miR-937-5p targets SOX17 to modulate breast cancer cell cycle and cell proliferation through the Wnt signaling pathway.

Breast cancer is one of the most frequent cancers in women and the globally leading cause of cancer-related deaths. Bioinformatics and experimental analyses found that miR-937-5p may play a proto-oncogenic role in breast cancer; however, the specific effects and the molecular mechanism need further investigation. GSEA-KEGG and GSEA-GO suggested that miR-937-5p might be related to cell cycle and DNA replication. The experimental data indicated that miR-937-5p inhibition significantly repressed the proliferation of breast carcinoma cells and elicited S-phase cell cycle arrest. Meanwhile, the protein levels of proliferating marker ki-67 and cell cycle regulators Cyclin A2, Cyclin B1, CDK1, and Cyclin D1 were also decreased by miR-937-5p inhibition. miR-937-5p could directly bind to and negatively regulate SOX17. SOX17 overexpression also significantly repressed the proliferation of breast carcinoma cells and elicited S-phase cell cycle arrest and decreased ki-67, ß-catenin, c-Myc, Cyclin A2, Cyclin B1, Cyclin D1, and CDK1 protein contents. More importantly, the effects of miR-937-5p were reversed by SOX17.

Sox17-mediated expression of adherent molecules is required for the maintenance of undifferentiated hematopoietic cluster formation in midgestation mouse embryos.

Hematopoietic stem cell-containing intra-aortic hematopoietic cell clusters (IAHCs) emerge in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation mouse embryos. We previously showed that transduction of Sox17 in CD45lowc-Kithigh cells, which are one component of IAHCs, maintained the cluster formation and the undifferentiated state, but the mechanism of the cluster formation by Sox17 has not been clarified. By microarray gene expression analysis, we found that genes for vascular endothelial-cadherin (VE-cad) and endothelial cell-selective adhesion molecule (ESAM) were expressed at high levels in Sox17-transduced c-Kit+ cells. Here we show the functional role of these adhesion molecules in the formation of IAHCs and the maintenance of the undifferentiated state by in vitro experiments. We detected VE-cad and ESAM expression in endothelial cells of dorsal aorta and IAHCs in E10.5 embryos by whole mount immunohistochemistry. Cells with the middle expression level of VE-cad and the low expression level of ESAM had the highest colony-forming ability. Tamoxifen-dependent nuclear translocation of Sox17-ERT fusion protein induced the formation of cell clusters and the expression of Cdh5 (VE-cad) and ESAM genes. We showed the induction of the Cdh5 (VE-cad) and ESAM expression and the direct interaction of Sox17 with their promoter by luciferase assay and chromatin immunoprecipitation assay, respectively. Moreover, shRNA-mediated knockdown of either Cdh5 (VE-cad) or ESAM gene in Sox17-transduced cells decreased the multilineage-colony forming potential. These findings suggest that VE-cad and ESAM play an important role in the high hematopoietic activity of IAHCs and cluster formation.

SOX18 exerts tumor-suppressive functions in papillary thyroid carcinoma through inhibition of Wnt/ß-catenin signaling.

Sex-determining region on the Y chromosome-related high mobility group box 18 (SOX18) has emerged as a key tumor-related protein in a wide range of human tumors. Yet, the involvement of SOX18 in papillary thyroid carcinoma has not been determined. This study aimed to explore the expression and biological function of SOX18 in papillary thyroid carcinoma. There was a significant decrease in SOX18 expression in papillary thyroid carcinoma tissues compared with that in normal tissues. Low expression of SOX18 was also detected in papillary thyroid carcinoma cell lines and upregulation of SOX18 effectively repressed the proliferative, colony-forming and invasive abilities of papillary thyroid carcinoma cells in vitro. In contrast, knockdown of SOX18 in papillary thyroid carcinoma cells was associated with a significant increase in cell proliferation and invasion. Further studies revealed that SOX18 upregulation was associated with the reduced nuclear accumulation of ß-catenin and the downregulation of Wnt/ß-catenin signaling in thyroid carcinoma cells. Moreover, inhibition of Wnt/ß-catenin signaling markedly attenuated SOX18 knockdown-evoked oncogenic effects in papillary thyroid carcinoma cells. In addition, SOX18 overexpression remarkably retarded the tumor growth of papillary thyroid carcinoma cell-derived xenograft tumors in nude mice. Taken together, these results demonstrate that SOX18 suppresses the proliferation and invasion of papillary thyroid carcinoma by inhibiting Wnt/ß-catenin signaling. Our study reveals a tumor-suppressive role of SOX18 in papillary thyroid carcinoma and suggests that SOX18 is an attractive candidate target for treatment of papillary thyroid carcinoma.

R-propranolol is a small molecule inhibitor of the SOX18 transcription factor in a rare vascular syndrome and hemangioma.

Propranolol is an approved non-selective ß-adrenergic blocker that is first line therapy for infantile hemangioma. Despite the clinical benefit of propranolol therapy in hemangioma, the mechanistic understanding of what drives this outcome is limited. Here, we report successful treatment of pericardial edema with propranolol in a patient with Hypotrichosis-Lymphedema-Telangiectasia and Renal (HLTRS) syndrome, caused by a mutation in SOX18. Using a mouse pre-clinical model of HLTRS, we show that propranolol treatment rescues its corneal neo-vascularisation phenotype. Dissection of the molecular mechanism identified the R(+)-propranolol enantiomer as a small molecule inhibitor of the SOX18 transcription factor, independent of any anti-adrenergic effect. Lastly, in a patient-derived in vitro model of infantile hemangioma and pre-clinical model of HLTRS we demonstrate the therapeutic potential of the R(+) enantiomer. Our work emphasizes the importance of SOX18 etiological role in vascular neoplasms, and suggests R(+)-propranolol repurposing to numerous indications ranging from vascular diseases to metastatic cancer.

LncRNA FTX inhibits hippocampal neuron apoptosis by regulating miR-21-5p/SOX7 axis in a rat model of temporal lobe epilepsy.

Emerging evidence has shown that long noncoding RNA (LncRNA) is involved in the development of epileptogenesis. However, the expression profile and the biological function of FTX in epilepsy remains unclear. This study aimed to provide functional evidence and elucidate the molecular mechanisms by which the FTX affects status epilepticus (SE) induced hippocampal apoptosis. SE rat model was introduced by intraperitoneal injection of lithium chloride and pilocarpine. Our results showed that FTX is notably reduced in the hippocampus. Moreover, the in vivo overexpression of FTX inhibited SE-induced hippocampus neuron apoptosis. Mechanically, we found that FTX negatively regulated miR-21-5p expression by targeting its 3'UTR to regulate neuron apoptosis. Upregulation of miR-21-5p attenuates anti-apoptosis property of FTX overexpression by regulating SOX7 expression in epileptiform hippocampal neurons. Collectively, our study for the first time demonstrated the anti-apoptosis ability of FTX during epileptogenesis and uncovered a novel FTX-mediated mechanism in SE-induced neural apoptosis by targeting miR-21-5p/SOX7 axis, which provides a new target in developing lncRNA-based strategies to reduce SE-induced hippocampal neuron apoptosis.

Dynamically and epigenetically coordinated GATA/ETS/SOX transcription factor expression is indispensable for endothelial cell differentiation.

Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine.

Small-Molecule Inhibitors of the SOX18 Transcription Factor.

Pharmacological modulation of transcription factors (TFs) has only met little success over the past four decades. This is mostly due to standard drug discovery approaches centered on blocking protein/DNA binding or interfering with post-translational modifications. Recent advances in the field of TF biology have revealed a central role of protein-protein interaction in their mode of action. In an attempt to modulate the activity of SOX18 TF, a known regulator of vascular growth in development and disease, we screened a marine extract library for potential small-molecule inhibitors. We identified two compounds, which inspired a series of synthetic SOX18 inhibitors, able to interfere with the SOX18 HMG DNA-binding domain, and to disrupt HMG-dependent protein-protein interaction with RBPJ. These compounds also perturbed SOX18 transcriptional activity in a cell-based reporter gene system. This approach may prove useful in developing a new class of anti-angiogenic compounds based on the inhibition of TF activity.

Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice.

Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics.

Structure and decoy-mediated inhibition of the SOX18/Prox1-DNA interaction.

The transcription factor (TF) SOX18 drives lymphatic vessel development in both embryogenesis and tumour-induced neo-lymphangiogenesis. Genetic disruption of Sox18 in a mouse model protects from tumour metastasis and established the SOX18 protein as a molecular target. Here, we report the crystal structure of the SOX18 DNA binding high-mobility group (HMG) box bound to a DNA element regulating Prox1 transcription. The crystals diffracted to 1.75Å presenting the highest resolution structure of a SOX/DNA complex presently available revealing water structure, structural adjustments at the DNA contact interface and non-canonical conformations of the DNA backbone. To explore alternatives to challenging small molecule approaches for targeting the DNA-binding activity of SOX18, we designed a set of five decoys based on modified Prox1-DNA. Four decoys potently inhibited DNA binding of SOX18 in vitro and did not interact with non-SOX TFs. Serum stability, nuclease resistance and thermal denaturation assays demonstrated that a decoy circularized with a hexaethylene glycol linker and terminal phosphorothioate modifications is most stable. This SOX decoy also interfered with the expression of a luciferase reporter under control of a SOX18-dependent VCAM1 promoter in COS7 cells. Collectively, we propose SOX decoys as potential strategy for inhibiting SOX18 activity to disrupt tumour-induced neo-lymphangiogenesis.

Silencing NKD2 by promoter region hypermethylation promotes gastric cancer invasion and metastasis by up-regulating SOX18 in human gastric cancer.

Naked cuticle homolog2 (NKD2) is located in chromosome 5p15.3, which is frequently loss of heterozygosity in human colorectal and gastric cancers. In order to understand the mechanism of NKD2 in gastric cancer development, 6 gastric cancer cell lines and 196 cases of human primary gastric cancer samples were involved. Methylation specific PCR (MSP), gene expression array, flow cytometry, transwell assay and xenograft mice model were employed in this study. The expression of NKD1 and NKD2 was silenced by promoter region hypermethylation. NKD1 and NKD2 were methylated in 11.7% (23/196) and 53.1% (104/196) in human primary gastric cancer samples. NKD2 methylation is associated with cell differentiation, TNM stage and distant metastasis significantly (all P < 0.05), and the overall survival time is longer in NKD2 unmethylated group compared to NKD2 methylated group (P < 0.05). Restoration of NKD2 expression suppressed cell proliferation, colony formation, cell invasion and migration, induced G2/M phase arrest, and sensitized cancer cells to docetaxel. NKD2 inhibits SOX18 and MMP-2,7,9 expression and suppresses BGC823 cell xenograft growth. In conclusion, NKD2 methylation may serve as a poor prognostic and chemo-sensitive marker in human gastric cancer. NKD2 impedes gastric cancer metastasis by inhibiting SOX18.

Sox17 inhibits hepatocellular carcinoma progression by downregulation of KIF14 expression.

Sox17, an antagonist of canonical Wnt/ß-catenin signaling, inhibits several malignant carcinogenesis and progression. However, little is known about Sox17 in hepatocellular carcinoma (HCC). Here, we found that Sox17 is downregulated in HCC tissue. Furthermore, Sox17 inhibits cell proliferation and migration in HCC. KIF14, a member of kinesin superfamily protein (KIFs), is an oncogene in a variety of malignant tumors including HCC. We demonstrated that Sox17 is negatively related to KIF14 expression in HCC tissue and Sox17 inhibits HCC cell proliferation and migration by transcriptional downregulation of KIF14 expression. Our results may provide a strategy for blocking HCC carcinogenesis and progression.

Genetic ablation of SOX18 function suppresses tumor lymphangiogenesis and metastasis of melanoma in mice.

The lymphatic vasculature provides a major route for tumor metastasis and inhibiting neolymphangiogenesis induced by tumors can reduce metastasis in animal models. Developmental biology studies have identified the transcription factor SOX18 as a critical switch for lymphangiogenesis in the mouse embryo. Here, we show that SOX18 is also critical for tumor-induced lymphangiogenesis, and we show that suppressing SOX18 function is sufficient to impede tumor metastasis. Immunofluorescence analysis of murine tumor xenografts showed that SOX18 is reexpressed during tumor-induced neolymphangiogenesis. Tumors generated by implantation of firefly luciferase-expressing B16-F10 melanoma cells exhibited a reduced rate of metastasis to the regional draining lymph node in Sox18-deficient mice, as assessed by live bioluminescence imaging. Lower metastatic rates correlated with reduced tumoral lymphatic vessel density and diameter and with impaired drainage of peritumoral injected liposomes specific for lymph vessels from the sentinel lymph nodes. Overall, our findings suggested that SOX18 induction is a key step in mediating tumor lymphangiogenesis and metastasis, and they identify SOX18 as a potential therapeutic target for metastatic blockade.

The transcription factor SOX18 regulates the expression of matrix metalloproteinase 7 and guidance molecules in human endothelial cells.

BACKGROUND: Mutations in the transcription factor SOX18 are responsible for specific cardiovascular defects in humans and mice. In order to gain insight into the molecular basis of its action, we identified target genes of SOX18 and analyzed one, MMP7, in detail. METHODOLOGY/PRINCIPAL FINDINGS: SOX18 was expressed in HUVEC using a recombinant adenoviral vector and the altered gene expression profile was analyzed using microarrays. Expression of several regulated candidate SOX18 target genes was verified by real-time PCR. Knock-down of SOX18 using RNA interference was then used to confirm the effect of the transcription factor on selected genes that included the guidance molecules ephrin B2 and semaphorin 3G. One gene, MMP7, was chosen for further analysis, including detailed promoter studies using reporter gene assays, electrophoretic mobility shift analysis and chromatin-immunoprecipitation, revealing that it responds directly to SOX18. Immunohistochemical analysis demonstrated the co-expression of SOX18 and MMP7 in blood vessels of human skin. CONCLUSIONS/SIGNIFICANCE: The identification of MMP7 as a direct SOX18 target gene as well as other potential candidates including guidance molecules provides a molecular basis for the proposed function of this transcription factor in the regulation of vessel formation.

SRY-box containing gene 17 regulates the Wnt/ß-catenin signaling pathway in oligodendrocyte progenitor cells.

The SRY-box (Sox) transcription factors regulate oligodendrocyte differentiation, but their signaling targets are largely unknown. We have identified a major signal transduction pathway regulated by Sox containing gene 17 (Sox17) in the oligodendrocyte lineage. Microarray analysis in oligodendrocyte progenitor cells (OPCs) after Sox17 attenuation revealed upregulated genes associated with cell cycle control and activation of the Wingless and integration site (Wnt)/ß-catenin pathway. Sox17 knockdown also increases the levels of cyclin D1, Axin2, and activated ß-catenin. In OPCs, the expression pattern of Sox17, cyclin D1, and secreted Frizzled-related protein-1 in the presence of platelet-derived growth factor (PDGF) was coordinately accelerated by addition of thyroid hormone, indicating differentiation-induced regulation of Sox17 targets. In developing white matter, decreased total ß-catenin, activated ß-catenin, and cyclin D1 levels coincided with the peak of Sox17 expression, and immunoprecipitates showed a developmentally regulated interaction among Sox17, T-cell transcription factor 4, and ß-catenin proteins. In OPCs, PDGF stimulated phosphorylation of glycogen synthase 3ß and the Wnt coreceptor LRP6, and enhanced ß-catenin-dependent gene expression. Sox17 overexpression inhibited PDGF-induced TOPFLASH and cyclin D1 promoter activity, and decreased endogenous cyclin D1, activated ß-catenin, as well as total ß-catenin levels. Recombinant Sox17 prevented Wnt3a from repressing myelin protein expression, and inhibition of Sox17-mediated proteasomal degradation of ß-catenin blocked myelin protein induction. These results indicate that Sox17 suppresses cyclin D1 expression and cell proliferation by directly antagonizing ß-catenin, whose activity in OPCs is stimulated not only by Wnt3a, but also by PDGF. Our identification of downstream targets of Sox17 thus defines signaling pathways and molecular mechanisms in OPCs that are regulated by Sox17 during cell cycle exit and the onset of differentiation in oligodendrocyte development.

SOX7, down-regulated in colorectal cancer, induces apoptosis and inhibits proliferation of colorectal cancer cells.

The sex-determining region Y-box 7 (Sox7) is a member of high mobility group (HMG) transcription factor family, essential for embryonic development and endoderm differentiation. Deregulation of Wnt signaling pathway is a hallmark of colorectal cancer. Our results showed that the expression level of SOX7 was frequently down-regulated in human colorectal cancer cell lines and in primary colorectal tumor tissues, and the SOX7 silencing was partially due to the aberrant DNA methylation of the gene. Restoration of SOX7 induced colorectal cancer cell apoptosis, inhibited cell proliferation and colony formation. In addition, SOX7 efficiently suppressed beta-catenin-mediated transcriptional activity.