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Targeting the Sphingosine-1-phosphate Signaling with FTY720 in Obesity Related Breast Cancer Progression
556
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
and cell lines. Apoptosis was confirmed by cleavage of caspases 3 and 7 and downstream PARP. Acetylated withanolides showed enhanced efficacy and induction of oxidation 30-50% above controls compared to 10-20% from non-acetylated withanolides (p<0.01). Biomarkers of oxidative stress,HSP32 and HSP70, were upregulated by 1mM for each agent with functional inhibition of Akt, mTOR, and p70 S6K by 1mM and complete inhibition by 3mM. Progressive induction of apoptosis with 0.3-3mM of all compounds was associated with phosphorylation of H2A.X that was completely abrogated by global caspase inhibitor pretreatment. Inhibition of known kinases for H2A.X including stress activators (JNK, p38, ERK) and DNA-damage response proteins (ATM, DNA-PK) failed to prevent phosphorylation.In vivo withanolide treatment yielded approximately 2- and 4-fold reduced tumor volumes 6 weeks post-treatment with non-acetylated and acetylated withanolides, respectively, without demonstrated toxicity by weight and histology. This was superior to standard cytotoxic therapy with cisplatin, doxorubicin, and docetaxel. Conclusions: Natural withanolides from Physalis are potent novel therapeutics against TNBC in vitro and in vivo and warrant further translation. This study uniquely defines their anticancer mechanism in TNBCs as primarily due to oxidation, caspase-dependent phosphorylation of H2A.X, and protein degradation that patterns HSP90 chaperone inhibition.
27.3. Targeting the Sphingosine-1-phosphate Signaling with FTY720 in Obesity Related Breast Cancer Progression. M. Nagahashi,1 A. Yamada,1 T. Aoyagi,1 W. Huang,1 J. C. Allegood,1 S. Milstien,1 S. Spiegel,1 K. Takabe1; 1 Virginia Commonwealth University - Surgical Oncology, Richmond, VA, USA Introduction: Obesity, the number one ranked health risk in the US by the CDC, is an established independent prognostic factor for breast cancer patients. While the link between obesity and worsened breast cancer mortality is well known, the underlying mechanisms are poorly understood. The pleiotropic bioactive lipid mediator sphingosine-1-phosphate (S1P) has emerged as a key regulatory molecule in cancer progression and inflammation. S1P is generated by two sphingosine kinases, SphK1 and SphK2, and exerts its functions by binding to specific G protein-coupled receptors (S1PR1-5). FTY720, a functional antagonist of S1P receptor-1 (S1PR1) when phosphorylated that was recently approved by FDA for multiple sclerosis, has been suggested to have anti-cancer actions. In this study, we found that obesity in animal breast cancer models increases SphK1 and levels of S1P in both tumor and its microenvironment. To examine the contribution of increased S1P to breast cancer progression and metastasis, mice were treated with FTY720 to disrupt the S1P signaling, which is strengthened by obesity. Methods: We utilized two different syngeneic breast cancer mouse models: 4T1-luc2 cells in BALB/c mice and E0771 cells in C57Bl/6 mice, both inoculated into mammary fat pads of mice fed with normal or high fat diets. FTY720 was given daily by gavage. Protein expression was determined by immunoblotting and levels of mRNA was determined by RT-qPCR. S1P and FTY720 were measured by mass spectrometry (LC-ESI-MS/MS). Results: We observed that breast tumors in obese animals were significantly larger, and expressed higher levels of SphK1 and S1P transporters, such as ABCC1 and Spns2, as compared to animals on a normal diet. The levels of S1P not only in the tumor, but also in the tumor interstitial fluid and serum were higher in the obese mice than in the lean mice, which appears to be a consequence of the higher production of S1P by SphK1 in the tumor and its microenvironment. FTY720 administration significantly suppressed tumor progression in both syngeneic mouse models. Interestingly, the cancer progression was suppressed more efficiently in the obese mice than the lean mice. LC-ESI-MS/MS analysis revealed that the ratio of FTY720-phosphate/FTY720 was higher in the obese mice than in the lean mice, which suggests that the higher SphK activity in the obese mice may activate more FTY720. Further, FTY720 decreased S1P levels not only in the blood, but also in the tumor and tumor interstitial fluid.
Conclusions: These results suggest that the S1P axis is strengthened by obesity, and has a role in cancer progression. Targeting the S1P axis with FTY720 may be useful for treating breast cancer in individuals with obesity. M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. This work was supported by NIH grants R37GM043880, R01CA61774 (to S.S.) and R01CA160688 (to K.T.).
27.4. Predictors of Hemodynamic Instability During Surgery for Pheochromocytoma. C. M. Kiernan,1 K. Fan,1 J. T. Broome,1 C. Shi,1 M. F. Peters,1 C. C. Solorzano1; 1Vanderbilt University Medical Center, Nashville, TN, USA Introduction: Resection of adrenal pheochromocytoma is often associated with hemodynamic instability (HDI). We examined patient and tumor factors that may influence HDI. The effect of pre-treatment with the traditional non-selective alpha-blocker phenoxybenzamine (PXB) vs. selective alpha-blockers (doxazosin, terazosin, prazosin) on HDI and patient outcomes was also evaluated. Methods: The records of eighty-six patients who underwent operations at a single academic center between 2002 and 2012 were retrospectively reviewed. Parameters studied were: patient demographics, catecholamine concentration, tumor size, blockade-type and duration, operative approach and presence of familial syndrome. HDI was determined by: the number and time periods the intraoperative systolic blood pressure (SBP) was greater or less than 200mmHg, the SBP was greater or less than 30% baseline, the heart rate (HR) was >110 or <50bpm, the need for postoperative fluids and/or pressors, and the degree of intraoperative blood loss (EBL). Chi-square, Fishers exact, Wilcoxon rank sum and Pearson test were performed. P<0.05 was considered significant. Results: The degree of preoperative metanephrine elevation was associated with a higher number of episodes of SBP>200mmHg (OR 1.23, CI 1.09-1.39, p¼0.001), episodes of SBP<30% baseline (OR1.04, CI 1.02-1.07, p¼0.001), episodes of HR>110bpm (OR 1.21, CI 1.14-1.28, p<0.001), episodes of HR<50bmp (OR 1.13, CI 1.03-1.24, p<0.001) and increased postoperative-pressor requirement (OR 1.76, CI 1.10-2.83, p¼0.019). Similar significant findings were observed for increasing tumor size (increased episodes of SBP>200mmHg and HR>110bpm and increased postoperative-pressor requirements). Of the 86 patients, 78% received PXB, 17% selective-alpha-blockers and 5% were not blocked. Patient demographics, co-morbidities, tumor factors and surgical approach to the adrenal gland were similar among the blockade groups. Patients who received selective alpha-blockers were more likely to experience a greater number and longer episodes of SBP >200mmHg (p ¼0.008) but there were no significant differences in other intraoperative HDI measurements, complications or readmission rates when compared to PXB. Conclusions: Preoperative catecholamine elevation and tumor size are associated with intraoperative HDI as determined by significant changes in intraoperative SBP, HR, and postoperative pressor requirements. Preoperative selective blockade is an effective and safe alternative to the classically used non-selective alpha-blocker phenoxybenzamine for adrenal-based pheochromocytoma resection. Selective alpha-blockers are more readily available and have a favorable administration schedule and sideeffect profile.
27.5. Primary Hyperparathyroidism in Young Patients. H. Wachtel,1 M. Gupta,1 E. K. Bartlett,1 R. R. Kelz,1 G. C. Karakousis,1 D. L. Fraker1; 1Hospital Of The University Of Pennsylvania - Department Of Surgery, Philadelphia, PA, USA Introduction: Primary hyperparathyroidism (PHPT) affects approximately 2% of Americans over the age of 55; it is less common in younger patients. Prior studies suggest that pediatric PHPT patients have higher rates of multiglandular disease (MGD) when compared to adults. Since patients with MGD are not eligible for minimally
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
and cell lines. Apoptosis was confirmed by cleavage of caspases 3 and 7 and downstream PARP. Acetylated withanolides showed enhanced efficacy and induction of oxidation 30-50% above controls compared to 10-20% from non-acetylated withanolides (p<0.01). Biomarkers of oxidative stress,HSP32 and HSP70, were upregulated by 1mM for each agent with functional inhibition of Akt, mTOR, and p70 S6K by 1mM and complete inhibition by 3mM. Progressive induction of apoptosis with 0.3-3mM of all compounds was associated with phosphorylation of H2A.X that was completely abrogated by global caspase inhibitor pretreatment. Inhibition of known kinases for H2A.X including stress activators (JNK, p38, ERK) and DNA-damage response proteins (ATM, DNA-PK) failed to prevent phosphorylation.In vivo withanolide treatment yielded approximately 2- and 4-fold reduced tumor volumes 6 weeks post-treatment with non-acetylated and acetylated withanolides, respectively, without demonstrated toxicity by weight and histology. This was superior to standard cytotoxic therapy with cisplatin, doxorubicin, and docetaxel. Conclusions: Natural withanolides from Physalis are potent novel therapeutics against TNBC in vitro and in vivo and warrant further translation. This study uniquely defines their anticancer mechanism in TNBCs as primarily due to oxidation, caspase-dependent phosphorylation of H2A.X, and protein degradation that patterns HSP90 chaperone inhibition.
27.3. Targeting the Sphingosine-1-phosphate Signaling with FTY720 in Obesity Related Breast Cancer Progression. M. Nagahashi,1 A. Yamada,1 T. Aoyagi,1 W. Huang,1 J. C. Allegood,1 S. Milstien,1 S. Spiegel,1 K. Takabe1; 1 Virginia Commonwealth University - Surgical Oncology, Richmond, VA, USA Introduction: Obesity, the number one ranked health risk in the US by the CDC, is an established independent prognostic factor for breast cancer patients. While the link between obesity and worsened breast cancer mortality is well known, the underlying mechanisms are poorly understood. The pleiotropic bioactive lipid mediator sphingosine-1-phosphate (S1P) has emerged as a key regulatory molecule in cancer progression and inflammation. S1P is generated by two sphingosine kinases, SphK1 and SphK2, and exerts its functions by binding to specific G protein-coupled receptors (S1PR1-5). FTY720, a functional antagonist of S1P receptor-1 (S1PR1) when phosphorylated that was recently approved by FDA for multiple sclerosis, has been suggested to have anti-cancer actions. In this study, we found that obesity in animal breast cancer models increases SphK1 and levels of S1P in both tumor and its microenvironment. To examine the contribution of increased S1P to breast cancer progression and metastasis, mice were treated with FTY720 to disrupt the S1P signaling, which is strengthened by obesity. Methods: We utilized two different syngeneic breast cancer mouse models: 4T1-luc2 cells in BALB/c mice and E0771 cells in C57Bl/6 mice, both inoculated into mammary fat pads of mice fed with normal or high fat diets. FTY720 was given daily by gavage. Protein expression was determined by immunoblotting and levels of mRNA was determined by RT-qPCR. S1P and FTY720 were measured by mass spectrometry (LC-ESI-MS/MS). Results: We observed that breast tumors in obese animals were significantly larger, and expressed higher levels of SphK1 and S1P transporters, such as ABCC1 and Spns2, as compared to animals on a normal diet. The levels of S1P not only in the tumor, but also in the tumor interstitial fluid and serum were higher in the obese mice than in the lean mice, which appears to be a consequence of the higher production of S1P by SphK1 in the tumor and its microenvironment. FTY720 administration significantly suppressed tumor progression in both syngeneic mouse models. Interestingly, the cancer progression was suppressed more efficiently in the obese mice than the lean mice. LC-ESI-MS/MS analysis revealed that the ratio of FTY720-phosphate/FTY720 was higher in the obese mice than in the lean mice, which suggests that the higher SphK activity in the obese mice may activate more FTY720. Further, FTY720 decreased S1P levels not only in the blood, but also in the tumor and tumor interstitial fluid.
Conclusions: These results suggest that the S1P axis is strengthened by obesity, and has a role in cancer progression. Targeting the S1P axis with FTY720 may be useful for treating breast cancer in individuals with obesity. M.N. is a Japan Society for the Promotion of Science Postdoctoral Fellow. This work was supported by NIH grants R37GM043880, R01CA61774 (to S.S.) and R01CA160688 (to K.T.).
27.4. Predictors of Hemodynamic Instability During Surgery for Pheochromocytoma. C. M. Kiernan,1 K. Fan,1 J. T. Broome,1 C. Shi,1 M. F. Peters,1 C. C. Solorzano1; 1Vanderbilt University Medical Center, Nashville, TN, USA Introduction: Resection of adrenal pheochromocytoma is often associated with hemodynamic instability (HDI). We examined patient and tumor factors that may influence HDI. The effect of pre-treatment with the traditional non-selective alpha-blocker phenoxybenzamine (PXB) vs. selective alpha-blockers (doxazosin, terazosin, prazosin) on HDI and patient outcomes was also evaluated. Methods: The records of eighty-six patients who underwent operations at a single academic center between 2002 and 2012 were retrospectively reviewed. Parameters studied were: patient demographics, catecholamine concentration, tumor size, blockade-type and duration, operative approach and presence of familial syndrome. HDI was determined by: the number and time periods the intraoperative systolic blood pressure (SBP) was greater or less than 200mmHg, the SBP was greater or less than 30% baseline, the heart rate (HR) was >110 or <50bpm, the need for postoperative fluids and/or pressors, and the degree of intraoperative blood loss (EBL). Chi-square, Fishers exact, Wilcoxon rank sum and Pearson test were performed. P<0.05 was considered significant. Results: The degree of preoperative metanephrine elevation was associated with a higher number of episodes of SBP>200mmHg (OR 1.23, CI 1.09-1.39, p¼0.001), episodes of SBP<30% baseline (OR1.04, CI 1.02-1.07, p¼0.001), episodes of HR>110bpm (OR 1.21, CI 1.14-1.28, p<0.001), episodes of HR<50bmp (OR 1.13, CI 1.03-1.24, p<0.001) and increased postoperative-pressor requirement (OR 1.76, CI 1.10-2.83, p¼0.019). Similar significant findings were observed for increasing tumor size (increased episodes of SBP>200mmHg and HR>110bpm and increased postoperative-pressor requirements). Of the 86 patients, 78% received PXB, 17% selective-alpha-blockers and 5% were not blocked. Patient demographics, co-morbidities, tumor factors and surgical approach to the adrenal gland were similar among the blockade groups. Patients who received selective alpha-blockers were more likely to experience a greater number and longer episodes of SBP >200mmHg (p ¼0.008) but there were no significant differences in other intraoperative HDI measurements, complications or readmission rates when compared to PXB. Conclusions: Preoperative catecholamine elevation and tumor size are associated with intraoperative HDI as determined by significant changes in intraoperative SBP, HR, and postoperative pressor requirements. Preoperative selective blockade is an effective and safe alternative to the classically used non-selective alpha-blocker phenoxybenzamine for adrenal-based pheochromocytoma resection. Selective alpha-blockers are more readily available and have a favorable administration schedule and sideeffect profile.
27.5. Primary Hyperparathyroidism in Young Patients. H. Wachtel,1 M. Gupta,1 E. K. Bartlett,1 R. R. Kelz,1 G. C. Karakousis,1 D. L. Fraker1; 1Hospital Of The University Of Pennsylvania - Department Of Surgery, Philadelphia, PA, USA Introduction: Primary hyperparathyroidism (PHPT) affects approximately 2% of Americans over the age of 55; it is less common in younger patients. Prior studies suggest that pediatric PHPT patients have higher rates of multiglandular disease (MGD) when compared to adults. Since patients with MGD are not eligible for minimally