Sex-determining Region Y-box 2 Expression Research Articles

The Sertoli cell marker FOXD1 regulates testis development and function in the chicken.

FOXD1, one of the transcription factors of the FOX family, has been shown to be important for mammalian reproduction but little is known about its function in avian species. In the present study, we identified the expression pattern and location of FOXD1 in chicken tissues and testis by performing quantitative polymerase chain reaction, immunohistochemistry and immunofluorescence, and further investigated the regulatory relationship of FOXD1 with genes involved in testis development by RNA interference. Our results showed that FOXD1 is confirmed to be significantly male-biased expressed in the brain, kidney and testis of adults as well as in embryonic gonads, and it is localised in the testicular Sertoli cell in chicken, consistent with its localisation in mammals. After knock-down of FOXD1 in chicken Sertoli cells, the expression of anti-Müllerian hormone (AMH), sex-determining region Y-box 9 (SOX9) and PKA regulatory subunits type I α (RIα) was significantly downregulated, expression of androgen receptor (AR) was notably increased whereas double-sex and MAB-3-related transcription factor 1 (DMRT1) showed no obvious change in expression. These results suggest that FOXD1 is an essential marker for Sertoli cells upstream of SOX9 expression and a potential regulator of embryonic testis differentiation and development and of normal testis function in the chicken.

MiR-145-5p Suppresses Breast Cancer Progression by Inhibiting SOX2

BackgroundIn this study, we aimed to investigate the expression and clinical significance of miR-145-5p and its tumor-suppressive effect in breast cancer patients. MethodsWe used luciferase reporter assay, real-time quantitative reverse transcription polymerase chain reaction and Western blot to identify sex-determining region Y-box2 (SOX2) as the target gene of miR-145-5p. Their expression levels in breast cancer tissues (n = 122) were detected by real-time quantitative polymerase chain reaction. We also applied 3-(4,5-dimethyl- 2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry to reveal the effect of miR-145-5p on proliferation in breast cancer. ResultsmiR-145-5p expression is downregulated in breast cancer tissues and negatively correlated with SOX2 expression. Decreased miR-145-5p expression was significantly associated with larger tumor size, distal metastasis, higher Ki67 expression level, and shorter overall survival. miR-145-5p inhibits breast cancer cell proliferation via targeting SOX2, and multivariate regression showed that both miR-145-5p and SOX2 were unfavorable prognostic factors. ConclusionsmiR-145-5p played a suppressive role in the proliferation of breast cancer cells by targeting SOX2, and miR-145-5p is a putative biomarker for risk assessment in patients with breast cancer.

Docetaxel promotes cell apoptosis and decreases SOX2 expression in CD133‑expressing hepatocellular carcinoma stem cells by suppressing the PI3K/AKT signaling pathway.

Docetaxel is a front‑line standard‑of‑care chemotherapeutic drug for the treatment of cancers. However, the underlying function and mechanism of docetaxel in human hepatocellular carcinoma (HCC) are uncertain. Therefore, the present study aimed to determine the effects of docetaxel on cell apoptosis and SOX2 expression in cultured human HCC stem cells. After human HCC stem cells were treated with docetaxel, cell proliferation was assessed by methyl thiazolyl tetrazolium (MTT) method, the cell apoptotic rate was evaluated by flow cytometry, the expression of CD133 and sex determining region Y‑box 2 (SOX2) was determined by RT‑PCR and immunohistochemistry, and the protein levels of CD133, SOX2, phosphoinositide 3‑kinases (PI3K), AKT and phosphorylated AKT (p‑AKT) were analyzed by western blotting. The results indicated that SOX2 and CD133 were highly expressed in patients with HCC while their expression was significantly decreased after patients with HCC were treated with docetaxel. In vitro, docetaxel inhibited the proliferation while it enhanced the apoptosis of human CD133‑expressing HCC stem cells. Furthermore, lower expression of p‑AKT and SOX2 were revealed in the presence of docetaxel. Notably, docetaxel‑inhibited SOX2 expression and growth of human CD133‑expressing HCC stem cells were partially restricted following the block of the PI3K/AKT signaling pathway using the inhibitor LY294002. The present study collectively indicated that docetaxel promoted apoptosis and upregulated SOX2 expression of human HCC stem cells through the suppression of the PI3K/AKT signaling pathway.

Basic Calcium Phosphate Crystals Induce Osteoarthritis-Associated Changes in Phenotype Markers in Primary Human Chondrocytes by a Calcium/Calmodulin Kinase 2-Dependent Mechanism.

Chondrocytes in osteoarthritis undergo a phenotype shift leading to increased production of cartilage-degrading enzymes. There are similarities between the phenotype of osteoarthritic chondrocytes and those of growth plate chondrocytes. Hydroxyapatite can promote chondrocyte differentiation in the growth plate. Basic calcium phosphate (BCP) crystals (which consist of hydroxyapatite, octacalcium apatite and tricalcium phosphate) are frequently found in osteoarthritic joints. The objective of this study was to determine whether BCP crystals induce disease-associated changes in phenotypic marker expression in chondrocytes. Primary human chondrocytes isolated from macroscopically normal cartilage were treated with BCP for up to 48h. Expression of indian hedgehog (IHH), matrix metalloproteinase 13 (MMP13), interleukin-6 (IL-6) and type X collagen (COLX) were higher, and expression of sry-box 9 (SOX9) lower, in BCP-treated chondrocytes (50µg/mL) compared to untreated controls. COLX protein was also present in BCP-treated chondrocytes. Intracellular calcium and levels of phosphorylated and total calcium/calmodulin kinase 2 (CaMK2) were elevated following BCP treatment due to BCP-induced release of calcium from intracellular stores. CaMK2 inhibition or knockdown ameliorated the BCP-induced changes in SOX9, IHH, COLX, IL-6 and MMP13 expression. BCP crystals induce osteoarthritis-associated changes in phenotypic marker expression in chondrocytes by calcium-mediated activation of CaMK2. The presence of BCP crystals in osteoarthritic joints may contribute to disease progression.

SOX2 regulates radioresistance in cervical cancer via the hedgehog signaling pathway

ObjectiveResistance to radiotherapy accounts for most treatment failures in cervical cancer patients who receive radical radiation therapy. To discover the possible mechanism of radioresistance and improve the 5-year survival rate, we focused on how sex-determining region Y-box 2 (SOX2) mediates radioresistance in cervical cancer as well as on the interaction between SOX2 and the hedgehog (Hh) signaling pathway in this study. MethodsWe established the acquired radioresistant subclone cells Hela-RR and Siha-RR. RT-qPCR, Western blot analysis, IHC, clonogenic survival assay, CCK-8 assay, apoptosis analysis, cell cycle analysis and xenograft models were used to explore the relationship between SOX2 expression and radiation resistance and to determine how SOX2 mediates radioresistance in cervical cancer. Furthermore, luciferase reporter and ChIP-PCR assays were utilized to assess the interaction between SOX2 and the Hh signaling pathway. ResultsOur research suggested that high expression of SOX2 was responsible for radioresistance in cervical cancer. SOX2 was observed to be closely related to irradiation-induced survival, proliferation, apoptosis, and cell cycle changes. The Hh signaling pathway was found to be activated in Hela-RR and Siha-RR, and the activation changed with SOX2 expression. IHC staining of SOX2 and Gli1 showed a close relationship between SOX2 and the Hh pathway. Luciferase reporter and ChIP-PCR assays demonstrated that SOX2 interacted with the Hh signaling pathway by occupying the HHAT promoter. ConclusionsSOX2 is a potential therapeutic target of irradiation resistance in cervical cancer. It mediates radioresistance in cervical cancer via the Hh signaling pathway.

Non-Canonical Regulation of Type I Collagen through Promoter Binding of SOX2 and Its Contribution to Ameliorating Pulmonary Fibrosis by Butylidenephthalide.

Pulmonary fibrosis is a fatal respiratory disease that gradually leads to dyspnea, mainly accompanied by excessive collagen production in the fibroblast and myofibroblast through mechanisms such as abnormal alveolar epithelial cells remodeling and stimulation of the extracellular matrix (ECM). Our results show that a small molecule, butylidenephthalide (BP), reduces type I collagen (COL1) expression in Transforming Growth Factor beta (TGF-β)-induced lung fibroblast without altering downstream pathways of TGF-β, such as Smad phosphorylation. Treatment of BP also reduces the expression of transcription factor Sex Determining Region Y-box 2 (SOX2), and the ectopic expression of SOX2 overcomes the inhibitory actions of BP on COL1 expression. We also found that serial deletion of the SOX2 binding site on 3′COL1 promoter results in a marked reduction in luciferase activity. Moreover, chromatin immunoprecipitation, which was found on the SOX2 binding site of the COL1 promoter, decreases in BP-treated cells. In an in vivo study using a bleomycin-induced pulmonary fibrosis C57BL/6 mice model, mice treated with BP displayed reduced lung fibrosis and collagen deposition, recovering in their pulmonary ventilation function. The reduction of SOX2 expression in BP-treated lung tissues is consistent with our findings in the fibroblast. This is the first report that reveals a non-canonical regulation of COL1 promoter via SOX2 binding, and contributes to the amelioration of pulmonary fibrosis by BP treatment.

Knockdown of miR-429 Attenuates Aβ-Induced Neuronal Damage by Targeting SOX2 and BCL2 in Mouse Cortical Neurons.

Accumulation of amyloid-β peptide (Aβ) and massive neuronal death due to apoptosis were the essential steps in the pathogenesis of Alzheimer's disease (AD). MiR-429 was reported to play an important role in the pathogenesis of AD. However, the detailed function and underlying molecular mechanism of miR-429 in the pathogenesis of AD remain elusive. Cortical neurons were stimulated with 20µM of Aβ25-35 for 24h to construct AD model in vitro. qRT-PCR assay was used to detect the expression of miR-429, and qRT-PCR or western blot analysis were performed to assess the levels of Sex-determining region Y-box 2 (SOX2) and B cell lymphoma-2 protein (BCL2) at mRNA or proteins levels in the AD mouse model and Aβ-induced treated cortical neurons. Luciferase reporter assay and western blot analysis were used to confirm the potential targets of miR-429. CCK-8 assay, flow cytometry analysis, and caspase3 activity assay were used to measure cell viability, cell apoptosis capacity and caspase3 activity, respectively. MiR-429 was upregulated and SOX2 and BCL2 were downregulated in the AD mouse model and Aβ-induced mouse cortical neurons. MiR-429 knockdown attenuated Aβ-induced cytotoxicity in mouse cortical neurons. SOX2 and BCL2 were direct targets of miR-429. Moreover, anti-miR-429-mediated neuroprotective effect was abated by the restoration of SOX2 or BCL2 expression. Knockdown of miR-429 might attenuate Aβ-induced cytotoxicity by targeting SOX2 and BCL2 in mouse cortical neurons, providing a novel prospect in AD therapy.

Downregulation of miR‑200c‑3p contributes to the resistance of breast cancer cells to paclitaxel by targeting SOX2.

Acquisition of resistance to paclitaxel is a major obstacle to successful treatment of breast cancer patients, but the molecular mechanisms underlying the development of drug resistance remain largely unclear. The aim of the present study was to investigate the role and mechanism of action of miR‑200c‑3p in the resistance of breast cancer to paclitaxel. It was observed that miR‑200c‑3p expression, as determined by reverse transcription‑quantitative polymerase chain reaction analysis, was significantly downregulated in paclitaxel‑resistant MCF‑7/Tax cells compared with parental MCF‑7 cells. Overexpression of miR‑200c‑3p increased the chemosensitivity to paclitaxel and enhanced apoptosis in MCF‑7/Tax cells, whereas the downregulation of miR‑200c‑3p exerted the opposite effect. In addition, upregulation of miR‑200c‑3p in MCF‑7/Tax cells suppressed the expression of sex‑determining region Y‑box 2 (SOX2) at the mRNA and protein levels. Dual‑luciferase reporter assay demonstrated that SOX2 is a target of miR‑200c‑3p in MCF‑7/Tax cells. Moreover, knockdown of SOX2 expression increased chemosensitivity to paclitaxel and upregulated miR‑200c‑3p expression in MCF‑7/Tax cells. Taken together, the results of the present study indicated that miR‑200c‑3p plays a key role in the development of paclitaxel resistance in breast cancer, possibly partially through regulating SOX2 expression, suggesting that the miR‑200c‑3p‑SOX2 loop may serve as a potential target for the reversal of paclitaxel resistance in breast cancer.

SFMBT2 positively regulates SOX9 and chondrocyte proliferation.

SRY‑box9(SOX9) is the master regulator of the chondrocyte phenotype, which is essential for differentiating chondrogenic mesenchymal condensations into chondrocytes, and is involved in regulating every stage of chondrocyte differentiation. SOX9 deletion in chondrocytes at the late stages of cartilage development results in decreased chondrocyte proliferation; inhibited expression of cartilage matrix genes, including Indian hedgehog and the downstream parathyroid hormone‑related protein; and premature conversion of proliferating chondrocytes into hypertrophic chondrocytes, which mineralize their matrix prematurely. Therefore, SOX9 is considered vital for the majority of phases of chondrocyte lineage, from early condensations to the differentiation of proliferating chondrocytes, leading to chondrocyte hypertrophy. It has been reported that SOX9 expression is decreased in osteoarthritis(OA) cartilage. Regeneration or repair of cartilage degradation in OA remains a challenge. Previous studies have indicated that overexpression of SOX9 can promote cartilage repair and can be used as a potential therapeutic agentat the early stages of human OA. The present study identified Scm‑like with four malignant brain tumor domains2(SFMBT2) as a novel regulator of SOX9 expression in human chondrocytes. Our previous study revealed that SFMBT2 is negatively regulated in OA cartilage, and decreased levels of SFMBT2 contribute to the catabolic phenotype of chondrocytes. The present study detected increased expression levels of SFMBT2 in early cartilage development and during the early phases of chondrogenesis. Overexpression of SFMBT2 in C28/I2 cells upregulated SOX9 expression in a dose‑dependent manner. Furthermore, SFMBT2 positively regulated C28/I2 cell proliferation and restored the decreased levels of SOX9 in chondrocytes following tumor necrosis factor‑α treatment. Additional studies may reveal novel insights into the molecular mechanism involved and the potential role of SFMBT2 in cartilage repair and OA management.

Incomplete radiofrequency ablation promotes the development of CD133+ cancer stem cells in hepatocellular carcinoma cell line HepG2 via inducing SOX9 expression

BackgroundCancer stem cells (CSCs) accelerate the growth of hepatocellular carcinoma (HCC) residual after incomplete radiofrequency ablation (In-RFA). The present study aimed to detect the effects of In-RFA on stemness transcription factors (STFs) expression which are important for the production and function of CSCs, and to find which STFs promote HCC stemness after In-RFA. MethodsHepG2 cells were used for in vitro and in vivo studies. Flow cytometry and sphere-formation assays were used to detect the level and function of CD133+CSCs in the models. PCR array and ELISA were applied to analyze the altered expression of 84 STFs in CD133+CSCs in two models. Specific lentiviral shRNA was used to knockdown STFs expression, followed by detecting In-RFA's effects on the levels and function of CD133+CSCs. ResultsIn-RFA was identified to induce CD133+CSCs and increase their tumorigenesis ability in vitro and in vivo. The mRNA levels of 84 STFs in CD133+CSCs were detected by PCR array, showing that 15 and 22 STFs were up-regulated in two models, respectively. Meanwhile, the mRNA levels of seven common STFs were up-regulated in both models. ELISA assay demonstrated that only the protein of sex determining region Y-box 9 (SOX9) was up-regulated in both models, the protein levels of the other 6 common STFs did not increase in both models. Finally, SOX9 was identified to play an important role in inducing, maintaining stemness and promoting tumorigenesis ability of CD133+CSCs in both models. ConclusionIn-RFA-induced SOX9 stimulates CD133+CSCs proliferation and increases their tumorigenesis ability, suggesting that SOX9 may be a good target for HCC treatment.

Low P16INK4A Expression Associated with High Expression of Cancer Stem Cell Markers Predicts Poor Prognosis in Cervical Cancer after Radiotherapy.

Previous studies have suggested that cancer stem cells (CSCs) resisted radiotherapy and chemotherapy. P16INK4A is a biomarker for cervical carcinogenesis and reduces proliferation of stem cells. We aimed to investigate the expression and clinical significance of cyclin-dependent kinase inhibitor 2A (P16INK4A), sex determining region Y-box 2 (SOX2), and Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) in cervical cancer treated with radiotherapy and cervical cell line models. The expressions of P16INK4A, SOX2, and ALDH1A1 were performed by immunohistochemical staining of tumor samples from 139 cervical cancer patients with International Federation of Gynecology and Obstetrics stages Ib to IV. The staining showed high expression in 100, 107, and 13 patients with P16INK4A (>80%), SOX2 (≥10%), and ALDH1A1 (50%), respectively. The high-P16INK4A group had a higher five-year overall survival (OS) rate and disease-free survival (DFS) than the low-P16INK4A group (OS: 62.0% and 35.2%, p = 0.016; DFS: 60.0% and 31.2%, p = 0.002). The low-P16INK4A/high-SOX2 and low-P16INK4A/high-ALDH1A1 groups had a worse five-year OS and DFS rate than the high-P16INK4A/low-SOX2 and high-P16INK4A/low-ALDH1A1 groups, respectively. Depletion of P16INK4A promoted chemoresistance and radioresistance of cervical cancer cells increased the expression of SOX2 and ALDH1A1 and exhibited higher self-renewal ability. These results suggest that lower P16INK4A expression associated with higher CSC markers predicts poor prognostic outcomes and is a promising target in patients with cervical cancer.

Combinatorial CRISPR/Cas9 Approach to Elucidate a Far-Upstream Enhancer Complex for Tissue-Specific Sox9 Expression

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation.

Cytoplasmic expression of SOX9 as a poor prognostic factor for oral squamous cell carcinoma.

Transcription factor SRY-box 9 (SOX9) is a key regulator of chondrocyte differentiation and sex determination, and it is also involved in the progression of various types of human cancer. However, its putative association with oral squamous cell carcinoma (OSCC) remains elusive. The aim of the present study was to investigate the expression profiles of SOX9 in various oral epithelial lesions, including OSCC. We performed immunohistochemical analysis of SOX9 expression in surgical specimens of OSCC, which simultaneously exhibited different grades of epithelial lesions, and analyzed the correlation between SOX9 expression and several clinicopathological factors. Moreover, we performed immunofluorescent staining, western blot analysis and real-time reverse transcription-polymerase chain reaction to assess SOX9 expression in OSCC HSC-3 (a metastatic cell line) and HSC-4 (a non-metastatic cell line) cell lines. In surgical specimens, SOX9 expression was detected in the nuclei of proliferating cells in areas with epithelial dysplasia and carcinoma in situ, but not in areas with normal epithelia. Nuclear SOX9 expression was observed in most SCC cells. Notably, cytoplasmic SOX9 expression was confirmed only in some SCC cells; however, cytoplasmic SOX9 expression was significantly and positively correlated with poor clinical outcomes. Both protein and mRNA expression of SOX9 were significantly higher in the HSC-3 cell line than that in the HSC-4 line. Notably, however, only HSC-3 cells exhibited cytoplasmic localization of SOX9 expression. Our findings indicate that SOX9 may be involved in the tumorigenesis and progression of OSCC. Furthermore, its cytoplasmic expression represents a potential predictive biomarker for tumor aggressiveness and OSCC prognosis.

Expression of PD-L1 and SOX2 during rectal tumourigenesis: Potential mechanisms for immune escape and tumour cell invasion.

Immunoediting is defined as a process whereby tumour cells develop the capacity to escape immune cell recognition. Accumulating evidence suggests that cancer stem-like cells (CSCs) have an enhanced capacity to interact with the immune system. The expression of CSCs and immune cell-associated markers has been demonstrated to change with disease progression from premalignant lesions to invasive cancer. The present study investigated the expression of putative CSC and immune cell-associated markers in different stages of progression from dysplasia to invasive malignancy in rectal lesions. Immunohistochemistry was performed for the CSC markers Lgr5 and SOX2 and the immune-associated markers CD8, Foxp3 and PD-L1 in 79 cases of endoscopically-excised rectal lesions, ranging from low grade adenoma (LG) to invasive adenocarcinoma (AdCa). CD8 and Foxp3 expression significantly increased with advances in disease progression [AdCa vs. LG: Odds ratio (OR) 4.33; 95% confidence interval (CI), 1.16–16.3; P=0.03 and OR, 40.5; 95% CI, 6.57–249.6; P<0.0001, respectively]. An increase in programmed death-ligand 1 (PD-L1) expression was also observed with disease progression (OR, 24.0; 95% CI, 4.23–136.2; P=0.0003). The expression of sex determining region Y-box 2 (SOX2) did not correlate with disease progression, although an elevated expression was observed in areas with high grade dysplasia. Increased PD-L1 expression may be a mechanism by which tumour cells evade immune recognition, facilitating tumour cell invasion in rectal cancer. The expression of SOX2 in areas with high grade dysplasia may indicate the de-differentiation of tumour cells, or the activation of migration pathways for invasion.

SOX9 regulated proliferation and apoptosis of human lung carcinoma cells by the Wnt/β-catenin signaling pathway.

Sex-determining region Y-box 9 (SOX9) is a transcription factor linked to stem cell maintenance and commonly over-expressed in solid cancers. In the present study, the effects of SOX9 on proliferation and apoptosis of human lung carcinoma cells and its mechanisms were investigated. Following over-expression or knock-down of SOX9 in human lung carcinoma cell line A549, cell viability was evaluated using XTT method, and cell apoptosis was measured by Flow cytometry. Caspase-3, Caspase-8, Caspase-9 and SOX9 expression was measured by RT-PCR, and Wnt, phosphorylated Wnt (p-Wnt) and β-catenin expression was detected by Western Blot. Results showed that SOX9 expression was elevated in human lung carcinoma cells. Knocking down cellular SOX9 by short hairpin RNA (shRNA) decreased cell proliferation while promoted apoptosis of A549 cells. Furthermore, down-regulation of p-Wnt and β-catenin expression levels was detected in A549 cells lack of SOX9. However, over-expression of SOX9 played the opposite roles in proliferation and apoptosis of human lung carcinoma cells. To further demonstrate the functions of the Wnt/β-catenin signaling pathway in SOX9 regulated-cell functions, the inhibitor IWP-2 was used to block the activation of Wnt/β-catenin signal. No significant differences between IWP-2-treated cells and SOX9 plus IWP-2-treated cells suggested the existence of a regulatory role for SOX9 through targeting the Wnt/β-catenin pathway. These findings establish the significance of SOX9 in lung cancer pathobiology and heterogeneity, with implications for targeting the Wnt/β-catenin-SOX9 signaling pathway as a rational therapeutic strategy.

Immunoexpression of SOX-2 in oral leukoplakia.

This study was conducted to correlate and compare the immunoexpression of sex-determining region Y-box 2 (SOX-2) in oral leukoplakia (OL) lesions with that in normal buccal mucosa (control). In this observational study, OL with low-risk (n=34) and high-risk (n=33) dysplasia and control samples (n=25) were subjected to immunohistochemical analysis for SOX-2. In the epithelium, SOX-2 positive and negative cells, as well as semiautomatic segmentation of the immunopositive nuclear area were counted. Statistical tests included chi-square, one-way analysis of variance, Tukey, and Games-Howell. The level of significance was 5%. Groups with OL lesions (low and high-risk) showed higher mean numbers of SOX-2 positive cells (63.47±25.70 and 68.18±21.17) compared to the control group (45.85±27.38) (p=0.00). Groups with OL lesions (low and high-risk) exhibited higher mean positive nuclear area (0.24±0.47 and 1.09±2.06) compared to the control group (0.00±0.01) (p=0.01). Oral leukoplakia lesions showed a higher expression of SOX-2, suggesting its contribution to the pathogenesis of OL.

Abstract 3061: The clinicopathologic roles of SOX2 and Oct4 expressions in stage 1 and 2 oral squamous cell carcinoma

Abstract [Background] Cancer stem cells (CSCs) exhibit self-replication, self-differentiation, drug resistance, and immune evasion activities. Recently, CSC has become increasingly important in the treatment of malignant tumors. Cancer stem cells express specific molecules termed CSC marker, including sex determining region Y-box2 (SOX2), and octamer-binding transcription factor 4 (Oct4), and their expression has been reported to be the potential prognostic values. However, the prognostic values of SOX2 and Oct4 expression in patients with stage I and II oral cancer are less understood. [Purpose] The aims of present study were to evaluate the expression of SOX2 and Oct4 in stage I and II oral squamous cell carcinoma (OSCC) and to elucidate the relationships among the CSC marker expression, clinical stages, histological differentiation, classification of invasion mode, cerebral lymph node metastasis, distant metastasis, and disease-free survival rate. [Materials and Methods] Tissue specimens were obtains from 83 patients with OSCC after surgery or biopsy. Immunohistochemistry was used to assess SOX2 and Oct4 protein using at least 10% staining-positive cells as the definition of positive staining. [Results] Immunohistochemical analysis of 83 cases showed that 31 cases (37%) expressed SOX2. There was no significant association between SOX2 expression and tumor size, invasion mode or histological differentiation. However, there was significant association between SOX2 expression and distant metastasis or disease-free survival rate at stage 1 and 2 patients. Otherwise, fifty three cases (64%) of 83 OSCC patients expressed Oct4. There was significant association between Oct4 expression and histological differentiation. There was no significant association between Oct4 expression and tumor size, invasion mode, metastasis, or disease-free survival rate. [Conclusions] These findings suggested that the expression of SOX2 may be good marker indicating survival in patients with stage 1 and 2 OSCC. Citation Format: Tetsuya Tamatani, Natsumi Takamaru, Go Ohe, Takayuki Nakagawa, Naito Kurio, Keiko Kudou, Yoshiko Yamamura, Takamitsu Mano, Youji Miyamoto. The clinicopathologic roles of SOX2 and Oct4 expressions in stage 1 and 2 oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3061.

Abstract 3059: SOX2 is a novel biomarker for poor long-term outcomes in oral cancer

Abstract Purpose: Sex determining region Y-BOX2 (SOX2), a key regulator of the self-renewal ability of stem cells, plays a role in the initiation and development of head and neck squamous cell carcinoma (HNSCC). However, the association between SOX2 expression and the outcomes of patients with oral squamous cell carcinoma (OSCC) has not been investigated. Thus, in this study, we investigated the possibility of predicting the survival outcomes of patients with OSCC by evaluating SOX2 expression. Experiment design: First, we analyzed SOX2 expression in patients with OSCC in The Cancer Genome Atlas (TCGA) dataset by a bioinformatics approach. In addition, samples of 219 patients with OSCC from two independent Chinese centers were collected. The median follow-up time and 95% confidence interval (CI) for the cohorts were 36 (25.06, 46.93) months and SOX2 expression in these samples was determined by immunostaining using tissue microarray. The Kaplan-Meier analysis, log-rank test, and Cox proportional hazards regression model were used to determine univariate and multivariate analyses, and hazard ratios for overall survival (OS), respectively. Results: In TCGA database, amplification of SOX2 was negatively correlated with the OS rate for patients who survived more than 300 days after the first diagnosis. Moreover, 39.7% of the samples from the cohorts showed high SOX2 expression. We found a significant difference in the survival rate between patients with high and low SOX2 expression ranging from 13 to 126 months. Conclusion: High SOX2 expression can predict poor long-term, but not short-term, survival outcomes in patients with OSCC. Our findings not only indicated that SOX2 may be a novel biomarker for the long-term prognosis of OSCC but also strongly supported the cancer stem cell theory that dysregulation of the self-renewal ability of stem cells may contribute to the development of OSCC. Citation Format: Xuefeng Zhang, Hao Xu, Chongkui Sun, Xinhua Liang, Peng Deng, Xin Zeng, Qianming Chen. SOX2 is a novel biomarker for poor long-term outcomes in oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3059.

MiR‑30a‑5p inhibits the proliferation, migration and invasion of melanoma cells by targeting SOX4.

MicroRNA (miR)‑30a‑5p has been reported to suppress the progression of hepatocellular cancer, renal cell carcinoma, oral cancer and gastric cancer. However, whether miR‑30a‑5p is involved in the regulation of melanoma remains unclear. The present study revealed that miR‑30a‑5p was downregulated in melanoma tissues and cell lines. Overexpression of miR‑30a‑5p significantly inhibited the proliferation, migration and invasion of melanoma cells invitro. In addition, ectopic expression of miR‑30a‑5p delayed tumor growth invivo. In terms of mechanism, miR‑30a‑5p targeted sex determining region Y‑box 4 (SOX4) and impeded the expression of SOX4 in melanoma cells. In addition, SOX4 was upregulated in melanoma tissues and cell lines when compared with normal tissues or cells. Furthermore, overexpression of SOX4 significantly rescued the proliferation, migration and invasion of melanoma cells transfected with miR‑30a‑5p mimics. Taken together, the results of the present study demonstrated that miR‑30a‑5p suppressed the proliferation, migration and invasion of melanoma cells in SOX4‑dependent manner.

PO-501 Loss of SOX9 expression is a predictive marker of relapse in gastric cancer

IntroductionGastric cancer is one of the most frequent tumours and the third leading cause of cancer-related death worldwide. The investigation of new biomarkers that can predict patient outcome more accurately and allow better treatment and follow-up decisions is of crucial importance. The transcription protein SRY-box 9 (SOX9) is a member of the high-mobility-group box class DNA-binding proteins. SOX9 is an important regulator of cell-fate decisions in embryogenesis and adulthood, playing critical roles in differentiation and proliferation, also in the gastrointestinal tract. SOX9 has been correlated to tumour behaviour in different tissues, including in gastric cancer, nevertheless with contradictory results. In this work we sought to ascertain the relevance of SOX9 transcription factor as a prognostic marker in gastric cancer.Material and methodsSOX9 expression was analysed by immunohistochemistry in a series of 333 cases of gastric adenocarcinoma, and its association with clinico-pathological and follow-up data was evaluated. A second gastric cancer validation cohort consisted of 354 cases from the cancer genome atlas (TCGA), showing high versus low SOX9 expression.Results and discussionsSOX9 expression was present in 83% of gastric cancer cases. Loss of SOX9 expression was significantly associated with relapse, however SOX9 expression was more frequent in stage IV cases. SOX9 loss of expression predicted worse disease-free survival but not overall survival, in this series. The prognostic value of SOX9 was independent of Laurén classification but it was more pronounced in tumours with expansive versus infiltrative growth (p=0.008). In patients that presented with disease in stage I to III, loss of SOX9 expression was significantly associated with venous invasion and lymph node metastases. In an independent series of gastric cancer, where SOX9 expression was assessed at the mRNA level (TCGA), low SOX9 expression levels were also associated with poor patient outcome. Functional studies, after down- and up-regulation of SOX9, are now being undertaken in order to better understand the clinico-pathological observations and the relevance of SOX9 as a potential biomarker in gastric cancer.ConclusionWe have identified SOX9 as a marker of relapse in gastric cancer. Further experiments are needed to elucidate its biological relevance at the cellular level and to test its potential role in invasion.