Fusobacterium nucleatum Promotes Chemoresistance to Colorectal Cancer by Modulating Autophagy

Abstract

Gut microbiota are linked to chronic inflammation and carcinogenesis. Chemotherapy failure is the major cause of recurrence and poor prognosis in colorectal cancer patients. Here, we investigated the contribution of gut microbiota to chemoresistance in patients with colorectal cancer. We found that Fusobacterium (F.) nucleatum was abundant in colorectal cancer tissues in patients with recurrence post chemotherapy, and was associated with patient clinicopathological characterisitcs. Furthermore, our bioinformatic and functional studies demonstrated that F. nucleatum promoted colorectal cancer resistance to chemotherapy. Mechanistically, F. nucleatum targeted TLR4 and MYD88 innate immune signaling and specific microRNAs to activate the autophagy pathway and alter colorectal cancer chemotherapeutic response. Thus, F. nucleatum orchestrates a molecular network of the Toll-like receptor, microRNAs, and autophagy to clinically, biologically, and mechanistically control colorectal cancer chemoresistance. Measuring and targeting F. nucleatum and its associated pathway will yield valuable insight into clinical management and may ameliorate colorectal cancer patient outcomes.

Keywords: Colorectal cancer; F.nucleatum; Toll-like receptor; autophagy; chemoresistance; miRNA; recurrence.

Figure 1. F. nucleatum Is Associated with Cancer Recurrence and Patient Outcome

(A) A cladogram representation of data in CRC patients with recurrence (16) versus no recurrence (15) by 16S rDNA sequencing. Taxa enriched in patients with recurrence (Red) and without recurrence (Blue). The brightness of each dot is proportional to its effect size. (B) Linear discriminant analysis (LDA) coupled with the effect size measurements identifies the significant abundance of data in A. Taxa enriched in recurrent (Red) and non-recurrent (Blue) patients are indicated with negative (Red) or positive (Blue) LDA scores, respectively. Only taxa greater than LDA threshold of 3.5 are shown. (C) Statistical analysis of the amount of F. nucleatum in Cohort 2, nonparametric Mann–Whitney test. (D) Recurrence-Free Survival (RFS) was compared between patients with low and high amount of F. nucleatum in Cohort 2, Log-rank test. (E) Receiver operating characteristic (ROC) analysis was conducted based on the amount of F. nucleatum and AJCC in colorectal cancer. (F) Univariate analysis was performed in Cohort 2. The bars correspond to 95% confidence intervals. (G) Multivariate analysis was performed in Cohort 2. The bars correspond to 95% confidence intervals. (H) Statistical analysis was conducted based on the amount of F. nucleatum and recurrence rate in Cohort 3 by the cut off value of F. nucleatum defined in Cohort 2, Chi-square test. (I) RFS was compared between patients with low and high abundance of F. nucleatum in 173 patients with colorectal cancer (Cohort 3) by the cut off value of F. nucleatum defined in Cohort 2, Log-rank test. See also Figure S1.

Figure 2. F. nucleatum Promotes Cancer Autophagy Activation

(A) ssGSEA analysis was conducted to show the relationship between the amount of F. nucleatum and autophagy-related pathways in CRC tissues. (B, C) Real-Time PCR was performed in HCT116 cells (B) and HT29 (C) cells cultured with F. nucleatum, nonparametric Mann–Whitney test. (D) Western blot was performed on autophagy element expression in HCT116 cells co-cultured with F. nucleatum, E. coli, E. faecalis or B. fragilis. (E) Western blot was performed in HCT116 cells co-cultured with F. nucleatum in the presence of CQ. (F and G) HCT116 cells (F) and HT29 cells (G) that stably expressed mRFP-EGFP-LC3 fusion protein were co-cultured with F. nucleatum. Confocal microscopic analysis is shown (2000 × magnification). Bar scale, 5 μm. (H) Autophagosomes were observed by transmission electron microscopy (17500 × magnification) in HCT116 cells (left) and HT29 cells (right) cultured with F. nucleatum. Bar scale, 1 μm. (I and J) Statistical analysis was performed to calculate the number of autophagosomes in HCT116 cells (I) and HT29 cells (J) shown by transmission electron microscopy, nonparametric Mann–Whitney test. See also Figure S2.

Figure 3. F. nucleatum Induces Chemoresistance in Colorectal Cancer Cells via Activation of the Autophagy Pathway

(A–D) Apoptosis was detected by flow cytometry in HCT116 cells (A, B) and HT29 cells (C, D). The cells were co-cultured with F. nucleatum or treated with CQ, and different concentrations of Oxaliplatin (A and C) and 5-FU (B, D). nonparametric Mann–Whitney test. (E and F) Cleaved caspases and p-H₂AX expression were detected by western blot in HCT116 cells (E) and HT29 cells (F). The cells were co-cultured with F. nucleatum or treated with CQ, and different concentrations of Oxaliplatin and 5-FU. (G and H) Apoptosis was detected by flow cytometry in HCT116 cells (G) and HT29 cells (H). The cells were transfected with ULK1 and ATG7 siRNAs, and subsequently co-cultured with F. nucleatum and different concentrations of Oxaliplatin, nonparametric Mann–Whitney test. See also Figure S3.

Figure 4. F. nucleatum Activates Cancer Autophagy via Downregulation of miR-18a* and miR-4802

(A) The predicted binding sequences for miR-18a* (left) and miR-4802 (right) within the human ULK1 and ATG7 3′UTR, respectively. Seed sequences are highlighted. (B) Luciferase activity was measured in HCT116 cells transfected with miR-18a* mimics or control miRNA. The luciferase reporters expressing wild-type or mutant human ULK1 3′UTRs were used. The luciferase activity was normalized based on the control miRNA transfection. n.s., not significant. (C) Luciferase activity was measured in HCT116 cells transfected with miR-4802 mimics or control miRNA. The luciferase reporters expressing wild-type or mutant human ATG7 3′UTRs were used. (D) Real-time PCR was performed in HCT116 cells to detect the expression of ULK1 gene after transfected with miR-18a* mimics or inhibitor, nonparametric Mann–Whitney test. (E) Real-time PCR was performed in HCT116 cells to detect the expression of ATG7 gene after transfected with miR-4802 mimics or inhibitor, nonparametric Mann–Whitney test. (F and G) HCT116 cells were transfected with mimics or inhibitor of miR-18a* (F) and miR-4802 (G), respectively. After culturing with F. nucleatum, autophagy and target proteins were detected by western blot in HCT116 cells. (H) HCT116 cells that stably expressed mRFP-EGFP-LC3 fusion protein were transfected with miR-18a* and miR-4802 mimics. After culturing with F. nucleatum, autophagosomes were observed under confocal microscope (2000 × magnification) in HCT116 cells. Bar scale, 5 μm. (I) Autophagosomes were observed by transmission electron microscopy (17500 × magnification) in HCT116 cells transfected with miR-18a* (left) and miR-4802 (right) mimics, and then co-cultured with F. nucleatum. Bar scale, 1 μm. See also Figure S4.

Figure 5. miR-18a* and miR-4802 Regulate F. nucleatum-Mediated Chemoresistance

(A–D) Apoptosis was detected by flow cytometry in HCT116 cells. HCT116 cells were transfected with mimics (A, B) or inhibitors (C, D) of miR-18a* and miR-4802, co-cultured with F. nucleatum, and treated with different concentrations of Oxaliplatin (A, C) and 5-FU (B, D). nonparametric Mann–Whitney test. (E) Western blot was performed in HCT116 cells. HCT116 cells were transfected with mimics or inhibitors of miR-18a* and miR-4802, co-cultured with F. nucleatum, and treated with different concentrations of Oxaliplatin (left) and 5-FU (right). (F) Representative data of tumors in mice under different conditions. Figure 5(F) and Figure S5G shared experimental controls. (G and H) Statistical analysis of tumor weights (G) and volumes (H) in different groups, n = 8/group nonparametric Mann–Whitney test. (I) TUNEL assays were performed to detect tumor cell apoptosis in xenograft tumor tissues. The mice received different treatments. (J) Transmission electron microscopy was performed to show the autophagosomes in xenograft tumor tissues. The mice received different treatments (17500 × magnification). Bar scale, 1 μm. (K) Statistical analysis of autophagosomes. Autophagosomes were detected by transmission electron microscopy in xenograft tumor tissues, nonparametric Mann–Whitney test. See also Figure S5–6.

Figure 6. TLR4 and MYD88 Pathway Is Involved in F. nucleatum-Mediated Chemoresistance

(A–D) Real-time PCR was performed to detect ATG7 (A), ULK1 (B), miR-18a* (C), and miR-4802 (D) expression in HCT116 cells. HCT116 cells were co-cultured with F. nucleatum and transfected with TLR4 and MYD88 siRNAs, respectively. (E and F) Apoptosis was detected by flow cytometry in HCT116 cells. The cells were co-cultured with F. nucleatum after TLR4 and MYD88 siRNAs transfection, and subsequently treated with different concentrations of Oxaliplatin (E) or 5-FU (F), nonparametric Mann–Whitney test. (G–J) Real-time PCR was performed to detect expression of ATG7 (G), ULK1 (H), miR-18a* (I), and miR-4802 (J) expression in HT29 cells. HT29 cells were co-cultured with F. nucleatum and transfected with TLR4 and MYD88 siRNAs, respectively. (K and L) Apoptosis was detected by flow cytometry in HT29 cells. The cells were co-cultured with F. nucleatum after TLR4 and MYD88 siRNAs transfection, and subsequently treated with different concentrations of Oxaliplatin (K) or 5-FU (L), nonparametric Mann–Whitney test. (M) Representative data of tumors in nude mice bearing HCT116 cells in different groups. Figure 6M and Figure S6A shared experimental controls. (N and O) Statistical analysis of mouse tumor weights (N) and volumes (O) in different groups, n = 8/group, nonparametric Mann–Whitney test. See also Figure S7.

Figure 7. The Levels of F.nucleatum, miR-18a*, miR-4802, and Autophagy Components Correlate and Are Relevant in CRC Patients

(A) Representative immunohistochemistry of p-ULK1 (upper), ULK1 (middle), and ATG7 (lower) proteins in CRC tissues from patients without recurrence and with recurrence (Cohort 2). NR, non-recurrence; R, recurrence. (B) Statistical analysis of immunohistochemical immunoreactive score of Remmele and Stegner (IRS) scores of pULK1 (upper), ULK1 (middle), and ATG7 (lower) proteins in Cohort 2. NR, non-recurrence; R, recurrence. (C) Statistical analysis of miR-18a* (left) and miR-4802 (right) expression by real-time PCR in Cohort 2. NR, non-recurrence; R, recurrence. (D) Correlations among F. nucleatum, miR-18a*, miR-4802, ULK1, and ATG7 levels in human colorectal cancer tissues (Cohort 2). (E) Schematic diagram of the relationship among F. nucleatum, autophagy and chemoresistance.