Sex Determining Region Y-box Protein Research Articles

METTL3 mediates SOX5 m6A methylation in bronchial epithelial cells to attenuate Th2 cell differentiation in T2 asthma

ObjectiveAsthma, a chronic inflammatory disease in which type 2 T helper cells (Th2) play a causative role in the development of T2 asthma. N6-methyladenosine (m6A) modification, an mRNA modification, and methyltransferase-like 3 (METTL3) is involved in the development of T2 asthma by inhibiting Th2 cell differentiation. Sex determining region Y-box protein 5 (SOX5) is involved in regulating T cell differentiation, but its role in T2 asthma was unclear. The objective of this study was to explore the role of METTL3 and SOX5 in T2 asthma and whether there is an interaction between the two. Materials and methodsAdults diagnosed with T2 asthma (n = 14) underwent clinical information collection and pulmonary function tests. In vivo and in vitro T2 asthma models were established using female C57BL/6 mice and human bronchial epithelial cells (HBE). The expressions of METTL3 and SOX5 were detected by Western blot and qRT-PCR and Western blot. Th2 cell differentiation was determined by flow cytometry and IL-4 level was detected by ELISA. m6A methylation level was determined by m6A quantitative assay. The relationship between METTL3 expression and clinical parameters was determined by Spearman rank correlation analysis. The function of METTL3 and SOX5 genes in asthma was investigated in vitro and in vivo. The RNA immunoprecipitation assay detected the specific interaction between METTL3 and SOX5. ResultsPatients with T2 asthma displayed lower METTL3 levels compared to healthy controls. Within this group, a negative correlation was observed between METTL3 and Th2 cells, while a positive correlation was noted between METTL3 and clinical parameters as well as Th1 cells. In both in vitro and in vivo models representing T2 asthma, METTL3 levels decreased significantly, while SOX5 levels showed the opposite trend. Overexpression of METTL3 gene in HBE cells significantly inhibited Th2 cell differentiation and increased m6A methylation activity. From a mechanism perspective, low METTL3 negatively regulates SOX5 expression through m6A modification dependence, while high SOX5 expression is positively associated with T2 asthma severity. Cell transfection experiments confirmed that METTL3 regulates Th2 cell differentiation and IL-4 release through SOX5. ConclusionsOverall, our results indicate that METTL3 alleviates Th2 cell differentiation in T2 asthma by modulating the m6A methylation activity of SOX5 in bronchial epithelial cells. This mechanism could potentially serve as a target for the prevention and management of T2 asthma.

Genetic and histological relationship between pheromone-secreting tissues of the musk gland and skin of juvenile Chinese forest musk deer (Moschus berezovskii Flerov, 1929).

BACKGROUND: The musk glands of adult male Chinese forest musk deer (Moschus berezovskii Flerov, 1929) (FMD), which are considered as special skin glands, secrete a mixture of sebum, lipids, and proteins into the musk pod. Together, these components form musk, which plays an important role in attracting females during the breeding season. However, the relationship between the musk glands and skin of Chinese FMD remains undiscovered. Here, the musk gland and skin of Chinese FMD were examined using histological analysis and RNA sequencing (RNA-seq), and the expression of key regulatory genes was evaluated to determine whether the musk gland is derived from the skin. METHODS: A comparative analysis of musk gland anatomy between juvenile and adult Chinese FMD was conducted. Then, based on the anatomical structure of the musk gland, skin tissues from the abdomen and back as well as musk gland tissues were obtained from three juvenile FMD. These tissues were used for RNA-seq, hematoxylin-eosin (HE) staining, immunohistochemistry (IHC), western blot (WB), and quantitative real-time polymerase chain reaction (qRT-PCR) experiments. RESULTS: Anatomical analysis showed that only adult male FMD had a complete glandular organ and musk pod, while juvenile FMD did not have any well-developed musk pods. Transcriptomic data revealed that 88.24% of genes were co-expressed in the skin and musk gland tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis found that the genes co-expressed in the abdomen skin, back skin, and musk gland were enriched in biological development, endocrine system, lipid metabolism, and other pathways. Gene Ontology (GO) enrichment analysis indicated that the genes expressed in these tissues were enriched in biological processes such as multicellular development and cell division. Moreover, the Metascape predictive analysis tool demonstrated that genes expressed in musk glands were skin tissue-specific. qRT-PCR and WB revealed that sex-determining region Y-box protein 9 (Sox9),Caveolin-1 (Cav-1), andandrogen receptor (AR) were expressed in all three tissues, although the expression levels differed among the tissues. According to the IHC results, Sox9 and AR were expressed in the nuclei of sebaceous gland, hair follicle, and musk gland cells, whereas Cav-1 was expressed in the cell membrane. CONCLUSIONS: The musk gland of Chinese FMD may be a derivative of skin tissue, and Sox9, Cav-1, and AR may play significant roles in musk gland development.

17-DMAG ameliorates neuroinflammation and BBB disruption via SOX5 mediated PI3K/Akt pathway after intracerebral hemorrhage in rats

Intracerebral hemorrhage (ICH) can result in secondary brain injury due to inflammation and breakdown of the blood–brain barrier (BBB), which are closely associated with patient prognosis. The potential of the heat shock protein 90 (Hsp90) inhibitor 17-DMAG in promoting neuroprotection has been observed in certain vascular diseases. However, the precise role of 17-DMAG treatment in ICH is not yet fully understood. In this study, we found that treatment with 17-DMAG (5 mg/kg) effectively reduced hematoma expansion and resulted in improved neurological outcomes. Meanwhile, the injection of 17-DMAG had a positive effect on reducing BBB disruption in rats with ICH. This effect was achieved by increasing the levels of BBB tight junction proteins (TJPs) such as zo-1, claudin-5, and occludin. As a result, the leakage of EB extravasation, brain edema and IgG in the peri-hematoma tissue were reduced. Furthermore, the injection of 17-DMAG decreased the infiltration of neutrophils into the brain tissues surrounding the hematoma in ICH rats and also reduced the production of proinflammatory cytokines IL-6 and TNF-α. Next, we used integrative mass spectrometry (MS) and molecular docking analysis to confirm that sex determining region Y-box protein 5 (SOX5) is a potential direct target of 17-DMAG in ICH. SOX5 encodes a positive regulator of the PI3K/Akt axis, and treatment with 17-DMAG resulted in a noticeable increase in SOX5 accumulation. To further investigate the role of SOX5, we employed virus-regulated SOX5 silencing and found that suppressing SOX5 blocked the ability of 17-DMAG to suppress neutrophil trafficking. Additionally, silencing SOX5 blocked the protective effects of 17-DMAG on the BBB by inhibiting PI3K, p-Akt, and BBB TJPs levels, which led to an increase in EB and IgG leakage in the peri-hematoma tissue after ICH. Similarly, when SOX5 was knocked down, the protective effects of 17-DMAG were lost. Overall, the results of our study indicate that the injection of 17-DMAG has the potential to mitigate neuroinflammation and prevent the disruption of the BBB caused by ICH, resulting in improved neurological outcomes in rats. These positive effects are attributed to the regulation of SOX5 and activation of the PI3K/Akt pathway. These findings highlight the possibility of targeting SOX5 and the PI3K/Akt pathway as a novel therapeutic approach for ICH.

CircWHSC1 serves as a prognostic biomarker and promotes malignant progression of non-small-cell lung cancer via miR-590-5p/SOX5 axis.

Dysregulated circWHSC1 has been shown to play potential roles in diverse cancer types, including ovarian cancer, endometrial cancer and hepatocellular carcinoma (HCC). The objective of this study was to investigate its expression, underlying role and regulatory mechanism in non-small-cell lung cancer (NSCLC). The expression of circWHSC1 was determined by real-time PCR. After knockdown of circWHSC1 expression in NSCLC cells, the proliferation, migration, and invasion were detected using CCK-8, colony formation, and Transwell assays, and the effects of circWHSC1 on NSCLC tumorigenesis in vivo was also investigated. With the help of luciferase reporter and pull-down assays, we further explored the downstream mechanism of circWHSC1 in NSCLC cells. CircWHSC1 was highly expressed in NSCLC tissues and cell lines. The inhibition of circWHSC1 suppressed the malignant properties of NSCLC cells, as evidenced by the reduction of proliferation, migration and invasion. CircWHSC1 sponged miR-590-5p and functioned as an oncogene in NSCLC by increasing sex determining region Y-boxprotein 5 (SOX5) expression. CircWHSC1 may contribute to the oncogenicity of NSCLC via the regulation of miR-590-5p/SOX5 axis, which might be a novel therapeutic target in NSCLC.

Noninvasive Assessment of Renal Fibrosis of Chronic Kidney Disease in Rats by [68Ga]Ga-FAPI-04 Small Animal PET/CT and Biomarkers

Renal fibrosis is the most common pathological feature and common pathway of progression in chronic kidney disease (CKD). We evaluated [68Ga]Ga-FAPI-04 small animal positron emission tomography/computed tomography (PET/CT) and biomarkers as noninvasive assessments of renal fibrosis (RF) in CKD rats to generate new ideas for clinical diagnosis. A rat model of renal fibrosis was administered adenine by gavage (n = 28), and the control group was given 0.9% NaCl by gavage (n = 20). At different time points (weeks 1, 2, 4, and 6), five rats were randomly selected from the two groups for [68Ga]Ga-FAPI-04 small animal PET/CT imaging. At the same time, the expression of Fibroblast activation protein (FAP) in renal tissue and the expression levels of type III procollagen N-terminal peptide (PIIINP), transforming growth factor (TGF-β1), Klotho, and sex-determining region Y-box protein 9 (SOX9) in blood and urine were determined. FAP was highly expressed in the renal tissue of rats in the CKD group and expression increased with the progression of renal fibrosis. [68Ga]Ga-FAPI-04 small animal PET/CT examination showed that the uptake of radioactive tracers in the CKD group was higher than that in the control group, and SUVmax (r = 0.9405) and target-to-background ratio (TBR) (r = 0.9392) were positively correlated with renal fibrosis. The serum levels of PIIINP, TGF-β1, and SOX9 in CKD rats were significantly higher than those in the control group and were positively correlated with RF (r = 0.8234, r = 0.7733, and r = 0.7135, respectively) and SUVmax (r = 0.8412, r = 0.7763, and r = 0.6814, respectively). Compared with the control group, the level of serum Klotho decreased and was negatively correlated with RF (r = -0.6925) and SUVmax (r = -0.6322). Compared with the control group, the levels of PIIINP and TGF-β1 in urine were positively correlated with RF (r = 0.8127 and r = 0.8077, respectively) and SUVmax (r = 0.8400 and r = 0.8177, respectively). Urine Klotho decreased compared with the control group and was negatively correlated with RF (r = -0.5919) and SUVmax (r = -0.5995). The change in urine SOX9 was not statistically significant. In conclusion, compared with renal biopsy, [68Ga]Ga-FAPI-04 small animal PET/CT shows renal fibrosis quickly and noninvasively. PIIINP, TGF-β1, and Klotho in serum and urine may be used as biomarkers of RF, and serum SOX9 is expected to become a new diagnostic biomarker of RF.

SOX5 promotes cell growth and migration through modulating the DNMT1/p21 pathway in bladder cancer.

Bladder cancer (BC) is one of the most prevalent and life-threatening cancers among the male population worldwide. Sex determining region Y-box protein 5 (SOX5) plays important roles in a variety of human cancers. However, little research has been conducted on the function and underlying mechanism of SOX5 in BC. In the present study, we first reveal the increased expression of SOX5 in BC tissues and in vitro cells lines. Second, we discover that inhibition of SOX5 inhibits cell growth and migration but promotes cell apoptosis. Meanwhile, ectopic SOX5 expression stimulates cell growth and migration in BC cells. Then, we show that suppressing SOX5 inhibits the expression of DNA methyltransferase 1 (DNMT1), and that overexpressing DNMT1 alleviates the cell progress of BC cells inhibited by SOX5. Furthermore, we demonstrate that DNMT1 inhibits p21 expression by affecting DNA methylation of the p21 promoter. Collectively, we demonstrate that SOX5 exerts its functions in BC cells by modulating the SOX5/DNMT1/p21 pathway. Finally, we demonstrate that SOX5 knockdown inhibits xenograft tumor growth in vivo. In conclusion, our study elucidates the oncogenic role of SOX5 and its underlying molecular mechanism in BC, and reveals a novel pathway which has the potential to serve as a diagnostic biomarker and therapeutic target for BC.

CircABCC3 knockdown inhibits glioblastoma cell malignancy by regulating miR-770-5p/SOX2 axis through PI3K/AKT signaling pathway

BackgroundCircular RNAs (circRNAs) are found to regulate glioblastoma evolution. However, the role of circ-ATP binding cassette subfamily C member 3 (circABCC3) in glioblastoma process is still unknown. In this study, the effects of circABCC3 on glioblastoma tumorigenesis and underlying mechanism were revealed. MethodsThe expression levels of circABCC3, microRNA-770-5p (miR-770-5p) and sex determining region Y-box protein 2 (SOX2) mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway-related proteins and SOX2 protein was detected by western blot analysis. Cell proliferation and invasion were severally investigated by cell colony formation and transwell invasion assays. Cell migration was demonstrated by transwell migration and wound-healing assays. Cell apoptosis was revealed by flow cytometry analysis. Tube formation was investigated by tube formation assay. The associated relationship between miR-770-5p and circABCC3 or SOX2 was predicted by starbase or targetscan online database, and identified by dual-luciferase reporter assay, RNA immunoprecipitation assay or RNA pull-down assay. The impacts of circABCC3 knockdown on glioblastoma growth in vivo were revealed by in vivo assay. ResultsCircABCC3 and SOX2 expression were dramatically upregulated, while miR-770-5p expression was apparently downregulated in glioblastoma tissues and cells compared with control groups. CircABCC3 expression was higher in stage III glioblastoma tissues than in stage I + II glioblastoma tissues with close correlation with tumor-node-metastasis (TNM) stage. CircABCC3 absence inhibited cell proliferation, migration, invasion, tube formation and the activation of PI3K/AKT pathway, whereas induced cell apoptosis in glioblastoma. Additionally, circABCC3 acted as a sponge for miR-770-5p, and miR-770-5p targeted SOX2. MiR-770-5p inhibitors impaired the impacts of circABCC3 silencing on glioblastoma progression, angiogenesis and PI3K/AKT pathway. Furthermore, circABCC3 knockdown repressed tumor growth in vivo. ConclusionCircABCC3 regulated glioblastoma development via miR-770-5p/SOX2 axis through PI3K/AKT pathway. This finding lays a theoretical foundation for studying circRNA-directed therapy for glioblastoma.

CircSEC24A promotes IL-1β-induced apoptosis and inflammation in chondrocytes by regulating miR-142-5p/SOX5 axis.

Osteoarthritis (OA) is a common joint disease. Currently, many studies have revealed that circular RNAs (circRNAs) are strongly related to the occurrence and development of diseases. Hence, we aimed to further elucidate the role and molecular mechanism of circRNA SEC24 homolog A, COPII coat complex component (circSEC24A) in OA. Chondrocytes were treated with interleukin-1β (IL-1β) to establish OA cell model in vitro. The expression levels of circSEC24A, microRNA-142-5p (miR-142-5p), and sex-determining region Y-box protein 5 (SOX5) were determined by quantitative real-time polymerase chain reaction. MTT and colony formation assays were used to determine cell proliferation. Cell apoptosis was detected by flow cytometry analysis. The protein levels of inflammatory factors and SOX5 were determined by western blot assay. The relationship between miR-142-5p and circSEC24A or SOX5 was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation assay. CircSEC24A and SOX5 expression were enhanced, while miR-142-5p level was reduced in OA cartilage tissues and chondrocytes. Overexpression of circSEC24A promoted IL-1β-induced injury through decreasing cell proliferation and increasing apoptosis and inflammation in chondrocytes. MiR-142-5p was a direct target of circSEC24A, and its upregulation ameliorated IL-1β-induced injury and abated the effect of oe-circSEC24A in IL-1β-induced chondrocytes. Additionally, SOX5 was a downstream target of miR-142-5p, and its overexpression had a similar role with oe-circSEC24A and reversed the impact of miR-142-5p in IL-1β-induced chondrocytes. CircSEC24A acted as a molecular sponge of miR-142-5p to regulate SOX5 expression in chondrocytes. CircSEC24A aggravated IL-1β-induced injury via modulating miR-142-5p/SOX5 axis, providing possible targets for the clinical diagnosis and treatment of OA.

Long non-coding RNA BLACAT1 expedites osteosarcoma cell proliferation, migration and invasion via up-regulating SOX12 through miR-608

BackgroundOsteosarcoma is the most common type of bone malignancy. Increasing evidence indicated that long non-coding RNAs (lncRNAs) possess multiple functions in the development of cancer and can be used as indicators of prognosis and diagnosis. LncRNA BLACAT1 has been found to promote the proliferation of breast cancer cells. However, the role of BLACAT1 in osteosarcoma remains largely unknown. MethodsQRT-PCR analysis was employed to evaluate mRNA expressions. Western blot was performed to measure relevant protein level. Colony formation and EdU assays were conducted to certify proliferative ability. TUNEL assay was finalized to assess apoptotic cells. Wound-healing and transwell assays were utilized for the exploration of migrating and invasive abilities. The subcellular distribution of BLACAT1 was studied by nucleus-cytoplasm separation assay. Relevant mechanical experiments were combined to elucidate molecular relationship between molecules. ResultsBLACAT1 was highly expressed in osteosarcoma. BLACAT1 promoted the proliferation and migration of osteosarcoma cells. BLACAT1 acted as a sponge for miR-608 to augment the expression of Sex determining region Y-box protein 12 (SOX12), the direct target of miR-608. Further, inhibiting miR-608 recovered the repressive effect of silenced BLACAT1 on the malignant behaviors of osteosarcoma cells. ConclusionThis study highlighted the contribution of BLACAT1/miR-608/SOX12 axis to the progression of osteosarcoma, suggesting novel targets for osteosarcoma therapy.

CircRNA CDR1as knockdown inhibits progression of non-small-cell lung cancer by regulating miR-219a-5p/SOX5 axis.

BackgroundCircular RNAs (circRNAs) participate in the development of human cancers by regulating multiple cell processes. CircRNA antisense to the cerebellar degeneration‐related protein 1 transcript (circCDR1as) expression is dysregulated in many cancers, including non‐small‐cell lung cancer (NSCLC). However, the mechanism by which circCDR1as mediates the development of NSCLC remains unknown.MethodsA total of 30 paired cancer and normal tissues were collected from patients with NSCLC. The expression levels of circCDR1as, microRNA (miR)‐219a‐5p and Sex determining region Y‐box protein 5 (SOX5) were measured in tissues or cells by quantitative real‐time polymerase chain reaction or western blot. Cell viability, apoptosis, migration and invasion were detected by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide, colony formation, flow cytometry and transwell assays, respectively. The target relationship between miR‐219a‐5p and circCDR1as or SOX5 was validated by dual‐luciferase reporter assay.ResultsCircCDR1as expression was elevated in NSCLC tissues and cells in comparison to the matched controls. Interference of circCDR1as led to obvious inhibition of cell viability, migration and invasion and increase of apoptosis in NSCLC cells. MiR‐219a‐5p acted as a target of circCDR1as and miR‐219a‐5p downregulation attenuated the regulatory effect of circCDR1as silencing on NSCLC progression. Moreover, miR‐219a‐5p targeted SOX5 to repress the progression of NSCLC in vitro. Besides, circCDR1as knockdown reduced the expression of SOX5 by increasing miR‐219a‐5p level.ConclusionKnockdown of circCDR1as inhibited the progression of NSCLC by decreasing cell viability, migration and invasion and increasing apoptosis by upregulating miR‐219a‐5p and downregulating SOX5.

NEAT1/miR-193a-3p/SOX5 axis regulates cartilage matrix degradation in human osteoarthritis.

Long non-coding RNAs (lncRNAs) were reported to be involved in the progression of osteoarthritis (OA). The aim of this work was to explore the functional role of lncRNA nuclear-enriched abundant transcript 1 (NEAT1) in OA. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was employed to analyze the expression of microRNA (miR-193a)-3p, NEAT1, and sex-determining region Y-box protein 5 (SOX5), as well as the levels of pro-inflammatory cytokines interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α), and IL-8 in OA cartilage tissue and chondrocytes. In addition, flow cytometry was used to measure the apoptosis of chondrocytes. The protein levels of extracellular matrix ACAN, collagen type II α1 chain (Col2a1), matrix metalloproteinase-3 (MMP-3), MMP-13, a disintegrin, and metalloproteinase with thrombospondin motifs (ADAMTS)-5 and SOX5 were determined using western blot analysis. Dual-luciferase reporter assay was performed to determine the target relationship among NEAT1, miR-193a-3p, and SOX5. We found that miR-193a-3p expression was downregulated, while NEAT1 and SOX5 were upregulated in OA cartilage tissue and chondrocytes. Both upregulation of miR-193a-3p and knockdown of NEAT1 suppressed inflammation, apoptosis, and reduced the protein levels of MMP-3, MMP-13, and ADAMTS-5, while elevating ACAN and Col2a1 expression in chondrocytes. NEAT1 targeted miR-193a-3p, and SOX5 was targeted by miR-193a-3p. Silencing of miR-193a-3p reversed the NEAT1 knockdown-mediated effect on the inflammation, apoptosis, and production of the extracellular matrix. The introduction of SOX5 abolished the impact of the upregulation of miR-193a-3p on inflammation, apoptosis, and production of extracellular matrix in chondrocytes. In conclusion, NEAT1/miR-193a-3p/SOX5 axis regulates cartilage matrix degradation in human OA.

FOX-A1 contributes to acquisition of chemoresistance in human lung adenocarcinoma via transactivation of SOX5

BackgroundChemoresistance is a major obstacle for the effective treatment of lung adenocarcinoma (LAD). Forkhead box (FOX) proteins have been demonstrated to play critical roles in promoting epithelial-mesenchymal transition (EMT) and chemoresistance. However, whether FOX proteins contribute to the acquisition of EMT and chemoresistance in LAD remains largely unknown. MethodsFOX-A1 expression was measured in LAD cells and tissues by qRT-PCR. The expression levels of EMT markers were detected by western blotting and immunofluorescence assay. The interaction between Sex determining region Y-box protein 5 (SOX5) and FOX-A1 was validated by chromatin immunoprecipitation sequence (ChIP-seq) and Chromatin immunoprecipitation (ChIP) assay. Kaplan-Meier analysis and multivariate Cox regression analysis were performed to analyze the significance of FOX-A1 and SOX5 expression in the prognosis of LAD patients. FindingsFOX-A1 was upregulated in docetaxel-resistant LAD cells. High FOX-A1 expression was closely associated with a worse prognosis. Upregulation of FOX-A1 in LAD samples indicated short progression-free survival (PFS) and overall survival (OS). SOX5 is a new and direct target of FOX-A1 and was positively regulated by FOX-A1 in LAD cell lines. Knockdown of FOX-A1 or SOX5 reversed the chemoresistance of docetaxel-resistant LAD cells by suppressing cell proliferation, migration and EMT progress. InterpretationThese data elucidated an original FOX-A1/SOX5 pathway that represents a promising therapeutic target for chemosensitizing LAD and provides predictive biomarkers for evaluating the efficacy of chemotherapies.

FOX-A1 Contributes to Acquisition of Chemoresistance in Human Lung Adenocarcinoma via Transactivation of SOX5

Background: Chemoresistance is a major obstacle for the effective treatment of lung adenocarcinoma (LAD). Forkhead box (FOX) proteins have recently been demonstrated to play critical roles in promoting epithelial-mesenchymal transition (EMT) and chemoresistance; however, it remains largely unknown whether FOX proteins contribute to the acquisition of EMT and chemoresistance in LAD. Methods: FOX-A1 expression were measured in LAD cells and tissues by qRT-PCR. The expression levels of EMT markers were detected by western blotting and immunofluorescence assay. Sex determining region Y-box protein 5 (SOX5) was identified as a novel and direct target of FOX-A1 via chromatin immunoprecipitation sequence (ChIP-seq) and Chromatin immunoprecipitation (ChIP) assay. Kaplan-Meier analysis and multivariate Cox regression analysis were performed to elucidate the significance of FOX-A1 and SOX5 expression in the prognosis of LAD patients. Findings: FOX-A1 was upregulated in docetaxel-resistant LAD cells that had been previously established, exhibited EMT-like characteristics and increased invasion phenotypes. High FOX-A1 expression was closely associated with a worse tumor response and poor prognosis and was significantly correlated with short progression-free survival (PFS) and overall survival (OS). SOX5 was identified as a new and direct target of FOX-A1 and also acted as an independent prognostic factor for poor PFS and OS in LAD patients. Knockdown of FOX-A1 and SOX5 reversed EMT tomesenchymal-epithelial transition (MET), attenuated metastatic characteristics and reversed the chemoresistance of docetaxel-resistant LAD cells. Interpretation: These data elucidate an original FOX-A1/SOX5 pathway that represents a promising therapeutic target for chemosensitizing LAD and provides predictive markers for evaluating the efficacy of chemotherapies. Funding: This work was supported by the National Natural Science Foundation of China (NO.81672928), Financial Grant from the China Postdoctoral Science Foundation (NO. 2018T110466 and NO.2017M621678), National Natural Science Foundation of China (NO.81702048, NO.81572345 and NO.81503528), the Suzhou Science and Technology Bureau project (No. SYS201609) and the Industrial Technology Innovation Project of Suzhou city (No. SYSD2016124). Declaration of Interest: The authors declare that there is no conflict of interest. Ethical Approval: This study was approved by the Review Board of Hospital Ethics Committee of Nanjing General Hospital of Nanjing Military Command (No. 2012-2-12-015, No. 2012-2-13-022, Nanjing General Hospital of Nanjing Military Command, Nanjing University, China) and conducted in accordance with the Declaration of Helsinki, and written informed consent was obtained from all patients before specimen collection.

SOX5 promotes cell invasion and metastasis via activation of Twist-mediated epithelial-mesenchymal transition in gastric cancer.

BackgroundSex-determining region Y-box protein 5 (SOX5) has been demonstrated to be implicated in oncogenic function in various types of cancers. However, the role of SOX5 in gastric cancer (GC) remains poorly elucidated. Herein, we investigated the role and the underlying mechanism of SOX5 in GC progression.MethodsSOX5 mRNA and protein expression were detected by quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry in human GC specimens, and their clinical significance was evaluated. The effects of SOX5 knockdown or overexpression on GC cell behavior were determined by proliferation, wound-healing and transwell assays in vitro, and metastasis assays in vivo; and epithelial–mesenchymal transition (EMT)-related markers were detected by qRT-PCR, Western blot and immunofluorescence staining.ResultsThe up-regulated expression of SOX5 in GC specimens was significantly correlated with clinical metastasis and poor prognosis for patients with GC. Besides, SOX5 promoted GC cell migration and invasion in vitro, as well as GC cell metastasis in vivo. Mechanically, Twist-mediated EMT was likely involved in SOX5-facilitated GC cell behavior.ConclusionSOX5 has an important function in GC progression. In addition, SOX5 promotes GC cell invasion and metastasis via activation of Twist-mediated EMT, thus providing a potential therapeutic target for GC metastasis.

SOX5 promotes breast cancer proliferation and invasion by transactivation of EZH2.

Sex determining region Y-box protein 5 (SOX5) is a transcriptional factor and serves important roles in various cancer types; however, the pathological role of SOX5 in patients with breast cancer remains unclear. In the present study, the expression and potential role of SOX5 in patients with breast cancer and in breast cancer cells was investigated. The data indicated that SOX5 was highly expressed in breast cancer tissues compared with adjacent healthy tissues, and overexpression of SOX5 was associated with a reduced overall survival rate in patients with breast cancer. Gain and loss of function studies with MTT, colony formation, wound healing and Matrigel invasion assays demonstrated that SOX5 significantly promoted breast cancer cell proliferation and invasion. The chromatin immunoprecipitation (ChIP) assay sequence, quantitative ChIP and luciferase reporter assays were used to identify enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) as a downstream target gene of SOX5. Furthermore, it was determined that ectopic expression of SOX5 increased EZH2 expression at the mRNA and protein level, while the knockdown of SOX5 decreased EZH2 expression. Additionally, the biological effect of SOX5 was investigated, and it was determined to be dependent on the regulation of EZH2 expression. The present results may provide important insights into the biological significance of SOX5 serving as a candidate therapeutic target in breast cancer progression.

SOX5 induces lung adenocarcinoma angiogenesis by inducing the expression of VEGF through STAT3 signaling.

Background and objectivesAngiogenesis is the main cause of lung adenocarcinoma (LAC) poor prognosis. This study aimed to investigate the effect of sex-determining region Y-box protein 5 (SOX5) expression on angiogenesis of LAC and explore its possible mechanism.Patients and methodsThe effect on angiogenesis was tested by tube formation assays using human umbilical vein endothelial cells cocultured with A549 cells. Lentivirus shRNA of SOX5 and lentivirus of SOX5 overexpression system were used to establish LAC cell lines, which expressed SOX5 of different levels. SOX5 downstream signaling targets were analyzed by real-time qPCR and Western blot. We collected 90 LAC cases and the tissues were examined by immunohistochemistry for SOX5 and vascular endothelial growth factor (VEGF).ResultsWe found that SOX5 overexpression in A549 cells significantly promoted tube formation capacity of the cocultured human umbilical vein endothelial cells. SOX5 increased VEGF expression and signal transducer activator of transcription 3 phosphorylation; however, SOX5 had no effect on extracellular signal-regulated kinase and protein kinase B pathway. Furthermore, the expression of SOX5 and VEGF had a significantly positive correlation (r=0.399, P=0.001) according to the tissue microarray data.ConclusionThese findings suggest that SOX5 induces angiogenesis by activating signal transducer activator of transcription 3/VEGF signaling and confer its candidacy as a potential therapeutic target in LAC.

Downregulation of SOX3 leads to the inhibition of the proliferation, migration and invasion of osteosarcoma cells

Sex determining regionY-box protein3(SOX3) is involved in embryonic development and tumorigenesis. However, the expression and precise role of SOX3 in osteosarcoma remain unclear. In this study, we reported that SOX3 expression was upregulated in osteosarcoma tissues compared with non-cancerous bone cyst tissues. To elucidate the cellular and molecular function of SOX3, we examined the consequences of SOX3 knockdown in osteosarcoma cells. We found that the downregulation of SOX3 inhibited the proliferation, migration and invasion of osteosarcoma cells. SOX3 downregulation also increased the cell population in the G1phase and induced cell apoptosis. SOX3 knockdown-mediated cell cycle arrest and cell apoptosis were associated with decreased levels of Cdc25A, cyclinD1, proliferating cell nuclear antigen(PCNA) and Bcl-2, as well as an increased Bax expression. We also found that the downregulation of SOX3 decreased the expression of Snail, Twist and matrix metalloproteinase-9(MMP-9), and increased E-cadherin expression, resulting in the inhibition of cell migration and invasion. Taken together, our data indicate that SOX3 may serve as an oncogene in osteosarcoma, and SOX3 downregulation may prove to be a novel approach for the inhibition of osteosarcoma progression.

SOX2 and SOX12 are predictive of prognosis in patients with clear cell renal cell carcinoma.

Sex-determining region Y-box protein (SOX) genes serve an important role in cancer growth and metastasis. The present study aimed to determine the predictive ability of SOX and associated genes identified through molecular network in clear cell renal cell carcinoma (RCC). A total of 505 patients with clear cell RCC from The Cancer Genome Atlas (TCGA) cohorts were collected in this study. The expression profile of SOX and associated genes were obtained from the TCGA RNAseq database. Clinicopathological characteristics, including age, gender, tumor grade, stage, laterality disease-free-survival and overall survival (OS) were collected. Cox's proportional hazards regression model, as well as Kaplan-Meier curves were used to assess the relative factors. Selected genes of SOXs that demonstrated significant associations with OS were further validated in 192 patients from the validation cohort. In the univariate Cox regression model, SOX1, SOX2, SOX6, SOX11, SOX12, SOX13, SOX15, SOX17 and SOX30 expression were predictive in the prognosis of clear cell RCC. Following adjustment for clinical factors, SOX2 [hazard ratio (HR), 1.130; 95% confidence interval (CI), 1.002-1.275), SOX12 (HR, 1.379; 95% CI, 1.060-1.793) and SOX15 (HR, 1.245; 95% CI, 1.063-1.459) remained statistically significant. Furthermore, POU class 5 homeobox 1 (POU5F1), POU2F1 and nuclear receptor subfamily 5 group A member 1 in the gene cluster network analysis associated with SOX2 did not reduce the statistical significance when added to the multivariate analysis. The findings were extended to the Fudan University Shanghai Cancer Center cohort. The results revealed that high SOX2 and SOX12 expression were associated with poor prognosis for OS (log-rank test, all P<0.05). SOX2 and SOX12 were identified as independent prognostic factors of OS in clear cell RCC.

SOX5 predicts poor prognosis in lung adenocarcinoma and promotes tumor metastasis through epithelial-mesenchymal transition

Lung cancer is the leading cause of cancer-related death worldwide. Epithelial-mesenchymal transition (EMT) promotes lung cancer progression and metastasis, especially in lung adenocarcinoma. Sex determining region Y-box protein 5 (SOX5) is known to stimulate the progression of various cancers. Here, we used immunohistochemical analysis to reveal that SOX5 levels were increased in 90 lung adenocarcinoma patients. The high SOX5 expression in lung adenocarcinoma and non-tumor counterparts correlated with the patients’ poor prognosis. Inhibiting SOX5 expression attenuated metastasis and progression in lung cancer cells, while over-expressing SOX5 accelerated lung adenocarcinoma progression and metastasis via EMT. An in vivo zebrafish xenograft cancer model also showed SOX5 knockdown was followed by reduced lung cancer cell proliferation and metastasis. Our results indicate SOX5 promotes lung adenocarcinoma tumorigenicity and can be a novel diagnosis and prognosis marker of the disease.

Abstract 4777: Cancer stemness and resistance: Napabucasin (BBI-608) sensitizes stemness-high cancer cells to Paclitaxel by inhibiting the STAT3-MUC1 pathway

Abstract Stemness-high cancer cells, or cancer stem cells (CSC) represent a subpopulation of cancer cells with enhanced tumorigenic capacity, metastasis-forming potential, and resistance to conventional chemotherapy and radiation. One such key CSC pathway is regulated by STAT3, a transcription factor that is downstream of several cytokines and growth factor receptors which controls the expression of a broad range of target genes and plays essential roles in CSC biology in many tumor types. Cancer stemness inhibitor Napabucasin (BBI-608), a small molecule that inhibits gene transcription driven by STAT3, can inhibit stemness gene expression, block spherogenesis, and kill CSCs. In vivo, BBI-608 effectively blocks cancer relapse and metastasis in xenograft models while sparing normal hematopoietic stem cells, suggesting that targeting stemness-high cancer cells is a feasible approach for developing next-generation cancer therapeutics to combat cancer recurrence. SOX2 (sex-determining region Y-box protein 2), a transcription factor that is essential for self-renewal and pluripotency, is amplified in various cancer types and has been shown to be required for CSC self-renewal and maintenance. Mucin 1 (MUC1), a glycoprotein normally expressed at the apical surface of epithelial cells, is overexpressed in most human epithelial cancers. MUC1 has been implicated in regulating tumor proliferation, metabolism, invasion, angiogenesis, chemoresistance, and inflammation. Here, we generated a stemness-high culture system based on the reporter activity of a SOX2-regulatory region construct in MKN28 gastric cancer cells (MKN28 SOX2-reporter GFP+ cells). BBI-608 treatment inhibited the STAT3-MUC1 pathway in stemness-high cells. High MUC1 status in stemness-high cells was associated with Paclitaxel resistance. Down-regulation of MUC1 sensitized stemness-high cells to Paclitaxel. Moreover, BBI-608 synergized with Paclitaxel in inhibiting spherogenesis of stemness-high cells. This study provides a new mechanism for the association of cancer stemness with drug resistance. Our findings support this combination therapy, which pairs a conventional chemotherapy (Paclitaxel) with a cancer stemness inhibitor (BBI-608), as a promising strategy to combat cancer. Citation Format: Harry A. Rogoff, Juying Li, Chiang Li. Cancer stemness and resistance: Napabucasin (BBI-608) sensitizes stemness-high cancer cells to Paclitaxel by inhibiting the STAT3-MUC1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4777. doi:10.1158/1538-7445.AM2017-4777