Analysis of regulatory cis-elements and FOXA1/GATA2 transcription factors binding behind MRP4/ABCC4 levels in pancreatic cancer

GOMEZ, NATALIA; GONZALEZ, BETINA; SAHORES, ANA; DAVIO, CARLOS; GANCEDO, SAMANTA N.; DE SOUSA FERRO, MAXIMILIANO

Congreso; Reunion conjunta SAIC SAI AAFE NANOMEDar; 2021

The multidrug resistance-associated protein 4 MRP4/ABCC4 is highly expressed in pancreatic ductal adenocarcinoma (PDAC), and linked to increased proliferation, a mesenchymal phenotype and poor prognosis. This study aimed to determine epigenetic and molecular mechanisms that control ABCC4 expression levels in PDAC. Supported by bibliographic data, we selected pioneer transcription factors (TFs) FOXA1 and GATA2 as candidates to regulate low vs high ABCC4 expression. We queried ChIP-seq and RNA-seq data available from PDAC cell lines in GEO, and from patients tumors at TCGA (PAAD database). ABCC4 showed negative correlation with FOXA1 and positive correlation with GATA2 in PDAC cell lines and patients tumors. Next, we searched for regulatory cis-elements in ABCC4 gene by looking for enhancer marks H3K27ac/H3K4me enrichment, indicative of active clusters of TF binding sites, and also analyzed FOXA1 and GATA2 enrichment available in PDAC and prostate cell lines. We detected two candidate clusters, one in the distal promoter (-13.5 kb) and one in the intron1 (29.9 kb), that showed positive enrichment for both FOXA1 and GATA2, and different epigenetic landscape in low vs high ABCC4-expressing PDAC cell lines. To test the in silico results, we generated low-grade HPAF2 and high-grade PANC1 xenografts in NGS mice, and evaluated FOXA1 and GATA2 immunostaining and enrichment at the selected clusters in ABCC4 gene (ChIP-qPCR). HPAF2 tumors showed glandular differentiation and high FOXA1 staining, whereas PANC1 tumors showed compact indifferentiated cells and high GATA2 staining. Consistently, we found specific FOXA1 enrichment in HPAF2, and specific GATA2 binding in PANC1, at the selected clusters. These findings characterize the role of pro-epithelial pioneer TF FOXA1 in maintaining low levels of ABCC4 expression in low-grade PDAC, and a novel role of GATA2, which could mediate the increased ABCC4 expression in high-grade PDAC and contribute to the mesenchymal phenotype.