Activin B Regulates Adhesion, Invasiveness, and Migratory Activities in Oral Cancer: a Potential Biomarker for Metastasis

Abstract

Activin B, a homodimer of inhibin beta b (INHBB), is a multifunctional cytokine belonging to the transforming growth factor-β (TGF-β) family. However, the molecular functions and clinical relevance of activin B have not been determined in oral cancer. We investigated the critical roles of activin B in oral squamous cell carcinoma (OSCC). We performed quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry to study INHBB expression in OSCC-derived cell lines and OSCC clinical samples. The INHBB expression levels were significantly (P < 0.05) overexpressed in OSCCs compared to normal counterparts in vitro and in vivo. Activin B-positivity in OSCC cases was significantly (P < 0.05) correlated with regional lymph node metastasis. The INHBB knockdown (shINHBB) cells promoted cellular adhesion and suppression of cellular invasiveness and migration. After treatment of shINHBB cells with activin B, those activities were restored similar to the shMock cells. In the processes of invasiveness and metastasis, the cells cause epithelial-mesenchymal transition (EMT). TGF-β and its family members are promoters of the EMT process. To investigate whether activin B is related to EMT, we examined the expressions of EMT-related genes and found that INHBB was related closely to EMT. Our results suggested for the first time that activin B indicates tumoral metastasis in OSCCs and might be a useful biomarker for OSCC metastasis.

Keywords: INHBB; activin B; inhibin beta b; metastasis.; oral squamous cell carcinoma.

Figure 1

Up-regulation of INHBB expression in OSCC-derived cell lines. (A) Quantitation of INHBB mRNA expression by qRT-PCR analysis in OSCC-derived cell lines. Significant (*P < 0.05, Student's t-test) up-regulation of INHBB mRNA is observed in nine OSCC-derived cell lines compared to the HNOKs. The data are expressed as the mean ± SEM of triplicate results. (B) Western blotting of INHBB protein in OSCC-derived cell lines and HNOKs. INHBB protein expression is up-regulated in OSCC-derived cell lines compared with that in the HNOKs. Densitometric INHBB protein data are normalized to the GAPDH protein levels. The values are expressed as a percentage of the HNOKs.

Figure 2

Evaluation of INHBB expression in primary OSCCs. Representative IHC results of activin B in normal oral tissues (A) and primary OSCCs (B). Original magnification, ×200. Scale bars, 50 μm. (C) The status of activin B protein expression in primary OSCCs (n=103) and normal counterparts. Activin B IHC scores for normal oral tissues and OSCCs range from 26.0 to 80.0 (median, 55.1) and 71.0 to 209.0 (median, 126.1), respectively. Activin B protein expression levels in OSCCs are significantly higher than in normal oral tissues (*P < 0.05, Student's t-test). (D) The area under the curve (AUC) of the ROC curve analysis is 0.71, and the cutoff value is 101.3 for the regional lymph node metastasis (P < 0.05).

Figure 3

Establishment of INHBB knockdown cells. (A) INHBB mRNA levels in shINHBB-transfected cells. qRT-PCR shows that INHBB is down-regulated significantly (*P < 0.05, Student's t-test) in shINHBB cells compared with Mock cells. (B) Representative Western blotting and densitometric data of INHBB protein levels in shINHBB cells and Mock cells. The INHBB protein is decreased markedly (*P < 0.05, Student's t-test) in shINHBB-transfected cells compared with Mock cells. Densitometric INHBB protein data are normalized to GAPDH protein levels.

Figure 4

Functional assays. (A) Adhesion assay of Mock and shINHBB cells (SAS and KOSC-2-derived transfectants). To evaluate the adhesion ability of shINHBB, the cells are seeded on collagen I-coated 96-well plates at a density of 2 ×10⁴ cells/well and allowed to adhere for 1 hour. After crystal violet staining, the numbers of stained cells are measured using a microplate spectrophotometer (absorbance at 540 nm and at 405 nm to subtract background). The cellular adhesion of the shINHBB cells are increased significantly (P < 0.05) compared with the Mock cells. The results are expressed as the means ± SEM of values from three assays (*P < 0.05, Student's t-test). (B) Invasiveness assay of Mock and shINHBB cells (SAS and KOSC-2-derived transfectants). To evaluate the effect of INHBB knockdown on invasiveness, the cells are seeded on Matrigel-coated Transwell inserts (8 μm pores) at a density of 2.5×10⁵ cells/well in serum-free medium. Serum-supplemented medium was added in the lower chamber as a chemoattractant. After incubation at 37°C for 48 hours, cells that penetrated through the pores are fixed, stained, and counted using a light microscope at ×100 magnification. The number of shINHBB cells penetrating through the pores is decreased significantly (*P < 0.05, Student's t-test) compared with the Mock cells. The mean value is calculated from data obtained from three separate chambers. (C) Migration assay of Mock and shINHBB cells (SAS and KOSC-2-derived transfectants). To evaluate the effect of INHBB knockdown on migration, uniform wounds are made in confluent cultures of the shINHBB and Mock cells and the extent of closure is monitored visually every 3 hours for 18 hours. The mean value is calculated from data obtained from three separate chambers. The wound area is decreased significantly (*P < 0.05, Student's t-test) in the Mock cell culture after 9 or 12 hours, whereas a gap remains in the shINHBB cells.

Figure 5

Functional assays after treatment with activin B. (A) Inhibition of cellular adhesion by treatment with activin B. The numbers of shINHBB cells attached to the dishes are decreased after treatment with activin B (*P < 0.05, Student's t-test). Cellular invasiveness (B) and cellular migration (C) after treatment with activin B. After treatment, invasiveness and migration of shINHBB cells are activated significantly (*P < 0.05, Student's t-test) compared with the untreated cells.

Figure 6

EMT in INHBB knockdown cells. (A) Western blotting of E-cadherin, Zo-1, and Snail proteins in shINHBB cells are treated with/without activin B. Treatment of shINHBB cells with activin B shows decreased levels of E-cad and Zo-1 and increased levels of Snail compared with the control. (B) Brightfield and immunofluorescence images of shINHBB cells are treated with/without activin B. The shINHBB cells treated with activin B shows loss of E-cadherin on the cell membrane. Scale bars: 20 μm (left panels); 5 μm (middle and right panels).