of the stem-like ovTICs and the impact of fission activity on the mitochondrial energetics of these cells have not been well characterized. Hypothesis: Modulation of mitochondrial energetics by fission, likely mediated by cyclic changes in Drp1 activity, maintains undifferentiated ovTICs. We studied mitochondrial properties in a derived chemoresistant EOC cell line previously shown to be enriched for ovTICs compared to its parental line. We found that the chemoresistant line has boosted mitochondrial energetics and repressed Drp1 activity compared to its parental line. We then FACS-sorted the chemoresistant line based on a TIC marker (Aldh) and mitochondrial membrane potential (ΔΨ) into 4 populations (Aldh-ΔΨlo, Aldh-ΔΨhi, Aldh þ ΔΨlo, and Aldh þ ΔΨhi). The sorted Aldh þ populations are dependent on mitochondrial energetics and cycle between ΔΨhi and ΔΨlo energetic states as they form in vitro tumorspheres, unlike the Aldh- populations. These energetic states have characteristic mitochondrial fission activity. Importantly, regulation of ATP production or redox state by fission is mutually exclusive for each energetic state. Understanding how fission can modulate the mitochondrial energetics of ovTICs that depend on mitochondria for ATP production and/or redox homeostasis will allow us to better identify and directly target TICs.
https://doi.org/10.1016/j.freeradbiomed.2018.10.160
158
Mn porphyrin/ascorbate sensitizes serous epithelial ovarian cancer to chemotherapy Ines Batinic-Haberle, Artak Tovmasyan*, Ashley Veade, Ivan Spasojevic, Shara Reihani, Angeles Secord Duke University School of Medicine, USA
Ovarian cancer is the most lethal gynecologic malignancy. Even with aggressive frontline treatment, the majority of patients with advanced disease will develop a recurrence within the first 3 years and ultimately die of disease. Serous epithelial ovarian cancers [high-grade serous ovarian cancers(HGSC) and low-grade serous ovarian cancers(LGSC)] are especially challenging to treat due to primary and/or secondary chemoresistance that ultimately result in death. Oxidative stress plays important role in the carcinogenesis, cancer progression and metastases. Mn porphyrin(MnP)based SOD mimics, when combined with ascorbate (Asc) and/or radiation (RT), largely suppress 4T1 mouse breast tumor growth through MnP-catalyzed H2O2/GSH-mediated oxidative modification of signaling proteins, with NF-kB/TAB3/p38MAPK/p38α(MAPK14)/HSP60 as a key S-glutathionylated network. Here we extended our studies of MnP/Asc tumor therapy to ovarian tumor. Genomic RNA microarray data from HGSC patients point to the key role of Bcl-2, which is transcriptionally regulated by NF-kB. Bcl-2