Thyroid Hormone Induces Oral Cancer Growth via the PD-L1-Dependent Signaling Pathway

Abstract

Oral cancer is a fatal disease, and its incidence in Taiwan is increasing. Thyroid hormone as L-thyroxine (T₄) stimulates cancer cell proliferation via a receptor on integrin αvβ3 of plasma membranes. It also induces the expression of programmed death-ligand 1 (PD-L1) and cell proliferation in cancer cells. Thyroid hormone also activates β-catenin-dependent cell proliferation in cancer cells. However, the relationship between PD-L1 and cancer proliferation is not fully understood. In the current study, we investigated the role of inducible thyroid hormone-induced PD-L1-regulated gene expression and proliferation in oral cancer cells. Thyroxine bound to integrin αvβ3 to induce PD-L1 expressions via activation of ERK1/2 and signal transducer and activator of transcription 3 (STAT3). Inactivated STAT3 inhibited PD-L1 expression and nuclear PD-L1 accumulation. Inhibition of PD-L1 expression reduced β-catenin accumulation. Furthermore, nuclear PD-L1 formed a complex with nuclear proteins such as p300. Suppression PD-L1 expression by shRNA blocked not only expression of PD-L1 and β-catenin but also signal transduction, proliferative gene expressions, and cancer cell growth. In summary, thyroxine via integrin αvβ3 activated ERK1/2 and STAT3 to stimulate the PD-L1-dependent and β-catenin-related growth in oral cancer cells.

Keywords: PD-L1; oral cancer; thyroxine; β-catenin.

Figure 1

Thyroid hormone induces abundances of PD-L1 in human oral cancer cells. OEC-M1 cells and SCC-25 cells were treated with different concentrations of thyroid hormone (T₄, 10^-8 to 10^-6 M) for 24 h. (A) Thyroid hormone-induced PD-L1 expression in both oral cancer cell. (B) OEC-M1 cells and (C) SCC-25 cells were treated with different concentrations of T₄ for 24 h. Cells were harvested. Total protein was extracted and Western blot analyses of pSTAT3, pERK1/2, PD-L1 were performed in Data were presented as the mean ± SD. Results are from three independent studies (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control. I.O.D., intensity of the optical density.

Figure 2

ERK1/2 activation plays a role in T₄-induced accumulation of PD-L1 and β-catenin in human oral cancer OEC-M1 cells. Oral cancer OEC-M1 cells were seeded in six-well trays and were treated with 10^-7 M T₄ in the presence or absence of PD98059 (10 μM) for 24 h. Western blot analyses of β-catenin, PD-L1, pERK1/2 and ERK1/2 were conducted. Data are presented as the mean ± SD. Results are from three independent studies (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control. I.O.D., intensity of the optical density.

Figure 3

Thyroxine (T₄)-induced PD-L1 accumulation is activated-STAT3-dependent in human oral cancer OEC-M1 cells. Oral cancer OEC-M1 cells were seeded on a cover glass. Cells were starved for 48 h, fed hormone-stripped serum-containing medium, and then treated with T₄ in the presence and absence of 40 μM S31-201 for another 24 h. Cells were fixed for confocal microscopic analysis of PD-L1 accumulation (red color). Nuclei were counterstained with DAPI (blue color). The field of view of the red square is magnified for easy viewing.

Figure 4

Thyroxine (T₄)-induced PD-L1 accumulation is activated- STAT3-dependent in human oral cancer SCC-25 cells. Oral cancer SCC-25 cells were seeded on a cover glass. Cells were starved for 48 h, fed hormone-stripped serum-containing medium, and then treated with T₄ in the presence and absence of 40 μM S31-201 for another 24 h. Cells were fixed for confocal microscopic analysis of PD-L1 accumulation (red color). Nuclei were counterstained with DAPI (blue color). The field of view of the red square is magnified for easy viewing.

Figure 5

Thyroxine (T₄)-induced PD-L1 complexes with nuclear proteins. Oral cancer (A) OEC-M1 cells and (B) SCC-25 cells were seeded on a cover glass. Cells were starved for 48 h, fed hormone-stripped serum-containing medium, and then treated with T₄ in the presence and absence of 40 μM S31-201 for another 24 h. Cells were fixed for confocal microscopic analysis of PD-L1 accumulation (green color) or P300 (red color). Nuclei were counterstained with DAPI (blue color). Red square is PD-L1 and P300 co-accumulation (white arrow.). Confocal microscopy was conducted as described.

Figure 6

Thyroid hormone (T₄)-induced PD-L1 controls β-catenin expression in human oral cancer cells. OEC-M1 cells were seeded in six-well trays and transfected with PD-L1 shRNA or a scrambled plasmid (0.5 μg/well) were treated with 10⁻⁷ M T₄ for 24 h. Cells were harvested, and RNA was extracted. A qPCR was conducted for proliferation-related genes. Data are presented as the mean ± SD. Results are from three independent studies (n = 4) ** p < 0.01, *** p < 0.001, compared to the control; ## p < 0.01, ### p < 0.001, compared to T₄, and $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, compared to PD-L1 shRNA group.

Figure 7

Thyroid hormone (T₄)-induced PD-L1-dependent protein expression and cell proliferation in oral cancer cells. (A). Oral cancer OEC-M1 cells were seeded in six-well trays and were transfected with either PD-L1 shRNA or a scrambled plasmid (0.5 μg/well). Prior to treatment, cells were refed with hormone-stripped FBS-containing medium and treated with T₄ (10⁻⁷ M) for 24 h. Cells were harvested, and Western blot analyses of total β-catenin, pβ-catenin, PD-L1, and pERK1/2 were conducted. Data are presented as the mean ± SD. Results are from three independent studies (n = 4) ** p < 0.01, compared to the control. I.O.D., intensity of the optical density. (B). Oral cancer OEC-M1 cells were seeded in 24-well trays and were transfected with either PD-L1 shRNA or a scrambled plasmid (0.1 μg/well). Prior to treatment, cells were refed with hormone-stripped FBS-containing medium and treated with T₄ (10⁻⁷ M) for 72 h. Cells were harvested for cell viability assay. Data are presented as the mean ± SD. Results are from three independent studies (n = 6) * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control; # p < 0.05, ### p < 0.001, compared to T₄, and $ p < 0.05, $$$ p < 0.001, compared to PD-L1 shRNA group.