Super-enhancers: novel target for pancreatic ductal adenocarcinoma

Abstract

Super-enhancers (SEs) are unique areas of the genome which drive high-level of transcription and play a pivotal role in the cell physiology. Previous studies have established several important genes in cancer as SE-driven oncogenes. It is likely that oncogenes may hack the resident tissue regenerative program and interfere with SE-driven repair networks, leading to the specific pancreatic ductal adenocarcinoma (PDAC) phenotype. Here, we used ChIP-Seq to identify the presence of SE in PDAC cell lines. Differential H3K27AC marks were identified at enhancer regions of genes including c-MYC, MED1, OCT-4, NANOG, and SOX2 that can act as SE in non-cancerous, cancerous and metastatic PDAC cell lines. GZ17-6.02 affects acetylation of the genes, reduces transcription of major transcription factors, sonic hedgehog pathway proteins, and stem cell markers. In accordance with the decrease in Oct-4 expression, ChIP-Seq revealed a significant decrease in the occupancy of OCT-4 in the entire genome after GZ17-6.02 treatment suggesting the possible inhibitory effect of GZ17-6.02 on PDAC. Hence, SE genes are associated with PDAC and targeting their regulation with GZ17-6.02 offers a novel approach for treatment.

Keywords: ChIP-Seq; PDAC; cancer stem cells; super-enhancers; therapeutics.

Figure 1. Binding activity of SEs in non-cancerous (HPNE) and in PDAC cells (MiaPaCa-2 and S2-007) using histone mark H3K27ac

PDAC cells demonstrated significant binding of different enhancers in PDAC cells than non-cancerous cells, (A) Binding with SOX2, FOXO-1, CDX-2, KLF-4, MED-1, PARD6B, KLF-7, MYC, OCT-4, SOX-9, USP12, INPP5D and (C) with SHH pathway using ChIP-Seq with the H3K27me3 antibody. (B and D) TCGA data also suggested a higher expression of those genes in PDAC compared with normal tissue.

Figure 1. Binding activity of SEs in non-cancerous (HPNE) and in PDAC cells (MiaPaCa-2 and S2-007) using histone mark H3K27ac

PDAC cells demonstrated significant binding of different enhancers in PDAC cells than non-cancerous cells, (A) Binding with SOX2, FOXO-1, CDX-2, KLF-4, MED-1, PARD6B, KLF-7, MYC, OCT-4, SOX-9, USP12, INPP5D and (C) with SHH pathway using ChIP-Seq with the H3K27me3 antibody. (B and D) TCGA data also suggested a higher expression of those genes in PDAC compared with normal tissue.

Figure 2. Detection of SEs related gene expression using heat map of RNAseq

(A and B) following treatment with GZ17-6.02, S2-007 cells demonstrated impairment of expressions SEs related genes including pluripotent stem cell and cancer stem cell markers after treatment. (C) The CSC markers, (D) SHH pathway and (E) pancosphere formation significantly affected by GZ17-6.02 treatment.

Figure 3. Effect on SEs following different doses of GZ17-6.02

(A) The treatment of S2-007 PDAC cells by GZ17-6.02 in higher doses (20 μg/ml) showing the TSS site of input using H3K27ac antibody, (B) whereas log curve showing SEs affected in higher doses. (C) The motif of KLF4 and JUN-AP1 were affected by the treatment.

Figure 4. Detection of reduction of binding of OCT-4 (enhancer) at different sites associated with SEs following treatment with GZ17-6.02

(A) Occupancy of OCT-4 throughout the genome and the effect of GZ17-6.02 treatment on it. (B) OCT-4 signal (peaks) associated with the oncogenes, master transcription factors and SHH pathway indicating its occupancy at these regions that is significantly affected by GZ17-6.02 in different doses. (C) Pie chart of the treated groups compared to untreated groups.

Figure 5. GZ17-6.02 inhibit tumor growth in vivo

(A and B) Treatment of GZ17-6.02 (100 mg/kg) on orthotopic mouse tumor showing fluorescence, there were significant differences of Td-tomato fluorescence in tumors of control and treated mice (N = 6) after 20 days of treatment, (C) Tumor ascites fluid decreased in treated group with comparison to control (arrow), control mouse has larger ascites. (D, E and F) Size of tumors is significantly inhibited by the treatment in comparison with control or untreated animals. Bar = 1 cm. (G and H) Effect of GZ17-06.12 on metastatic markers Matrix metallopeptidase (MMP)-9 and MMP-2 in primary pancreatic tumor and metastatic spleen, liver and lung tissues of control and treated groups. (G) Western blots showing a decrease in expression of active MMP-9 and MMP-2 in the treated group than control, (H) Zymography, showing the clear bands, which depicts the reaction of active MMPs with their substrate gelatin showing by arrows.

Figure 6. Schematic representation of effects of GZ17-6.02 inhibiting SE following tumorigenesis in PDAC