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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 enhances tumor invasion through JNK activation.(A–E) Confocal cross sections of cephalic complexes from third instar larvae with GFP-marked tumors stained with an antibody against ElaV to mark terminally differentiated cells and phalloidin to reveal f-actin structure. VNCs are outlined in panels showing GFP expression, and arrows highlight invading tumor tissue. Expressing RasV12 alone in whole eye clones (A) causes a mild invasive phenotype characterized by either no invasion or migration of tumor cells from one optic lobe. Coexpression of CagA with RasV12 (B) dramatically enhances the extent of VNC invasion from both optic lobes, while coexpression of CagAEPISA with RasV12 (C) shows a milder enhancement of invasion. Coexpression of BskDN with RasV12 (D) does not significantly affect the invasive capacity of tumor cells, while coexpression of BskDN with RasV12 and CagA (E) suppresses the VNC invasion phenotype. Scale bar, 50 µm. (F) Projections of several confocal cross sections from third instar larval cephalic complexes with GFP-marked tumors showing different classes of invasiveness: (0) noninvasive, (1) invasion from one optic lobe, (2) invasion from both optic lobes, (3) significant invasion of the VNC. Brain lobes and ventral nerve cords are outlined. Scale bar, 50 µm. (G) Quantitation of the percentage of cephalic complexes classified into each category. The number of samples analyzed is shown above each column. * indicates a distribution that differs significantly compared to RasV12; † indicates a distribution that differs significantly compared to RasV12, CagA; p<0.0001.
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ppat-1002939-g005: CagA enhances tumor invasion through JNK activation.(A–E) Confocal cross sections of cephalic complexes from third instar larvae with GFP-marked tumors stained with an antibody against ElaV to mark terminally differentiated cells and phalloidin to reveal f-actin structure. VNCs are outlined in panels showing GFP expression, and arrows highlight invading tumor tissue. Expressing RasV12 alone in whole eye clones (A) causes a mild invasive phenotype characterized by either no invasion or migration of tumor cells from one optic lobe. Coexpression of CagA with RasV12 (B) dramatically enhances the extent of VNC invasion from both optic lobes, while coexpression of CagAEPISA with RasV12 (C) shows a milder enhancement of invasion. Coexpression of BskDN with RasV12 (D) does not significantly affect the invasive capacity of tumor cells, while coexpression of BskDN with RasV12 and CagA (E) suppresses the VNC invasion phenotype. Scale bar, 50 µm. (F) Projections of several confocal cross sections from third instar larval cephalic complexes with GFP-marked tumors showing different classes of invasiveness: (0) noninvasive, (1) invasion from one optic lobe, (2) invasion from both optic lobes, (3) significant invasion of the VNC. Brain lobes and ventral nerve cords are outlined. Scale bar, 50 µm. (G) Quantitation of the percentage of cephalic complexes classified into each category. The number of samples analyzed is shown above each column. * indicates a distribution that differs significantly compared to RasV12; † indicates a distribution that differs significantly compared to RasV12, CagA; p<0.0001.

Mentions: Generating whole eye clones that express RasV12 alone most commonly caused either a mildly invasive phenotype characterized by the migration of a small number of GFP-positive cells along one edge of the ventral nerve cord (VNC), or a noninvasive phenotype in which cells within the optic lobe approached but did not migrate into the VNC (Figure 5A). Whole eye clones expressing either GFP alone (Figure S5A) or with CagA (Figure S5B) were not invasive, but coexpression of CagA with RasV12 resulted in a much larger number of GFP-positive tumor cells migrating from both optic lobes into the VNC (Figure 5B). These cells were not terminally differentiated, as indicated by a lack of staining with the neuron-specific ElaV antibody, and phalloidin staining showed a morphology distinct from other cells in the VNC (Figure S5D). Expressing CagAEPISA in whole eye clones also did not produce an invasive phenotype (Figure S5C), and coexpression of CagAEPISA with RasV12 caused a less pronounced enhancement of the mild invasion caused by expression of RasV12 alone (Figure 5C), suggesting that the phosphorylation-resistant form of CagA is less effective at promoting tumor progression. Coexpression of BskDN did not affect the invasive phenotype generated by RasV12 expression alone (Figure 5D), but BskDN expression caused a dramatic reduction in the invasive capacity of tumors expressing both RasV12 and CagA (Figure 5E). These data show that CagA expression can enhance the invasion of RasV12-expressing tumor cells through JNK 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 enhances tumor invasion through JNK activation.(A–E) Confocal cross sections of cephalic complexes from third instar larvae with GFP-marked tumors stained with an antibody against ElaV to mark terminally differentiated cells and phalloidin to reveal f-actin structure. VNCs are outlined in panels showing GFP expression, and arrows highlight invading tumor tissue. Expressing RasV12 alone in whole eye clones (A) causes a mild invasive phenotype characterized by either no invasion or migration of tumor cells from one optic lobe. Coexpression of CagA with RasV12 (B) dramatically enhances the extent of VNC invasion from both optic lobes, while coexpression of CagAEPISA with RasV12 (C) shows a milder enhancement of invasion. Coexpression of BskDN with RasV12 (D) does not significantly affect the invasive capacity of tumor cells, while coexpression of BskDN with RasV12 and CagA (E) suppresses the VNC invasion phenotype. Scale bar, 50 µm. (F) Projections of several confocal cross sections from third instar larval cephalic complexes with GFP-marked tumors showing different classes of invasiveness: (0) noninvasive, (1) invasion from one optic lobe, (2) invasion from both optic lobes, (3) significant invasion of the VNC. Brain lobes and ventral nerve cords are outlined. Scale bar, 50 µm. (G) Quantitation of the percentage of cephalic complexes classified into each category. The number of samples analyzed is shown above each column. * indicates a distribution that differs significantly compared to RasV12; † indicates a distribution that differs significantly compared to RasV12, CagA; p<0.0001.
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ppat-1002939-g005: CagA enhances tumor invasion through JNK activation.(A–E) Confocal cross sections of cephalic complexes from third instar larvae with GFP-marked tumors stained with an antibody against ElaV to mark terminally differentiated cells and phalloidin to reveal f-actin structure. VNCs are outlined in panels showing GFP expression, and arrows highlight invading tumor tissue. Expressing RasV12 alone in whole eye clones (A) causes a mild invasive phenotype characterized by either no invasion or migration of tumor cells from one optic lobe. Coexpression of CagA with RasV12 (B) dramatically enhances the extent of VNC invasion from both optic lobes, while coexpression of CagAEPISA with RasV12 (C) shows a milder enhancement of invasion. Coexpression of BskDN with RasV12 (D) does not significantly affect the invasive capacity of tumor cells, while coexpression of BskDN with RasV12 and CagA (E) suppresses the VNC invasion phenotype. Scale bar, 50 µm. (F) Projections of several confocal cross sections from third instar larval cephalic complexes with GFP-marked tumors showing different classes of invasiveness: (0) noninvasive, (1) invasion from one optic lobe, (2) invasion from both optic lobes, (3) significant invasion of the VNC. Brain lobes and ventral nerve cords are outlined. Scale bar, 50 µm. (G) Quantitation of the percentage of cephalic complexes classified into each category. The number of samples analyzed is shown above each column. * indicates a distribution that differs significantly compared to RasV12; † indicates a distribution that differs significantly compared to RasV12, CagA; p<0.0001.
Mentions: Generating whole eye clones that express RasV12 alone most commonly caused either a mildly invasive phenotype characterized by the migration of a small number of GFP-positive cells along one edge of the ventral nerve cord (VNC), or a noninvasive phenotype in which cells within the optic lobe approached but did not migrate into the VNC (Figure 5A). Whole eye clones expressing either GFP alone (Figure S5A) or with CagA (Figure S5B) were not invasive, but coexpression of CagA with RasV12 resulted in a much larger number of GFP-positive tumor cells migrating from both optic lobes into the VNC (Figure 5B). These cells were not terminally differentiated, as indicated by a lack of staining with the neuron-specific ElaV antibody, and phalloidin staining showed a morphology distinct from other cells in the VNC (Figure S5D). Expressing CagAEPISA in whole eye clones also did not produce an invasive phenotype (Figure S5C), and coexpression of CagAEPISA with RasV12 caused a less pronounced enhancement of the mild invasion caused by expression of RasV12 alone (Figure 5C), suggesting that the phosphorylation-resistant form of CagA is less effective at promoting tumor progression. Coexpression of BskDN did not affect the invasive phenotype generated by RasV12 expression alone (Figure 5D), but BskDN expression caused a dramatic reduction in the invasive capacity of tumors expressing both RasV12 and CagA (Figure 5E). These data show that CagA expression can enhance the invasion of RasV12-expressing tumor cells through JNK 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