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Hedgehog-mediated regulation of PPARγ controls metabolic patterns in neural precursors and shh-driven medulloblastoma.

Bhatia B, Potts CR, Guldal C, Choi S, Korshunov A, Pfister S, Kenney AM, Nahlé ZA - Acta Neuropathol. (2012)

Bottom Line: Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis.This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index.These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

ABSTRACT
Sonic hedgehog (Shh) signaling is critical during development and its aberration is common across the spectrum of human malignancies. In the cerebellum, excessive activity of the Shh signaling pathway is associated with the devastating pediatric brain tumor medulloblastoma. We previously demonstrated that exaggerated de novo lipid synthesis is a hallmark of Shh-driven medulloblastoma and that hedgehog signaling inactivates the Rb/E2F tumor suppressor complex to promote lipogenesis. Indeed, such Shh-mediated metabolic reprogramming fuels tumor progression, in an E2F1- and FASN-dependent manner. Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis. Our data show that in primary cerebellar granule neural precursors (CGNPs), proposed medulloblastoma cells-of-origin, Shh stimulation elicits a marked induction of PPARγ alongside major glycolytic markers. This is also documented in the actively proliferating Shh-responsive CGNPs in the developing cerebellum, and PPARγ expression is strikingly elevated in Shh-driven medulloblastoma in vivo. Importantly, pharmacological blockade of PPARγ and/or Rb inactivation inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals in vivo. This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index. This also reveals a dominant role of Shh in the etiology of glucose metabolism in medulloblastoma and underscores the function of the Shh → E2F1 → PPARγ axis in altering substrate utilization patterns in brain cancers in favor of tumor growth. These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors.

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Treatment of medulloblastoma-bearing mice with PPARγ antagonist GW9662 impairs glycolysis in tumor in vivo. a Western blot analysis for E2F1, PPARγ, glycolysis (HKI, HKII, PKM2, and Glut4), lipid synthesis (FASN) and proliferation (cyclin D2) in samples of adjacent cerebella and medulloblastomas from vehicle (DMSO)-treated (−) or GW9662-treated (+) mice. The results shown are typical for several control and experimental sets of mice. b Immunostaining in adjacent cerebellum and medulloblastomas in vehicle- or GW9662-treated mice. Slides from the tissue of several untreated and treated NeuroD2-SmoA mice exhibiting medulloblastomas were analyzed and the presented results were typical. Magnification, ×40. Bars 16 μM. c Effects of increasing doses of the PPARγ antagonist GW9662 (0, 0.01, 0.02, 0.05, 0.07, 0.1 μM) for 24 h on pzp53med cell viability as determined by cell Titer-Glo assay, a bioluminescent analysis based on the presence of ATP. Each bar in the bioluminescence graph represents the average of separate quadruplicate determinations with error bars showing the standard deviation of the mean. Bars not sharing common letter are significantly different at P < 0.05
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Fig5: Treatment of medulloblastoma-bearing mice with PPARγ antagonist GW9662 impairs glycolysis in tumor in vivo. a Western blot analysis for E2F1, PPARγ, glycolysis (HKI, HKII, PKM2, and Glut4), lipid synthesis (FASN) and proliferation (cyclin D2) in samples of adjacent cerebella and medulloblastomas from vehicle (DMSO)-treated (−) or GW9662-treated (+) mice. The results shown are typical for several control and experimental sets of mice. b Immunostaining in adjacent cerebellum and medulloblastomas in vehicle- or GW9662-treated mice. Slides from the tissue of several untreated and treated NeuroD2-SmoA mice exhibiting medulloblastomas were analyzed and the presented results were typical. Magnification, ×40. Bars 16 μM. c Effects of increasing doses of the PPARγ antagonist GW9662 (0, 0.01, 0.02, 0.05, 0.07, 0.1 μM) for 24 h on pzp53med cell viability as determined by cell Titer-Glo assay, a bioluminescent analysis based on the presence of ATP. Each bar in the bioluminescence graph represents the average of separate quadruplicate determinations with error bars showing the standard deviation of the mean. Bars not sharing common letter are significantly different at P < 0.05

Mentions: Our data in CGNPs show that PPARγ is associated with a proliferative state, and that PPARγ positively regulates glycolytic markers. Many tumor cells are known to exhibit high levels of glycolysis, and NeuroD2-SmoA1 medulloblastomas are consistently highly proliferative. We wished to determine whether PPARγ inhibition could impair glycolysis and affect proliferation in these tumors. We treated medulloblastoma-bearing NeuroD2-SmoA1 mice with GW9662 or its vehicle DMSO for up to 10 days, then harvested the tumors for western blot and immunofluorescence analysis. As shown in Fig. 5a, GW9662 treatment had no impact on the level of E2F1 or FASN, but sharply reduced PPARγ protein levels (Fig. 5a, b), consistent with PPARγ lying downstream of E2F1 and in a parallel pathway to FASN. GW9662 treatment also reduced levels of glycolytic markers and cyclin D2 protein, but had no effect on HKI, in keeping with our in vitro results in CGNPs. When tested in the pzp53 MB cell line, GW9662 treatment was very effective in killing cancer cells in a dose-dependent manner (Fig. 5c), as show by a luminescence viability assay (Cell Titer-Glo).Fig. 5


Hedgehog-mediated regulation of PPARγ controls metabolic patterns in neural precursors and shh-driven medulloblastoma.

Bhatia B, Potts CR, Guldal C, Choi S, Korshunov A, Pfister S, Kenney AM, Nahlé ZA - Acta Neuropathol. (2012)

Treatment of medulloblastoma-bearing mice with PPARγ antagonist GW9662 impairs glycolysis in tumor in vivo. a Western blot analysis for E2F1, PPARγ, glycolysis (HKI, HKII, PKM2, and Glut4), lipid synthesis (FASN) and proliferation (cyclin D2) in samples of adjacent cerebella and medulloblastomas from vehicle (DMSO)-treated (−) or GW9662-treated (+) mice. The results shown are typical for several control and experimental sets of mice. b Immunostaining in adjacent cerebellum and medulloblastomas in vehicle- or GW9662-treated mice. Slides from the tissue of several untreated and treated NeuroD2-SmoA mice exhibiting medulloblastomas were analyzed and the presented results were typical. Magnification, ×40. Bars 16 μM. c Effects of increasing doses of the PPARγ antagonist GW9662 (0, 0.01, 0.02, 0.05, 0.07, 0.1 μM) for 24 h on pzp53med cell viability as determined by cell Titer-Glo assay, a bioluminescent analysis based on the presence of ATP. Each bar in the bioluminescence graph represents the average of separate quadruplicate determinations with error bars showing the standard deviation of the mean. Bars not sharing common letter are significantly different at P < 0.05
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Fig5: Treatment of medulloblastoma-bearing mice with PPARγ antagonist GW9662 impairs glycolysis in tumor in vivo. a Western blot analysis for E2F1, PPARγ, glycolysis (HKI, HKII, PKM2, and Glut4), lipid synthesis (FASN) and proliferation (cyclin D2) in samples of adjacent cerebella and medulloblastomas from vehicle (DMSO)-treated (−) or GW9662-treated (+) mice. The results shown are typical for several control and experimental sets of mice. b Immunostaining in adjacent cerebellum and medulloblastomas in vehicle- or GW9662-treated mice. Slides from the tissue of several untreated and treated NeuroD2-SmoA mice exhibiting medulloblastomas were analyzed and the presented results were typical. Magnification, ×40. Bars 16 μM. c Effects of increasing doses of the PPARγ antagonist GW9662 (0, 0.01, 0.02, 0.05, 0.07, 0.1 μM) for 24 h on pzp53med cell viability as determined by cell Titer-Glo assay, a bioluminescent analysis based on the presence of ATP. Each bar in the bioluminescence graph represents the average of separate quadruplicate determinations with error bars showing the standard deviation of the mean. Bars not sharing common letter are significantly different at P < 0.05
Mentions: Our data in CGNPs show that PPARγ is associated with a proliferative state, and that PPARγ positively regulates glycolytic markers. Many tumor cells are known to exhibit high levels of glycolysis, and NeuroD2-SmoA1 medulloblastomas are consistently highly proliferative. We wished to determine whether PPARγ inhibition could impair glycolysis and affect proliferation in these tumors. We treated medulloblastoma-bearing NeuroD2-SmoA1 mice with GW9662 or its vehicle DMSO for up to 10 days, then harvested the tumors for western blot and immunofluorescence analysis. As shown in Fig. 5a, GW9662 treatment had no impact on the level of E2F1 or FASN, but sharply reduced PPARγ protein levels (Fig. 5a, b), consistent with PPARγ lying downstream of E2F1 and in a parallel pathway to FASN. GW9662 treatment also reduced levels of glycolytic markers and cyclin D2 protein, but had no effect on HKI, in keeping with our in vitro results in CGNPs. When tested in the pzp53 MB cell line, GW9662 treatment was very effective in killing cancer cells in a dose-dependent manner (Fig. 5c), as show by a luminescence viability assay (Cell Titer-Glo).Fig. 5

Bottom Line: Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis.This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index.These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

ABSTRACT
Sonic hedgehog (Shh) signaling is critical during development and its aberration is common across the spectrum of human malignancies. In the cerebellum, excessive activity of the Shh signaling pathway is associated with the devastating pediatric brain tumor medulloblastoma. We previously demonstrated that exaggerated de novo lipid synthesis is a hallmark of Shh-driven medulloblastoma and that hedgehog signaling inactivates the Rb/E2F tumor suppressor complex to promote lipogenesis. Indeed, such Shh-mediated metabolic reprogramming fuels tumor progression, in an E2F1- and FASN-dependent manner. Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis. Our data show that in primary cerebellar granule neural precursors (CGNPs), proposed medulloblastoma cells-of-origin, Shh stimulation elicits a marked induction of PPARγ alongside major glycolytic markers. This is also documented in the actively proliferating Shh-responsive CGNPs in the developing cerebellum, and PPARγ expression is strikingly elevated in Shh-driven medulloblastoma in vivo. Importantly, pharmacological blockade of PPARγ and/or Rb inactivation inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals in vivo. This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index. This also reveals a dominant role of Shh in the etiology of glucose metabolism in medulloblastoma and underscores the function of the Shh → E2F1 → PPARγ axis in altering substrate utilization patterns in brain cancers in favor of tumor growth. These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors.

Show MeSH
Related in: MedlinePlus