Epithelial Transcription Factor FOXA1 Regulates Prostate Cancer Immune Response

Abstract

Background: While localized prostate cancer (PCa) can be mitigated by surgery and radiation, metastatic PCa remains a challenge to treat. Androgen deprivation therapies and androgen receptor (AR) pathway inhibitors are mainstay treatments for advanced PCa. Yet, resistance often develops leading to castration-resistant prostate cancer (CRPC). Forkhead Box A1 (FOXA1) is a pioneer transcription factor that plays pivotal roles in regulating AR activity and promoting epithelial differentiation. Studies have shown that FOXA1 is frequently downregulated in CRPC tumors. Congruently, FOXA1 loss is reported to induce aberrant AR signaling, epithelial-mesenchymal transition, and PCa de-differentiation. However, the role of FOXA1 in regulating PCa immune response, an area of much interest recently, has not been reported. CRPC has shown poor response to immune checkpoint inhibitors, due to its immunosuppressive nature. A better understanding of the tumor intrinsic mechanisms regulating PCa tumor immunity will inform the design of better targeted immunotherapeutic approaches. Methods: We performed RNA-seq, ChIP-seq, qPCR, western blot, and ELISA analyses to evaluate how FOXA1 regulates inflammatory response genes. We utilized an in vitro macrophage infiltration transwell assay, in which M2-like macrophages were added to the upper chamber and PCa cells were plated in the lower chamber, to examine how perturbations to PCa cells affect macrophage migration. Finally, we performed bioinformatic analyses of patient datasets to confirm the clinical relevance of FOXA1 repression of inflammatory genes in PCa. Results: Through integration of RNA-seq and ChIP-seq data, we uncovered a novel function of FOXA1 in suppressing inflammatory response pathways. In accordance, patient data analyses revealed that inflammatory response genes were upregulated in FOXA1-low PCa tumors. Mechanistically, we showed that FOXA1 proteins bound an intragenic enhancer of Hypoxia-inducible factor 1-alpha (HIF1A) gene to directly repress its expression, such that FOXA1 loss induced HIF1A upregulation. We further showed that Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) became upregulated upon FOXA1 depletion in a HIF1A-dependent manner. This led to infiltration by immunosuppressive, tumor promoting M2-like macrophages. Inhibiting this HIF1A-CCL2 axis with a HIF1A inhibitor or CCL2 neutralizing antibody blocked macrophage infiltration. Future studies using immunocompetent mouse models are needed to confirm the effect of FOXA1 on macrophage infiltration in vivo and evaluate the preclinical potential of targeting the FOXA1-HIF1A-CCL2 axis in CRPC. Conclusion: This study proposes a novel role for FOXA1 loss in promoting macrophage infiltration via the HIF1A-CCL2 axis. Moreover, our findings suggest that targeting this axis may be a promising approach for the treatment of FOXA1-low CRPC tumors.