- Email: [email protected]
DPP3 in NRF2 Signaling and Breast Cancer
(DEH), T32 HL076122 (ACL), and LCCRO Juckett Award.
doi: 10.1016/j.freeradbiomed.2016.10.345 305 High-Dose Ascorbate Administration Increases Tumor Ascorbate Levels and Decreases Hypoxia-Inducible Factor-1 Activity and Tumor Growth in Mice Elizabeth Campbell1, Margreet Vissers1, Christina Wohlrab1, Kevin Hicks2, Matthew Strother3, Bridget Robinson1,3, Stephanie Bozonet1, and Gabi Dachs1 1 University of Otago, Christchurch, New Zealand, 2University of Auckland, New Zealand, 3Christchurch Hospital, New Zealand The activation of the transcription factor hypoxia-inducible factor (HIF)-1 is associated with an aggressive tumor phenotype. HIF-1 is regulated by hydroxylase enzymes that require vitamin C (ascorbate) as cofactor and analyses of clinical tumor samples and dietary supplementation studies in mice have demonstrated an inverse correlation between tumor ascorbate levels and HIF-1 activity. In this study we investigated whether pharmacological doses of ascorbate can affect tumor ascorbate levels and exert an anti-tumor effect in tumor-bearing Gulo-/- mice, a model of human ascorbate dependency. Ascorbate concentrations peaked within an hour after a single intra-peritoneal injection (1g/kg), with millimolar levels measureable in plasma, and increased levels in liver and tumor tissues. Plasma and liver ascorbate returned to baseline within 16h, but tumor levels remained elevated for 48h. Modelling ascorbate uptake and stability in tumors suggests that increased stability under hypoxia may account for its prolonged presence within the tumor tissue. The expression of HIF-1 and its target proteins was down-regulated during the first 4h postinjection, and increased again after 16–48h. Increased tumor ascorbate levels could be maintained with repeated daily or alternate day administration. HIF-1 and vascular endothelial growth factor proteins were inversely correlated with tumor ascorbate and were significantly down-regulated in tumors when ascorbate was administered daily. Increased tumor ascorbate was associated with reduced microvessel density, tumor hypoxia and tumor growth. These results support the suppression of the hypoxic response as being a possible anti-tumor activity of ascorbate that may be useful in a clinical setting.
doi: 10.1016/j.freeradbiomed.2016.10.346 306 Preliminary Results of a Phase I Study of Pharmacological Ascorbate (P-AscH–) with Gemcitabine Chemoradiotherapy in Pancreatic Cancer Justin Wilkes1, Bryan Allen1, Daniel J Berg1, Brett Wagner1, Juan Du1, Kellie Bodeker1, Logan Ahmann1, Sandy Vollstedt1, Heather Brown1, Matthew Alexander1, and Joseph Cullen1 1 University of Iowa, Iowa City, USA
Trials.gov NCT0152890). Patients were given P-AscH– (50-100 g/daily), 5 days/week and Gemcitabine as prescribed per standard of care guidelines. Intensity modulated radiation therapy was also administered at either 50.4 Gy in 28 fractions or 50 Gy in 25 fractions. The primary endpoints were safety and tolerability of PAscH– other co-primary endpoints were dose-limiting toxicity and maximum tolerated dose. Since June, 2014, 14 patients have enrolled and 11 have completed therapy. Both 75 gram and 100 gram infusions achieved plasma ascorbate levels of 20 mM. The adverse event most commonly attributable to P-AscH– was dry mouth during infusion. No other adverse events were observed to be greater in intensity or frequency than with gemcitabine and radiation alone. Grade 3 or greater adverse events were hematologic in nature which occurred in all patients to varying severity. One P-AscH–-related toxicity was reported with noncardiac chest pain which did not alter ongoing treatment. Common grade 1 and 2 adverse events included chills, abdominal pain, dry mouth, fatigue, nausea, vomiting, and thrombocytopenia, all in similar frequency and intensity to chemoradiotherapy alone. Progression Free and overall survival is 10.8 months and 13.1 months, respectively, compared to 6.0 months and 11.1 months, for historical controls. Our preliminary results demonstrate that PAscH– in combination with gemcitabine/radiation is safe and tolerable.
doi: 10.1016/j.freeradbiomed.2016.10.347 307 DPP3 in NRF2 Signaling and Breast Cancer Kevin Lu1, Allen L Alcivar1, Jianglin Ma1, Tzeh Keong Foo1, Susan Zywea1, Yanying Huo1, Thomas W Kensler2, Michael L Gatza1, and Bing Xia1 1 Rutgers Cancer Institute of New Jersey, New Brunswick, USA, 2 University of Pittsburgh, USA NRF2 is a transcription factor serving as a master regulator of the expression of many genes involved in cellular responses to oxidative and other stresses. In the absence of stress, NRF2 is constantly synthesized but maintained at low levels as it is targeted by KEAP1 for ubiquitination and proteasome-mediated degradation. NRF2 binds KEAP1 mainly through a conserved “ETGE” motif that has also been found in several other proteins, such as DPP3, which has been shown to bind KEAP1 and enhance NRF2 function upon overexpression. Here we demonstrate the interaction between endogenous DPP3 and endogenous KEAP1. We further show that the DPP3-KEAP1 interaction is strongly induced by hydrogen peroxide and that DPP3 is required for hydrogen peroxide-induced NRF2 nuclear accumulation in the estrogen receptor (ER)-positive MCF7 breast cancer cells. Finally, we present evidence that DPP3 is overexpressed in breast cancer and that elevated levels of DPP3 mRNA correlate with increased NRF2 downstream gene expression and poor prognosis, particularly for ER-positive breast cancer. Our studies reveal novel insights into the regulation of NRF2 and identify DPP3 and an NRF2 transcriptional signature as potential biomarkers for breast cancer prognosis and treatment.
doi: 10.1016/j.freeradbiomed.2016.10.348
P-AscH– radiosensitizes pancreatic cancer (PDAC) in vitro and in vivo. The purpose of this Phase I trial is to determine the safety and tolerability of P-AscH- when combined with gemcitabine and radiation. Patients with locally advanced PDAC who were to undergo gemcitabine and radiotherapy were enrolled (Clinical
S132
SfRBM / SFRRI 2016
doi: 10.1016/j.freeradbiomed.2016.10.345 305 High-Dose Ascorbate Administration Increases Tumor Ascorbate Levels and Decreases Hypoxia-Inducible Factor-1 Activity and Tumor Growth in Mice Elizabeth Campbell1, Margreet Vissers1, Christina Wohlrab1, Kevin Hicks2, Matthew Strother3, Bridget Robinson1,3, Stephanie Bozonet1, and Gabi Dachs1 1 University of Otago, Christchurch, New Zealand, 2University of Auckland, New Zealand, 3Christchurch Hospital, New Zealand The activation of the transcription factor hypoxia-inducible factor (HIF)-1 is associated with an aggressive tumor phenotype. HIF-1 is regulated by hydroxylase enzymes that require vitamin C (ascorbate) as cofactor and analyses of clinical tumor samples and dietary supplementation studies in mice have demonstrated an inverse correlation between tumor ascorbate levels and HIF-1 activity. In this study we investigated whether pharmacological doses of ascorbate can affect tumor ascorbate levels and exert an anti-tumor effect in tumor-bearing Gulo-/- mice, a model of human ascorbate dependency. Ascorbate concentrations peaked within an hour after a single intra-peritoneal injection (1g/kg), with millimolar levels measureable in plasma, and increased levels in liver and tumor tissues. Plasma and liver ascorbate returned to baseline within 16h, but tumor levels remained elevated for 48h. Modelling ascorbate uptake and stability in tumors suggests that increased stability under hypoxia may account for its prolonged presence within the tumor tissue. The expression of HIF-1 and its target proteins was down-regulated during the first 4h postinjection, and increased again after 16–48h. Increased tumor ascorbate levels could be maintained with repeated daily or alternate day administration. HIF-1 and vascular endothelial growth factor proteins were inversely correlated with tumor ascorbate and were significantly down-regulated in tumors when ascorbate was administered daily. Increased tumor ascorbate was associated with reduced microvessel density, tumor hypoxia and tumor growth. These results support the suppression of the hypoxic response as being a possible anti-tumor activity of ascorbate that may be useful in a clinical setting.
doi: 10.1016/j.freeradbiomed.2016.10.346 306 Preliminary Results of a Phase I Study of Pharmacological Ascorbate (P-AscH–) with Gemcitabine Chemoradiotherapy in Pancreatic Cancer Justin Wilkes1, Bryan Allen1, Daniel J Berg1, Brett Wagner1, Juan Du1, Kellie Bodeker1, Logan Ahmann1, Sandy Vollstedt1, Heather Brown1, Matthew Alexander1, and Joseph Cullen1 1 University of Iowa, Iowa City, USA
Trials.gov NCT0152890). Patients were given P-AscH– (50-100 g/daily), 5 days/week and Gemcitabine as prescribed per standard of care guidelines. Intensity modulated radiation therapy was also administered at either 50.4 Gy in 28 fractions or 50 Gy in 25 fractions. The primary endpoints were safety and tolerability of PAscH– other co-primary endpoints were dose-limiting toxicity and maximum tolerated dose. Since June, 2014, 14 patients have enrolled and 11 have completed therapy. Both 75 gram and 100 gram infusions achieved plasma ascorbate levels of 20 mM. The adverse event most commonly attributable to P-AscH– was dry mouth during infusion. No other adverse events were observed to be greater in intensity or frequency than with gemcitabine and radiation alone. Grade 3 or greater adverse events were hematologic in nature which occurred in all patients to varying severity. One P-AscH–-related toxicity was reported with noncardiac chest pain which did not alter ongoing treatment. Common grade 1 and 2 adverse events included chills, abdominal pain, dry mouth, fatigue, nausea, vomiting, and thrombocytopenia, all in similar frequency and intensity to chemoradiotherapy alone. Progression Free and overall survival is 10.8 months and 13.1 months, respectively, compared to 6.0 months and 11.1 months, for historical controls. Our preliminary results demonstrate that PAscH– in combination with gemcitabine/radiation is safe and tolerable.
doi: 10.1016/j.freeradbiomed.2016.10.347 307 DPP3 in NRF2 Signaling and Breast Cancer Kevin Lu1, Allen L Alcivar1, Jianglin Ma1, Tzeh Keong Foo1, Susan Zywea1, Yanying Huo1, Thomas W Kensler2, Michael L Gatza1, and Bing Xia1 1 Rutgers Cancer Institute of New Jersey, New Brunswick, USA, 2 University of Pittsburgh, USA NRF2 is a transcription factor serving as a master regulator of the expression of many genes involved in cellular responses to oxidative and other stresses. In the absence of stress, NRF2 is constantly synthesized but maintained at low levels as it is targeted by KEAP1 for ubiquitination and proteasome-mediated degradation. NRF2 binds KEAP1 mainly through a conserved “ETGE” motif that has also been found in several other proteins, such as DPP3, which has been shown to bind KEAP1 and enhance NRF2 function upon overexpression. Here we demonstrate the interaction between endogenous DPP3 and endogenous KEAP1. We further show that the DPP3-KEAP1 interaction is strongly induced by hydrogen peroxide and that DPP3 is required for hydrogen peroxide-induced NRF2 nuclear accumulation in the estrogen receptor (ER)-positive MCF7 breast cancer cells. Finally, we present evidence that DPP3 is overexpressed in breast cancer and that elevated levels of DPP3 mRNA correlate with increased NRF2 downstream gene expression and poor prognosis, particularly for ER-positive breast cancer. Our studies reveal novel insights into the regulation of NRF2 and identify DPP3 and an NRF2 transcriptional signature as potential biomarkers for breast cancer prognosis and treatment.
doi: 10.1016/j.freeradbiomed.2016.10.348
P-AscH– radiosensitizes pancreatic cancer (PDAC) in vitro and in vivo. The purpose of this Phase I trial is to determine the safety and tolerability of P-AscH- when combined with gemcitabine and radiation. Patients with locally advanced PDAC who were to undergo gemcitabine and radiotherapy were enrolled (Clinical
S132
SfRBM / SFRRI 2016