Inhibin B suppresses anoikis resistance and migration through the transforming growth factor-β signaling pathway in nasopharyngeal carcinoma

Abstract

Inhibin B (INHBB), a heterodimer of a common α-subunit and a βB-subunit, is a glycoprotein belonging to the transforming growth factor-β (TGF-β) family. In this study, we observed INHBB expression was reduced in nasopharyngeal carcinoma (NPC) tissues compared to non-tumor nasopharyngeal epithelium tissues, and INHBB was associated with lymph node metastasis, stage of disease, and clinical progress. Positive expression of INHBB in NPC predicted a better prognosis (overall survival, P = 0.038). However, the molecular mechanisms of INHBB have not been addressed in NPC. We induced anoikis-resistant cells in NPC cell lines under anchorage-independent conditions, then found epithelial-mesenchymal transition markers changed, cell apoptosis decreased, cell cycle was modified, and invasion strengthened in anoikis-resistant NPC cells. These anoikis-resistant NPC cells showed decreased expression of INHBB compared with adhesion cells. Furthermore, INHBB was found to influence the above-mentioned changes. In the anoikis-resistant NPC cells with INHBB overexpression, apoptotic cells increased, S phase cells weakened, vimentin, matrix metallopeptidase-9, and vascular endothelial growth factor A expression were downregulated, and E-cadherin expression was upregulated, and vice versa in knockdown of INHBB (INHBB shRNA) anoikis-resistant NPC cells. Diminished INHBB expression could activate the TGF-β pathway to phosphorylate Smad2/3 and form complexes in the nucleus, which resulted in the above changes. Thus, our results revealed for the first time that INHBB could suppress anoikis resistance and migration of NPC cells by the TGF-β signaling pathway, decrease p53 overexpression, and could serve as a potential biomarker for NPC metastasis and prognosis as well as a therapeutic application.

Keywords: TGF-β; anoikis; inhibin B; metastasis; nasopharyngeal carcinoma.

Figure 1

Inhibin B (INHBB) is downregulated in nasopharyngeal carcinoma (NPC) tissues and is associated with clinical prognosis. A, INHBB protein was localized in the nucleus of columnar epithelial cells (brown denotes a positive signal). B, INHBB staining was negative in the differentiated non‐keratinizing NPC. C, D, Kaplan‐Meier survival curves in NPC patients with varying INHBB expression. The difference in INHBB expression levels was not statistically significant between the event‐free survival (EFS) of the two groups, but was statistically significant between the overall survival (OS) of the two groups. Blue, negative expression; green, positive expression. EFS, P = 0.295; OS, P = 0.038, log‐rank test

Figure 2

Establishment of anoikis‐resistant nasopharyngeal carcinoma (NPC) cells. A, B, Suspension culture of NPC HNE1 and 5‐8F cells over 72 hours of MTT test (A) and showing the number of cells (B). C, HNE1 and 5‐8F cell morphology after 48 hours in 3 different culture conditions: adhesion (ad), suspension (sus), and re‐adhesion after suspension for 48 hours (re‐ad). D, Cell apoptosis flow cytometry analysis. X‐axes, propidium iodide (PI)‐A staining; Y‐axes, cell counts. MMP‐9, matrix metallopeptidase‐9; VEGF‐A, vascular endothelial growth factor‐A. E, Apoptotic cell comparison of HNE1 and 5‐8F cells, and that of 3 different culture condition cells. F, Epithelial‐mesenchymal transition marker changes and migration of 3 different culture condition cells. G, Cell cycle flow cytometry analysis. X‐axes, PI‐A staining; Y‐axes, cell counts. H, G₀/G₁ and S cell comparison of HNE1 cells in three different culture conditions. HNE1^sus and HNE1^re‐ad G₀/G₁ cells were reduced, and S cells were increased. I, G₀/G₁ and S cells comparison of 5‐8F cells in three different culture conditions. 5‐8F^sus and 5‐8F^re‐ad G₀/G₁ cells were reduced, and S cells were increased. J, Inhibin B (INHBB) expression for HNE1 and 3 different cultured conditions of 5‐8F cells

Figure 3

Inhibin B (INHBB) induces apoptosis in anoikis‐resistant nasopharyngeal carcinoma cells. A, Overexpression of INHBB cells for apoptosis flow cytometry analysis. X‐axes, propidium iodide (PI)‐A staining; Y‐axes, cell counts. B, Overexpression of INHBB promoted apoptosis. Number of apoptotic cells from HNE1 adhesion culture (HNE1^ad) and suspension culture (HNE1^sus) treated with INHBB increased. C, INHBB induced apoptosis of anoikis‐resistant cells. Cells from HNE1 re‐adhesion culture (HNE1^re‐ad) and HNE1^sus treated with INHBB had stronger resistance than HNE1^ad. D, INHBB knockdown cells for apoptosis flow cytometry analysis. X‐axes, PI‐A staining; Y‐axes, cell counts. E, INHBB knockdown cells lowering apoptosis. Apoptotic cells of 5‐8F^ad and 5‐8F^sus with shRNA‐INHBB were decreased. F, Knockdown of INHBB inhibited apoptosis of anoikis‐resistant cells. 5‐8F^re‐ad and 5‐8F^sus cells with shRNA‐INHBB showed stronger resistance than 5‐8F^ad cells

Figure 4

Inhibin B (INHBB) inhibits DNA synthesis in anoikis‐resistant nasopharyngeal carcinoma cells. A, Overexpression of INHBB cells for cell cycle flow cytometry analysis. X‐axes, propidium iodide (PI)‐A staining; Y‐axes, cell counts. B, INHBB suppressed S phase cell growth. S phase cells of adhesion culture (HNE1^ad) and suspension culture (HNE1^sus) treated with INHBB decreased. C, INHBB weakened the S phase cell increase of anoikis‐resistant cells, and there was no change in G₀/G₁ phase cells. D, INHBB knockdown cells for cell cycle flow cytometry analysis. X‐axes, PI‐A staining; Y‐axes, cell counts. E, 5‐8F^sus with shRNA‐INHBB S phase cells increased. F, Anoikis‐resistant cells with shRNA‐INHBB G₀/G₁ phase cells decreased, and S phase cells increased

Figure 5

Inhibin B (INHBB) is involved in epithelial‐mesenchymal transition (EMT) and metastasis by influencing the transforming growth factor‐β (TGF‐β) pathway. A, Western blot analysis of EMT marker protein (vimentin and E‐cadherin) and metastasis marker protein (matrix metallopeptidase‐9 [MMP‐9] and vascular endothelial growth factor‐A [VEGF‐A]) expression for HNE1 and 3 different culture conditions of 5‐8F cells with INHBB and shRNA‐INHBB. B, Western blot analysis of TGF‐β pathway protein (TGF‐β1, Smad2/3, p‐Smad2/3(T8), Smad4) expression for HNE1 and 3 different culture conditions of 5‐8F cells with INHBB and shRNA‐INHBB. ad, adhesion; re‐ad, re‐adhesion after suspension for 48 hours; sus, suspension

Figure 6

Diminished inhibin B (INHBB) expression activates the transforming growth factor‐β (TGF‐β) pathway to promote anoikis resistance and migration. Schematic of diminished INHBB reprogramming by activation of the TGF‐β pathway in anoikis‐resistant cells, accelerating the anoikis sensibility and DNA synthesis of cells, promoting the proliferation of cells and the growth of the tumor, inducing epithelial‐mesenchymal transition (EMT) changes to enhance motor abilities of tumor cells, upregulating matrix metallopeptidase‐9 (MMP‐9) and vascular endothelial growth factor‐A (VEGF‐A), and improving tumor invasion and metastasis