Targets for Renal Carcinoma Growth Control Identified by Screening FOXD1 Cell Proliferation Pathways

Simple Summary FOXD1 regulates the proliferation of clear cell renal cell carcinoma (ccRCC) cells, and ccRCC cells in which FOXD1 has been inactivated do not form tumors efficiently in an animal model. Reproducing growth inhibition in tumor cells by inhibiting FOXD1 pathways presents a possible therapeutic approach for ccRCC and other cancers. We have established an analysis strategy to identify FOXD1-regulated target pathways that may be therapeutically tractable, and compounds that modulate these pathways were selected for testing. Targets in three pathways were identified: FOXM1, PME1, and TMEM167A, which were inhibited by compounds FDI-6, AMZ-30, and silibinin, respectively. The effects of these compounds on the growth of tumor cells from patients cultured in a novel 3D tumor-replica culture environment revealed that FDI-6 and silibinin had strong growth inhibitory effects. This investigation informs new therapeutic targets to control ccRCC tumor growth, and provides a strategy to compare the responsiveness of individual patient tumor replicas to growth-inhibitory compounds. Clinical association studies suggest that FOXD1 is a determinant of patient outcome in clear cell renal cell carcinoma (ccRCC), and laboratory investigations have defined a role for this transcription factor in controlling the growth of tumors through regulation of the G2/M cell cycle transition. We hypothesized that the identification of pathways downstream of FOXD1 may define candidates for pharmacological modulation to suppress the G2/M transition in ccRCC. We developed an analysis pipeline that utilizes RNA sequencing, transcription factor binding site analysis, and phenotype validation to identify candidate effectors downstream from FOXD1. Compounds that modulate candidate pathways were tested for their ability to cause growth delay at G2/M. Three targets were identified: FOXM1, PME1, and TMEM167A, which were targeted by compounds FDI-6, AMZ-30, and silibinin, respectively. A 3D ccRCC tumor replica model was used to investigate the effects of these compounds on the growth of primary cells from five patients. While silibinin reduced 3D growth in a subset of tumor replicas, FDI-6 reduced growth in all. This study identifies tractable pathways to target G2/M transition and inhibit ccRCC growth, demonstrates the applicability of these strategies across patient tumor replicas, and provides a platform for individualized patient testing of compounds that inhibit tumor growth.