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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

Persistent Salmonella infection in mice. Mice were infected with the indicated Salmonella strains by oral gavage and treated with AOM-DSS to induce colon cancer. (a) Schema of experimental design. Day-1: Mice were infected by Salmonella gavage 24 h after streptomycin pretreatment (7.5 mg/mouse), ↓azoxymethane (AOM), 10 mg/kg body weight, intraperitoneal injection; ▪ 1% dextran sodium sulfate (DSS) in drinking water, ↓Salmonella gavage; ▴ mice were killed. Experimental groups: Control, normal WT mice (no Salmonella, no AOM/DSS); AOM/DSS, only (no Salmonella), PhoPC, AOM/DSS-treated mice infected with Salmonella with WT AvrA; PhoPCAvrA−, AOM/DSS-treated mice infected with AvrA− mutant strain derived from PhoPC; PhoPCAvrA−/AvrA+, AOM/DSS-treated mice infected with PhoPCAvrA− transfected with plasmid encoding AvrA. (b) Chronic Salmonella infection was assessed by fecal cultures at week 45. On a BBL CHROMagar plate, intestinal Salmonella species appear mauve (rose to purple) in color, due to metabolic differences in the presence of selected chromogens. Other bacteria are either inhibited or produce blue–green or colorless colonies. (c) Salmonella detected in fecal microbial DNA by 16S rRNA PCR 45 weeks postinfection. Mice were infected with PhoPC expressing AvrA protein or PhoPC AvrA− (AvrA deficient) or AvrA-deficient bacteria transfected with AvrA-expressing plasmid. (d) AvrA western blotting. Protein lysates from colonic tissue were probed for AvrA levels. (e) Immunofluorescence confocal microscopy. Salmonella invading the colonic epithelium 45 weeks postinfection appear green.
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fig1: Persistent Salmonella infection in mice. Mice were infected with the indicated Salmonella strains by oral gavage and treated with AOM-DSS to induce colon cancer. (a) Schema of experimental design. Day-1: Mice were infected by Salmonella gavage 24 h after streptomycin pretreatment (7.5 mg/mouse), ↓azoxymethane (AOM), 10 mg/kg body weight, intraperitoneal injection; ▪ 1% dextran sodium sulfate (DSS) in drinking water, ↓Salmonella gavage; ▴ mice were killed. Experimental groups: Control, normal WT mice (no Salmonella, no AOM/DSS); AOM/DSS, only (no Salmonella), PhoPC, AOM/DSS-treated mice infected with Salmonella with WT AvrA; PhoPCAvrA−, AOM/DSS-treated mice infected with AvrA− mutant strain derived from PhoPC; PhoPCAvrA−/AvrA+, AOM/DSS-treated mice infected with PhoPCAvrA− transfected with plasmid encoding AvrA. (b) Chronic Salmonella infection was assessed by fecal cultures at week 45. On a BBL CHROMagar plate, intestinal Salmonella species appear mauve (rose to purple) in color, due to metabolic differences in the presence of selected chromogens. Other bacteria are either inhibited or produce blue–green or colorless colonies. (c) Salmonella detected in fecal microbial DNA by 16S rRNA PCR 45 weeks postinfection. Mice were infected with PhoPC expressing AvrA protein or PhoPC AvrA− (AvrA deficient) or AvrA-deficient bacteria transfected with AvrA-expressing plasmid. (d) AvrA western blotting. Protein lysates from colonic tissue were probed for AvrA levels. (e) Immunofluorescence confocal microscopy. Salmonella invading the colonic epithelium 45 weeks postinfection appear green.

Mentions: Mice were infected with the indicated Salmonella strains by one oral gavage and then treated with an AOM-DSS protocol to induce colon cancer. The treatment protocol is summarized in Figure 1a. Colonic Salmonella persistence was confirmed by Salmonella culture (pink colonies) and 16S RNA PCR of fecal bacteria DNA (Figures 1b and c). AvrA persistence was also confirmed by western blotting of colon tissue from mice infected with AvrA-expressing bacteria 45 weeks postinfection (Figure 1d). Furthermore, we confirmed persistent colonization of Salmonella in the colon by immunostaining (Figure 1e). Note that Salmonella was identified at the base and surface of the crypts in the infected colon (indicated by arrows) (Figure 1e). We found no significant changes in body weight associated with infection (Supplementary Figure S1). Salmonella levels in the intestine, gallbladder, spleen and liver also correlated with expression of AvrA in infected mice (Supplementary Figure S2).


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)

Persistent Salmonella infection in mice. Mice were infected with the indicated Salmonella strains by oral gavage and treated with AOM-DSS to induce colon cancer. (a) Schema of experimental design. Day-1: Mice were infected by Salmonella gavage 24 h after streptomycin pretreatment (7.5 mg/mouse), ↓azoxymethane (AOM), 10 mg/kg body weight, intraperitoneal injection; ▪ 1% dextran sodium sulfate (DSS) in drinking water, ↓Salmonella gavage; ▴ mice were killed. Experimental groups: Control, normal WT mice (no Salmonella, no AOM/DSS); AOM/DSS, only (no Salmonella), PhoPC, AOM/DSS-treated mice infected with Salmonella with WT AvrA; PhoPCAvrA−, AOM/DSS-treated mice infected with AvrA− mutant strain derived from PhoPC; PhoPCAvrA−/AvrA+, AOM/DSS-treated mice infected with PhoPCAvrA− transfected with plasmid encoding AvrA. (b) Chronic Salmonella infection was assessed by fecal cultures at week 45. On a BBL CHROMagar plate, intestinal Salmonella species appear mauve (rose to purple) in color, due to metabolic differences in the presence of selected chromogens. Other bacteria are either inhibited or produce blue–green or colorless colonies. (c) Salmonella detected in fecal microbial DNA by 16S rRNA PCR 45 weeks postinfection. Mice were infected with PhoPC expressing AvrA protein or PhoPC AvrA− (AvrA deficient) or AvrA-deficient bacteria transfected with AvrA-expressing plasmid. (d) AvrA western blotting. Protein lysates from colonic tissue were probed for AvrA levels. (e) Immunofluorescence confocal microscopy. Salmonella invading the colonic epithelium 45 weeks postinfection appear green.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig1: Persistent Salmonella infection in mice. Mice were infected with the indicated Salmonella strains by oral gavage and treated with AOM-DSS to induce colon cancer. (a) Schema of experimental design. Day-1: Mice were infected by Salmonella gavage 24 h after streptomycin pretreatment (7.5 mg/mouse), ↓azoxymethane (AOM), 10 mg/kg body weight, intraperitoneal injection; ▪ 1% dextran sodium sulfate (DSS) in drinking water, ↓Salmonella gavage; ▴ mice were killed. Experimental groups: Control, normal WT mice (no Salmonella, no AOM/DSS); AOM/DSS, only (no Salmonella), PhoPC, AOM/DSS-treated mice infected with Salmonella with WT AvrA; PhoPCAvrA−, AOM/DSS-treated mice infected with AvrA− mutant strain derived from PhoPC; PhoPCAvrA−/AvrA+, AOM/DSS-treated mice infected with PhoPCAvrA− transfected with plasmid encoding AvrA. (b) Chronic Salmonella infection was assessed by fecal cultures at week 45. On a BBL CHROMagar plate, intestinal Salmonella species appear mauve (rose to purple) in color, due to metabolic differences in the presence of selected chromogens. Other bacteria are either inhibited or produce blue–green or colorless colonies. (c) Salmonella detected in fecal microbial DNA by 16S rRNA PCR 45 weeks postinfection. Mice were infected with PhoPC expressing AvrA protein or PhoPC AvrA− (AvrA deficient) or AvrA-deficient bacteria transfected with AvrA-expressing plasmid. (d) AvrA western blotting. Protein lysates from colonic tissue were probed for AvrA levels. (e) Immunofluorescence confocal microscopy. Salmonella invading the colonic epithelium 45 weeks postinfection appear green.
Mentions: Mice were infected with the indicated Salmonella strains by one oral gavage and then treated with an AOM-DSS protocol to induce colon cancer. The treatment protocol is summarized in Figure 1a. Colonic Salmonella persistence was confirmed by Salmonella culture (pink colonies) and 16S RNA PCR of fecal bacteria DNA (Figures 1b and c). AvrA persistence was also confirmed by western blotting of colon tissue from mice infected with AvrA-expressing bacteria 45 weeks postinfection (Figure 1d). Furthermore, we confirmed persistent colonization of Salmonella in the colon by immunostaining (Figure 1e). Note that Salmonella was identified at the base and surface of the crypts in the infected colon (indicated by arrows) (Figure 1e). We found no significant changes in body weight associated with infection (Supplementary Figure S1). Salmonella levels in the intestine, gallbladder, spleen and liver also correlated with expression of AvrA in infected mice (Supplementary Figure S2).

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