ONCOLOGY REPORTS 45: 129-138, 2021 SOX12 contributes to the activation of the JAK2/STAT3 pathway and malignant transformation of esophageal squamous cell carcinoma CHUNGUANG LI1*, MAOLING ZHU2*, JI ZHU1, QIJUE LU1, BOWEN SHI1, BIN SUN3 and HEZHONG CHEN1 1Department of Thoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200438; 2Department of Gastroenterology, Yangpu Hospital, Tongji University School of Medicine, Shanghai 200090; 3Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, P.R. China Received May 15, 2020; Accepted October 6, 2020 DOI: 10.3892/or.2020.7863 Abstract. As a crucial transcription factor, sex-determining cells in vitro. Recombinant protein of SOX12 could restore the region Y box 12 (SOX12) is closely related with tumorigenesis aggressive phenotype of ESCC cells. Furthermore, knockdown and malignant transformation in various malignant tumor of the expression of SOX12 inhibited the activation of the Janus types. To date, the specific function of SOX12 in esophageal kinase 2 (JAK2)/signal transducer and activator of transcrip- squamous cell carcinoma (ESCC) has remained largely tion 3 (STAT3) signaling pathway by decreasing the expression elusive and requires further investigation. The present study of the JAK2/STAT3 signaling pathway. Recombinant protein aimed to determine whether aberrant expression of SOX12 is of SOX12 could recover the activation of the JAK2/STAT3 associated with malignant development of ESCC. The expres- signaling pathway. Analysis of the clinical data revealed that sion level of SOX12 in ESCC cells and tissues was analyzed overexpression of SOX12 indicated shorter overall survival by RT-qPCR and western blotting. Short hairpin RNA time (OS; P=0.0341) and disease-free survival time (DFS; (shRNA) targeting SOX12 was transfected into ESCC cells to P=0.04). Univariate and multivariate analysis revealed that knock down the expression of SOX12. Colony formation and overexpression of SOX12 was an independent factor for ESCC. Transwell assays were used to detect viability and mobility of In conclusion, SOX12 was revealed to serve a crucial function ESCC cells. Signaling pathway-related proteins were assessed in sustaining the viability, as well as enhancing the motility of using western blot analysis and cellular immunofluorescence. ESCC cells via activating the JAK2/STAT3 signaling pathway. Clinical prognosis data was analyzed by Kaplan-Meier and Cox Thus, SOX12 may potentially serve as a novel biomarker and logistic regression. The present results revealed that SOX12 candidate for the targeted treatment of ESCC. was overexpressed in ESCC cells and tissues. Knockdown of the expression of SOX12 by shRNA inhibited the colony Introduction forming efficiency, migration and invasion capacities of ESCC Esophageal cancer (EC) is a common human malignant tumor type with high incidence and mortality and the sixth leading cause of cancer-associated deaths worldwide (1-3). While esophageal adenocarcinoma (EAC) is the main histologic Correspondence to: Dr Bin Sun, Molecular Oncology Laboratory, subtype in Western countries, esophageal squamous cell Eastern Hepatobiliary Surgery Hospital, Second Military Medical carcinoma (ESCC) remains the predominant form in Asian University, 225 Changhai Road, Shanghai 200438, P.R. China E-mail: [email protected] countries, accounting for more than 90% of EC (4). The devel- opment of early diagnostic and adjuvant treatment methods has Dr Hezhong Chen, Department of Thoracic Surgery, Changhai provided a benefit for certain ESCC patients in recent decades. Hospital, Second Military Medical University, 225 Changhai Road, However, the poor prognosis of patients remains a major clinical Shanghai 200438, P.R. China problem (5). Recurrence and the ability to form metastasis are E-mail: [email protected] crucial characteristics of malignant compared with benign *Contributed equally tumors, and are accountable for the mortality of a considerable proportion of affected patients. Tumor metastasis is multistep Key words: sex-determining region Y box 12, transcription factor, and complex biological process, including the spread of tumor esophageal squamous cell carcinoma, short hairpin RNA, Janus cells from their primary location and disseminating through the kinase 2/signal transducer and activator of transcription 3 signaling bloodstream to eventually colonize in situ or in distant organs (6). pathway Identification of effective regulators in every key regulatory step of malignant progression is anticipated to provide improvement for the diagnosis and treatment of ESCC. 130 LI et al: SOX12 PROMOTES MALIGNANT TRANSFORMATION OF ESCC Sex-determining region Y-box 12 (SOX12) is well-known immunopositive cells was determined in 10 randomly selected as a member of a transcription factor superfamily belonging to high‑power fields and immunoreactive scoring was performed. the high-mobility group, and has been revealed to perform key A score of >4 was considered as high expression. functions in embryonic development and the determination of cell fate (7,8). Previous studies have also revealed that aberrant Cell lines culture and vectors. The human ESCC cell lines expression of SOX12 affects the biological behaviors of tumor Eca109, TE1 and the human normal esophageal epithelial cells and has a potential prognostic value in various type of cell line HEEC (used as control) were obtained from the Cell tumors, such as liver (9), breast (10), and lung cancer (11), as Bank of the Shanghai Institutes for Biological Sciences. The well as renal carcinoma (12) and acute myeloid leukemia (13). cells were maintained in DMEM medium supplemented with However, the potential roles and specific regulatory mecha- 10% FBS (HyClone; Cytiva) at 37˚C in a 5% CO2 incubator. nisms of SOX12 in ESCC remain unknown. The plasmids expressing short hairpin (sh)RNAs targeting The purpose of this study was to investigate the role of SOX12 or scrambled shRNA (shRNA1: 5'-CAT GGC GGA SOX12 in the biological behavior of ESCC cells and its TTA CCC GGA CTA; shRNA2: 5'-TCC GCA GTC TTA CGA predictive value for clinical prognosis GGA GTC; scrambled shRNA: 5'-GAG CTC TCT CTG CAC ATT CTT;) were purchased from Guangzhou RiboBio Co., Materials and methods Ltd. (product nos. stB0007967A-1-5, stB0007967B-1-5 and stB0007967C-1-5). Cell culture was performed in six Patients and collection of tissue samples. Esophageal squa- well plates transfected with a total of 4 µg shRNA using mous cell carcinoma tissue and paired adjacent non-cancerous Lipofectamine® 2000 transfection reagent (Invitrogen; tissues (n=56) were collected from patients who were Thermo Fisher Scientific, Inc.), used according to the manu- diagnosed with primary ESCC and received radical esophageal facturer's protocol, that was added in each well when the cell surgery without preoperative radiotherapy, chemotherapy or density reached 50‑60%, and culture was continued at 37˚C for biotherapy from August 2012 to September 2013 at Changhai 48-72 h according to the experimental requirements. hospital, Second Military Medical University (Shanghai, Recombinant human SOX12 protein (cat. China). The median age of patients at the time of admission no. H00006666-Q01; Abnova) and WP1066 (cat. no. HY‑­15312; was 58 years, range 43-72 years. In total, 15 patients were MCE), a potent inhibitor of the JAK2/STAT3 signaling women, and 41 were men. Total RNA from 56 pairs of tissue pathway were purchased and used according to the manu- samples were extracted for RT-qPCR detection. Total protein facturer's instructions. Recombinant human SOX12 protein from 6 pairs of tissue samples were extracted for western blot (10 µl) was co-cultured with shRNA1 in the colony formation, detection. All tissues (tumor tissue and paired para-cancerous Transwell and cellular immunofluorescence assays. WP1066 tissue) were embedded in paraffin immediately after surgery. (5 µl), an inhibitor of the JAK2/STAT3 signaling pathway, Each sample was confirmed by pathological diagnosis and was was added to the cell culture medium in the colony formation, followed up until March 2018. The use of esophageal cancer Transwell and western blotting assays. DMSO was used as a tissues and clinical data was approved by the Biomedical solvent control. Ethics Committee of the Second Military Medical University (Shanghai, China). Informed consent from each patient before Colony formation assay. After transfection with the indi- surgery was obtained. cated plasmids, Eca109 and TE1 cells were collected and re-suspended in 1.5 ml complete culture medium supplemented Immunohistochemistry (IHC). IHC was used to evaluate the with 0.45% low-melting point agarose (Invitrogen; Thermo expression level of SOX12 in paraffin-embedded tissues. Fisher Scientific, Inc.). The cells (1x103) were inoculated All specimens had been fixed in 10% buffered formalin and in 35-mm tissue culture dishes containing 1.5 ml culture embedded in paraffin. The embedded tissues were cut into medium and agarose (0.75%) on the bottom layer. The dishes 4‑µm‑thick serial sections and stored at 4˚C. After deparaf- were cultured for two weeks. Cell colonies were fixed with finization, rehydration and antigen retrieval, each slide was 4% formaldehyde solution for 15 min and stained with 0.005% blocked with 10% (w/v) normal goat serum (product no. SP crystal violet for 10-30 min at room temperature. The number KIT-B1; Fuzhou Maixin Biotech Co., Ltd.) at room