Characterization of FOXF1 as a novel regulator of nodal metastasis in bladder cancer.

Abstract

480 Background: Members of the forkhead transcription factor (FOX) family are important mediators of embryonic development and are known to be altered in a variety of cancers. The functional role of FOXF1 in bladder tumorigenesis and progression has not been clearly characterized thus far. This study investigated the clinical implications of differential FOXF1 expression in bladder cancer, and potential mechanisms by which its alteration can lead to tumor metastasis. Methods: Whole genome expression profiling was performed on paired primary tumors and nodal metastases from a radical cystectomy discovery cohort using Illumina HT12 v3-4 BeadChip arrays to identify FOXF1 as a top differentially expressed gene. Prognostic role of differential FOXF1 expression was validated on two independent cystectomy cohorts. Differential FOXF1 expression was also evaluated in murine orthotopic xenografts. Small interfering RNA was used to knock down FOXF1 in RT112 and UC6 bladder cancer cell lines to develop an in vitro model for assessment of metastatic potential. Next-generation sequencing and hierarchical clustering analysis were used to identify differentially altered genes secondary to FOXF1 knockdown. 186 biologically curated pathways were interrogated with internal validation to elucidate the downstream biologic mechanisms of metastasis. Results: In the discovery cohort, FOXF1 was a top differentially expressed gene with 3.6-fold lower expression in nodal metastases than paired primary tumors (n = 33, p < 0.001). Multivariable analyses in two validation cohorts (total n = 128) indicated that FOXF1 underexpression was associated with worse cancer-specific (p = 0.046) and overall survival (p = 0.006). Murine orthotopic xenografts (n = 13) established from human bladder cancer cell lines (UC3, UC6, UC14) showed FOXF1 underexpression in metastatic deposits compared with primary tumors (p = 0.004). Hierarchical clustering identified 40 differentially expressed genes between FOXF1-knockdown bladder cancer cell lines and their corresponding controls. Biological pathway interrogation showed differential enrichment for genes associated with mitogen-activated protein kinase signaling, focal adhesion and other carcinogenic pathways in FOXF1-knockdown cells compared with controls (normalized enrichment score ≥ 1.3). Conclusions: We identify and characterize FOXF1 as a novel regulatory molecule that potentially drives bladder cancer metastasis. This may be modulated through alterations in intracellular signaling and cellular adhesion. FOXF1 may serve as a prognostic biomarker that can identify patients at impending risk for metastasis who may benefit from more aggressive management.