Abstract

Article4 March 2019free access Source DataTransparent process Fusobacterium nucleatum promotes colorectal cancer by inducing Wnt/β-catenin modulator Annexin A1 Mara Roxana Rubinstein Division of Periodontics, College of Dental Medicine, Columbia University, New York, NY, USA Search for more papers by this author Jung Eun Baik Division of Periodontics, College of Dental Medicine, Columbia University, New York, NY, USA Search for more papers by this author Stephen M Lagana Department of Pathology and Cell Biology, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Richard P Han The Horace Mann School, Bronx, NY, USA Search for more papers by this author William J Raab Department of Pathology and Cell Biology, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Debashis Sahoo Department of Computer Science and Engineering, University of California San Diego, San Diego, CA, USA Search for more papers by this author Piero Dalerba Department of Pathology and Cell Biology, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA Columbia Stem Cell Initiative, Columbia University, New York, NY, USA Division of Digestive and Liver Diseases, Columbia University, New York, NY, USA Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA Search for more papers by this author Timothy C Wang Division of Digestive and Liver Diseases, Columbia University, New York, NY, USA Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA Search for more papers by this author Yiping W Han Corresponding Author [email protected] orcid.org/0000-0002-8444-499X Division of Periodontics, College of Dental Medicine, Columbia University, New York, NY, USA Division of Digestive and Liver Diseases, Columbia University, New York, NY, USA Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA Department of Microbiology and Immunology, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Mara Roxana Rubinstein Division of Periodontics, College of Dental Medicine, Columbia University, New York, NY, USA Search for more papers by this author Jung Eun Baik Division of Periodontics, College of Dental Medicine, Columbia University, New York, NY, USA Search for more papers by this author Stephen M Lagana Department of Pathology and Cell Biology, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Richard P Han The Horace Mann School, Bronx, NY, USA Search for more papers by this author William J Raab Department of Pathology and Cell Biology, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Debashis Sahoo Department of Computer Science and Engineering, University of California San Diego, San Diego, CA, USA Search for more papers by this author Piero Dalerba Department of Pathology and Cell Biology, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA Columbia Stem Cell Initiative, Columbia University, New York, NY, USA Division of Digestive and Liver Diseases, Columbia University, New York, NY, USA Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA Search for more papers by this author Timothy C Wang Division of Digestive and Liver Diseases, Columbia University, New York, NY, USA Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA Search for more papers by this author Yiping W Han Corresponding Author [email protected] orcid.org/0000-0002-8444-499X Division of Periodontics, College of Dental Medicine, Columbia University, New York, NY, USA Division of Digestive and Liver Diseases, Columbia University, New York, NY, USA Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA Department of Microbiology and Immunology, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Author Information Mara Roxana Rubinstein1,‡, Jung Eun Baik1,‡, Stephen M Lagana2, Richard P Han3, William J Raab2, Debashis Sahoo4, Piero Dalerba2,5,6,7, Timothy C Wang6,7 and Yiping W Han *,1,6,7,8 1Division of Periodontics, College of Dental Medicine, Columbia University, New York, NY, USA 2Department of Pathology and Cell Biology, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA 3The Horace Mann School, Bronx, NY, USA 4Department of Computer Science and Engineering, University of California San Diego, San Diego, CA, USA 5Columbia Stem Cell Initiative, Columbia University, New York, NY, USA 6Division of Digestive and Liver Diseases, Columbia University, New York, NY, USA 7Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA 8Department of Microbiology and Immunology, Vagelos College of Physicians & Surgeons, Columbia University, New York, NY, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 212 342 1790; E-mail: [email protected] EMBO Rep (2019)20:e47638https://doi.org/10.15252/embr.201847638 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Fusobacterium nucleatum, a Gram-negative oral anaerobe, is a significant contributor to colorectal cancer. Using an in vitro cancer progression model, we discover that F. nucleatum stimulates the growth of colorectal cancer cells without affecting the pre-cancerous adenoma cells. Annexin A1, a previously unrecognized modulator of Wnt/β-catenin signaling, is a key component through which F. nucleatum exerts its stimulatory effect. Annexin A1 is specifically expressed in proliferating colorectal cancer cells and involved in activation of Cyclin D1. Its expression level in colon cancer is a predictor of poor prognosis independent of cancer stage, grade, age, and sex. The FadA adhesin from F. nucleatum up-regulates Annexin A1 expression through E-cadherin. A positive feedback loop between FadA and Annexin A1 is identified in the cancerous cells, absent in the non-cancerous cells. We therefore propose a “two-hit” model in colorectal carcinogenesis, with somatic mutation(s) serving as the first hit, and F. nucleatum as the second hit exacerbating cancer progression after benign cells become cancerous. This model extends the “adenoma-carcinoma” model and identifies microbes such as F. nucleatum as cancer “facilitators”. Synopsis Fusobacterium nucleatum specifically stimulates colorectal carcinoma cells by inducing the Wnt/β-catenin modulator Annexin A1. This supports a model where driver mutations serve as the first, and microbial infection as the second “hit” to promote cancer progression. Fusobacterium nucleatum stimulates colorectal cancer cells without affecting precancerous adenoma cells. Annexin A1 is specifically expressed in colorectal cancer cells and is a predictor of poor prognosis. The Fusobacterium nucleatum FadA adhesin up-regulates Annexin A1 expression through E-cadherin. A positive feedback loop between FadA and Annexin A1 is specifically present in cancer cells. Introduction Colorectal cancer (CRC) is the second leading cause of cancer death in the United States, affecting 1 in 20 individuals 1. CRC has long been recognized to result from host mutations that accumulate over time, developing from pre-cancerous adenomatous polyps into adenocarcinoma over approximately 10 years 2. Advancements in microbial detection technology and human microbiome research have revolutionized our understanding of a wide spectrum of diseases, including CRC 3-7. Numerous recent studies have reported that the anaerobic Gram-negative oral commensal bacterium, Fusobacterium nucleatum, plays a significant role in colorectal carcinogenesis 8-14. F. nucleatum has been detected in ~10–90% CRC tissues, with higher prevalence in the proximal than distal colon 15-17. It is often associated with advanced disease, chemo-resistance, metastasis, and poor prognosis 14, 18-20. A few studies have supported a causal role of F. nucleatum in CRC 10, 12, 14, 21, but detailed mechanistic investigations are scarce. We have reported previously that F. nucleatum promotes CRC growth through its unique FadA adhesin, which binds to E-cadherin (CDH1) and activates Wnt/β-catenin signaling, causing nuclear translocation of β-catenin and overexpression of inflammatory genes, Wnt genes, and oncogenes c-Myc and Cyclin D1 (CCND1) 12. Binding of FadA to E-cadherin requires both the intact pre-FadA consisting of 129 amino acid residues and mFadA of 111 amino acid residues without the signal peptide. Together, they form the FadAc complex 12, 22. FadA was unable to promote growth of non-cancerous HEK293 cells even in the presence of E-cadherin 12. This observation raised the question whether F. nucleatum-induced growth stimulation is specific for CRC. We report here that F. nucleatum selectively stimulates the growth of colorectal cancerous cells through activation of Annexin A1 (ANXA1), a member of the Annexin family of Ca2+-dependent phospholipid-binding proteins 23. Annexin A1 has a molecular weight of 35–40 kDa and can be found in nucleus, cytoplasm, and plasma membrane of various cell types. ANXA1 is involved in a variety of biological processes and has been postulated to be either a tumor suppressor or promoter depending on the tumor types 24, 25. Increased expression of Annexin A1 in CRC has been reported 26, 27, but its role remains unclear. We discover that Annexin A1 is a previously unrecognized Wnt/β-catenin modulator and a critical growth stimulator of CRC, whose expression is increased in proliferating CRC cells, but not in non-proliferating or non-cancerous cells. Given the lack of stimulation of non-cancerous cells by F. nucleatum, we propose a “two-hit” model, whereby F. nucleatum becomes a “facilitator” of cancer progression only after the benign cells progress to a malignant phenotype. Results Fusobacterium nucleatum selectively stimulates the growth of colorectal cancerous cells In order to determine the specificity of F. nucleatum-mediated growth stimulation, we tested the effects of F. nucleatum strain WAL12230 on the PC-9 lung cancer cells, 22RV1 prostate cancer cells, and MCF7 breast cancer cells, all of which expresses E-cadherin, as well as UMUC3 bladder cancer cells, which does not express E-cadherin 28-31 (Fig EV1A). No growth stimulation was detected; on the contrary, F. nucleatum inhibited the proliferation of PC-9, 22RV1, and UMUC3 cells, presumably due to toxic effects (Fig 1A). Click here to expand this figure. Figure EV1. Expression of E-cadherin, Annexin A1, inflammatory genes and oncogene Cyclin D1 in different cell lines Western blot analysis of E-cadherin and Annexin A1 expression in lung cancer cells PC-9, prostate cancer cells 22RV1, bladder cancer cells UMUC3, and breast cancer cells MCF-7. β-Actin was included as an internal control. Real-time qPCR analysis of Il-1β, Nfkb2, Rantes, CCL20, and CCND1 mRNA in MCF-7, AA/C1, AA/C1/SB (aka SB), and AA/C1/SB/10C (aka 10C) either untreated or following incubation with wild-type F. nucleatum 12230. Results obtained from untreated controls were designated as 1. Data were mean values ± SD. The experiment was performed in duplicates and repeated twice. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t-test). Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Fusobacterium nucleatum preferentially binds, invades, and stimulates the growth of cancerous colorectal cells via Annexin A1 Lung cancer cells PC-9, prostate cancer cells 22RV1, bladder cancer cells UMUC3, breast cancer cells MCF-7, colonic adenoma-derived non-cancerous cells AA/C1 (aka C1) and AA/C1/SB (aka SB), or cancerous cells AA/C1/SB/10C (aka 10C) were incubated with wild-type F. nucleatum 12230 (Fn), the fadA-deletion mutant US1 (US1), or E. coli DH5α (E. coli) at multiplicity of infection (MOI) of 1,000:1. Cell numbers are mean values ± SEM. The experiment was performed in triplicates and repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001, compared to untreated controls; #P < 0.05, ###P < 0.001, compared to US1-treated cells (two-way ANOVA). Attachment (left panel) and invasion (right panel) of wild-type F. nucleatum 12230 (Fn) to the non-cancerous SB cells, either untreated or transfected with antisense or sense ANXA1, and to the cancerous 10C cells, either untreated or transfected with control or ANXA1- or CDH1-specific siRNA or both (MOI 50:1). F. nucleatum attachment and invasion to the untreated SB cells were designated as 100%, respectively; all other values were expressed as relative to those obtained with untreated SB. Data are mean values ± SEM. The experiment was performed in triplicates and repeated four times. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA). Left panel: qPCR analysis of Villin 1 (VIL1) mRNA levels in 10C cells treated with control siRNA or VIL1-specific siRNA, demonstrating knockdown of Villin 1. Right panel: Attachment of F. nucleatum 12230 to 10C cells treated with control siRNA or VIL1-specific siRNA. Data are mean values ± SD. The experiment was performed in triplicates and repeated twice. *P < 0.05 (Student's t-test). Download figure Download PowerPoint We then tested F. nucleatum stimulation of the colonic cells, utilizing a CRC progression model consisting of a series of cell lines sequentially derived from a human colonic adenoma 32. AA/C1 is a slow-growing non-cancerous adenoma cell line with low colony-forming efficiency. Following treatment with 1 mM sodium butyrate, it gave rise to the AA/C1/SB cell line, which grows faster with increased colony-forming efficiency, but remains non-tumorigenic in mice. The AA/C1/SB cells were further mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine to produce a tumorigenic (cancerous) cell line, AA/C1/SB/10C 32. F. nucleatum 12230 accelerated the growth of the AA/C1/SB/10C cells (from now on referred to as “10C”), but not of the non-tumorigenic AA/C1 or AA/C1/SB (from now on referred to as “SB”) cells (Fig 1A). As with our previous report, the growth stimulation was mediated predominantly through FadA although the fadA-deletion mutant US1 retained a weak stimulatory effect, suggesting additional component(s) may be involved 12. Although all cell lines tested with F. nucleatum 12230 expressed increased levels of proinflammatory markers, only the cancerous 10C cells exhibited elevated expression of the oncogene Cyclin D1, consistent with growth stimulation (Fig EV1B). Fusobacterium nucleatum binds and invades cancerous cells more efficiently due to Annexin A1 F. nucleatum 12230 bound 75% more and invaded 150% more efficiently to the cancerous 10C cells, as compared to its non-cancerous predecessor SB (Fig 1B). These results were consistent with our previous finding that the fadA gene levels (and F. nucleatum) were significantly higher in the human colorectal carcinoma tissues than in the adenoma tissues 12. The results also suggest that 10C and SB may differ in their membrane components, which might explain the differential binding by F. nucleatum. Previous comparative proteomic analysis revealed two membrane proteins that were increased in 10C compared to SB: Annexin A1 and Villin 1 (VIL1) 33. Down-regulation of Annexin A1 in the 10C cells by siRNA effectively reduced F. nucleatum binding and invasion, in a similar manner as suppression of CDH1 (Fig 1B), whereas knockdown of VIL1 had no effect (Fig 1C). Transfection of ANXA1 into SB cells significantly increased F. nucleatum binding and invasion (Fig 1B). These results indicate that Annexin A1 plays an important role in F. nucleatum interaction with the cancerous 10C cells. Hence, from this point onward, our investigation focused on Annexin A1. Annexin A1 is an independent CRC prognosis biomarker To examine ANXA1 expression in CRC biology in humans, we tested for their association with recurrence rates in a clinical database annotated with microarray gene-expression measurements from 466 primary colon carcinomas 34. The association was first tested using Kaplan–Meier survival curves, using three different approaches for patient stratification: (i) based on the median of ANXA1 mRNA expression levels (Fig 2A); (ii) based on the quartile distribution of ANXA1 mRNA expression levels (Fig 2B); and (iii) based on expression thresholds calculated using the StepMiner algorithm (Fig 2C), as previously described 35, 36. High levels of ANXA1 mRNA expression were associated with a statistically significant reduction in disease-free survival (DFS) rates, irrespective of the method used for the stratification (P < 0.001, log-rank test). Differences in ANXA1 mRNA expression levels did not appear to correlate with differences in each tumor's relative content of epithelial cells (i.e., tumor cell density) as revealed by the lack of visual correlations with the epithelial cell marker Desmoplakin (DSP). Importantly, the association between high levels of ANXA1 mRNA expression and increased risk of recurrence remained statistically significant in a series of multivariate analyses (Cox proportional hazards method) that excluded clinical stage, pathological grade, age, and sex as possible confounding variables, even after modeling ANXA1 expression as a continuous variable (Table 1). The hazard ratio (HR) for disease recurrence associated with increased ANXA1 expression levels was 1.44 (95% CI 1.24–1.68; P < 0.001) when tested alongside clinical stage, age, and sex across the full patient cohort (n = 466), and 1.56 (95% CI 1.21–2.02; P < 0.001) when tested alongside clinical stage, pathological grade, age, and sex in the patient subgroup annotated with pathological grade (n = 216). These analyses demonstrate that ANXA1 is a novel colon cancer prognostic biomarker. Figure 2. Annexin A1 is a novel colon cancer prognosis marker A–C. Relationship between ANXA1 mRNA expression levels and disease-free survival (DFS) in colon cancer patients. The relationship between ANXA1 mRNA expression levels and DFS was investigated in a database of 466 primary colon carcinomas, assembled by pooling four independent gene-expression array datasets from the NCBI-GEO online repository (GSE14333, GSE17538, GSE31595, GSE37892), as previously described 34. The association between ANXA1 expression levels and DFS was tested using Kaplan–Meier survival curves, after patient stratification in groups with high, medium, and low ANXA1 expression, using three different methods: (A) based on the median of ANXA1 mRNA expression levels (high 50% versus low 50%); (B) based on the quartile distribution of ANXA1 mRNA expression levels (high 25% versus middle 50% versus low 25%); and (C) based on ANXA1 mRNA expression thresholds calculated using the StepMiner algorithm (low versus high), as previously described 35, 36. Overall, high ANXA1 mRNA expression levels were associated with a statistically significant reduction in DFS (P < 0.001, log-rank test), irrespective of the method used for the stratification. Differences in ANXA1 mRNA expression levels did not appear to correlate with differences in each tumor's relative content of epithelial cells in the analyzed biospecimens (i.e., tumor cell density) as revealed by the lack of visual correlations with the epithelial cell marker Desmoplakin (DSP). Download figure Download PowerPoint Table 1. Relationship between ANXA1 mRNA expression levels and risk of recurrence in colon cancer patients Cox proportional hazards model Univariate Multivariate HR 95% CI P-value HR 95% CI P-value All patients (n = 466) ANXA1aa ANXA1 mRNA levels and age modeled as a continuous variable. 1.46 1.25–1.71 < 0.001****** P < 0.001. 1.44 1.24–1.68 < 0.001****** P < 0.001. Stage (I–IV) 3.47 2.62–4.59 < 0.001****** P < 0.001. 3.73 2.74–5.09 < 0.001****** P < 0.001. Ageaa ANXA1 mRNA levels and age modeled as a continuous variable. 0.99 0.97–1.00 0.06 1.00 0.98–1.01 0.75 Sex (M/F)bb M/F: male versus female. 1.07 0.89–1.28 0.49 1.05 0.88–1.27 0.58 Patients annotated with information on tumor grade (n = 216) ANXA1aa ANXA1 mRNA levels and age modeled as a continuous variable. 1.48 1.18–1.87 < 0.001****** P < 0.001. 1.56 1.21–2.02 < 0.001****** P < 0.001. Stage (I–IV) 3.13 2.14–4.60 < 0.001****** P < 0.001. 3.63 2.31–5.70 < 0.001****** P < 0.001. Grade (G1–G3) 1.63 0.94–2.82 0.08 1.02 0.58–1.79 0.95 Ageaa ANXA1 mRNA levels and age modeled as a continuous variable. 0.99 0.97–1.01 0.20 1.00 0.98–1.02 0.85 Sex (M/F)bb M/F: male versus female. 1.15 0.88–1.51 0.32 1.16 0.86–1.55 0.33 CI, confidence interval; HR, hazard ratio. The relationship between ANXA1 mRNA expression levels and risk of recurrence was investigated in a database of 466 primary colon carcinomas, obtained by assembling four independent gene-expression array datasets downloaded from the NCBI-GEO online repository (GSE14333, GSE17538, GSE31595, GSE37892), as previously described 34. The association between ANXA1 mRNA expression levels and risk of recurrence was tested using both univariate and multivariate analyses based on the Cox proportional hazards method, where ANXA1 mRNA expression levels were modeled as a continuous variable. Overall, higher ANXA1 mRNA expression levels were associated with a statistically significant increase in the risk of recurrence when using univariate analysis, in both the whole database (n = 466; P < 0.001) and the subset of patients annotated with information on the pathological grading of the tumors (n = 216; P < 0.001). Importantly, the association remained significant also in a multivariate analysis aimed at excluding possible confounding effects from clinical stage, pathological grade, age, or sex, both when tested across the whole database (n = 466; P < 0.001) and when tested in the subset of patients annotated for the pathological grading of the respective tumors (n = 216; P < 0.001). Hazard ratios (HRs) were tested for statistical significance (null hypothesis: HR=1) using the Wald test. a ANXA1 mRNA levels and age modeled as a continuous variable. b M/F: male versus female. *** P < 0.001. Annexin A1 is a CRC growth factor specifically expressed in proliferating cancerous cells We found Annexin A1 was selectively expressed in proliferating cancerous cells. In 10C and human CRC cells, HCT116, DLD1, and RKO, ANXA1 gene expression was significantly higher in the non-confluent than confluent state, despite that very little was expressed in RKO (Fig 3A). Immunofluorescence staining revealed that Annexin A1 was expressed on the outer layer of the growing mass of cancerous 10C cells (Fig 3B; Movie EV1). In contrast, Annexin A1 expression in SB remained at low levels, unaffected by cell density (Fig 3B). Figure 3. Annexin A1 is a novel CRC growth factor selectively expressed in proliferating cancerous colorectal cells Real-time qPCR analysis of ANXA1 expression using mRNA extracted from the non-cancerous SB, cancerous 10C, and human CRC cell lines HCT116, DLD1, and RKO, each grown to 50 or 100% confluency. All results were normalized to the ANXA1 mRNA levels in SB cells of 100% confluency, which was designated as 1. Data are mean values ± SEM. The experiment was performed in triplicates and repeated twice. **P < 0.01 and ***P < 0.001 (Student's t-test). Confocal microscopy analysis of SB and 10C cells grown to 20% (top panels) or 100% (bottom panels) confluency followed by immunofluorescence staining of Annexin A1 (green). The nuclei were stained with DAPI (blue). A series of 20–50 consecutive images in the z-axis were stacked together to generate the 3D figure at 400× magnification. Annexin A1 is most abundantly expressed on the outer layer of 20% confluent 10C (also see Movie EV1), compared to 100% confluent 10C, or SB of either confluency. Scale bars, x = 1 μm, y = 1 μm, z = 1.6 μm. The experiment was repeated at least twice. Cell proliferation assay of adenoma-derived non-cancerous SB and cancerous 10C and human CRC cell lines HCT116, DLD1, SW480, HT29, and RKO either untreated (black lines) or following treatment with control siRNA (gray lines) or ANXA1-specific siRNA (green lines). Data are mean values ± SEM. The experiment was performed in triplicates and repeated three times. *P < 0.05, **P < 0.01, and ***P < 0.001, compared to the untreated cells (two-way ANOVA). Cell proliferation assay of SB (left panel) and RKO (right panel) cells transfected with ANXA1 (red line), as compared to the control cells (black lines). Data are mean values ± SEM. The experiment was performed in triplicates and repeated three times. *P < 0.05, **P < 0.01, and ***P < 0.001 (two-way ANOVA). Xenografted tumor growth in nude mice following subcutaneous and bilateral inoculation of HCT116 cells transfected with control siRNA or ANXA1-specific siRNA (n = 4). The tumor volumes were measured after 7 days postinjection (left panel). For each mouse, the tumor resulting from ANXA1-specific siRNA-treated cells was normalized to that from control siRNA-treated cells, which was designated 100%. The line represents the average. *P < 0.05 (paired t-test). The individual tumor pairs are shown on the right panel: top, tumors arising from control siRNA-treated cells; bottom, tumors arising from ANXA1-specific siRNA-treated cells. Download figure Download PowerPoint Using ANXA1-specific siRNA, we were able to inhibit its expression (Fig EV2A). This led to inhibition of the growth of cancerous 10C, HCT116, DLD1, SW480, and HT29, but not the non-cancerous SB, or the CRC cells RKO, which expressed the lowest level of ANXA1 (Fig 3C). Transfection of ANXA1 into SB and RKO significantly stimulated their growth (Figs 3D and EV2B). Equal numbers of HCT116 cells transfected with control or ANXA1-specific siRNA were then injected subcutaneously and bilaterally into nude mice. Suppression of ANXA1 significantly attenuated tumor growth compared to controls (Fig 3E). Similar results were obtained with DLD1 (Fig EV2C). As no F. nucleatum was included in these experiments, the results demonstrate that Annexin A1 is a critical growth factor for CRC, regardless of F. nucleatum. Click here to expand this figure. Figure EV2. Annexin A1 knockdown in 10C, HCT116 and DLD1 cells and knock-in in RKO cells, and the effect of knockdown in DLD1 xenograft tumor growth qPCR analysis of ANXA1 mRNA levels in 10C, HCT116, DLD1 cells transfected with control siRNA or ANXA1-specific siRNA, demonstrating knockdown of ANXA1. The experiment was performed in triplicates. Data are mean values ± SEM. *P < 0.05 (Student's t-test). qPCR analysis of ANXA1 mRNA levels in RKO cells transfected with control vector or ANXA1, demonstrating knock-in of ANXA1. The experiment was performed in triplicates. Data are mean values ± SEM. *P < 0.05 (Student's t-test). Xenografted tumor growth in nude mice following subcutaneous and bilateral inoculation of DLD1 cells transfected with control siRNA or ANXA1-specific siRNA (n = 4). The tumor volumes were measured after 8 days postinjection (left panel). For each mouse, the tumor resulting from ANXA1-specific siRNA-treated cells was normalized to that from control siRNA-treated cells, which was designated as 100%. The line represents the average. *P < 0.05 (paired t-test). The individual tumor pairs are shown on the right panel: top, tumors arising from control siRNA treated cells; bottom, tumors arising from ANXA1-specific siRNA-treated cells. Download figure Download PowerPoint Fusobacterium nucleatum induces Annexin A1 expression in cancerous cells through FadA and E-cadherin We investigated the relationship between F. nucleatum and Annexin A1 and found F. nucleatum induced ANXA1 gene expression in the cancerous 10C, HCT116, and DLD1 cells, but not in the non-cancerous SB cells (Fig 4A). The induction required FadA, because the fadA-deletion mutant US1 was defective, and purified FadAc induced Annexin A1 in a dose-dependent manner (Fig 4A and B). Of note, similar induction was detected upon re-analysis of a recently published, publicly available RNA-seq dataset 14 that contains gene-expression data from human CRC cells HT29 incubated with a different strain, F. nucleatum ATCC 25586 (Fig EV3A). Taken together, these consistent observations indicate that ANXA1 overexpression can be interprete