Targeting FOXA1-mediated repression of TGF-β signaling suppresses castration-resistant prostate cancer progression
Bing Song, … , Ximing Yang, Jindan Yu
Bing Song, … , Ximing Yang, Jindan Yu
Published December 4, 2018
Citation Information: J Clin Invest. 2019;129(2):569-582. https://doi.org/10.1172/JCI122367.
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Research Article Genetics Oncology

Targeting FOXA1-mediated repression of TGF-β signaling suppresses castration-resistant prostate cancer progression

Abstract

Prostate cancer (PC) progressed to castration resistance (CRPC) is a fatal disease. CRPC tumors develop resistance to new-generation antiandrogen enzalutamide through lineage plasticity, characterized by epithelial-mesenchymal transition (EMT) and a basal-like phenotype. FOXA1 is a transcription factor essential for epithelial lineage differentiation. Here, we demonstrate that FOXA1 loss leads to remarkable upregulation of transforming growth factor beta 3 (TGFB3), which encodes a ligand of the TGF-β pathway. Mechanistically, this is due to genomic occupancy of FOXA1 on an upstream enhancer of the TGFB3 gene to directly inhibit its transcription. Functionally, FOXA1 downregulation induces TGF-β signaling, EMT, and cell motility, which is effectively blocked by the TGF-β receptor I inhibitor galunisertib (LY2157299). Tissue microarray analysis confirmed reduced levels of FOXA1 protein and a concordant increase in TGF-β signaling, indicated by SMAD2 phosphorylation, in CRPC as compared with primary tumors. Importantly, combinatorial LY2157299 treatment sensitized PC cells to enzalutamide, leading to synergistic effects in inhibiting cell invasion in vitro and xenograft CRPC tumor growth and metastasis in vivo. Therefore, our study establishes FOXA1 as an important regulator of lineage plasticity mediated in part by TGF-β signaling, and supports a novel therapeutic strategy to control lineage switching and potentially extend clinical response to antiandrogen therapies.

Authors

Bing Song, Su-Hong Park, Jonathan C. Zhao, Ka-wing Fong, Shangze Li, Yongik Lee, Yeqing A. Yang, Subhasree Sridhar, Xiaodong Lu, Sarki A. Abdulkadir, Robert L. Vessella, Colm Morrissey, Timothy M. Kuzel, William Catalona, Ximing Yang, Jindan Yu

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Figure 2

FOXA1 protein binds to an upstream enhancer of TGFB3 to inhibit its transcription.

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FOXA1 protein binds to an upstream enhancer of TGFB3 to inhibit its tran...
(A) Genome browser view showing FOXA1 binding at an upstream TGFB3 enhancer. FOXA1 ChIP-seq was performed in LNCaP shCtr and shFOXA1 cells as previously described (12). The magnitudes of FOXA1 binding peak at this enhancer in shCtr and shFOXA1 were 189.5 and 139.2, respectively. (B) FOXA1 and IgG ChIP were performed in shCtrl and shFOXA1 LNCaP cells and subjected to qPCR using primers flanking the promoter (prom), enhancer (enh), and an intermediate (inter) region (as a negative control) of the TGFB3 gene. KIAA0066 was used as a negative control (n = 3, *P < 0.05). (C and D) ChIP of RNA Pol II (phosphorylated at Ser5) (C) and H3K4me3 (D) was performed in LNCaP cells with shCtr or shFOXA1 knockdown and subjected to qPCR analysis with primers for TGFB3 enhancer and KIAA0066 control gene. (E) Schema of CRISPR/Cas9 editing of FOXA1-bound TGFB3 enhancer region. Distance to TSS of the TGFB3 gene is labeled. The sgRNA sequences are shown in green and FKHD motifs in red. (F and G) LNCaP cells were infected with CRISPR/Cas9 along with control (ctr) or TGFB3 enhancer-targeting sgRNA1 and 2 (gRNA1+2), which were then subjected to control (shCtr) or FOXA1 (shFOXA1) knockdown. Total RNA and genomic DNA were isolated from a pooled population of cells under each condition. Genomic DNA were subjected to PCR using primers F and R as labeled in panel E. The top PCR band indicates the full-length WT PCR amplicons, whereas the lower band indicates the CRISPR-edited PCR amplicon, with about 25% editing rate (F). RNA were subjected to qRT-PCR to measure TGFB3 gene expression, and normalized to GAPDH (G).