Inhibition of SOX2 induces cell apoptosis and G1/S arrest in Ewing’s sarcoma through the PI3K/Akt pathway

Chongmin Ren  ^{1   2} , Tingting Ren  ^{1   2} , Kang Yang  ^{1   2} , Shidong Wang  ^{1   2} , Xing Bao  ^{1   2} , Fan Zhang  ^{1   2} , Wei Guo  ^{3   4}

Affiliations

• ¹ Musculoskeletal Tumor Center, Peking University People’s Hospital, No. 11 Xizhimen South Street, Beijing, 100044, People’s Republic of China.
• ² Beijing Key Laboratory of Musculoskeletal Tumor, Beijing, 100044, People’s Republic of China.
• ³ Musculoskeletal Tumor Center, Peking University People’s Hospital, No. 11 Xizhimen South Street, Beijing, 100044, People’s Republic of China. bonetumor@163.com.
• ⁴ Beijing Key Laboratory of Musculoskeletal Tumor, Beijing, 100044, People’s Republic of China. bonetumor@163.com.

Abstract

Background: Ewing’s sarcoma is an aggressive bone and soft tissue tumor with a high incidence in children and adolescents. Due to its high malignancy and poor prognosis, identification of novel biomarkers for intervention therapies is necessary to improve outcome. The EWS/FLI1 fusion gene is a characteristic of Ewing’s sarcoma in most cases. Sex determining region Y-box 2 (SOX2) is a primary target of EWS/FLI1. It has been identified as an oncogene and linked to apoptotic resistance in several types of cancer. However, its role and regulatory mechanisms in Ewing’s sarcoma are largely unknown.

Methods: We systematically investigated the role of SOX2 in Ewing’s sarcoma cell lines, human tissue samples and xenograft models. The expression of SOX2 was detected in Ewing’s sarcoma samples by WB and IHC. siRNAs were used to knockdown EWS/FLI1 and SOX2 in A673 and RD-ES cell lines with the efficiencies tested by qRT-PCR and WB. The effect of SOX2 on cell cycle and apoptosis was determined by Flow cytometric and TUNEL assays. Akt overexpression was performed with plasmid. The protein expression of the corresponding factors was examined by WB analysis. Inhibition of SOX2 in vivo was performed by siRNA against SOX2 in xenograft models, and the protein expression of the regulators testified in vitro was examined in xenograft tumors by IHC and WB.

Results: The results confirmed that SOX2 was highly expressed in Ewing’s sarcoma and was the target of EWS/FLI1. SOX2 advanced Ewing’s sarcoma cell survival and proliferation by regulating p21, p27 and cyclin-E to facilitate G1/S phase transition and mediating caspase-3, PARP via both extrinsic (Fas and caspase-8) and intrinsic (caspase-9, Bad, Bcl-2 and XIAP) apoptotic pathways to restrain cell apoptosis. Additionally, SOX2 regulated the cell-cycle progression and apoptosis via activation of the PI3K/Akt signaling pathway. The mechanisms were proved both in vitro and in vivo.

Conclusions: The results demonstrate that SOX2 played a central role in promoting Ewing’s sarcoma cell proliferation in vitro and in vivo with the underlying mechanisms expounded. These findings suggest that SOX2 may serve as a potential biomarker for targeted intervention in Ewing’s sarcoma.

Keywords: Apoptotsis; Cell cycle; Ewing’s sarcoma; PI3K/Akt signaling; SOX2.