F. Nucleatum enhances oral squamous cell carcinoma proliferation via E-cadherin/β-Catenin pathway

Abstract

Background: Fusobacterium nucleatum (F. nucleatum) is a microbial risk factor whose presence increases the risk of oral squamous cell carcinoma (OSCC) progression. However, whether it can promote the proliferation of OSCC cells remains unknown.

Methods: In this study, we investigated F. nucleatum effect on OSCC cell proliferation using in vitro and in vivo experiments.

Results: Our results showed that F. nucleatum promoted OSCC cell proliferation, doubling the cell count after 72 h (CCK-8 assay). Cell cycle analysis revealed G2/M phase arrest. F. nucleatum interaction with CDH1 triggered phosphorylation, upregulating downstream protein β-catenin and activating cyclinD1 and Myc. Notably, F. nucleatum did not affect noncancerous cells, unrelated to CDH1 expression levels in CAL27 cells. Overexpression of phosphorylated CDH1 in 293T cells did not upregulate β-catenin and cycle-related genes. In vivo BALB/c nude experiments showed increased tumor volume and Ki-67 proliferation index after F. nucleatum intervention.

Conclusion: Our study suggests that F. nucleatum promotes OSCC cell proliferation through the CDH1/β-catenin pathway, advancing our understanding of its role in OSCC progression and highlighting its potential as a therapeutic target.

Keywords: Fusobacterium nucleatum; CDH1(E-Cadherin); Cell cycle; Cell proliferation; Oral squamous cell carcinoma; Phosphorylation; β-catenin.

Fig. 1

Methodological approach for studying cell proliferation in OSCC cells, with a focus on CAL27, conducted in three replicates. (A) Utilizing the CCK-8 assay, the influence of F. nucleatum and its supernatant on CAL27 cell proliferation was assessed. (B) Method to determine cell clone formation post-F. nucleatum intervention. (C) A 72-hour cocultivation followed by a cell counting procedure revealed an approximately 100% acceleration in cell growth, though the supernatant displayed negligible impact. (D) Control observation was employed to assess the proliferation effects of S. mutans on the cells, indicating no induction of cell proliferation. (E) Post-exposure effects of genistein on CAL27 cells in relation to (F) nucleatum was analyzed, highlighting genistein’s nullifying effects on F. nucleatum’s pro-proliferative attributes. F. A comparative cell cycle analysis encompassing the control, the F. nucleatum-treated, and the genistein-treated groups was performed, accompanied by statistical evaluations. (*, P<0.05; **, P<0.01; ***, P<0.001.)

Fig. 2

Analytical procedures and findings pertaining to CDH1 expression and its downstream molecules following diverse treatments, utilizing Western blot and qPCR techniques. (A) Western blot (WB) was employed to monitor the modulation of CDH1 and its downstream proteins after F. nucleatum exposure. Notably, there was an upregulation in the cytoplasmic protein β-catenin, subsequently elevating the levels of Myc and Cyclin D1. (B) Post-genistein treatment analysis revealed an inhibition in CDH1 phosphorylation, ensuring stable expression of CDH1 and its downstream entities. (C) For oncogene expression post F. nucleatum treatment, qPCR was the chosen technique. This assessment highlighted an elevation in oncogene expression after F. nucleatum exposure. (D) Following genistein administration, qPCR outcomes indicated no marked disparities in oncogene expression levels. (*, P<0.05; **, P<0.01; ***, P<0.001.)

Fig. 3

The effect of CDH1/β-catenin on cell proliferation. (A) Changes in the expression of CDH1 and its downstream proteins after transient transfection. After knocking down β-catenin, downstream protein expression was inhibited. After CDH1 overexpression, oncogene expression increased slightly. When cells overexpressed CDH1 and β-catenin were silenced simultaneously, the expression of downstream Myc and Cyclin D1 decreased, indicating that β-catenin was the key protein that activated downstream signaling. (B) Cell cycle analysis following knockdown β-catenin, the cell cycle was not different from the control group

Fig. 4

Detection of F. nucleatum on the proliferation of noncancerous cells. (A) Detection of CDH1 expression in four cell lines revealed differential expression in tumor cell lines, human oral keratinocytes (HOK), 293T cells, and human gingival fibroblasts (HGF). (B) After F. nucleatum was cocultured with 293T, HGF and HOK cells, the CCK-8 assay showed no obvious promotion of proliferation. (C) There was no difference in the plate clone formation observation after culturing F. nucleatum with HOK cells present. (D) In 293T cells overexpressing CDH1, phosphorylated CDH1 was detected, but downstream signaling was not activated. (E) No difference in oncogene expression was detected using qPCR in 293T cells overexpressing CDH1 and stimulated with (F) nucleatum

Fig. 5

Xenografts in nude mice. (A) After the F. nucleatum injection, nude mice had larger tumors, and the difference was obvious. (B) IHC showed the changes in CDH1 and p-CDH1 expression at different magnifications. CDH1 was expressed in both the control group and the F. nucleatum group. Because this protein is mainly located in the cell membrane, IHC revealed a diffuse expression pattern. The fibroblasts in the tumor were not stained, indicating that they did not express CDH1. IHC showed obvious increases in p-CDH1 expression in the F. nucleatum intervention group. Compared with the control group, the protein was upregulated and appeared in the cytoplasm. Scale bars, 80, 40 and 20 μm at 10x, 20x and 40x magnification, respectively. (C) The IHC analysis of changes in Ki-67 expression showed a higher positive rate in the F. nucleatum intervention group

Fig. 6

The pattern diagram of F. nucleatum promotes OSCC cell proliferation. F. nucleatum interacts with CDH1 on the membrane, and CDH1 is phosphorylated and enters the cytoplasm. β-Catenin is isolated and subsequently activates the transcription of downstream oncogenes. And OSCC cell proliferation is increased