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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 genetically interacts with nTSGs, Eiger and Rho1.(A–E) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression with bx-GAL4 and stained with anti-active caspase-3 antibody to mark apoptotic cells. Dorsal wing expression of CagA with bx-GAL4 (A) causes formation of apoptotic clusters. RNAi-mediated knockdown of the nTSG Dlg alone (B) does not cause significant apoptosis, but enhances apoptosis induced by CagA expression (C). The apoptosis phenotype is enhanced when CagA is expressed in an egr mutant background (D). Coexpression of Rho1 with CagA (E) also enhances apoptosis. Scale bars, 50 µm. (F) Quantitation of apoptosis as a percentage of the expression domain showing active caspase-3 staining, n = 10 or 15 wing discs per genotype; bar indicates average value for each group. * indicates values that show significant enhancement compared to CagA, whose quantitation (from Figure 2) is provided for comparison; p<0.0001. (G) A model showing the localization of polarity protein complexes in an epithelial cell, their known interactions with other upstream activators of JNK signaling in Drosophila, and the downstream effects of these interactions.
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ppat-1002939-g003: CagA genetically interacts with nTSGs, Eiger and Rho1.(A–E) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression with bx-GAL4 and stained with anti-active caspase-3 antibody to mark apoptotic cells. Dorsal wing expression of CagA with bx-GAL4 (A) causes formation of apoptotic clusters. RNAi-mediated knockdown of the nTSG Dlg alone (B) does not cause significant apoptosis, but enhances apoptosis induced by CagA expression (C). The apoptosis phenotype is enhanced when CagA is expressed in an egr mutant background (D). Coexpression of Rho1 with CagA (E) also enhances apoptosis. Scale bars, 50 µm. (F) Quantitation of apoptosis as a percentage of the expression domain showing active caspase-3 staining, n = 10 or 15 wing discs per genotype; bar indicates average value for each group. * indicates values that show significant enhancement compared to CagA, whose quantitation (from Figure 2) is provided for comparison; p<0.0001. (G) A model showing the localization of polarity protein complexes in an epithelial cell, their known interactions with other upstream activators of JNK signaling in Drosophila, and the downstream effects of these interactions.

Mentions: JNK signaling is activated by a complex set of signals including TNF and loss of epithelial polarity (Figure 2M). To examine the mechanism through which CagA activates JNK signaling, we used the bx-GAL4 driver to express CagA in combination with RNAi-mediated knockdown of known epithelial polarity determinants and examined wing imaginal discs for enhancement of the apoptosis phenotype (Figure 3A). We tested a panel of polarity proteins, many of which caused apoptosis when knocked down in the absence of CagA expression (Table S1). We chose to target a protein from each of the previously described complexes whose localization and function establish epithelial cell polarity [27], and to simplify our analysis we selected polarity proteins that did not cause an apoptosis phenotype when knocked down on their own (Figure S3A–S3C). When tested in combination with CagA expression, we found that RNAi-mediated knockdown of neither the junctional protein Bazooka (Baz), nor the apical protein Crumbs (Crb) enhanced apoptosis (Figure S3D and S3E). In addition, knockdown of Par1, which has been shown to interact with CagA in tissue culture cells [28], did not enhance the apoptosis phenotype caused by CagA expression in this context (Figure S3F).


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 genetically interacts with nTSGs, Eiger and Rho1.(A–E) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression with bx-GAL4 and stained with anti-active caspase-3 antibody to mark apoptotic cells. Dorsal wing expression of CagA with bx-GAL4 (A) causes formation of apoptotic clusters. RNAi-mediated knockdown of the nTSG Dlg alone (B) does not cause significant apoptosis, but enhances apoptosis induced by CagA expression (C). The apoptosis phenotype is enhanced when CagA is expressed in an egr mutant background (D). Coexpression of Rho1 with CagA (E) also enhances apoptosis. Scale bars, 50 µm. (F) Quantitation of apoptosis as a percentage of the expression domain showing active caspase-3 staining, n = 10 or 15 wing discs per genotype; bar indicates average value for each group. * indicates values that show significant enhancement compared to CagA, whose quantitation (from Figure 2) is provided for comparison; p<0.0001. (G) A model showing the localization of polarity protein complexes in an epithelial cell, their known interactions with other upstream activators of JNK signaling in Drosophila, and the downstream effects of these interactions.
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ppat-1002939-g003: CagA genetically interacts with nTSGs, Eiger and Rho1.(A–E) Confocal cross sections of male third instar larval wing imaginal discs showing mGFP expression with bx-GAL4 and stained with anti-active caspase-3 antibody to mark apoptotic cells. Dorsal wing expression of CagA with bx-GAL4 (A) causes formation of apoptotic clusters. RNAi-mediated knockdown of the nTSG Dlg alone (B) does not cause significant apoptosis, but enhances apoptosis induced by CagA expression (C). The apoptosis phenotype is enhanced when CagA is expressed in an egr mutant background (D). Coexpression of Rho1 with CagA (E) also enhances apoptosis. Scale bars, 50 µm. (F) Quantitation of apoptosis as a percentage of the expression domain showing active caspase-3 staining, n = 10 or 15 wing discs per genotype; bar indicates average value for each group. * indicates values that show significant enhancement compared to CagA, whose quantitation (from Figure 2) is provided for comparison; p<0.0001. (G) A model showing the localization of polarity protein complexes in an epithelial cell, their known interactions with other upstream activators of JNK signaling in Drosophila, and the downstream effects of these interactions.
Mentions: JNK signaling is activated by a complex set of signals including TNF and loss of epithelial polarity (Figure 2M). To examine the mechanism through which CagA activates JNK signaling, we used the bx-GAL4 driver to express CagA in combination with RNAi-mediated knockdown of known epithelial polarity determinants and examined wing imaginal discs for enhancement of the apoptosis phenotype (Figure 3A). We tested a panel of polarity proteins, many of which caused apoptosis when knocked down in the absence of CagA expression (Table S1). We chose to target a protein from each of the previously described complexes whose localization and function establish epithelial cell polarity [27], and to simplify our analysis we selected polarity proteins that did not cause an apoptosis phenotype when knocked down on their own (Figure S3A–S3C). When tested in combination with CagA expression, we found that RNAi-mediated knockdown of neither the junctional protein Bazooka (Baz), nor the apical protein Crumbs (Crb) enhanced apoptosis (Figure S3D and S3E). In addition, knockdown of Par1, which has been shown to interact with CagA in tissue culture cells [28], did not enhance the apoptosis phenotype caused by CagA expression in this context (Figure S3F).

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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Related in: MedlinePlus