Increased expression of FOXD1 is associated with cervical node metastasis and unfavorable prognosis in oral squamous cell carcinoma.

BACKGROUND: Previous studies suggest that FOXD1 is involved in tumorigenesis and closely related to the patients' poor outcome in human cancer. However, its expression pattern in primary oral squamous cell carcinoma (OSCC) remains uncovered. In this study, we tried to explore the expression pattern of FOXD1 and its clinicopathological significance in primary OSCC. METHODS: Data mining and analysis on FOXD1 mRNA expression in OSCC samples were performed using publicly available databases. Its protein expression was supervised by immunohistochemistry in a retrospective cohort containing 58 primary OSCC samples. Furthermore, the potential associations between FOXD1 expression and various clinicopathological characteristics and patients' survival were further investigated. RESULTS: Bioinformatic analysis indicated that FOXD1 mRNA abundance was obviously up-regulated in OSCC cohorts. Immunohistochemical staining results showed that FOXD1 protein was significantly up-regulated in OSCC specimens as compared to normal counterparts and its aberrant up-regulation was remarkably related to cervical lymph node metastasis (P = .0198) and decreased overall survival (P = .0281) and disease-free survival (P = .0312). Both univariate and multivariate Cox regression analysis further revealed the expression pattern of FOXD1 as an independent prognostic factor for overall survival of patients. CONCLUSION: Taken together, these findings indicate that the aberrant up-regulation of FOXD1 is related to cervical node metastasis and unfavorable prognosis in OSCC and it also may play a key role during OSCC tumorigenesis and regard as a novel diagnostic and prognostic biomarker for OSCC.

Sox, Fox, and Lmx1b binding sites differentially regulate a Gdf5-Associated regulatory region during elbow development.

Introduction: The articulating ends of limb bones have precise morphology and asymmetry that ensures proper joint function. Growth differentiation factor 5 (Gdf5) is a secreted morphogen involved in cartilage and bone development that contributes to the architecture of developing joints. Dysregulation of Gdf5 results in joint dysmorphogenesis often leading to progressive joint degeneration or osteoarthritis (OA). The transcription factors and cis-regulatory modules (CRMs) that regulate Gdf5 expression are not well characterized. We previously identified a Gdf5-associated regulatory region (GARR) that contains predicted binding sites for Lmx1b, Osr2, Fox, and the Sox transcription factors. These transcription factors are recognized factors involved in joint morphogenesis and skeletal development. Methods: We used in situ hybridization to Gdf5, Col2A1, and the transcription factors of interest in developing chicken limbs to determine potential overlap in expression. We further analyzed scRNA-seq data derived from limbs and knees in published mouse and chicken datasets, identifying cells with coexpression of Gdf5 and the transcription factors of interest. We also performed site-directed mutatgenesis of the predicted transcription factor binding sites in a GARR-reporter construct and determined any change in activity using targeted regional electroporation (TREP) in micromass and embryonic chicken wing bioassays. Results: Gdf5 expression overlapped the expression of these transcription factors during joint development both by in situ hybridization (ISH) and scRNA-seq analyses. Within the GARR CRM, mutation of two binding sites common to Fox and Sox transcripstion factors reduced enhancer activity to background levels in micromass cultures and in ovo embryonic chicken wing bioassays, whereas mutation of two Sox-only binding sites caused a significant increase in activity. These results indicate that the Fox/Sox binding sites are required for activity, while the Sox-only sites are involved in repression of activity. Mutation of Lmx1b binding sites in GARR caused an overall reduction in enhancer activity in vitro and a dorsal reduction in ovo. Despite a recognized role for Osr2 in joint development, disruption of the predicted Osr2 site did not alter GARR activity. Conclusion: Taken together, our data indicates that GARR integrates positive, repressive, and asymmetrical inputs to fine-tune the expression of Gdf5 during elbow joint development.