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Cited by 206Bacterial Cancer TherapyWnt/β-catenin signaling in development and cancerSalmonella and Campylobacter epidemiology

The DOI matches its Crossref record; 47 of the 47 references it cites were found in a registry or an index.

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

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Lu, R, Wu, S, Zhang, Y-g, Xia, Y, Liu, X, Zheng, Y, Chen, H, Schaefer, K L, Zhou, Z, Bissonnette, M, Li, L, Sun, J·2014·Oncogenesis
DOI 10.1038/oncsis.2014.20PMC PMC4150214
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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.

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Figure 1 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.
Figure 1 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.CC BY-NC-SA · PubMed Central
Figure 2 AvrA-expressing bacteria significantly increase colonic tumor incidence. (a) Colonic tumors in situ. Representative colons from the indicated groups 45 weeks after bacterial infection. Tumors are indicated by red arrows. (b) Tumor multiplicity. Average number of tumors in each experimental group. *P<0.05, **P<0.01. (c) Tumor location. The distance of each tumor from the anus was measured. *P<0.05. (d) Swiss rolls of representative colons from the indicated groups. Rectangles in the middle panel are shown at higher magnification in the lower panel. (e) Salmonella invasion. Localization of Salmonella in tumor tissue was assessed by immunofluorescence staining.
Figure 2 AvrA-expressing bacteria significantly increase colonic tumor incidence. (a) Colonic tumors in situ. Representative colons from the indicated groups 45 weeks after bacterial infection. Tumors are indicated by red arrows. (b) Tumor multiplicity. Average number of tumors in each experimental group. *P<0.05, **P<0.01. (c) Tumor location. The distance of each tumor from the anus was measured. *P<0.05. (d) Swiss rolls of representative colons from the indicated groups. Rectangles in the middle panel are shown at higher magnification in the lower panel. (e) Salmonella invasion. Localization of Salmonella in tumor tissue was assessed by immunofluorescence staining.CC BY-NC-SA · PubMed Central
Figure 3 AvrA regulates β-catenin activation as assessed by phospho (Ser552) β-catenin in AOM/DSS-induced tumors. (a) β-Catenin localization in control mucosa and colonic tumors. (b) P-β-catenin Ser552 localization in control mucosa and colonic tumors. (c) Quantitation of nuclear P-β-catenin Ser552 in control mucosa and colonic tumors. Data are means±s.d. of n=3 control mice or AOM/DSS tumors in each group. *P<0.05, **P<0.01 (d) Expression levels of p-β-catenin, total β-catenin and Akt and P-Akt as assessed by western blotting in control and AOM/DSS-induced tumors. Shown are control mucosa and tumors representative of three mice in each group. (e) Immunostaining of p-Myc, MMP-7, Cyclin D1 and Bmi1 in control colons and AOM/DSS-induced tumors (left panel). Quantitative imaging of nuclear Bmi1 in control mucosa and mucosa adjacent to tumors (right panel). Data are means±s.d. of n=3 control mice or AOM/DSS mice in each group. *P<0.05, **P<0.01.
Figure 3 AvrA regulates β-catenin activation as assessed by phospho (Ser552) β-catenin in AOM/DSS-induced tumors. (a) β-Catenin localization in control mucosa and colonic tumors. (b) P-β-catenin Ser552 localization in control mucosa and colonic tumors. (c) Quantitation of nuclear P-β-catenin Ser552 in control mucosa and colonic tumors. Data are means±s.d. of n=3 control mice or AOM/DSS tumors in each group. *P<0.05, **P<0.01 (d) Expression levels of p-β-catenin, total β-catenin and Akt and P-Akt as assessed by western blotting in control and AOM/DSS-induced tumors. Shown are control mucosa and tumors representative of three mice in each group. (e) Immunostaining of p-Myc, MMP-7, Cyclin D1 and Bmi1 in control colons and AOM/DSS-induced tumors (left panel). Quantitative imaging of nuclear Bmi1 in control mucosa and mucosa adjacent to tumors (right panel). Data are means±s.d. of n=3 control mice or AOM/DSS mice in each group. *P<0.05, **P<0.01.CC BY-NC-SA · PubMed Central
Figure 4 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.
Figure 4 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.CC BY-NC-SA · PubMed Central
Figure 5 Bacterial AvrA activates β-catenin signaling. (a) AvrA induces β-catenin signaling in HEK293 cells. HEK293 cells were transiently co-transfected with a pcmv-myc-AvrA plasmid and a pGL3-OT plasmid (TCF-responsive reporter with WT TCF binding site) or pGL3-OF (mutant TCF binding site), using lipofectin reagent according to the manufacturer's instructions. LiCl treatment (20 mmol/l) was used as the positive control. Each experiment was assayed in triplicate. N=3 independent platings *P<0.05. (b) Colonic BrdU was detected in normal mucosa and tumors in control mice (AOM/DSS alone) or mice also gavaged with the indicated bacteria 45 weeks postinfection. (c) Quantitation of BrdU staining in normal tissue and tumors from the indicated groups. *P<0.05, **P<0.01. (d) Model of AvrA regulation of β-catenin signaling in colonic tumorigenesis. In mice chronically infected with Salmonella, AvrA activates β-catenin, which upregulates its target genes to promote colonic tumorigenesis.
Figure 5 Bacterial AvrA activates β-catenin signaling. (a) AvrA induces β-catenin signaling in HEK293 cells. HEK293 cells were transiently co-transfected with a pcmv-myc-AvrA plasmid and a pGL3-OT plasmid (TCF-responsive reporter with WT TCF binding site) or pGL3-OF (mutant TCF binding site), using lipofectin reagent according to the manufacturer's instructions. LiCl treatment (20 mmol/l) was used as the positive control. Each experiment was assayed in triplicate. N=3 independent platings *P<0.05. (b) Colonic BrdU was detected in normal mucosa and tumors in control mice (AOM/DSS alone) or mice also gavaged with the indicated bacteria 45 weeks postinfection. (c) Quantitation of BrdU staining in normal tissue and tumors from the indicated groups. *P<0.05, **P<0.01. (d) Model of AvrA regulation of β-catenin signaling in colonic tumorigenesis. In mice chronically infected with Salmonella, AvrA activates β-catenin, which upregulates its target genes to promote colonic tumorigenesis.CC BY-NC-SA · PubMed Central

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Authors
Lu, R., Wu, S., Zhang, Y.-G., Xia, Y., Liu, X., Zheng, Y., Chen, H., Schaefer, K. L., Zhou, Z., Bissonnette, M., Li, L., & Sun, J.
Affiliations
  • Rush University (US)
  • University of Rochester (US)
  • University of Rochester Medicine (US)
  • University of Chicago (US)
  • Stowers Institute for Medical Research (US)
Corresponding author
Sun, J
ORCID
Venue
Oncogenesis
Publisher
Springer Science and Business Media LLC
Published
2014-06-09
Volume
3
Issue
6
Pages
e105-e105
ISSN
2157-9024 (ISSN-L)
DOI
10.1038/oncsis.2014.20
PMC
PMC4150214
OpenAlex
W1968540478
Cited by
206
References
47
Citations by year
2026: 18 · 2025: 22 · 2024: 21 · 2023: 27 · 2022: 16 · 2021: 26 · 2020: 18 · 2019: 9 · 2018: 11 · 2017: 13 · 2016: 10 · 2015: 13 · 2014: 2
FWCI
10.74
Type
journal article
Language
en
Open access
Licence
Locations
  • Oncogenesis (publishedVersion, cc-by-nc-sa)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
  • American Cancer Society
  • National Cancer Institute (R01CA164124)
  • National Institute of Diabetes and Digestive and Kidney Diseases (K01DK075386, DK075386, KO1-DK-075386)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Bacterial Cancer Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Salmonella and Campylobacter epidemiology
Keywords
avrA, Salmonella typhimurium, colon carcinogenesis, β-catenin signaling, chronic bacterial infection, cancer stem cells, BMI1, matrix metalloproteinase-7, cyclin D1, stem/progenitor cells, gut microbiota

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