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The G protein α subunit Gαs is a tumor suppressor in Sonic hedgehog-driven medulloblastoma.

He X, Zhang L, Chen Y, Remke M, Shih D, Lu F, Wang H, Deng Y, Yu Y, Xia Y, Wu X, Ramaswamy V, Hu T, Wang F, Zhou W, Burns DK, Kim SH, Kool M, Pfister SM, Weinstein LS, Pomeroy SL, Gilbertson RJ, Rubin JB, Hou Y, Wechsler-Reya R, Taylor MD, Lu QR - Nat. Med. (2014)

Bottom Line: Gαs is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components.Elevation in levels of a Gαs effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas-ablated mice.Thus, our gain- and loss-of-function studies identify a previously unrecognized tumor suppressor function for Gαs that can be found consistently across Shh-group medulloblastomas of disparate cellular and anatomical origins, highlighting G protein modulation as a potential therapeutic avenue.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Forensic Medicine, State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, School of Preclinical and Forensic Medicine, West China Second Hospital, Sichuan University, Chengdu, China. [2] Department of Pediatrics, Brain Tumor Center, Division of Experimental Hematology and Cancer Biology, Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

ABSTRACT
Medulloblastoma, the most common malignant childhood brain tumor, exhibits distinct molecular subtypes and cellular origins. Genetic alterations driving medulloblastoma initiation and progression remain poorly understood. Herein, we identify GNAS, encoding the G protein Gαs, as a potent tumor suppressor gene that, when expressed at low levels, defines a subset of aggressive Sonic hedgehog (SHH)-driven human medulloblastomas. Ablation of the single Gnas gene in anatomically distinct progenitors in mice is sufficient to induce Shh-associated medulloblastomas, which recapitulate their human counterparts. Gαs is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components. Elevation in levels of a Gαs effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas-ablated mice. Thus, our gain- and loss-of-function studies identify a previously unrecognized tumor suppressor function for Gαs that can be found consistently across Shh-group medulloblastomas of disparate cellular and anatomical origins, highlighting G protein modulation as a potential therapeutic avenue.

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Loss of Gnas in neural stem/progenitor cells induces MB formation(a) Sagittal brain sections from hGFAPCre:Gnaslox/lox (GFAP:Gnas) and hGFAPCre:Gnaslox/+ (Ctrl) mice at indicated stages were stained with hematoxylin and eosin (H/E).(b) Brain appearance of control and Gnas mutants at P67. The arrows indicate the cerebellum.(c) Tumors from Gnas mutants (left) displays similar histology to human MB (right; SHH group). Insets are shown at high magnification.(d) The cerebella of control and Gnas mutants at P60 were stained with anti-Ki67 and DAPI.(e) Kaplan-Meier survival curves for control and GFAP:Gnas mice (n = 52).(f) Heatmap shows expression of Shh pathway components in control cerebella and GFAP:Gnas tumor tissues. The color bar shows expression intensity.(g) qRT-PCR quantification of Gnas and Shh pathway genes in control and GFAP:Gnas cerebella at P30. Data represent the mean ± SEM (n = six animals). ** P < 0.01; Student's t test.(h) mRNA expression of Shh target genes as indicated in control and GFAP:Gnas brain sections at P60. Arrow and arrowhead indicate the cerebellum and pontine grey nucleus, respectively.(i) qRT-PCR analysis of Gnas, Gli1 and Ptch1 in GNPs from control and GFAP:Gnas mice at P7. Data represent the mean ± SEM (n = five animals). ** P < 0.01; Student's t test.(j) The cerebellar EGL region of GFAP:Gnas mice carrying the Atoh1-GFP reporter at P50 was immunostained with anti-Zic1, Olig2 and GFAP as indicated.Scale bars in a, 300 μm; b, 5 mm; c, d, h, 200 μm; inset in c, 10 μm; j, 100 μm.
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Figure 2: Loss of Gnas in neural stem/progenitor cells induces MB formation(a) Sagittal brain sections from hGFAPCre:Gnaslox/lox (GFAP:Gnas) and hGFAPCre:Gnaslox/+ (Ctrl) mice at indicated stages were stained with hematoxylin and eosin (H/E).(b) Brain appearance of control and Gnas mutants at P67. The arrows indicate the cerebellum.(c) Tumors from Gnas mutants (left) displays similar histology to human MB (right; SHH group). Insets are shown at high magnification.(d) The cerebella of control and Gnas mutants at P60 were stained with anti-Ki67 and DAPI.(e) Kaplan-Meier survival curves for control and GFAP:Gnas mice (n = 52).(f) Heatmap shows expression of Shh pathway components in control cerebella and GFAP:Gnas tumor tissues. The color bar shows expression intensity.(g) qRT-PCR quantification of Gnas and Shh pathway genes in control and GFAP:Gnas cerebella at P30. Data represent the mean ± SEM (n = six animals). ** P < 0.01; Student's t test.(h) mRNA expression of Shh target genes as indicated in control and GFAP:Gnas brain sections at P60. Arrow and arrowhead indicate the cerebellum and pontine grey nucleus, respectively.(i) qRT-PCR analysis of Gnas, Gli1 and Ptch1 in GNPs from control and GFAP:Gnas mice at P7. Data represent the mean ± SEM (n = five animals). ** P < 0.01; Student's t test.(j) The cerebellar EGL region of GFAP:Gnas mice carrying the Atoh1-GFP reporter at P50 was immunostained with anti-Zic1, Olig2 and GFAP as indicated.Scale bars in a, 300 μm; b, 5 mm; c, d, h, 200 μm; inset in c, 10 μm; j, 100 μm.

Mentions: To determine whether Gnas inactivation could lead to brain tumorigenesis, we deleted Gnas in neural stem/progenitor cells by breeding floxed Gnas mice (Gnaslox/lox) with an hGFAP-Cre line 13,14. Strikingly, all resulting hGFAP-Cre+/−:Gnaslox/lox conditional knockout mice (designated as GFAP:Gnas) developed MB-like tumors at adult stages (Fig. 2a,b). We observed expansion of granule neuron progenitors (GNPs) in the cerebellar external granular layer (EGL) beginning at neonatal stages, when the control cerebellum contained only a few rows of GNPs on its surface. Diffuse, continuous GNP expansion continued to increase at postnatal stages. Gnas mutant cerebella were exophytic and delineated by a thick and disorganized EGL (Fig. 2a). By six weeks, GFAP:Gnas mice developed a diffuse MB-like tumor exhibiting the densely-packed, “small round blue” GNP-like histology (Fig. 2c; inset), resembling the histological features of human MB 15. In the mutants at P60, the neoplastic cells were highly proliferative as indicated by extensive expression of Ki67, a proliferative marker, which was barely detectable in controls (Fig. 2d). 100% of animals succumbed to the tumor around 3-4 months of age (Fig. 2e). Although the hGFAP-Cre-recombined cells appear in most brain regions 13, tumor formation was confined only to the cerebellum during the lifespan of Gnas mutants.


The G protein α subunit Gαs is a tumor suppressor in Sonic hedgehog-driven medulloblastoma.

He X, Zhang L, Chen Y, Remke M, Shih D, Lu F, Wang H, Deng Y, Yu Y, Xia Y, Wu X, Ramaswamy V, Hu T, Wang F, Zhou W, Burns DK, Kim SH, Kool M, Pfister SM, Weinstein LS, Pomeroy SL, Gilbertson RJ, Rubin JB, Hou Y, Wechsler-Reya R, Taylor MD, Lu QR - Nat. Med. (2014)

Loss of Gnas in neural stem/progenitor cells induces MB formation(a) Sagittal brain sections from hGFAPCre:Gnaslox/lox (GFAP:Gnas) and hGFAPCre:Gnaslox/+ (Ctrl) mice at indicated stages were stained with hematoxylin and eosin (H/E).(b) Brain appearance of control and Gnas mutants at P67. The arrows indicate the cerebellum.(c) Tumors from Gnas mutants (left) displays similar histology to human MB (right; SHH group). Insets are shown at high magnification.(d) The cerebella of control and Gnas mutants at P60 were stained with anti-Ki67 and DAPI.(e) Kaplan-Meier survival curves for control and GFAP:Gnas mice (n = 52).(f) Heatmap shows expression of Shh pathway components in control cerebella and GFAP:Gnas tumor tissues. The color bar shows expression intensity.(g) qRT-PCR quantification of Gnas and Shh pathway genes in control and GFAP:Gnas cerebella at P30. Data represent the mean ± SEM (n = six animals). ** P < 0.01; Student's t test.(h) mRNA expression of Shh target genes as indicated in control and GFAP:Gnas brain sections at P60. Arrow and arrowhead indicate the cerebellum and pontine grey nucleus, respectively.(i) qRT-PCR analysis of Gnas, Gli1 and Ptch1 in GNPs from control and GFAP:Gnas mice at P7. Data represent the mean ± SEM (n = five animals). ** P < 0.01; Student's t test.(j) The cerebellar EGL region of GFAP:Gnas mice carrying the Atoh1-GFP reporter at P50 was immunostained with anti-Zic1, Olig2 and GFAP as indicated.Scale bars in a, 300 μm; b, 5 mm; c, d, h, 200 μm; inset in c, 10 μm; j, 100 μm.
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Related In: Results  -  Collection

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Figure 2: Loss of Gnas in neural stem/progenitor cells induces MB formation(a) Sagittal brain sections from hGFAPCre:Gnaslox/lox (GFAP:Gnas) and hGFAPCre:Gnaslox/+ (Ctrl) mice at indicated stages were stained with hematoxylin and eosin (H/E).(b) Brain appearance of control and Gnas mutants at P67. The arrows indicate the cerebellum.(c) Tumors from Gnas mutants (left) displays similar histology to human MB (right; SHH group). Insets are shown at high magnification.(d) The cerebella of control and Gnas mutants at P60 were stained with anti-Ki67 and DAPI.(e) Kaplan-Meier survival curves for control and GFAP:Gnas mice (n = 52).(f) Heatmap shows expression of Shh pathway components in control cerebella and GFAP:Gnas tumor tissues. The color bar shows expression intensity.(g) qRT-PCR quantification of Gnas and Shh pathway genes in control and GFAP:Gnas cerebella at P30. Data represent the mean ± SEM (n = six animals). ** P < 0.01; Student's t test.(h) mRNA expression of Shh target genes as indicated in control and GFAP:Gnas brain sections at P60. Arrow and arrowhead indicate the cerebellum and pontine grey nucleus, respectively.(i) qRT-PCR analysis of Gnas, Gli1 and Ptch1 in GNPs from control and GFAP:Gnas mice at P7. Data represent the mean ± SEM (n = five animals). ** P < 0.01; Student's t test.(j) The cerebellar EGL region of GFAP:Gnas mice carrying the Atoh1-GFP reporter at P50 was immunostained with anti-Zic1, Olig2 and GFAP as indicated.Scale bars in a, 300 μm; b, 5 mm; c, d, h, 200 μm; inset in c, 10 μm; j, 100 μm.
Mentions: To determine whether Gnas inactivation could lead to brain tumorigenesis, we deleted Gnas in neural stem/progenitor cells by breeding floxed Gnas mice (Gnaslox/lox) with an hGFAP-Cre line 13,14. Strikingly, all resulting hGFAP-Cre+/−:Gnaslox/lox conditional knockout mice (designated as GFAP:Gnas) developed MB-like tumors at adult stages (Fig. 2a,b). We observed expansion of granule neuron progenitors (GNPs) in the cerebellar external granular layer (EGL) beginning at neonatal stages, when the control cerebellum contained only a few rows of GNPs on its surface. Diffuse, continuous GNP expansion continued to increase at postnatal stages. Gnas mutant cerebella were exophytic and delineated by a thick and disorganized EGL (Fig. 2a). By six weeks, GFAP:Gnas mice developed a diffuse MB-like tumor exhibiting the densely-packed, “small round blue” GNP-like histology (Fig. 2c; inset), resembling the histological features of human MB 15. In the mutants at P60, the neoplastic cells were highly proliferative as indicated by extensive expression of Ki67, a proliferative marker, which was barely detectable in controls (Fig. 2d). 100% of animals succumbed to the tumor around 3-4 months of age (Fig. 2e). Although the hGFAP-Cre-recombined cells appear in most brain regions 13, tumor formation was confined only to the cerebellum during the lifespan of Gnas mutants.

Bottom Line: Gαs is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components.Elevation in levels of a Gαs effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas-ablated mice.Thus, our gain- and loss-of-function studies identify a previously unrecognized tumor suppressor function for Gαs that can be found consistently across Shh-group medulloblastomas of disparate cellular and anatomical origins, highlighting G protein modulation as a potential therapeutic avenue.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Forensic Medicine, State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, School of Preclinical and Forensic Medicine, West China Second Hospital, Sichuan University, Chengdu, China. [2] Department of Pediatrics, Brain Tumor Center, Division of Experimental Hematology and Cancer Biology, Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

ABSTRACT
Medulloblastoma, the most common malignant childhood brain tumor, exhibits distinct molecular subtypes and cellular origins. Genetic alterations driving medulloblastoma initiation and progression remain poorly understood. Herein, we identify GNAS, encoding the G protein Gαs, as a potent tumor suppressor gene that, when expressed at low levels, defines a subset of aggressive Sonic hedgehog (SHH)-driven human medulloblastomas. Ablation of the single Gnas gene in anatomically distinct progenitors in mice is sufficient to induce Shh-associated medulloblastomas, which recapitulate their human counterparts. Gαs is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components. Elevation in levels of a Gαs effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas-ablated mice. Thus, our gain- and loss-of-function studies identify a previously unrecognized tumor suppressor function for Gαs that can be found consistently across Shh-group medulloblastomas of disparate cellular and anatomical origins, highlighting G protein modulation as a potential therapeutic avenue.

Show MeSH
Related in: MedlinePlus