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Transgenic expression of the Helicobacter pylori virulence factor CagA promotes apoptosis or tumorigenesis through JNK activation in Drosophila.

Wandler AM, Guillemin K - PLoS Pathog. (2012)

Bottom Line: This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells.We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis.In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.

ABSTRACT
Gastric cancer development is strongly correlated with infection by Helicobacter pylori possessing the effector protein CagA. Using a transgenic Drosophila melanogaster model, we show that CagA expression in the simple model epithelium of the larval wing imaginal disc causes dramatic tissue perturbations and apoptosis when CagA-expressing and non-expressing cells are juxtaposed. This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells. We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis. In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner. These data suggest a potential role for CagA-mediated JNK pathway activation in promoting gastric cancer progression.

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CagA-induced apoptosis occurs through JNK pathway activation.(A–E) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression with bx-GAL4 and stained with an anti-active caspase-3 antibody to mark apoptotic cells. Ectopic overexpression of wild type Bsk in the dorsal wing disc (A) causes a mild apoptosis phenotype that is strongly enhanced by coexpression with CagA (B). Coexpression of Bsk with CagAEPISA (C) also enhances the apoptosis phenotype. Expression of BskDN alone (D) does not cause apoptosis, and coexpression with CagA (E) strongly suppresses apoptosis induced by CagA expression. Scale bars, 50 µm. (F–J) Adult wing images from male flies expressing different forms of Bsk alone or in combination with CagA. Ectopic overexpression of Bsk with bx-GAL4 (F) causes only subtle vein defects in the adult wing, while coexpression with CagA (G) enhances epithelial disruption. Coexpression of Bsk with CagAEPISA (H) does not significantly affect formation of the adult wing structure. Expression of BskDN with bx-GAL4 (I) also causes only subtle vein defects in the adult wing, while coexpression with CagA (J) enhances epithelial disruption. Arrowheads highlight ectopic veins in adult wings expressing different forms of Bsk alone. Scale bar, 500 µm. (K) Quantitation of apoptosis as a percentage of the expression domain showing active caspase-3 staining, n = 15 wing discs per genotype; bar indicates average value for each group. * indicates values that differ significantly from the control with expression of a single transgene; † indicates values that show significant enhancement or suppression compared to CagA; ‡ indicates values that show significant enhancement compared to CagAEPISA; p<0.0001. (L) Confocal cross section of a male wing imaginal disc epithelium carrying the puc-lacZ reporter allele and expressing mGFP and CagA with bx-GAL4. Staining with antibodies against active caspase-3 and β-galactosidase (β-gal) shows that apoptotic cells lie adjacent to those in which JNK signaling has been activated. Scale bar, 50 µm. (M) A model of the JNK pathway depicting the multiple upstream activators known to induce JNK-dependent apoptosis in Drosophila, and indicating human homologs for each pathway component.
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ppat-1002939-g002: CagA-induced apoptosis occurs through JNK pathway activation.(A–E) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression with bx-GAL4 and stained with an anti-active caspase-3 antibody to mark apoptotic cells. Ectopic overexpression of wild type Bsk in the dorsal wing disc (A) causes a mild apoptosis phenotype that is strongly enhanced by coexpression with CagA (B). Coexpression of Bsk with CagAEPISA (C) also enhances the apoptosis phenotype. Expression of BskDN alone (D) does not cause apoptosis, and coexpression with CagA (E) strongly suppresses apoptosis induced by CagA expression. Scale bars, 50 µm. (F–J) Adult wing images from male flies expressing different forms of Bsk alone or in combination with CagA. Ectopic overexpression of Bsk with bx-GAL4 (F) causes only subtle vein defects in the adult wing, while coexpression with CagA (G) enhances epithelial disruption. Coexpression of Bsk with CagAEPISA (H) does not significantly affect formation of the adult wing structure. Expression of BskDN with bx-GAL4 (I) also causes only subtle vein defects in the adult wing, while coexpression with CagA (J) enhances epithelial disruption. Arrowheads highlight ectopic veins in adult wings expressing different forms of Bsk alone. Scale bar, 500 µm. (K) Quantitation of apoptosis as a percentage of the expression domain showing active caspase-3 staining, n = 15 wing discs per genotype; bar indicates average value for each group. * indicates values that differ significantly from the control with expression of a single transgene; † indicates values that show significant enhancement or suppression compared to CagA; ‡ indicates values that show significant enhancement compared to CagAEPISA; p<0.0001. (L) Confocal cross section of a male wing imaginal disc epithelium carrying the puc-lacZ reporter allele and expressing mGFP and CagA with bx-GAL4. Staining with antibodies against active caspase-3 and β-galactosidase (β-gal) shows that apoptotic cells lie adjacent to those in which JNK signaling has been activated. Scale bar, 50 µm. (M) A model of the JNK pathway depicting the multiple upstream activators known to induce JNK-dependent apoptosis in Drosophila, and indicating human homologs for each pathway component.

Mentions: Several aspects of the apoptosis phenotype caused by CagA expression in the wing imaginal disc suggested an interaction between CagA and the JNK pathway. In order to determine the nature of this potential interaction, we examined the effects of expressing several forms of Bsk, the Drosophila homolog of JNK, on the CagA-induced wing phenotype. Ectopic overexpression of wild type Bsk with the bx-GAL4 dorsal wing driver generated small apoptotic clusters (Figure 2A), indicating that the presence of excess JNK in the wing can phenocopy CagA expression. Furthermore, the cell death phenotype caused by CagA expression in the wing was dramatically enhanced by coexpression with wild type Bsk (Figure 2B). Coexpression of Bsk with CagAEPISA also caused a substantial amount of apoptosis in the wing imaginal disc, suggesting that this interaction is not dependent on phosphorylated CagA (Figure 2C). As expected, expression of a dominant-negative form of Bsk (BskDN) alone did not cause apoptosis in the wing imaginal disc (Figure 2D). Significantly, coexpression of BskDN with CagA almost completely suppressed the apoptosis phenotype caused by CagA expression (Figure 2E), indicating that blocking JNK signaling suppresses CagA-dependent cell death in the wing. These data suggest that CagA expression triggers wing imaginal disc apoptosis through JNK pathway activation.


Transgenic expression of the Helicobacter pylori virulence factor CagA promotes apoptosis or tumorigenesis through JNK activation in Drosophila.

Wandler AM, Guillemin K - PLoS Pathog. (2012)

CagA-induced apoptosis occurs through JNK pathway activation.(A–E) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression with bx-GAL4 and stained with an anti-active caspase-3 antibody to mark apoptotic cells. Ectopic overexpression of wild type Bsk in the dorsal wing disc (A) causes a mild apoptosis phenotype that is strongly enhanced by coexpression with CagA (B). Coexpression of Bsk with CagAEPISA (C) also enhances the apoptosis phenotype. Expression of BskDN alone (D) does not cause apoptosis, and coexpression with CagA (E) strongly suppresses apoptosis induced by CagA expression. Scale bars, 50 µm. (F–J) Adult wing images from male flies expressing different forms of Bsk alone or in combination with CagA. Ectopic overexpression of Bsk with bx-GAL4 (F) causes only subtle vein defects in the adult wing, while coexpression with CagA (G) enhances epithelial disruption. Coexpression of Bsk with CagAEPISA (H) does not significantly affect formation of the adult wing structure. Expression of BskDN with bx-GAL4 (I) also causes only subtle vein defects in the adult wing, while coexpression with CagA (J) enhances epithelial disruption. Arrowheads highlight ectopic veins in adult wings expressing different forms of Bsk alone. Scale bar, 500 µm. (K) Quantitation of apoptosis as a percentage of the expression domain showing active caspase-3 staining, n = 15 wing discs per genotype; bar indicates average value for each group. * indicates values that differ significantly from the control with expression of a single transgene; † indicates values that show significant enhancement or suppression compared to CagA; ‡ indicates values that show significant enhancement compared to CagAEPISA; p<0.0001. (L) Confocal cross section of a male wing imaginal disc epithelium carrying the puc-lacZ reporter allele and expressing mGFP and CagA with bx-GAL4. Staining with antibodies against active caspase-3 and β-galactosidase (β-gal) shows that apoptotic cells lie adjacent to those in which JNK signaling has been activated. Scale bar, 50 µm. (M) A model of the JNK pathway depicting the multiple upstream activators known to induce JNK-dependent apoptosis in Drosophila, and indicating human homologs for each pathway component.
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ppat-1002939-g002: CagA-induced apoptosis occurs through JNK pathway activation.(A–E) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression with bx-GAL4 and stained with an anti-active caspase-3 antibody to mark apoptotic cells. Ectopic overexpression of wild type Bsk in the dorsal wing disc (A) causes a mild apoptosis phenotype that is strongly enhanced by coexpression with CagA (B). Coexpression of Bsk with CagAEPISA (C) also enhances the apoptosis phenotype. Expression of BskDN alone (D) does not cause apoptosis, and coexpression with CagA (E) strongly suppresses apoptosis induced by CagA expression. Scale bars, 50 µm. (F–J) Adult wing images from male flies expressing different forms of Bsk alone or in combination with CagA. Ectopic overexpression of Bsk with bx-GAL4 (F) causes only subtle vein defects in the adult wing, while coexpression with CagA (G) enhances epithelial disruption. Coexpression of Bsk with CagAEPISA (H) does not significantly affect formation of the adult wing structure. Expression of BskDN with bx-GAL4 (I) also causes only subtle vein defects in the adult wing, while coexpression with CagA (J) enhances epithelial disruption. Arrowheads highlight ectopic veins in adult wings expressing different forms of Bsk alone. Scale bar, 500 µm. (K) Quantitation of apoptosis as a percentage of the expression domain showing active caspase-3 staining, n = 15 wing discs per genotype; bar indicates average value for each group. * indicates values that differ significantly from the control with expression of a single transgene; † indicates values that show significant enhancement or suppression compared to CagA; ‡ indicates values that show significant enhancement compared to CagAEPISA; p<0.0001. (L) Confocal cross section of a male wing imaginal disc epithelium carrying the puc-lacZ reporter allele and expressing mGFP and CagA with bx-GAL4. Staining with antibodies against active caspase-3 and β-galactosidase (β-gal) shows that apoptotic cells lie adjacent to those in which JNK signaling has been activated. Scale bar, 50 µm. (M) A model of the JNK pathway depicting the multiple upstream activators known to induce JNK-dependent apoptosis in Drosophila, and indicating human homologs for each pathway component.
Mentions: Several aspects of the apoptosis phenotype caused by CagA expression in the wing imaginal disc suggested an interaction between CagA and the JNK pathway. In order to determine the nature of this potential interaction, we examined the effects of expressing several forms of Bsk, the Drosophila homolog of JNK, on the CagA-induced wing phenotype. Ectopic overexpression of wild type Bsk with the bx-GAL4 dorsal wing driver generated small apoptotic clusters (Figure 2A), indicating that the presence of excess JNK in the wing can phenocopy CagA expression. Furthermore, the cell death phenotype caused by CagA expression in the wing was dramatically enhanced by coexpression with wild type Bsk (Figure 2B). Coexpression of Bsk with CagAEPISA also caused a substantial amount of apoptosis in the wing imaginal disc, suggesting that this interaction is not dependent on phosphorylated CagA (Figure 2C). As expected, expression of a dominant-negative form of Bsk (BskDN) alone did not cause apoptosis in the wing imaginal disc (Figure 2D). Significantly, coexpression of BskDN with CagA almost completely suppressed the apoptosis phenotype caused by CagA expression (Figure 2E), indicating that blocking JNK signaling suppresses CagA-dependent cell death in the wing. These data suggest that CagA expression triggers wing imaginal disc apoptosis through JNK pathway activation.

Bottom Line: This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells.We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis.In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA.

ABSTRACT
Gastric cancer development is strongly correlated with infection by Helicobacter pylori possessing the effector protein CagA. Using a transgenic Drosophila melanogaster model, we show that CagA expression in the simple model epithelium of the larval wing imaginal disc causes dramatic tissue perturbations and apoptosis when CagA-expressing and non-expressing cells are juxtaposed. This cell death phenotype occurs through activation of JNK signaling and is enhanced by loss of the neoplastic tumor suppressors in CagA-expressing cells or loss of the TNF homolog Eiger in wild type neighboring cells. We further explored the effects of CagA-mediated JNK pathway activation on an epithelium in the context of oncogenic Ras activation, using a Drosophila model of metastasis. In this model, CagA expression in epithelial cells enhances the growth and invasion of tumors in a JNK-dependent manner. These data suggest a potential role for CagA-mediated JNK pathway activation in promoting gastric cancer progression.

Show MeSH
Related in: MedlinePlus