Significance of the TGF-β1/IL-6 axis in oral cancer

Abstract

The aim of the present study was to explore specific molecular markers that could lead to new insights into the identification of innovative treatments in oral cancer. The role of TGF-β1 (transforming growth factor-β1) and its predictive power in the prognosis of oral cancer has been identified. Human oral cancer cell lines, including SCC4 and SCC25, were selected for cellular experiments. Changes in tumour aggressiveness, responses to treatment and the signalling pathway responsible were investigated in vitro. Furthermore, 125 oral cancer tissue specimens were constructed into tissue microarray blocks for immunohistochemical analysis to correlate the expression of TGF-β1 with clinical outcome. Using in vitro experiments, our results revealed that activated TGF-β1 signalling resulted in more aggressive tumour growth, augmented the epithelial-mesenchymal transition and more resistance to treatment. Activated IL-6 (interleukin-6) signalling could be the mechanism underlying the effects of TGF-β1 on oral cancer. Regarding clinical data, the incidence of TGF-β1 immunoreactivity in oral cancer specimens was significantly higher than in non-malignant epithelium and positively linked to IL-6 staining. Furthermore, expression of TGF-β1 was significantly correlated with the risk of lymph node involvement, disease recurrence and shorter survival in patients with pathological stage III-IV oral cancer. In conclusion, the TGF-β1/IL-6 axis had predictive power in the prognosis of oral cancer, and targeting TGF-β1 could represent a promising treatment strategy.

Figure 1. Immunohistochemical staining of human oral cancer TMA specimens

(A) Representative images of TGF-β1- and IL-6-positive staining by immunohistochemistry using the Good Speed scan slide scanning platform and Image Pro Plus 6.3. Upper row, adjacent non-malignant epithelium; lower panel, oral cancer tissue. Scale bars, 100 μm. (B) Representative data analysed by Image Pro Plus 6.3 are shown for each molecular marker. Left-hand panel, original immunohistochemical images; right-hand panel, Image Pro Plus 6.3 analysis with positive events marked in red. (C) Representative images of TGF-βRI- and TGF-βRII-positive staining by immunohistochemistry using the Good Speed slide scanning platform.

Figure 2. Effect of TGF-β1 in cancer cell proliferation

(A) Effects of the TGF-β1–RFP-silencing vector and TGF-β1–GFP-expression vector on the level of TGF-β1 in SCC4 cells as determined by immunofluorescence. Expression of TGF-β1 in SCC4 transfectants was also examined by Western blot analysis. W or Wild, wild-type; R-V, cells with control–RFP vector; TG-, cells with the TGF-β1–RFP-silencing vector; G-V, cells with control–GFP vector; TG⁺, cells with the TGF-β1–GFP-expression vector. (B) Effect of TGF-β1 on the proliferation rate of cancer cells. The same number of cells (10⁴) were plated on to each plate on day (D) 0 and were allowed to grow in their respective cultures. The number of viable cells after incubation for 2, 4 and 6 days were counted. *P<0.05. (C) Effect of TGF-β1 inhibition on SCC25 cancer cell proliferation in situ as detected by immunofluorescence. Upper panel, nuclei stained with propidium iodine (PI); lower panel, slides were stained with BrdU–FITC. Scale bars, 100 μm.

Figure 3. Effect of TGF-β1 on tumour aggressiveness and EMT changes

(A) The invasive capacities of oral cancer cells with or without TGF-β1 regulation were evaluated using an invasion assay in SCC25 cells (left-hand panels) and a migration assay in SCC4 cells (right-hand panels). Representative images and quantification are shown. For the invasion assay, the relative number of invading cells normalized to that under control conditions was calculated. For the migration assay, the relative ratio, normalized to the distance under control conditions at 24 h after scratching, was calculated. Results are means±S.D. for three separate experiments; *P<0.05. Ab, antibody; Wild, wild-type. (B) E-cadherin levels in SCC25 cells was evaluated by immunofluorescence and quantified. Representative images are shown (left-hand panel, immunofluorescence image stained with DAPI for the nucleus; middle panel, immunofluorescence image stained with an anti-E-cadherin antibody). Scale bars, 100 μm. Right-hand panel, quantification of E-cadherin expression. Levels of E-cadherin were determined by dividing the number of cells positive for E-cadherin immunofluorescence by the total cell number for each condition. *P<0.05. (C) Change in EMT-associated proteins in cells with regulated TGF-β1 levels was evaluated by Western blot analysis. C, cells under control conditions; TG-Ab, cells treated with the anti-TGF-β1 antibody; TG, cells treated with TGF-β1; W, wild-type; G-V, cells transfected with control–GFP vector; TG⁺, cells transfected with TGF-β1–GFP-expression vector.

Figure 4. Effect of TGF-β1 expression on IL-6 signalling

(A) Effect of TGF-β1 on IL-6 signalling was evaluated by Western blotting in SCC25 and SCC4 cells. p-, phospho-. (B) Influence of TGF-β1 on the level of IL-6 in cells was examined by real-time RT–PCR. The IL-6/β-actin ratio in cancer cells normalized to cells without treatment was calculated. Values are means±S.D. for three separate experiments; *P<0.05. (C) Influence of TGF-β1 on the level of IL-6 in the supernatants of cells was examined by ELISA. Values are means±S.D. for three separate experiments; *P<0.05. (D) Effect of TGF-β1 on the expression of IL-6 in SCC25 cells was examined by immunofluorescence. Representative images are shown. Top row, cells stained with an anti-TGF-β1 antibody; middle row, cells stained with an anti-IL-6 antibody; bottom row, merged images with DAPI used to stain nuclei. Wild, wild-type.

Figure 5. Effect of IL-6 on TGF-β1 expression and the proliferation rate in oral cancer cells

(A) Effect of IL-6 on the level of TGF-β1 was examined by Western blotting. Representative blots from experiments performed in triplicate are shown. (B) Effect of IL-6 signalling on the levels of HIF-1α, TGF-β1, vimentin and MMP-9 in oral cancer cells. Representative blots from experiments performed in triplicate are shown. Ab, antibody; W, wild-type. (C) Effects of IL-6 on the proliferation rate of oral cancer cells. The same number of cells (10⁴) were plated on day 0 and were allowed to grow in their respective cultures. The number of viable cells after incubation for 2, 4 and 6 days was counted. The relative viable cell number normalized to that under control conditions was calculated. Values are means±S.D. for three separate experiments; *P<0.05.

Figure 6. Effects of TGF-β1 on the treatment response

(A) Cells were treated with 0 or 6 μg/ml cisplatin in the presence or absence of the anti-TGF-β1 antibody, and the survival rate was determined at 48 h using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. The relative ratio normalized to that in SCC25 cells under control conditions for 48 h was calculated. (B) Cells were treated with 0 or 4 Gy in the presence or absence of the anti-TGF-β1 antibody, and the survival rate was determined at 48 h using the XTT assay. The relative ratio normalized to that in SCC25 cells under control conditions was calculated. (C) SCC4 transfectants were irradiated with 0, 2, 4 and 6 Gy, and the survival fractions were determined by measuring the colonies after irradiation and dividing by the plating efficiency. Values are means±S.D.; P<0.05. (D) Effect of TGF-β1 on radiation (RT)-induced cell death was demonstrated using annexin V–propidium iodide (PI) staining in cells 24 h after 6 Gy irradiation following pre-incubation with or without the anti-TGF-β1 antibody (Ab). Scale bars, 100 μm.

Figure 7. Effect of TGF-β1 on radiation-induced 8-oxoG, phospho-ATM and phosho-H2AX levels

(A) Influence of TGF-β1 on radiation (RT)-induced 8-oxoG and phospho-H2AX was evaluated by immunofluorescence in cells 30 min after irradiation following pre-incubation with or without the anti-TGF-β1 antibody (Ab). Representative images and quantitative results are shown. The quantification of 8-oxoG and phospho-H2AX expression was calculated by dividing the number of cells positive for the target protein by the total cell number for each condition. Values are means±S.D. for three separate experiments. (B) The influence of TGF-β1 on radiation-induced nuclear accumulation of phospho-ATM was evaluated by immunofluorescence and Western blot analysis in cells 30 min after irradiation. p-, phospho-.

Figure 8. Effect of the levels of TGF-β1 and IL-6 in the clinical outcome of oral cancer

(A) Positive immunohistochemical (IHC) staining for IL-6 was significantly linked with TGF-β1 expression in human oral cancer specimens. Representative images of a selected tumour specimen positive for TGF-β1 and IL-6 staining are shown. (B) Overall survival differences according to the positive staining of TGF-β1 and IL-6, and disease failure.