Steroidogenic factor-1 regulates a core set of target genes to promote malignancy in adrenocortical carcinoma

Abstract Objective Gene dosage is at the core of the biological activity of the steroidogenic factor-1 (SF-1/NR5A1) transcription factor. Its overexpression in adrenocortical carcinoma (ACC) is associated with enhanced proliferation and invasive capacities, steroid modulation, immune suppression, and poor prognosis. Surprisingly, 3 independent studies showed less than 10% agreement in identifying SF-1–regulated genes in the same ACC cell line, raising concerns about technical reproducibility and methodological consistency. This study aimed to reconcile discrepancies in SF-1–regulated gene identification across independent studies using a systematic approach. Design and Methods We reanalysed datasets from those studies using an in silico SF-1 regulon obtained from ACC TCGA data as an external reference to evaluate transcriptional patterns. Additionally, we assessed how threshold selection impacts the overlap between experiments and optimized this process. Furthermore, we performed functional experiments to evaluate how variations in SF-1 dosage impact target gene expression. Results Our analysis revealed comparable transcriptional patterns across all studies, reconciling transcriptional signatures and phenotypes. Threshold optimization identified consensus sets of genes responsive to SF-1 perturbations. Functional experiments confirmed that variations in SF-1 dosage significantly impact gene expression, explaining discrepancies in previous studies, and evidenced negative autoregulation of the SF-1 transcript by its encoded protein both in ACC cells and in a mouse model of Sf-1 overexpression in the adrenal cortex. Conclusions Our findings deepen our understanding of SF-1 regulatory activity in ACC and demonstrate that dosage is critical for observed gene expression patterns. Our integrative approach improves reproducibility and biological interpretation, offering a framework to reconcile cross-study findings.