Porphyromonas gingivalis promotes progression of esophageal squamous cell cancer via TGFβ-dependent Smad/YAP/TAZ signaling

Abstract

Microbial dysbiosis in the upper digestive tract is linked to an increased risk of esophageal squamous cell carcinoma (ESCC). Overabundance of Porphyromonas gingivalis is associated with shorter survival of ESCC patients. We investigated the molecular mechanisms driving aggressive progression of ESCC by P. gingivalis. Intracellular invasion of P. gingivalis potentiated proliferation, migration, invasion, and metastasis abilities of ESCC cells via transforming growth factor-β (TGFβ)-dependent Drosophila mothers against decapentaplegic homologs (Smads)/Yes-associated protein (YAP)/Transcriptional coactivator with PDZ-binding motif (TAZ) activation. Smads/YAP/TAZ/TEA domain transcription factor1 (TEAD1) complex formation was essential to initiate downstream target gene expression, inducing an epithelial-mesenchymal transition (EMT) and stemness features. Furthermore, P. gingivalis augmented secretion and bioactivity of TGFβ through glycoprotein A repetitions predominant (GARP) up-regulation. Accordingly, disruption of either the GARP/TGFβ axis or its activated Smads/YAP/TAZ complex abrogated the tumor-promoting role of P. gingivalis. P. gingivalis signature genes based on its activated effector molecules can efficiently distinguish ESCC patients into low- and high-risk groups. Targeting P. gingivalis or its activated effectors may provide novel insights into clinical management of ESCC.

Fig 1. P. gingivalis predicts poor clinical outcome and promotes ESCC cell aggressive progression.

(A) Representative images of IHC and RNAscope of P. gingivalis in ESCC. Scale bars, 50 μm. (B) Kaplan–Meier survival curves for 190 patients with ESCC were compared between patients with high and low amounts of P. gingivalis. (C) The cell growth rates of NE6-T and KYSE30 cells in vitro treated with P. gingivalis, LPS or heat-killed P. gingivalis or untreated for indicated times were evaluated by an MTT assay. **P < 0.01 by Student t test. (D) The haptotactic migration assay and matrigel chemoinvasion assay of NE6-T and KYSE30 cells treated with P. gingivalis or LPS, heat-killed P. gingivalis or untreated. **P < 0.01 by Student t test. (E) Accumulation of P. gingivalis in NE6-T and KYSE30 cells after 24 h of P. gingivalis infection were shown by confocal immunofluorescence microscopy (upper panel) and RNAscope assay (lower panel). Scale bar, 50 μm. (F) The left panel shows representative fluorescent images of GFP signals captured from subcutaneous tumors. The middle and right panels show the tumor growth curve (**P < 0.01 by one-way ANOVA and Bonferroni multiple comparison test) and tumor weight (*P < 0.05 by one-way ANOVA). (G) Left panel, representative bioluminescent images of photon flux show the lung metastasis. Middle panel, bioluminescent quantification of metastatic cells in lung (**P < 0.01, Mann–Whitney U test). Results represent means ± SD. Right panel, representative HE staining shows ESCC cell lung metastasis. ANOVA, analysis of variance; ESCC, esophageal squamous cell carcinoma; GFP, green fluorescent protein; HE, hematoxylin–eosin; IHC, immunohistochemistry; LPS, Lipopolysaccharide; MTT, 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide; RNAscope, RNA in situ hybridization.

Fig 2. P. gingivalis activates TGFβ/Smad signaling in ESCC cells.

(A) The heatmap illustrates the differentially expressed genes in response to P. gingivalis in KYSE30 cells from 3 independent experiments. Red and green indicate up-regulated and down-regulated genes, respectively. (B) Two-hundred forty-five genes with differential expression were used to build a subnetwork that consisted of 74 linker genes and 86 seed genes. Bigger size and red color direction indicate higher degree and higher betweenness, respectively. (C) Pathway enrichment analyses (left, Reactome; right, KEGG) were conducted to identify pathways affected by P. gingivalis. Star denotes overlapped biological pathways. (D) ELISA was performed to measure the total and active TGFβ secreted from NE6-T and KYSE30 cells cultured with or without P. gingivalis. (E) Dual luciferase assay of Smad reporter was measured in NE6-T and KYSE30 cells with different treatments. Renilla luciferase activity was normalized to firefly activity as relative luciferase activity. *P< 0.05, **P < 0.05, by Student t test. (F) Western blot was performed to detect the indicated proteins in NE6-T and KYSE30 cells with different treatments. GAPDH served as the loading control. (G) Immunofluorescence microscopy of Smad2/3 and Oct4 was observed in NE6-T and KYSE30 cells with different treatments. Scale bar, 50 μm. (H) Representative data of xenograft tumors from NE6-T cells receiving different treatments, the tumor weight (*P < 0.05 by one-way ANOVA), and the tumor growth curve (*P < 0.05 by one-way ANOVA and Bonferroni multiple comparison test). (I) Representative bioluminescence images and quantification of photon flux (***P < 0.05; **P < 0.01, Mann–Whitney U test) in different groups of mice. Results represent means ± SD. Akt, v-akt murine thymoma viral oncogene homolog; ANOVA, analysis of variance; CDKN1A, cyclin-dependent kinase inhibitor 1; ECM, extracellular matrix proteins; ESCC, esophageal squamous cell carcinoma; FGFR3, fibroblast growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCM, hypertrophic cardiomyopathy; KEGG, Kyoto Encyclopedia of Genes and Genomes; MH2, mad-homology domain 2; NCAM, neural cell adhesion molecule; PI3K, phosphatidylinositol 3 kinase; pSmad, phosphorylated Smad; RUNX, runt-related transcription factor; Smad, Drosophila mothers against decapentaplegic homolog; TGFβ, transforming growth factor-β; TGFβ1-N, TGFβ1 neutralizing antibody.

Fig 3. P. gingivalis activates YAP/TAZ through TGFβ noncanonical signaling in ESCC cells.

(A) Indicated proteins were detected by western blot in NE6-T and KYSE30 cells with different treatments. (B) Subcellular localization of YAP/TAZ was detected by immunofluorescence microscopy in NE6-T cells with different treatments. Scale bar, 50 μm. (C) NE6-T and KYSE30 cells were transfected with control siRNA or siRNA targeting Smad2/3 and then treated with P. gingivalis. Western blots were used to detect the indicated protein levels in NE6-T and KYSE30 cells with different treatments. (D) NE6-T and KYSE30 cells were treated with PBS control or P. gingivalis for 24 h. Hippo pathway components pLats1/2, pMst1/2, and pMerlin, together with their total levels, were detected by western blots. (E) NE6-T and KYSE30 cells were transfected with control plasmid, Lats1/2, or Merlin S51A mutant expression plasmids and then were treated with P. gingivalis. Western blots were used to detect the indicated protein levels in NE6-T and KYSE30 cells with different treatments. Representatives of 3 independent experiments. CTGF, connective tissue growth factor; ESCC, esophageal squamous cell carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Lats, large tumor suppressor homolog; Merlin, moesin-ezrin-radixin-like protein; Mst, mammalian STE20-like protein kinase; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate-buffered saline; pLats, phosphorylated Lats; pMst, phosphorylated Mst; pMerlin, phosphorylated Merlin; siCON, scramble control for short interfering RNA; siRNA, short interfering RNA; Smad, Drosophila mothers against decapentaplegic homolog; TAZ, Transcriptional coactivator with PDZ-binding motif; TGFβ, transforming growth factor-β; TGFβ1-N, TGFβ1 neutralizing antibody; YAP, Yes-associated protein.

Fig 4. P. gingivalis induces Smads/YAP/TAZ/TEAD1 complex formation.

(A) Western blots were used to detect the indicated protein levels in NE6-T and KYSE30 cells with different treatments. Representative of 3 independent experiments. (B) NE6-T and KYSE30 cells were transfected with control siRNA or siRNA targeting YAP/TAZ and treated with P. gingivalis for 24 h and dual luciferase assay of Smad reporter was performed. R. luciferase activity was normalized to firefly activity as relative luciferase activity. *P < 0.05, **P < 0.01 by Student t test. n = 3 independent experiments performed in triplicate. (C) Dual luciferase assays for YAP/TAZ transcriptional activity using pGL3 luciferase reporter vector carrying CTGF promoter or CYR61 promoter were performed in NE6-T and KYSE30 cells treated with PBS control or P. gingivalis. R. luciferase activity was normalized to firefly activity as relative luciferase activity. *P < 0.05, **P < 0.01 by Student t test. n = 3 independent experiments performed in triplicate. (D) ChIP assay was performed in NE6-T cells with different treatments using different antibodies for CTGF promoters by PCR assay with GAPDH as the internal control. (E) NE6-T cells were cocultured with PBS control or P. gingivalis, and coimmunoprecipitation was performed using different antibodies. (F) Representative data of xenograft tumors, the tumor weight (**P < 0.01 by one-way ANOVA), and the tumor growth curves (**P < 0.01 by one-way ANOVA and Bonferroni multiple comparison test) from NE6-T cells receiving different treatments. (G) Representative bioluminescence images and quantification of photon flux (*P < 0.05, Mann–Whitney U test) in different groups of mice. Results represent means ± SD. ANOVA, analysis of variance; ChIP, chromatin immunoprecipitation; CTGF, connective tissue growth factor; CYR61, cysteine-rich angiogenic inducer 61; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitation; PBS, phosphate-buffered saline; pSmad, phosphorylated Smad; SBE, Smad-binding element; siCON, scramble control for short interfering RNA; siRNA, short interfering RNA; Smads, Drosophila mothers against decapentaplegic homologs; TAZ, Transcriptional coactivator with PDZ-binding motif; TEAD1, TEA domain transcription factor1; WB, western blot; YAP, Yes-associated protein.

Fig 5. P. gingivalis-induced GARP up-regulation promotes TGFβ bioactivity and drives aggressiveness of ESCC.

(A) Western blots were used to detect the indicated protein levels in ESCC cells with different treatments. Representative of 3 independent experiments. (B) Representative immunostaining of GARP proteins in xenograft tumor tissues from NE6-T. Scale bar, 200 μm. (C) Subcellular localization of GARP was detected by immunofluorescence microscopy in NE6-T and KYSE30 cells treated with PBS control or P. gingivalis. Scale bar represents 50 μm. **P < 0.01 by Student t test. (D) NE6-T and KYSE30 cells were transfected with control siRNA or siRNA targeting GARP and treated with P. gingivalis for 24 h and dual luciferase assay of Smad reporter was performed. R. luciferase activity was normalized to firefly activity as relative luciferase activity. *P < 0.05, **P < 0.01 by Student t test. n = 3 independent experiments performed in triplicate. (E) NE6-T and KYSE30 cells were transfected with control siRNA or siRNA targeting TLR4 or MYD88 and then treated with P. gingivalis. Western blots were used to detect the indicated protein levels in NE6-T and KYSE30 cells with different treatments. Representative of 3 independent experiments. CTGF, connective tissue growth factor; ESCC, esophageal squamous cell carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GARP, glycoprotein A repetitions predominant; MYD88, myeloid differentiation primary response protein 88; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate-buffered saline; pSmad, phosphorylated Smad; siCON, scramble control for short interfering RNA; siRNA, short interfering RNA; Smad, Drosophila mothers against decapentaplegic homolog; TGFβ, transforming growth factor-β; TLR4, toll-like receptor 4.

Fig 6. P. gingivalis-activated effectors correlate and are relevant in ESCC patients.

(A) Representative IHC of GARP, pSmad2, YAP/TAZ, Snail, and Oct4 proteins in ESCC tissues from patients with low and high levels of P. gingivalis in ESCC. Scale bars represent 200 μm and 100 μm, respectively. (B) Correlations among P. gingivalis, GARP, TGFβ, pSmad2, YAP/TAZ, Snail, and Oct4 levels in 190 ESCCs. (C & D) The P. gingivalis signature gene sets predict overall survival of patients with ESCC. Kaplan–Meier survival curves of patients in the training set (C) and test set (D) were classified into low- and high-risk groups. (E) Comparison of clinicopathological features between the low- and high-risk groups classified by P. gingivalis signature-gene–based classifier. Statistical significance was performed by chi-square test. (F) A proposed model for P. gingivalis-induced aggressive progression of ESCC. ESCC, esophageal squamous cell carcinoma; GARP, glycoprotein A repetitions predominant; IHC, immunohistochemistry; LAP, latency-associated protein; Lats, large tumor suppressor homolog; Merlin, moesin-ezrin-radixin-like protein; pS, phosphorylated serine/threonine; pSmad, phosphorylated Smad; SARA, Smad anchor receptor activation; Smad, Drosophila mothers against decapentaplegic homolog; TAZ, Transcriptional coactivator with PDZ-binding motif; TEAD1, TEA domain transcription factor1; TGFβ, transforming growth factor-β; TNM, tumor-node metastasis; YAP, Yes-associated protein.