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The mouse model for Glut-1 transporter deficiency syndrome: spared from postnatal apoptosis, but not from reactive astrogliosis. Paivi M. Ullner*, Alessia DiNardo, Leslie Bagay, Hong Yang, Kristin Engelstad, Dong Wang, James E. Goldman, Mustafa Sahin and Darryl C. De Vivo *Invited author BACKGROUND: Brain depends on glucose as the preferred substrate for its energy production. Glucose transporter type 1 (Glut-1) is the predominant glucose transporter facilitating glucose penetration through the vascular endothelial cells on the blood-brain-barrier. In humans, Glut-1 transporter deficiency syndrome is known to cause acquired microcephaly, seizures and ataxia, of variable severity. Our previously generated Glut-1 deficiency mouse model exhibits decreased CSF glucose, microcephaly, EEG-abnormality, and compromised motor performance. As mTOR-pathway is affected by environmental stress and energy deprivation, and is an important regulator for cell size and survival, we studied this and apoptosis (programmed cell death) in our Glut-1+/- mouse model, as a potential pathophysiologic mechanism for the microcephaly. METHODS: Glut-1+/- and wild type female and male mouse brains were weighed and processed for histology and western blotting (WB) analysis (min. 3-4 per age/gender matched group) at ages 1-3 weeks (N=72), 3-6 months (N=44) and 9-12 / 21-24 months (N=12). Histological quantification was performed at the time of developmental apoptosis on cortex, cerebellum (TUNEL) and hippocampus (Nissl). Reactive astrogliosis (GFAP) was quantified on neonates and adults, and neuron size (SMI311) on adults. Apoptosis, mTOR-pathway and synaptic proteins (WB: activated caspase-3, PARP-1, TSC1, TSC2, pACC, pmTOR, pAMPK-, pAkt1, pS6, pGSK3-synapsin, PSD95) were explored in adults, with/without 6-hour-fasting. General histology (H&E, Nissl, LFB for myelination, GFAP) was reviewed in aged mice. RESULTS: Glut-1+/- mice exhibited decreased mean brain weight (females P=0.003, males P=0.024), and reactive cortical astrogliosis (P=0.01) already at P21, presumably indicative of metabolic stress. Increased cell death was absent in neonates and adults. mTOR signaling, cortical neuron size, and synaptic proteins were unaffected. 6-hour-fasting mildly elevated levels of pAkt1-growth signal, glucogen synthesis inhibitor pGSK3-, fatty acid synthesis inhibitor pACC, and ribosomal pS6, indicative for increased translation, in females (4 of 5; males unaffected). General histology was otherwise normal. CONCLUSIONS: Signs of metabolic stress, reduced brain weight and reactive astrogliosis, were present already at the weaning age, in the absence of increased apoptosis or degenerative changes. Fasting females appeared to resort to energy reserves sooner than males. The Glut-1+/- mouse (129S6/SvEvTac) tolerates the Glut-1 transporter deficit relatively well. Favorable genetic background may play a role in reducing the impact of the metabolic compromise, and phenotypic severity, in the Glut-1+/- mouse, phenomenon relevant perhaps also in humans. Further studies with increased metabolic stress are implicated to elucidate the vulnerability in Glut-1+/- mice. |
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New use of an established agent: rapamycin and Ewing’s sarcoma Authors: Samuel Soffer MD*, Aaron Lipskar MD, Richard Glick MD, Jason Fisher MD, Morris Edelman MD, Bettie Steinberg PhD , Darrell Yamashiro MD, PhD and Jessica Kandel MD *presenting author Background: Rapamycin can inhibit tumor growth in a variety of human cancers. At traditional, cytotoxic doses, rapamycin binds to the mammalian target of rapamycin (mTOR) protein complex and inhibits the phosphorylation of p70s6k causing high-potency cell growth inhibition. Low, daily dosing of chemotherapeutic agents is thought to target tumor angiogenesis. Rapamycin can also inhibit angiogenesis, but the anti-angiogenic mechanism of rapamycin is incompletely understood. Hypothesis: We hypothesized that low-dose rapamycin, as compared to cytotoxic dosing, would target the endothelial cells (anti-angiogenesis) via a p70s6k-independent pathway and result in tumor suppression. Methods: Ewing’s sarcoma (ES) cells were surgically implanted in athymic mice and treated with either vehicle (n=15), low-dose rapamycin (1.5mg/kg, n=15), or cytotoxic (high-dose) rapamycin (15mg/kg, n=15) daily for 5 weeks. Tumor vascularity was assessed by lectin perfusion angiography and immunostaining with antibodies to vascular markers (PECAM-1, type IV-collagen, αSMA). Double immunostaining for type IV collagen and TUNEL was performed to assess endothelial cell apoptosis. Phosphorylation of p70s6k was determined by western blot analysis and normalized to total levels of p70s6k. A rapamycin dose-dependant experiment was performed on ES cells and the phosphorylation of p70s6k at each treatment concentration was determined by western blot analysis. Results: Mean tumor weights were significantly reduced in the treated groups (4.1g +/- 0.81 SEM, control; 0.9g +/- 0.15 SEM, low-dose (p<0.0001); 1.0g +/- 0.22 SEM high-dose (p=0.0002)). Grossly, control and high-dose tumors were vascular and hemorrhagic, whereas tumors in the low-dose group were avascular and pale. Lectin angiography and immunostaining for endothelial cell markers demonstrated abundant vasculature in the untreated and high-dose groups and blunted, sparse vascularity in low-dose treated xenografts. Marked endothelial cell apoptosis was noted in the low-dose treated group on double immunostaining. Western blot analysis demonstrated inhibition of the phosphorylation of p70s6k in the high-dose treated xenografts, while the phosphorylation of p70s6k in the low-dose treated tumors were similar to controls. This correlated with the dose-dependant experiment, which revealed minimal inhibition of the phosphorylation of p70s6k in the untreated and low-dose rapamycin-treated cells and complete suppression of the phosphorylation of p70s6k in the high-dose rapamycin-treated cells. Conclusions: Low-dose rapamycin effectively blocks angiogenesis and leads to tumor suppression in ES without affecting the phosphorylation of p70s6k. This suggests that low-dose rapamycin may be acting via a p70s6k-independent pathway. Low-dose rapamycin may be an attractive alternative to conventional dosing with less toxicity and resistance to therapy. |
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COMBINED CYCLOOXYGENASE-2 AND VEGF INHIBITION DISRUPTS TUMOR VASCULAR ARCHITECTURE AND SUPPRESSES XENOGRAFT METASTASIS Jason Fisher*, MJ Haley, Jason Frischer, Jeff Gander, Jay Huang, Sonia Hernandez, Darrell Yamashiro, and Jessica Kandel
*presenting author BACKGROUND: While most children with Wilms’ tumor are cured, a subset develop life-threatening pulmonary metastases. We have demonstrated that VEGF blockade disrupts tumor vessel endothelium, resulting in decreased metastasis and increased recruitment of vascular mural cells (VMC). We have additionally shown that cyclooxygenase-2 (COX-2) inhibition impairs VMC recruitment, with recent data also linking COX-2 expression to metastasis. Collectively, these data suggest that combined COX-2/VEGF blockade may inhibit tumor growth and metastasis more effectively than either approach alone. METHODS: Intrarenal xenografts were induced by injection of 106 cultured human Wilms’ tumor cells (SKNEP-1) in athymic mice (N=81). At Day 7, animals were divided into groups: (1) control, N=19; (2) COX-2 blockade, N=20; (3) VEGF blockade, N=22; and (4) COX-2+VEGF blockade, N=20, and received water with vehicle or COX-2 inhibitor (SC236, 30μg/mL). Mice were injected biweekly with vehicle or anti-VEGF antibody (bevazicumab, 250μg/dose) beginning at Day 21. Tumors were harvested at Day 42, weights compared by Kruskal-Wallis analysis, and vessels analyzed by immunohistochemistry. H&E slides of lung tissue were examined by a surgical pathologist to determine metastasis. RNA isolated from primary and metastatic tumor cells cultured in 0.1% oxygen was analyzed by quantitative-PCR. Tumor cell invasion was measured using trans-Matrigel migration assays normalized to proliferation. Matrix-metalloproteinase (MMP) activity was assessed by gelatin-zymography of tumor protein extracts. Gene expression analysis was performed using U133A-Affymetrix GeneChips with cRNA synthesized from xenografts. RESULTS: Tumor growth was significantly suppressed in SC236 (3.99 0.73g; P=0.04), bevazicumab (1.58 0.26g; P<0.001), and SC236+bevacizumab groups (1.72 0.34g; P<0.001). Tumors treated with SC236+bevacizumab displayed reduced VMC recruitment with defective vascular branching hierarchy. Combined COX-2/VEGF blockade significantly reduced metastasis compared to monotherapy with bevacizumab (13% vs 47%; P=0.046) or SC236 (13% vs 65%; P=0.004; Figure 1). Hypoxia-mediated increases in COX-2 expression were greater in cultured lung metastasis cells than in primary tumor cells (P<0.001). SC236 suppressed tumor cell invasion through Matrigel by 78% (P=0.02; Figure 2). Tumor MMP-9 enzymatic-activity was suppressed in all treatment groups. Gene-set expression analysis of microarray data using a validated lung-metastasis signature gene-set significantly distinguished bevacizumab-only from SC236+bevacizumab-treated tumors (P<0.001) CONCLUSIONS: Dual COX-2/VEGF blockade perturbs tumor vasculature through differential effects on VMCs. Suppression of metastasis by combination therapy may result from: (1) blunting of hypoxia-mediated increases in COX-2 expression; (2) impaired invasion through basement membrane; and/or (3)differential MMP-9 activation. The addition of SC-236 to bevacizumab therapy broadly alters expression of lung metastasis signature genes implicated in these mechanisms. Co-targeting tumor vasculature and metastatic potential may offer sustained therapeutic responses for children with treatment-refractory cancers. |
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TUMOR-SPECIFIC INHIBITION OF PLACENTA-LIKE GROWTH FACTOR INCREASES METASTASIS IN A MURINE MODEL OF NEUROBLASTOMA MJ Haley*, Jason Fisher, Sonia Hernandez, Jay Huang, Jessica Kandel, and Darrell Yamashiro *presenting author Background: Blockade of
vascular endothelial growth factor (VEGF) is a validated strategy in the
treatment of pediatric solid tumors.
VEGF signaling occurs primarily through the VEGFR2 receptor, while
mediators such as placenta-like growth
factor (PlGF) preferentially signal via VEGFR1. We have previously demonstrated that neuroblastoma
is initially inhibited by VEGF blockade but ultimately recurs, implying recruitment of alternative
pathways (i.e., the PlGF–VEGFR1 axis).
We hypothesized that simultaneous blockade of VEGF and PlGF may inhibit
tumor growth greater than either treatment alone. Methods: We utilized lentivirus-delivered shRNA to
obtain a stable knockdown of PlGF in cultured SY5Y human neuroblastoma cells engineered
to express luciferase. Xenografts were subsequently
induced by implantation of 106 transfected cells into athymic mice (PlGF
knockdown,N=36; controls,N=42). Cohorts were
further randomized at 1 week to receive injections of bevacizumab (anti-huVEGF
antibody,250 mcg/dose) or vehicle for 5 weeks.
Tumor growth was monitored through bioluminescence using a
charge-coupled device (CCD) camera. Mice
were sacrificed when tumor flux reached 3.0x109 photons/second, tumors
harvested, and metastasis assessed by CCD signal and culture of bone marrow. Differences in tumor growth were measured by
Kaplan-Meir survival analysis. Results: Vehicle-treated PlGF knockdown xenografts
demonstrated a trend towards decreased tumor growth compared to vehicle-treated
controls (P=0.08). Addition of VEGF
blockade slowed tumor growth in control xenografts (P=0.001), but did not affect
the growth of PlGF knockdown tumors (P=0.89).
Tumor metastasis was significantly increased in PlGF knockdown
xenografts compared to untransfected controls (76% vs. 16%; P=0.03; Figure 1). Addition of bevacizumab did not reduce the
overall metastasis rate (76% vs. 50%; P=0.16), but did significantly reduce the
bone marrow metastasis burden (33% vs. 17% of total metastatic photon-flux;
P<0.001; Figure 2). Conclusions: Tumor-specific PlGF inhibition may delay
tumor growth in human neuroblastoma xenografts.
Isolated PlGF blockade in neuroblastoma cells markedly increases their
metastatic potential, an effect partially reversed by the addition of bevacizumab
through a reduction in bone marrow metastasis.
These data warrant further investigation to determine whether unopposed
VEGF signaling through VEGFR1 in the absence of PlGF is responsible for this paradoxical
increase in tumor metastasis. |
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Cyclooxygenase-2 mediates the anti-angiogenic effect
of rapamycin in Ewing’s sarcoma Aaron M Lipskar MD*,
Richard D Glick MD, Jianzhong Huang MD, Jason C Fisher MD, James DeVoti PhD, Ray
Pica BS, Morris Edelman MD, Bettie M Steinberg PhD, and Samuel Z Soffer MD *presenting author Background: Rapamycin, an inhibitor
of the mammalian target of rapamycin (mTOR), inhibits tumor growth and angiogenesis
in a variety of human cancers.
Cyclooxygenase-2 (COX-2) is an enzyme induced in inflammatory cells and
human tumors that is involved in the angiogenic process. In
vitro studies have linked downstream activation of COX-2 to the mTOR
pathway. We hypothesized that the
anti-angiogenic effect of rapamycin is mediated by suppression of COX-2. Methods: Ewing’s sarcoma (ES) cells were intrarenally
implanted in athymic mice (n=30). At 1 week, selected animals were treated with
rapamycin for 5 weeks (n-15). Tumor
vascularity was assessed by lectin perfusion angiography and
immunohistochemistry. Phosphorylation of
mTOR pathway proteins was determined by western blot analysis. Staining of COX-2 protein was determined by
immunohistochemistry and expression of COX-2 messenger RNA levels was assessed
with quantitative RT-PCR. Results: Mean tumor weights were significantly reduced
in the treated group (4.1g±0.81 SEM v 0.9g±0.15 SEM, P<0.0001). There was abundant
vasculature in the control group and blunted vascularity in the treated
xenografts. The phosphorylation of
p70s6k and Akt was not inhibited in the rapamycin-treated tumors. COX-2 was suppressed in the treated
xenografts at both the protein and messenger RNA levels. Conclusion: Low-dose rapamycin inhibits tumor growth and
angiogenesis in human ES without inhibiting the phosphorylation of p70s6k and
Akt. COX-2 levels are inhibited by
low-dose treatment of ES with rapamycin.
COX-2 suppression may mediate the anti-angiogenic effect of rapamycin in
Ewing’s sarcoma. |
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