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Enteric bacterial protein AvrA promotes colonic tumorigenesis and activates colonic beta-catenin signaling pathway.

Lu R, Wu S, Zhang YG, Xia Y, Liu X, Zheng Y, Chen H, Schaefer KL, Zhou Z, Bissonnette M, Li L, Sun J - Oncogenesis (2014)

Bottom Line: In the current study, we colonized mice with Salmonella AvrA-sufficient or AvrA-deficient Salmonella typhimirium strains and induced inflammation-associated colon cancer by azoxymethane/dextran sulfate sodium (AOM/DSS).Tumor incidence in the AvrA+infected group was 100% compared with 51.4% in the AOM/DSS group without bacterial gavage and 56.3% in mice infected with the AvrA- strain.Our observations also raise a note of caution regarding the use of mutant Salmonella organisms as vectors for anti-cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Rush University, Chicago, IL, USA.

ABSTRACT
Salmonella infections can become chronic and increase the risk of cancer. The mechanisms by which specific Salmonella organisms contribute to cancer, however, are still unknown. Live and attenuated Salmonella are used as vectors to target cancer cells, but there have been no systematic studies of the oncogenic potential of chronic Salmonella infections in cancer models. AvrA, a pathogenic product of Salmonella, is inserted into host cells during infection and influences eukaryotic cell pathways. In the current study, we colonized mice with Salmonella AvrA-sufficient or AvrA-deficient Salmonella typhimirium strains and induced inflammation-associated colon cancer by azoxymethane/dextran sulfate sodium (AOM/DSS). We confirmed Salmonella persisted in the colon for up to 45 weeks. Salmonella was identified not only in epithelial cells on the colonic luminal surface and base of the crypts but also in invading tumors. Tumor incidence in the AvrA+infected group was 100% compared with 51.4% in the AOM/DSS group without bacterial gavage and 56.3% in mice infected with the AvrA- strain. Infection with AvrA+ strain also altered tumor distribution from the distal to proximal colon that might reflect changes in the microbiome. AvrA-expressing bacteria also upregulated beta-catenin signaling as assessed by decreased beta-catenin ubiquitination, increased nuclear beta-catenin and increased phosphorylated-beta-catenin (Ser552), a marker of proliferating stem-progenitor cells. Other β-catenin targets increased by AvrA included Bmi1, a cancer stem cell marker, matrix metalloproteinase-7, and cyclin D1. In summary, AvrA-expressing Salmonella infection activates β-catenin signals and enhances colonic tumorigenesis. Our findings provide important new mechanistic insights into how a bacterial protein targets proliferating stem-progenitor cells and contributes to cancer development. Our observations also raise a note of caution regarding the use of mutant Salmonella organisms as vectors for anti-cancer therapy. Finally, these studies could suggest biomarkers (such as AvrA level in gut) to assess cancer risk in susceptible individuals and infection-related dysregulation of β-catenin signaling in cancer.

No MeSH data available.


Related in: MedlinePlus

Bacterial effector protein AvrA modulates β-catenin ubiquitination in colonic epithelial cells. (a) AvrA expression decreased the ub-β-catenin in the colonic epithelial cells. At 6 weeks after Salmonella infection, colonic epithelial cells were collected and immunoprecipitated with anti-β-catenin antibody. Membranes were probed with anti-β-catenin and anti-ubiquitin antibody and visualized using enhanced chemiluminescence. Protein input was normalized by the colonic epithelial marker villin. Ubiquitinated (ub) β-catenin is shown as smear. Colon infected by the PhoPC lacked the ub-β-catenin bands. (b) AvrA expression decreased the ub-β-catenin through ubiquitination in the human colonic epithelial HCT116 cells. Cells were transiently cotransfected with AvrA and the indicated ubiquitin plasmids; 24 h later, the cells were treated with MG262 for 3 h. The ubiquitin plasmids included pRK5-HA-Ub-KO (all lysines mutated), -Ub WT, -Ub-K48 (K48 only, other lysines mutated) and -Ub-K63 (K63 only, other lysines mutated). Equal amounts of total cell lysates were analyzed for protein levels by immunoblot with anti-β-catenin antibody. (c) AvrA expression mostly removes k48-link ubiquitins in the human colonic epithelial HCT116 cells. Cells were transiently cotransfected with AvrA and the indicated ubiquitin plasmids. Equal amounts of total cell lysates were analyzed for protein levels by immunoblot with anti-ub-48 antibody.
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fig4: Bacterial effector protein AvrA modulates β-catenin ubiquitination in colonic epithelial cells. (a) AvrA expression decreased the ub-β-catenin in the colonic epithelial cells. At 6 weeks after Salmonella infection, colonic epithelial cells were collected and immunoprecipitated with anti-β-catenin antibody. Membranes were probed with anti-β-catenin and anti-ubiquitin antibody and visualized using enhanced chemiluminescence. Protein input was normalized by the colonic epithelial marker villin. Ubiquitinated (ub) β-catenin is shown as smear. Colon infected by the PhoPC lacked the ub-β-catenin bands. (b) AvrA expression decreased the ub-β-catenin through ubiquitination in the human colonic epithelial HCT116 cells. Cells were transiently cotransfected with AvrA and the indicated ubiquitin plasmids; 24 h later, the cells were treated with MG262 for 3 h. The ubiquitin plasmids included pRK5-HA-Ub-KO (all lysines mutated), -Ub WT, -Ub-K48 (K48 only, other lysines mutated) and -Ub-K63 (K63 only, other lysines mutated). Equal amounts of total cell lysates were analyzed for protein levels by immunoblot with anti-β-catenin antibody. (c) AvrA expression mostly removes k48-link ubiquitins in the human colonic epithelial HCT116 cells. Cells were transiently cotransfected with AvrA and the indicated ubiquitin plasmids. Equal amounts of total cell lysates were analyzed for protein levels by immunoblot with anti-ub-48 antibody.

Mentions: Our previous study showed that AvrA protein acts as a deubiquitinase to stabilize β-catenin.14 To determine whether AvrA is responsible for the activation of β-catenin signaling through ubiquitination, we investigated the ubiquitinated β-catenin levels in the AOM/DSS mice with or without Salmonella (Figure 4a). The ubiquitinated proteins results in the appearance of multiple higher molecular weight bands above the regular protein band in western blotting. We found that mice colonized with Salmonella strain PhoPC and PhoPC AvrA−/AvrA+ lacked detectable ubiquitinated β-catenin bands (Figure 4a). In contrast, in the PhoPC AvrA− group, ubiquitinated β-catenin levels were similar to the control AOM/DSS mice without Salmonella (Figure 4a).


Enteric bacterial protein AvrA promotes colonic tumorigenesis and activates colonic beta-catenin signaling pathway.

Lu R, Wu S, Zhang YG, Xia Y, Liu X, Zheng Y, Chen H, Schaefer KL, Zhou Z, Bissonnette M, Li L, Sun J - Oncogenesis (2014)

Bacterial effector protein AvrA modulates β-catenin ubiquitination in colonic epithelial cells. (a) AvrA expression decreased the ub-β-catenin in the colonic epithelial cells. At 6 weeks after Salmonella infection, colonic epithelial cells were collected and immunoprecipitated with anti-β-catenin antibody. Membranes were probed with anti-β-catenin and anti-ubiquitin antibody and visualized using enhanced chemiluminescence. Protein input was normalized by the colonic epithelial marker villin. Ubiquitinated (ub) β-catenin is shown as smear. Colon infected by the PhoPC lacked the ub-β-catenin bands. (b) AvrA expression decreased the ub-β-catenin through ubiquitination in the human colonic epithelial HCT116 cells. Cells were transiently cotransfected with AvrA and the indicated ubiquitin plasmids; 24 h later, the cells were treated with MG262 for 3 h. The ubiquitin plasmids included pRK5-HA-Ub-KO (all lysines mutated), -Ub WT, -Ub-K48 (K48 only, other lysines mutated) and -Ub-K63 (K63 only, other lysines mutated). Equal amounts of total cell lysates were analyzed for protein levels by immunoblot with anti-β-catenin antibody. (c) AvrA expression mostly removes k48-link ubiquitins in the human colonic epithelial HCT116 cells. Cells were transiently cotransfected with AvrA and the indicated ubiquitin plasmids. Equal amounts of total cell lysates were analyzed for protein levels by immunoblot with anti-ub-48 antibody.
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fig4: Bacterial effector protein AvrA modulates β-catenin ubiquitination in colonic epithelial cells. (a) AvrA expression decreased the ub-β-catenin in the colonic epithelial cells. At 6 weeks after Salmonella infection, colonic epithelial cells were collected and immunoprecipitated with anti-β-catenin antibody. Membranes were probed with anti-β-catenin and anti-ubiquitin antibody and visualized using enhanced chemiluminescence. Protein input was normalized by the colonic epithelial marker villin. Ubiquitinated (ub) β-catenin is shown as smear. Colon infected by the PhoPC lacked the ub-β-catenin bands. (b) AvrA expression decreased the ub-β-catenin through ubiquitination in the human colonic epithelial HCT116 cells. Cells were transiently cotransfected with AvrA and the indicated ubiquitin plasmids; 24 h later, the cells were treated with MG262 for 3 h. The ubiquitin plasmids included pRK5-HA-Ub-KO (all lysines mutated), -Ub WT, -Ub-K48 (K48 only, other lysines mutated) and -Ub-K63 (K63 only, other lysines mutated). Equal amounts of total cell lysates were analyzed for protein levels by immunoblot with anti-β-catenin antibody. (c) AvrA expression mostly removes k48-link ubiquitins in the human colonic epithelial HCT116 cells. Cells were transiently cotransfected with AvrA and the indicated ubiquitin plasmids. Equal amounts of total cell lysates were analyzed for protein levels by immunoblot with anti-ub-48 antibody.
Mentions: Our previous study showed that AvrA protein acts as a deubiquitinase to stabilize β-catenin.14 To determine whether AvrA is responsible for the activation of β-catenin signaling through ubiquitination, we investigated the ubiquitinated β-catenin levels in the AOM/DSS mice with or without Salmonella (Figure 4a). The ubiquitinated proteins results in the appearance of multiple higher molecular weight bands above the regular protein band in western blotting. We found that mice colonized with Salmonella strain PhoPC and PhoPC AvrA−/AvrA+ lacked detectable ubiquitinated β-catenin bands (Figure 4a). In contrast, in the PhoPC AvrA− group, ubiquitinated β-catenin levels were similar to the control AOM/DSS mice without Salmonella (Figure 4a).

Bottom Line: In the current study, we colonized mice with Salmonella AvrA-sufficient or AvrA-deficient Salmonella typhimirium strains and induced inflammation-associated colon cancer by azoxymethane/dextran sulfate sodium (AOM/DSS).Tumor incidence in the AvrA+infected group was 100% compared with 51.4% in the AOM/DSS group without bacterial gavage and 56.3% in mice infected with the AvrA- strain.Our observations also raise a note of caution regarding the use of mutant Salmonella organisms as vectors for anti-cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Rush University, Chicago, IL, USA.

ABSTRACT
Salmonella infections can become chronic and increase the risk of cancer. The mechanisms by which specific Salmonella organisms contribute to cancer, however, are still unknown. Live and attenuated Salmonella are used as vectors to target cancer cells, but there have been no systematic studies of the oncogenic potential of chronic Salmonella infections in cancer models. AvrA, a pathogenic product of Salmonella, is inserted into host cells during infection and influences eukaryotic cell pathways. In the current study, we colonized mice with Salmonella AvrA-sufficient or AvrA-deficient Salmonella typhimirium strains and induced inflammation-associated colon cancer by azoxymethane/dextran sulfate sodium (AOM/DSS). We confirmed Salmonella persisted in the colon for up to 45 weeks. Salmonella was identified not only in epithelial cells on the colonic luminal surface and base of the crypts but also in invading tumors. Tumor incidence in the AvrA+infected group was 100% compared with 51.4% in the AOM/DSS group without bacterial gavage and 56.3% in mice infected with the AvrA- strain. Infection with AvrA+ strain also altered tumor distribution from the distal to proximal colon that might reflect changes in the microbiome. AvrA-expressing bacteria also upregulated beta-catenin signaling as assessed by decreased beta-catenin ubiquitination, increased nuclear beta-catenin and increased phosphorylated-beta-catenin (Ser552), a marker of proliferating stem-progenitor cells. Other β-catenin targets increased by AvrA included Bmi1, a cancer stem cell marker, matrix metalloproteinase-7, and cyclin D1. In summary, AvrA-expressing Salmonella infection activates β-catenin signals and enhances colonic tumorigenesis. Our findings provide important new mechanistic insights into how a bacterial protein targets proliferating stem-progenitor cells and contributes to cancer development. Our observations also raise a note of caution regarding the use of mutant Salmonella organisms as vectors for anti-cancer therapy. Finally, these studies could suggest biomarkers (such as AvrA level in gut) to assess cancer risk in susceptible individuals and infection-related dysregulation of β-catenin signaling in cancer.

No MeSH data available.


Related in: MedlinePlus