Overexpression of ERβ inhibits the proliferation through regulating TNG-β signaling pathway in osteosarcoma

Highlights

• The expression of ERβ is low in osteosarcoma cells.

• ERβ overexpression inhibits osteosarcoma cell proliferation and blocks cell cycle.

• ERβ overexpression promotes osteosarcoma cell apoptosis and autophagy.

• ERβ overexpression inhibits osteosarcoma cell migration and invasion.

• ERβ plays an anti-tumor role through TNG-β signaling pathway.

Abstract

The present study aimed to explore the potential anti-tumor effect of ERβ overexpression and investigate its related mechanism in osteosarcoma. Cell cycle and apoptosis rates were measured by flow cytometry. Cell proliferation and formation of autophagosome were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and dansylcadaverine (MDC) staining assay. Cell migration and invasion were detected by wound healing assay and transwell assay. Western blot analysis was designed to detect the protein expressions of surviving, Bax, LC-3 П, Beclin-1, ERβ, TβRⅠ, TβRⅡ, Smad2, Smad3 and Smad7. Real-Time fluorogenic PCR was designed to examine the mRNA expressions of surviving, Bax, ERβ, TβRⅠ, TβRII, Smad2, Smad3 and Smad7. The results showed that ERβ overexpression inhibited cell proliferation, migration and invasion, blocked cell cycle, and induced apoptosis and autophagy. Additionally, ERβ overexpression significantly inhibited the expression of surviving, TβRⅠ, TβRⅡ, Smad2 and Smad3. Meanwhile, the expressions of Bax, LC-3 П, Beclin-1 and Smad7 were dramatically upregulated by ERβ overexpression. In conclusion, ERβ overexpression could inhibit cell proliferation, migration and invasion, block cell cycle, and promote apoptosis and autophagy in OS by downregulating TNG-β signaling pathway.

Introduction

Osteosarcoma (OS) is the most common malignant primary bone tumor affecting children, adolescents or young adults with an average age of eighteen years [1], with a high mortality rate and poor prognosis [2]. Although recent treatment strategies such as neoadjuvant chemotherapy combined with surgery is efficient for osteosarcoma, most chemotherapeutics carry the risk of severe side effects and treatment outcomes are still unsatisfied [3]. The development of osteosarcoma is a complex process which involving various factors. Therefore, in-depth exploration of the pathogenesis of osteosarcoma will definitely improve the treatment of this disease.

Several researches have confirmed that the transforming growth factor-β (TGF-β) as a tumor suppressor gene primarily, inhibited proliferation ability at the early stage of tumor through participating in cell cycle arrest, apoptosis or autophagy [4,5]. TGF-β family contains 33 members in humans, such as TGF-β1, TGF-β2 and TGF-β3 [6]. After activated, TGF-β moves to the nucleus through combination with two serine/threonine kinase receptor cell surface complexes, named type I (TβRI) and type II (TβRII). Ligand binding induces the formation of the heteromeric complex between specific type I and type II receptors, in which TβRII phosphorylates and activates TβRI [[7], [8], [9]].

The Smad proteins appear to be the primary mediators of the canonical TGF-β signaling pathway. TβRI phosphorylates and activates receptor-regulated Smads (R-Smads), including Smad2 and Smad3. Activated R-Smads then recruit the common-mediator Smad (co-Smad), Smad4. The R-Smad/Smad4 complex translocates into the nucleus to regulate gene expression. The inhibitory Smad (Smad7) regulates the canonical TGF-β signaling pathway by different means: (i) Smad7 binds TβRI and prevents R-Smad phosphorylation [10], (ii) Smad7 recruits E3-type ubiquitin ligases to activate TβRI ultimately leading to their degradation [4,11], and (iii) Smad7 recruits protein phosphatases to the R-Smad/Smad4 complex, leading to its dephosphorylation and inactivation [12].

Estrogen receptor β (ERβ), a hormone-regulated transcription factor, has been considered as a potential target for the diagnosis and treatment of OS [13]. Many researches have demonstrated that ERβ overexpression could suppress breast cancer through regulating TGF-β signaling pathway [14]. However, few studies on the anti-tumor effects by ERβ-mediated TGF-β signaling pathway in OS had been reported. Therefore, the present study was performed to assess the anti-tumor effects and mechanisms of ERβ-mediated TGF-β signaling pathway in OS.