Volume 152, Issue 6, Pages 1419-1433.e5 (May 2017) Peptostreptococcus anaerobius Induces Intracellular Cholesterol Biosynthesis in Colon Cells to Induce Proliferation and Causes Dysplasia in Mice  Ho Tsoi, Eagle S.H. Chu, Xiang Zhang, Jianqiu Sheng, Geicho Nakatsu, Siew C. Ng, Anthony W.H. Chan, Francis K.L. Chan, Joseph J.Y. Sung, Jun Yu  Gastroenterology  Volume 152, Issue 6, Pages 1419-1433.e5 (May 2017) DOI: 10.1053/j.gastro.2017.01.009 Copyright © 2017 AGA Institute Terms and Conditions

Figure 1 P anaerobius is enriched in stool and tissue samples of patients with CRC. (A) The level of P anaerobius in stool samples from normal subjects (N = 54) and patients with CRC (N = 58). (B) The level of P anaerobius in mucosal samples of normal, polyps, and CRC from 2 cohorts of Hong Kong and Beijing. qPCR was performed to determine the level of P anaerobius in the clinical samples. Each spot represents 1 subject. Statistical significance was determined by Mann−Whitney U test and nonparametric 1-way analysis of variance. Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Figure 2 P anaerobius promotes the development of high-grade dysplasia in mice. (A) Schematic diagram showing the experimental design and timeline of mouse models. (B) The level of total bacteria in stool samples of mice was determined by real-time qPCR (left panel). The level of P anaerobius in stool samples of mice during the antibiotic treatment period (right panel) by qPCR. (C) The level of P anaerobius in stool samples of mice during P anaerobius feeding period by qPCR. (D) Representative colonic morphologies of mice under different treatment. (E) P anaerobius promoted the formation of colonic dysplasia. Representative histologic images of colon tissues of mice by H&E staining and statistical analysis of colon samples according to the histologic score. Colon tissue samples used for evaluation include 32 tissues from 9 mice treated with AOM only; 32 tissues from 8 mice treated with AOM + E coli and 37 tissues from 10 mice treated with AOM + P anaeronius. Data are expressed as mean ± SD. HGD, high-grade dysplasia; LGD, low-grade dysplasia. Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Figure 3 P anaerobius promotes proliferation and enriches pathways in colon cells. (A) Colon normal epithelial cell line NCM460 and colon cancer cell lines HT-29 and Caco-2 were exposed to P anaerobius (multiplicity of infection = 100) for 2 h/d under anaerobic condition. The procedure was repeated for 4 consecutive days (left panel). The survival rates of P anaerobius were obtained according to their physical attachment ability in the colon cell lines (middle panel). Viabilities of colon cell lines under anaerobic incubation condition for 2 hours were not altered (right panel). (B) P anaerobius promoted cell proliferation in normal colonic epithelial cell line (NCM460) and in colon cancer cell lines (HT-29 and Caco-2) as determined by MTT cell viability assay. (C) P anaerobius promoted cell proliferation in NCM460, HT-29, and Caco-2 cells as determined by fluorescent intensity assay after BrdU labeling. Data are expressed as mean ± SD from 3 independent experiments. (D) Differentiated gene expression pattern induced by P anaerobius by PCR arrays. (E) P anaerobius regulated genes enrichment in a numbers of pathways in NCM460, HT-29, and Caco-2 cell lines. Eight pathways were commonly enriched in the 3 cell lines. Statistical significance was determined by unpaired Student t test and 2-way analysis of variance. KEGG, Kyoto Encyclopedia of Genes and Genomes. Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Figure 4 P anaerobius promotes cell proliferation by modulating cholesterol biosynthesis. (A) P anaerobius induced intracellular total cholesterol levels in NCM460, HT-29, and Caco-2. (B) SREBP2 activity was induced by P anaerobius in NCM460, HT-29, and Caco-2. (C) The effect of SREBP2 inhibitor, fatostatin on cholesterol biosynthesis mediated by P anaerobius in NCM460, HT-29, and Caco-2 cells. The effect of fatostatin on cell proliferation was demonstrated by PCNA protein expression (D) and by BrdU labeling assay (E) after exposure to P anaerobius. Tubulin was used as loading control. Band intensity was measured by the intensity ratio of PCNA to tubulin. All samples were collected after 48-hour exposure to P anaerobius. Data are expressed as mean ± SD from 3 independent experiments. Statistical significance was determined by unpaired Student t test. DMSO, dimethyl sulfoxide. Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Figure 5 Up-regulation of ROS is essential for P anaerobius−enhanced cell proliferation. (A) The levels of ROS were determined in NCM460, HT-29, and Caco-2 cells by fluorescent intensity of 2′,7′-dichlorodihydrofluorescein diacetate. (B) Depletion of ROS by its scavenger NAC (10 mM) suppressed the cholesterol biosynthesis induced by P anaerobius in NCM460, HT-29, and Caco-2 cells and blunted the effect of P anaerobius on cell proliferation as evidenced by PCNA Western blot (C) and by BrdU labeling assay in all 3 cell lines (D). Band intensities measured by the intensity ratio of PCNA to tubulin are shown. All samples were collected after 48-hour exposure to P anaerobius. Data are expressed as mean ± SD from 3 independent experiments. Statistical significance was determined by unpaired Student t test and 1-way analysis of variance. Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Figure 6 Toll-Like receptor TLR2 and TLR4 are involved in P anaerobius−mediated cellular responses. (A) Knockdown of TLR2 and TLR4 could significantly reduce the effect of P anaerobius on the up-regulation of ROS in NCM460, HT-29, and Caco-2 cells. (B) Knockdown of both TLR2 and TLR4 could abolish the effect of P anaerobius on the up-regulation of cholesterol biosynthesis in NCM460, HT-29, and Caco-2 cells. (C) Apocynin (4 μM, an inhibitor of NADPH oxidase) reduced the effect of P anaerobius on ROS up-regulation significantly. (D) Apocynin reduced the effect of P anaerobius on cholesterol biosynthesis in NCM460, HT-29, and Caco-2 cells significantly. (E) Knockdown of TLR2 and TLR4 could abolish the effect of P anaerobius on cell proliferation as determined by PCNA protein expression by Western blot. Tubulin was used as loading control. Band intensities measured by the intensity ratio of PCNA to tubulin are shown. All samples were collected after 48-hour exposure to P anaerobius. Data are expressed as mean ± SD from 3 independent experiments. *P < .05; **P < .01; ***P < .001. Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Figure 7 The effects of P anaerobius in vitro are verified in mouse models. Consistent with in vitro results, mice depleted of bacteria and exposed to AOM and P anaerobius showed significantly increased (A) PCNA protein expression, (B) cholesterol level, and (C) SREBP-2 activity compared with AOM only treated mice with or without administration of apocynin (2 mg/kg, an inhibitor of NADPH oxidase). (D) qPCR was performed in mouse colon tissues to validate a subset of the gene targets identified by PCR array in colon cell lines. β-actin served as internal control. (E) Protein expression of key cholesterol biosynthesis factor was confirmed by Western blot. Tubulin was used as loading control. Ratio of band intensity of candidate protein to tubulin are shown. Eight mice were used in each of the groups. Statistical significance was determined by 1-way analysis of variance and unpaired Student t test. Data are expressed as mean ± SD. *P < .05; **P < .01; ***P < .001. (F) Proposed mechanistic scheme of P anaerobius promoting colorectal carcinogenesis. P anaerobius interacts with TLR2/4 on colonic cells to induce intracellular ROS, and increase cholesterol biosynthesis; P anaerobius also enhances cholesterol biosynthesis by modulating SREBP2 and, therefore, activates pro-oncogenic pathways. P anaerobius and its induced ROS, cholesterol, and oncogenic signaling promote cell proliferation and dysplasia and, ultimately, CRC. Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 1 Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 2 Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 3 Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 4 Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 5 Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 6 Gastroenterology 2017 152, 1419-1433.e5DOI: (10.1053/j.gastro.2017.01.009) Copyright © 2017 AGA Institute Terms and Conditions