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Aurora A kinase regulates N-myc activity through phosphorylation of GSK3-beta and beta-catenin
PEDIATRIC SURGERY II METHODS: After IACUC approval, mice were assigned to four groups: control, lysate vaccine, lysate vaccine with CpG and tumor challenge. Vaccine groups received heat shocked NB lysate with and without CpG and were boosted at two weeks. The tumor challenge group was inoculated with live NB cells. Spleens were harvested two weeks after the boost or when the tumor reached 20 mm in size in the challenge group. Splenocytes were subsequently stimulated in-vitro with Survivin peptides and response determined by IFN-gamma levels. CD69 staining was performed to confirm T-cell activation.
Sequential VEGF and EGFR inhibition improves survival in a neuroblastoma model Artur Chernoguz MD, Kelly M Crawford BS, Timothy P Cripe MD, PhD, Jason S Frischer MD, FACS Cincinnati Children’s Hospital Medical Center, Cincinnati, OH INTRODUCTION: Neuroblastoma, a common pediatric malignancy, is often resistant to multimodal therapy. Anti-angiogenic therapy, primarily focused on Vascular Endothelial Growth Factor (VEGF) inhibition is prone to rapid development of resistance. Based on observations of its increased expression in VEGF therapyresistant neuroblastoma, we hypothesized that Epidermal Growth Factor Receptor (EGFR) blockade would contribute to tumor regression by suppressing angiogenesis.
RESULTS: IFN-gamma response to Survivin was significantly higher in vaccinated mice compared to control (p⫽0.002) and tumor bearing mice (p⫽0.023). This response was amplified when CpG was added to the vaccine (p⫽0.007). CD69 staining showed a substantial rise of activated lymphocytes in the vaccinated group.
METHODS: SH-SY5Y neuroblastoma cells were incubated with gradient concentrations of gefitinib (EGFR-inhibitor) or bevacizumab (anti-VEGF antibody) for in vitro cytotoxicity assays. Xenografts were established by intrarenal SH-SY5Y cell injections in athymic mice (n⫽76). Animals with established tumors received gefitinib, bevacizumab, combination, or placebo for 20-days. Cohorts were weight-matched and re-assigned to receive a complementary therapy for 50-days. Analysis of microvessel and perivascular compartments was performed. Expression of pro-angiogenic signals was determined using qPCR.
CONCLUSIONS: In-vitro response to Survivin is a marker of effective in-vivo priming of CD8⫹ T-cells against NB vaccine. This immune response is greatly enhanced by the adjuvant effect of TLR9 stimulation. These findings present a valuable method for tracking T-cell responses to vaccine strategies against neuroblastoma.
Aurora A kinase regulates N-myc activity through phosphorylation of GSK3-beta and beta-catenin Nadja C Colon MD, Cameron E Schlegel BS, Jingbo Qiao PhD, Dai H Chung MD, FACS Vanderbilt University Medical Center, Nashville, TN
RESULTS: Gefitinib and bevacizumab had a limited cytostatic effect (⬍50%) on SH-SY5Y cells. Interval addition of gefitinib to bevacizumab conferred the longest median survival compared to bevacizumab (p⫽0.03), gefitinib (p⫽0.01), or combination (p⫽0.01). Refractory increase in tumor microvessel density caused by prolonged exposure to bevacizumab was offset by the addition of gefitinib (bevacizumab: 2,2735⫾3,178sq.micrometer vs. Bevacizumab-to-Combination: 11,620⫾1,991sq.micrometer; p⫽0.04). This regimen preserved relative immaturity of tumor vessels, consistent with apoptotic changes observed in pericytes exposed to gefitinib. Levels of human, but not murine VEGF mRNA, correlated with EGFR expression (p⫽0.02).
INTRODUCTION: Aurora A kinase (AurkA) is a serine/threonine kinase required for G2/M phase progression and centrosome maturation. Overexpression promotes aneuploidy and malignant transformation through unknown mechanisms. As a negative prognostic factor in neuroblastoma, AurkA has been shown to directly stabilize N-myc to prevent its ubiquitin-mediated degradation. We hypothesized that N-myc expression is further induced by AurkA’s downregulation of GSK3-beta with subsequent upregulation of betacatenin and its downstream targets.
CONCLUSIONS: EGFR inhibition augments anti-angiogenic therapy by targeting pericytes. This strategy may be a useful adjunct to current neuroblastoma treatment and is more effective when administered after a prolonged anti-VEGF therapy course.
METHODS: Human neuroblastoma BE(2)-C cells were transientlyand stably-transfected with plasmid to overexpress and silence AurkA, respectively. Western blot was used to determine alterations in protein levels of N-myc, GSK3-beta, phospho-GSK3-beta, betacatenin, and phospho-beta-catenin. Immunofluorescence and immunoprecipitation studies will ascertain AurkA and GSK3-beta colocalization and complex formation. Cell proliferation and anchorage-independent growth will be measured by BrdU and soft agar assays, while a luciferase assay will assess transcriptional activity of beta-catenin. Treatment with a PI3K/Akt inhibitor will ensure findings are a result of altering AurkA, and overexpression of GSK3beta will be used to rescue the wild-type phenotype.
CD8ⴙ T-cell response to survivin is a marker of effective immune priming in a neuroblastoma vaccine strategy Felix C Blanco MD, Priya Srinivasan PhD, Ryan M Walk MD, Jason A Snyder MD, Lina Chakrabarti PhD, Stanislav Vukmanovic MD, PhD, Anthony D Sandler MD, FACS Children’s National Medical Center, Washington, DC INTRODUCTION: Survivin, an inhibitor of apoptosis protein promotes proliferation and progression of neuroblastoma (NB) and is a potential target for immunotherapy as it is expressed primarily in tumors. This study was designed to investigate whether mice vaccinated with NB lysate were able to induce a CD8⫹ T-cell response against Survivin and compared it with that of naïve and tumor bearing mice. In addition, we studied whether CpG-oligonucleotide, a TLR9-agonist could enhance this response.
© 2011 by the American College of Surgeons Published by Elsevier Inc.
RESULTS: AurkA silencing resulted in significant decreases in phospho-GSK3-beta and beta-catenin protein levels relative to GSK3-beta and phospho-beta-catenin. There was also a decrease in N-myc protein levels. Western blot and RT-PCR confirmed overexpression and silencing.
S82
ISSN 1072-7515/11/$36.00 doi:10.1016/j.jamcollsurg.2011.06
Vol. 213, No. 3S, September 2011
CONCLUSIONS: Our results suggest that in neuroblastoma, AurkA inactivates GSK3-beta via phosphorylation to promote nuclear translocation of active beta-catenin and induction of N-myc transcription. This provides insight into the mechanism by which in vivo studies on the efficacy of Aurora A kinase inhibitors have expedited current clinical trials.
Phosphatidylinositol 3-kinase-independent rapid activation of vascular endothelial growth factor in neuroblastoma Cameron E Schlegel BS, Jingbo Qiao PhD, Nadja C Colon MD, Dai H Chung MD, FACS Vanderbilt University Medical Center, Nashville, TN INTRODUCTION: Activation of gastrin-releasing peptide (GRP) receptor-mediated cell signaling leads to increased neuroblastoma cell growth, invasion, and metastasis. Neovascularization has been associated with stimulation of GRP receptor signaling, yet the mechanism and kinetics of this tumorigenic function has not been fully elucidated. We sought to determine the role of GRP receptor in the regulation of vascular endothelial growth factor (VEGF) in neuroblastoma. METHODS: Human neuroblastoma cell lines, SK-N-SH and BE(2)-C, were treated with GRP with or without inhibitor (LY294002) of PI3K, a critical cell survival pathway. We also used Actinomycin-D to further examine the effects on transcriptional regulation of VEGF. Cell supernatant and lysates were collected over a time course. ELISA was performed to determine VEGF levels. Immunoblotting was used to assess for the critical role of vascularrelated proteins. RESULTS: There was an increase in VEGF levels at 30 min after GRP treatment; this returned to baseline, followed by a delayed VEGF peak. Interestingly, levels of hypoxia-inducing factor (HIF)1alpha, a transcription factor for VEGF, also increased at 2-24 hrs after GRP. LY294002 compound had no effects on 30 min VEGF peak after GRP; however, it significantly attenuated delayed activation of HIF1-alpha and VEGF secretion. Actinomycin D inhibited the HIF1-alpha-dependent second peak of VEGF activation. CONCLUSIONS: We describe a novel, biphasic stimulation of VEGF by GRP receptor signaling in neuroblastoma. Further, GRP induces a delayed activation of HIF1-alpha and VEGF production through non-hypoxic pathways in PI3K/Akt-dependent manner. GRP serves a crucial role in the tumorigenesis of neuroblastoma and may provide a target for antiangiogenic therapy.
Dysregulation of upstream and downstream TGF-beta transcripts in livers of children with biliary atresia and fibrogenic gene signatures Tatiana Iordanskaia PhD, Monica J Hubal PhD, Evan P Nadler MD, FACS Children’s National Medical Center, Washington, DC and The George Washington University School of Medicine & Health Sciences, Washington, DC
Surgical Forum Abstracts
S83
INTRODUCTION: Our previous work has shown that the transforming-growth factor (TGF) beta pathway plays a central role in liver fibrosis associated with experimental biliary atresia (BA). To confirm these findings in humans, we performed an in silico microarray analysis of liver specimens from children with BA with the hypothesis that mediators of the TGF beta pathway would be dysregualted in patients with fibrotic gene signatures. METHODS: Liver specimens were obtained from 47 infants with BA at portoenterostomy in a prospective study published by Moyer et al. (Genome Med, 2010). Total RNA was profiled using Affymetrix Human 133 plus 2.0 microarrays. We used the publicly available image (CEL) files and meta data to compare gene expression differences between the fibrogenic and inflammatory cohorts predicted by the study. ANOVA was performed using Partek Genomics Suite. RESULTS: We focused on 24 TGF beta-related transcripts. Expression for procollagen transcripts assessed was increased in the fibrogenic group (1.2-fold to 1.36 fold). Expression of matrix metalloproteinase (MMP)-7 was similarly increased 2-fold, while that for MMP-9 and plasminogen activator inhibitor-1 was decreased 2-fold and 3-fold respectively. One probe for integrin beta 5 (1.23-fold) and one for integrin beta 8 (1.75-fold) showed statistically increased expression in the fibrogenic group. CONCLUSIONS: Gene transcripts for known upstream and downstream TGF-beta mediators are differentially expressed in liver specimens from children with BA and a fibrogenic gene signature. Further investigation into whether these mediators may be attractive targets for future therapy in children with BA is warranted.
Postnatal assembly of microbial communities across multiple body habitats in low birthweight infants Erica M Carlisle MD, Elizabeth K Costello PhD, David A Relman MD, Michael J Morowitz MD University of Chicago Medical Center, Chicago, IL, Stanford University School of Medicine, Palo Alto, CA and University of Pittsburgh Medical Center, Pittsburgh, PA INTRODUCTION: Aberrant bacterial colonization likely contributes to necrotizing enterocolitis (NEC), yet little is known about microbiome assembly in low birthweight infants. Here, we investigate the spatiotemporal dynamics of infant-associated bacterial communities during the first month of life. METHODS: We enrolled infants ⬍2kg not receiving antibiotics on day of life (DOL) 8, and collected fecal, oral, and skin microbiotas on DOL 8,10,12,15,18,21. We surveyed bacteria using multiplexed 16S rRNA gene pyrosequencing. Results were analyzed in QIIME and compared to corresponding samples from adults. RESULTS: Six infants (including two twin pairs) were delivered the same week and cared for in a common NICU (weights 0.75-1.82 kg, gestational age 24-38 wks). Using alpha-and beta-diversity rarefaction, PCoA, and ANOSIM, we found that infant microbiotas, compared to adults, were less diverse, less niche-differentiated, and compositionally distinct: a limited set of taxa were abundant, but
Sequential VEGF and EGFR inhibition improves survival in a neuroblastoma model Artur Chernoguz MD, Kelly M Crawford BS, Timothy P Cripe MD, PhD, Jason S Frischer MD, FACS Cincinnati Children’s Hospital Medical Center, Cincinnati, OH INTRODUCTION: Neuroblastoma, a common pediatric malignancy, is often resistant to multimodal therapy. Anti-angiogenic therapy, primarily focused on Vascular Endothelial Growth Factor (VEGF) inhibition is prone to rapid development of resistance. Based on observations of its increased expression in VEGF therapyresistant neuroblastoma, we hypothesized that Epidermal Growth Factor Receptor (EGFR) blockade would contribute to tumor regression by suppressing angiogenesis.
RESULTS: IFN-gamma response to Survivin was significantly higher in vaccinated mice compared to control (p⫽0.002) and tumor bearing mice (p⫽0.023). This response was amplified when CpG was added to the vaccine (p⫽0.007). CD69 staining showed a substantial rise of activated lymphocytes in the vaccinated group.
METHODS: SH-SY5Y neuroblastoma cells were incubated with gradient concentrations of gefitinib (EGFR-inhibitor) or bevacizumab (anti-VEGF antibody) for in vitro cytotoxicity assays. Xenografts were established by intrarenal SH-SY5Y cell injections in athymic mice (n⫽76). Animals with established tumors received gefitinib, bevacizumab, combination, or placebo for 20-days. Cohorts were weight-matched and re-assigned to receive a complementary therapy for 50-days. Analysis of microvessel and perivascular compartments was performed. Expression of pro-angiogenic signals was determined using qPCR.
CONCLUSIONS: In-vitro response to Survivin is a marker of effective in-vivo priming of CD8⫹ T-cells against NB vaccine. This immune response is greatly enhanced by the adjuvant effect of TLR9 stimulation. These findings present a valuable method for tracking T-cell responses to vaccine strategies against neuroblastoma.
Aurora A kinase regulates N-myc activity through phosphorylation of GSK3-beta and beta-catenin Nadja C Colon MD, Cameron E Schlegel BS, Jingbo Qiao PhD, Dai H Chung MD, FACS Vanderbilt University Medical Center, Nashville, TN
RESULTS: Gefitinib and bevacizumab had a limited cytostatic effect (⬍50%) on SH-SY5Y cells. Interval addition of gefitinib to bevacizumab conferred the longest median survival compared to bevacizumab (p⫽0.03), gefitinib (p⫽0.01), or combination (p⫽0.01). Refractory increase in tumor microvessel density caused by prolonged exposure to bevacizumab was offset by the addition of gefitinib (bevacizumab: 2,2735⫾3,178sq.micrometer vs. Bevacizumab-to-Combination: 11,620⫾1,991sq.micrometer; p⫽0.04). This regimen preserved relative immaturity of tumor vessels, consistent with apoptotic changes observed in pericytes exposed to gefitinib. Levels of human, but not murine VEGF mRNA, correlated with EGFR expression (p⫽0.02).
INTRODUCTION: Aurora A kinase (AurkA) is a serine/threonine kinase required for G2/M phase progression and centrosome maturation. Overexpression promotes aneuploidy and malignant transformation through unknown mechanisms. As a negative prognostic factor in neuroblastoma, AurkA has been shown to directly stabilize N-myc to prevent its ubiquitin-mediated degradation. We hypothesized that N-myc expression is further induced by AurkA’s downregulation of GSK3-beta with subsequent upregulation of betacatenin and its downstream targets.
CONCLUSIONS: EGFR inhibition augments anti-angiogenic therapy by targeting pericytes. This strategy may be a useful adjunct to current neuroblastoma treatment and is more effective when administered after a prolonged anti-VEGF therapy course.
METHODS: Human neuroblastoma BE(2)-C cells were transientlyand stably-transfected with plasmid to overexpress and silence AurkA, respectively. Western blot was used to determine alterations in protein levels of N-myc, GSK3-beta, phospho-GSK3-beta, betacatenin, and phospho-beta-catenin. Immunofluorescence and immunoprecipitation studies will ascertain AurkA and GSK3-beta colocalization and complex formation. Cell proliferation and anchorage-independent growth will be measured by BrdU and soft agar assays, while a luciferase assay will assess transcriptional activity of beta-catenin. Treatment with a PI3K/Akt inhibitor will ensure findings are a result of altering AurkA, and overexpression of GSK3beta will be used to rescue the wild-type phenotype.
CD8ⴙ T-cell response to survivin is a marker of effective immune priming in a neuroblastoma vaccine strategy Felix C Blanco MD, Priya Srinivasan PhD, Ryan M Walk MD, Jason A Snyder MD, Lina Chakrabarti PhD, Stanislav Vukmanovic MD, PhD, Anthony D Sandler MD, FACS Children’s National Medical Center, Washington, DC INTRODUCTION: Survivin, an inhibitor of apoptosis protein promotes proliferation and progression of neuroblastoma (NB) and is a potential target for immunotherapy as it is expressed primarily in tumors. This study was designed to investigate whether mice vaccinated with NB lysate were able to induce a CD8⫹ T-cell response against Survivin and compared it with that of naïve and tumor bearing mice. In addition, we studied whether CpG-oligonucleotide, a TLR9-agonist could enhance this response.
© 2011 by the American College of Surgeons Published by Elsevier Inc.
RESULTS: AurkA silencing resulted in significant decreases in phospho-GSK3-beta and beta-catenin protein levels relative to GSK3-beta and phospho-beta-catenin. There was also a decrease in N-myc protein levels. Western blot and RT-PCR confirmed overexpression and silencing.
S82
ISSN 1072-7515/11/$36.00 doi:10.1016/j.jamcollsurg.2011.06
Vol. 213, No. 3S, September 2011
CONCLUSIONS: Our results suggest that in neuroblastoma, AurkA inactivates GSK3-beta via phosphorylation to promote nuclear translocation of active beta-catenin and induction of N-myc transcription. This provides insight into the mechanism by which in vivo studies on the efficacy of Aurora A kinase inhibitors have expedited current clinical trials.
Phosphatidylinositol 3-kinase-independent rapid activation of vascular endothelial growth factor in neuroblastoma Cameron E Schlegel BS, Jingbo Qiao PhD, Nadja C Colon MD, Dai H Chung MD, FACS Vanderbilt University Medical Center, Nashville, TN INTRODUCTION: Activation of gastrin-releasing peptide (GRP) receptor-mediated cell signaling leads to increased neuroblastoma cell growth, invasion, and metastasis. Neovascularization has been associated with stimulation of GRP receptor signaling, yet the mechanism and kinetics of this tumorigenic function has not been fully elucidated. We sought to determine the role of GRP receptor in the regulation of vascular endothelial growth factor (VEGF) in neuroblastoma. METHODS: Human neuroblastoma cell lines, SK-N-SH and BE(2)-C, were treated with GRP with or without inhibitor (LY294002) of PI3K, a critical cell survival pathway. We also used Actinomycin-D to further examine the effects on transcriptional regulation of VEGF. Cell supernatant and lysates were collected over a time course. ELISA was performed to determine VEGF levels. Immunoblotting was used to assess for the critical role of vascularrelated proteins. RESULTS: There was an increase in VEGF levels at 30 min after GRP treatment; this returned to baseline, followed by a delayed VEGF peak. Interestingly, levels of hypoxia-inducing factor (HIF)1alpha, a transcription factor for VEGF, also increased at 2-24 hrs after GRP. LY294002 compound had no effects on 30 min VEGF peak after GRP; however, it significantly attenuated delayed activation of HIF1-alpha and VEGF secretion. Actinomycin D inhibited the HIF1-alpha-dependent second peak of VEGF activation. CONCLUSIONS: We describe a novel, biphasic stimulation of VEGF by GRP receptor signaling in neuroblastoma. Further, GRP induces a delayed activation of HIF1-alpha and VEGF production through non-hypoxic pathways in PI3K/Akt-dependent manner. GRP serves a crucial role in the tumorigenesis of neuroblastoma and may provide a target for antiangiogenic therapy.
Dysregulation of upstream and downstream TGF-beta transcripts in livers of children with biliary atresia and fibrogenic gene signatures Tatiana Iordanskaia PhD, Monica J Hubal PhD, Evan P Nadler MD, FACS Children’s National Medical Center, Washington, DC and The George Washington University School of Medicine & Health Sciences, Washington, DC
Surgical Forum Abstracts
S83
INTRODUCTION: Our previous work has shown that the transforming-growth factor (TGF) beta pathway plays a central role in liver fibrosis associated with experimental biliary atresia (BA). To confirm these findings in humans, we performed an in silico microarray analysis of liver specimens from children with BA with the hypothesis that mediators of the TGF beta pathway would be dysregualted in patients with fibrotic gene signatures. METHODS: Liver specimens were obtained from 47 infants with BA at portoenterostomy in a prospective study published by Moyer et al. (Genome Med, 2010). Total RNA was profiled using Affymetrix Human 133 plus 2.0 microarrays. We used the publicly available image (CEL) files and meta data to compare gene expression differences between the fibrogenic and inflammatory cohorts predicted by the study. ANOVA was performed using Partek Genomics Suite. RESULTS: We focused on 24 TGF beta-related transcripts. Expression for procollagen transcripts assessed was increased in the fibrogenic group (1.2-fold to 1.36 fold). Expression of matrix metalloproteinase (MMP)-7 was similarly increased 2-fold, while that for MMP-9 and plasminogen activator inhibitor-1 was decreased 2-fold and 3-fold respectively. One probe for integrin beta 5 (1.23-fold) and one for integrin beta 8 (1.75-fold) showed statistically increased expression in the fibrogenic group. CONCLUSIONS: Gene transcripts for known upstream and downstream TGF-beta mediators are differentially expressed in liver specimens from children with BA and a fibrogenic gene signature. Further investigation into whether these mediators may be attractive targets for future therapy in children with BA is warranted.
Postnatal assembly of microbial communities across multiple body habitats in low birthweight infants Erica M Carlisle MD, Elizabeth K Costello PhD, David A Relman MD, Michael J Morowitz MD University of Chicago Medical Center, Chicago, IL, Stanford University School of Medicine, Palo Alto, CA and University of Pittsburgh Medical Center, Pittsburgh, PA INTRODUCTION: Aberrant bacterial colonization likely contributes to necrotizing enterocolitis (NEC), yet little is known about microbiome assembly in low birthweight infants. Here, we investigate the spatiotemporal dynamics of infant-associated bacterial communities during the first month of life. METHODS: We enrolled infants ⬍2kg not receiving antibiotics on day of life (DOL) 8, and collected fecal, oral, and skin microbiotas on DOL 8,10,12,15,18,21. We surveyed bacteria using multiplexed 16S rRNA gene pyrosequencing. Results were analyzed in QIIME and compared to corresponding samples from adults. RESULTS: Six infants (including two twin pairs) were delivered the same week and cared for in a common NICU (weights 0.75-1.82 kg, gestational age 24-38 wks). Using alpha-and beta-diversity rarefaction, PCoA, and ANOSIM, we found that infant microbiotas, compared to adults, were less diverse, less niche-differentiated, and compositionally distinct: a limited set of taxa were abundant, but