key: cord-0003607-enrs71hc authors: Li, Zhuo-Ran; Jiang, Yong; Hu, Jian-Zhong; Chen, Yang; Liu, Quan-Zhong title: SOX2 knockdown inhibits the migration and invasion of basal cell carcinoma cells by targeting the SRPK1-mediated PI3K/AKT signaling pathway date: 2018-12-07 journal: Oncol Lett DOI: 10.3892/ol.2018.9810 sha: d6da8802866acb6cd35ee1c4a0ec6c64d06b8345 doc_id: 3607 cord_uid: enrs71hc Basal cell carcinoma (BCC) is the most common type of human skin cancer, which is driven by the aberrant activation of Hedgehog signaling. Previous evidence indicated that sex determining region Y-box 2 (SOX2) is associated with the tumor metastasis. However, the expression and role of SOX2 in BCC remain unknown. Therefore, the aim of the current study was to analyze the possible mechanism of SOX2 in the progression of BCC. The levels of SOX2 in BCC cells were detected by reverse transcription-quantitative polymerase chain reaction. Transwell assays were also used to determine the migration and invasion of BCC cells. Immunoblotting and immunofluorescence were used for analyzing the role of SOX2 knockdown in the serine-arginine protein kinase 1 (SRPK1)-mediated phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway in BCC cells. The results demonstrated that SOX2 is overexpressed in BCC tissues and cells. In addition, SOX2 knockdown inhibited the migration and invasion of BCC cells, and the epithelial-mesenchymal transition (EMT) progress of BCC cells. It was also observed that SOX2 knockdown decreased SRPK1 expression, which further led to the downregulation of PI3K and AKT expression levels in BCC cells. Furthermore, SRPK1 transfection or PI3K/AKT pathway activation abolished the inhibitory effects of SOX2 knockdown on the migration, invasion and EMT progress of BCC cells. In conclusion, these results indicated that SOX2 may potentially serve as a target for BCC therapy by targeting the SRPK1-mediated PI3K/AKT signaling pathway. Basal cell carcinoma (BCC) is one of the most common types of human skin cancer, which is characterized by mutations in the patched and/or smoothened genes, and aberrant expression of the Hedgehog signaling pathway (1) . Epidemiological studies have reported that BCC is associated with exposure to radiation in various populations, including atomic bomb survivors, radiologists and interventional cardiologists (2, 3) . Currently, exposure to UV rays is the most common cause of BCC onset in the general population, and numerous possible mechanisms have been proposed for the pathogenesis of BC, such as DNA damage and certain cell signaling pathways (4) (5) (6) . However, various regulatory factors involved in the chronological progression of BCC lesions also require further investigation. Sex determining region Y (SRY)-box 2 (SOX2) is a member of the SRY-related high mobility group box family that has been widely reported to be involved in human carcinogenesis and several malignancies (7, 8) . More recently, it has been observed that SOX2 presented completely distinct effects for different tumor types (9) (10) (11) . Previous studies have indicated that SOX2 promoted the development of certain types of cancer, including osteosarcomas (12) , colorectal cancer (13) , glioblastomas (14) , prostate cancer (15) , breast cancer (16) and ovarian cancer (17) , while it exhibited inhibitory roles in gastric cancer (18) and squamous cell lung cancer (19) . These findings suggested that SOX2 may be regarded as a tumor-associated gene in human cancer. In addition, multivariate analysis further demonstrated that the SOX2 expression may be a prognostic factor in cancer patients (20, 21) . However, the regulatory roles and potential mechanisms of SOX2 remain elusive in BCC. A previous study has indicated that epithelial-mesenchymal transition (EMT) signaling is implicated in the invasion and metastasis of various types of human cancer (22, 23) . In addition, it has been reported that inhibition of serine-arginine protein kinase 1 (SRPK1) suppressed prostate cancer progression by modulation of VEGF alternative splicing (24) . Furthermore, phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway participates in the signal transduction in tumor cells (25, 26) . Therefore, the present study investigated the role of SOX2 in the invasion and metastasis of BCC cells, and hypothesized that the regulatory effect of SOX2 was exerted through the SRPK1-mediated PI3K/AKT signaling pathway. Clinical tissues. A total of 12 (male/female, 6/6) BCC tissues and matched adjacent non-tumor tissues were collected at the Department of Dermatology, The Second Hospital of Tianjin Medical University (Tianjin, China) between May 2008 and June 2016. The pathological diagnosis of the BCC patients (mean age, 48.6 years; range, 42.2-54.5) was performed according to the World Health Organization criteria (27) . Patients with a history of cancer history were excluded from the present study. The BCC tissues were stored at -80˚C and then embedded in paraffin. None of the patients received chemotherapy, radiotherapy or other treatments prior to tumor resection. All patients were required to provide written informed consent. This study also approved by Ethics committee of Tianjin Medical University General Hospital. Cell culture. BCC and normal cells were obtained from the 12 BCC tissues and matched adjacent non-tumor tissues, respectively, as described previously (28) . Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA), 100 U/ml penicillin, 100 µg/ml streptomycin, sodium pyruvate and L-glutamine at 37˚C in an atmosphere with 5% CO 2 . Immunohistochemical (IHC) analysis. The BCC and normal tissue sections (4 µm) obtained from patients were deparaffinized in xylene and rehydrated through graded ethanol. Following blocking of endogenous peroxidase activity in 3% hydrogen peroxide for 10 min at room temperature, the samples were analyzed for SOX2 expression using immunostaining, as previously described (29) . Briefly, tumor sections were incubated with specific primary antibodies for anti-SOX2 (1:1,000; ab92494; Abcam, Cambridge, MA, USA) for 12 h at 4˚C. Tumor tissues were then incubated with a monoclonal horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (PV-6001; OriGene Technologies, Inc., Beijing, China). A Ventana Benchmark automated staining system was used for the examination of protein expression in the tumor tissues (Tucson, AZ, USA). The staining results were semi-quantitatively evaluated based on the percentage of positive staining cells (magnification, x400). RNA extraction and quantitative polymerase chain reaction (qPCR). Total cellular RNA was extracted from the BCC and normal cells using the RNeasy Mini kit (Qiagen Sciences, Inc., Gaithersburg, MD, USA) according to the manufacturer's protocol, and then 1 µg total RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (product code, 4368814; Applied Biosystems). Next, for mRNA expression determination, 1 µg cDNA was subjected to qPCR using an iQ SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All the primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and were as follows: SOX-2 forward, 5'-CAG GAG TTG TCA AGG CAG AGA-3', and reverse, 5'-CAG GAG TTG TCA AGG CAG AGA-3'; β-actin forward, 5'-CGG AGT CAA CGG ATT TGG TC-3' , and reverse, 5'-AGC CTT CTC CAT GGT CGT GA-3'. The PCR thermocycling conditions included 45 amplification cycles, of denaturation at 95˚C for 15 sec, primer annealing at 66˚C for 20 sec and primer extension at 72˚C for 15 sec. Relative levels of mRNA expression were calculated using the 2 -ΔΔCq method (30) . The results were expressed as a fold change relative to the normal controls by comparing the levels of target mRNA expression to that of the β-actin control group. Western blotting. BCC cells were homogenized in lysis buffer containing protease inhibitor (Sigma-Aldrich; Merck KGaA) and centrifuged at 8,000 x g at 4˚C for 10 min. SDS assays were performed as described previously (31) . Subsequent to blocking in 5% skimmed milk for 1 h at 37˚C, the following primary antibodies at a dilution of 1:1,000 were added to membranes: PI3K (ab86714), AKT (ab8805), SOX2 (ab92494), SRPK1 (ab90527), E-cadherin (ab40772), Vimentin (ab92547), Fibronectin (ab2413) and β-actin (ab5694; all purchased from Abcam). Membranes were then incubated with monoclonal HRP-conjugated goat anti-rabbit IgG secondary antibodies (PV-6001; OriGene Technologies, Inc.) for 24 h at 4˚C. A Ventana Benchmark automated staining system (Tucson, AZ, USA) was used for analyzing the protein expression. Small interfering RNA (siRNA) transfection. BCC cells (4x10 5 cells/well) were seeded into 6-well and incubated for 24 h at 37˚C. Next, the medium was removed and Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc.) was added for 24 h at 37˚C. The siRNA sequences corresponding to each gene were designed and synthesized by GenePharma Co., Ltd. (Shanghai, China), and were as follows: siRNA-SOX2, 5'-CUG CAG UAC AAC UCC AUG ATT-3'; siRNA-SRPK1, 5'-CCA UTG GUU CGG UGG UCA ATT-3'; siRNA-vector (control), 5'-CUC GUC UCA UUG ATG ACA GTT-3'. A total of 100 pmol siRNA was transfected into cultured BCC cells, respectively, using 5 µl Lipofectamine RNAiMax reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 48 h at 37˚C, cells were used for further analysis. Lentivirus production and cell transduction. The packaging plasmid psPAX2 and the envelope plasmid pMD2.G were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). pWPXL-SOX2 (pSOX2), pWPXL-SRPK1 (pSRPK1) or pWPXL-vector (pvector) vector was transfected with psPAX2 and pMD2.G into BCC cells or siRNA-SOX2-transfected BCC cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 48 h at 37˚C, the treated cells were used to investigate the effect of gene depletion using proliferation, migration, invasion and western blot assays. Cell proliferation assays. BCC cells transfected with pSOX2, pvector, siRNA-vector or siRNA-SOX2 vector were seeded into 6-well plates (2x10 3 cells/well) and cultured at 37˚C for 14 days. Following incubation, the medium was removed, and the cells were fixed with 100% methanol and stained with 0.1% (w/v) crystal violet (Sigma-Aldrich; Merck KGaA). Cell colonies were counted using Image-Pro Plus version 5.0 software (Media Cybernetics, Inc., Bethesda, MD, USA). Cell migration and invasion analysis. BCC cells were transfected with pSOX2, pSRPK1, siRNA-SOX2 or siRNA-SRPK1, and BCC or transfected BCC cells were treated with MK2206 (2 mg, Sigma-Aldrich; Merck KGaA) for 12 h at 37˚C. For the migration and invasion assay, BCC cells with 150 µl serum-free DMEM were placed into the upper chamber of 6-well plates at a density of 1x10 4 cells/well, and DMEM in the lower chamber supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA). Transwell inserts (pore size, 8 µm; EMD Millipore, Billerica, MA, USA) that were uncoated or coated with Matrigel were used to evaluate the cell migration and invasion, respectively. After 24 h, BCC cells in the lower chamber were fixed in 4% paraformaldehyde for 15 min at 37˚C and stained with 0.1% crystal violet dye (Sigma-Aldrich; Merck KGaA) for 20 min at 37˚C. Subsequently, the cells were counted in 3 randomly selected fields of view using a light microscope (Olympus BX51; Olympus Corporation). Immunofluorescence assay. Immunofluorescence was performed as previously described (32) . Brief ly, the siRNA-SOX2-transfected BCC cells were cultured in 6-well plates for 24 h at 37˚C. The BCC cells were then washed with phosphate-buffered saline and fixed with 4% paraformaldehyde (Sigma-Aldrich) at 4˚C for 10 min. The cells were again rinsed with phosphate-buffered saline and permeabilized with 1% Triton X-100 at 4˚C for 10 min. Subsequently, the cells were incubated for 2 h at 37˚C with the indicated antibodies as follows: E-cadherin (1:1,000; ab40772), Vimentin (1:1,000; ab92547), Fibronectin (1:1,000; ab2413; all purchased from Abcam). Cells were then treated with fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG polyclonal antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 2 h at 37˚C. Finally, the cells were examined using a microscope (Leica DMI4000 B; Leica Microsystems GmbH, Wetzlar, Germany). Statistical analysis. Data are expressed as the mean ± standard deviation of at least three independent replicates. All data were analyzed by SPSS software, version 19.0 (IBM Corp., Armonk, NY, USA) and Graphpad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) using one-way analysis of variance, followed by Tukey's multiple comparison post hoc tests. P<0.05 and P<0.01 values were considered to indicate differences that were statistically significant. Expression of SOX2 in BCC tissues and cell lines. BCC tissue samples (12 patients) and BCC cell lines were used to analyze the expression of SOX2. It was demonstrated that SOX2 was overexpressed in BCC tissue samples when compared with normal basal tissue, as determined by IHC staining (Fig. 1A) . As shown in Fig. 1B , it was demonstrated that SOX2 expression was upregulated in BCC tissue compared with normal tissue. SOX2 gene and protein expression levels were also upregulated in BCC cell lines as compared with the normal basal cell line ( Fig. 1C and D) . These results suggested that SOX2 expression levels were upregulated in BCC tissues and cells. SOX2 knockdown inhibits BCC cell migration and invasion in vitro. The role of SOX2 in BCC cells was next analyzed. It was observed that SOX2 knockdown by siRNA-SOX2 transfection markedly downregulated the expression of SOX2, while SOX2 overexpression by pSOX2 transfection markedly upregulated SOX2 expression in BCC cells, indicating that the transfections were successfully performed ( Fig. 2A and B) . The results then demonstrated that SOX2 overexpression promoted the cell migration and invasion ( Fig. 2C and D) , while SOX2 knockdown inhibited the migration and invasion of BCC cells (Fig. 2E and F) . These results suggest that SOX2 expression is associated with BCC cell migration and invasion. A previous study has suggested that the EMT process is a critical regulator in the progression of cancer metastasis (33) . Therefore, the effects of SOX2 on EMT processes in BCC cells were further analyzed. The results revealed that overexpression of SOX2 promoted BCC cell proliferation, whereas knockdown of SOX2 inhibited BCC cell proliferation ( Fig. 3A and B ). In addition, SOX2 knockdown upregulated the expression of the epithelial marker E-cadherin, and inhibited the levels of the mesenchymal markers Vimentin and Fibronectin in BCC cells. By contrast, SOX2 overexpression caused the reverse effects on the EMT process markers in BCC cells (Fig. 3C and D) . The immunofluorescence assay also confirmed the effects of SOX2 knockdown and overexpression on the epithelial and mesenchymal marker expression levels in BCC cells (Fig. 3E and F) . These results indicate that SOX2 expression regulates the EMT processes and proliferation of BCC cells. A previous study has indicated that SRPK1 is associated with human cancer metastasis (34) . To elucidate the potential molecular mechanisms mediated by SOX2, SRPK1 expression was analyzed in BCC cells. The results demonstrated that SOX2 transfection promoted SRPK1 expression, while SOX2 knockdown inhibited SRPK1 expression in BCC cells ( Fig. 4A and B ). In addition, SRPK1 knockdown was observed to result in a marked reduction of cell migration and invasion ( Fig. 4C and D) . However, SRPK1 overexpression prominently canceled the SOX2 knockdown-inhibited migration and invasion of BCC cells compared to control ( Fig. 4E and F) . Furthermore, SRPK1 knockdown downregulated the EMT processes of BCC cells as observed by the increased E-cadherin and decreased the levels of Vimentin and Fibronectin (Fig. 4G) . By contrast, SRPK1 overexpression canceled the SOX2 knockdown-inhibited EMT processes of BCC cells (Fig. 4H) . These data suggest that SRPK1 is a direct target of SOX2-induced EMT processes in BCC cells. A previous study has indicated that SRPK1 promotes the activation of PI3K/AKT signaling in the metastasis of human cancer (35) . In the present study, the association between SOX2 and the PI3K/AKT signaling pathway was explored in BCC cells. As shown in Fig. 5A and B, SOX2 knockdown markedly decreased the expression levels of PI3K and AKT in BCC cells, while SOX2 overexpression significantly increased these levels. It was also observed that SRPK1 knockdown markedly decreased the expression levels of PI3K and AKT in BCC cells, whereas SRPK1 overexpression increased these levels ( Fig. 5C and D) . However, activation of the PI3K/AKT pathway by MK2206 abrogated the inhibitory effects of SOX2 knockdown on BCC cell migration and invasion ( Fig. 5E and F) . Thus, these results suggest that SOX2 knockdown inhibits BCC cell invasion through the PI3K/AKT signaling pathway. Several studies have indicated that SOX2 expression is markedly increased in human cancer tissues compared with that in normal tissues, including in ovarian and prostate cancer (36, 37) . In the present study, SOX2 expression in BCC tissues and cell lines was determined, and the results indicated that SOX2 is overexpressed in BCC tissues and cell lines. More recently, SOX2 expression was reported to be associated with lymph node metastases and distant invasion in right-sided colon cancer, suggesting that SOX2 also regulates tumorigenesis (38) . Findings in the current study indicated that SOX2 regulated BCC cell migration and invasion through targeting the SRPK1-mediated EMT and the PI3K/AKT signaling pathway. Although the association of SOX2 expression with the progression of several human cancer cells has been reported (39) (40) (41) , the functional roles and potential mechanisms in BCC have not been examined previously. Currently, the role of SOX2 remains controversial in different cancer types (42) . In the present study, it was observed that SOX2 is upregulated in BCC. The differences between SOX gene and protein expression may have resulted from the translational efficiency of SOX in BCC tissue and cells. Yang et al (43) reported that SOX2 promoted the migration and invasion of laryngeal cancer cells by induction of matrix metalloproteinase-2 via the PI3K/AKT/mechanistic target of rapamycin pathway. However, Yoon et al (44) indicated that overexpression of SOX2 is associated with better overall survival in squamous cell lung cancer patients treated with adjuvant radiotherapy. The present study reported that SOX2 knockdown inhibited BCC cell migration and invasion by downregulation of the SRPK1-induced EMT signaling pathway. SRPK1 is a highly conserved protein that is dysregulated in different types of cancer. SRPK1 inhibition has been regarded as a potential targeted therapeutic strategy for prostate cancer therapy (45) . A previous study has indicated that SRPK1 serves a critical role in the EMT process of human glioblastoma (46) . In the current study, it was demonstrated that SRPK1 is a potential target of SOX2, and its expression was decreased by SOX2 knockdown in BCC cells. In addition, this SRPK1 downregulation mediated by SOX2 knockdown further regulated the EMT process in BCC cells, resulting in inhibition of migration and invasion. Furthermore, it also reported that SRPK1 downregulation inhibited the PI3K/AKT signaling pathway. Therefore, reduced expression of SOX2 may lead to suppression of BCC metastasis. A previous study has demonstrated that SRPK1 functions as an oncogene via promoting the activation of PI3K/AKT signaling (35) . Numerous reports have also suggested that the activation of PI3K/AKT signaling pathway is involved in the development and progression of human cancer, and regulates the invasion of cancer cells (47) (48) (49) . The current study results demonstrated that SOX2 knockdown downregulated the PI3K/AKT signaling pathway in BCC cells. Notably, PI3K/AKT serves a crucial role in the EMT process and in the proliferation and invasion of lung cancer cells (50) . SOX2 was also involved in paclitaxel resistance of the prostate cancer cell line PC-3 via the PI3K/AKT pathway (51) . The current study reported that activation of PI3K/AKT signaling pathway abrogated the effects of SOX2 knockdown on BCC cell migration and invasion. Therefore, the SRPK1/PI3K/AKT pathway may be involved in the role of SOX2 in the migration and invasion of BCC cells. In conclusion, the present study findings indicated that SOX2 served a pivotal role in BCC cell migration and invasion through targeting the SRPK1-mediated PI3K/AKT signaling pathway, suggesting that SOX2 may be a novel potential therapeutic target for BCC. Not applicable. Not funding was received. The analyzed data sets generated during the study are available from the corresponding author on reasonable request. ZL and YJ performed all experiments in the present study. JH and YC analyzed the data, and QL designed all experiments in the present study. The present study was approved by the Ethics Committee of Tianjin Medical University General Hospital. The study participants provided consent for the publication of this data. 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