G-quadruplex structures regulate long-range transcriptional reprogramming to promote drug resistance in ovarian cancer cells

Abstract

Background: Epigenetic evolution is a common mechanism used by cancer cells to evade the therapeutic effects of drug treatment. In ovarian cancers, epigenetically driven resistance is thought to be responsible for many late-stage patient deaths. DNA secondary structures called G-quadruplexes (G4s) are emerging as potential epigenetic marks of relevance to cancer evolution, but their prevalence and distribution in ovarian cancer models have never been investigated before.

Results: Here, we describe the first investigation of the role of G4s in the epigenetic regulation of drug-resistant ovarian cancer cells. Through genome-wide mapping of G4s in paired drug-sensitive and drug-resistant cell lines, we find that increased G4 accumulation is associated with enhanced transcription of signalling pathways previously established to promote drug-resistant states, including genes involved in the epithelial to mesenchymal transition and WNT signalling. In contrast to previous studies, the expression-enhancing effects of G4s are not found at gene promoters, but intergenic and intronic regions, indicating that G4s can promote long-range transcriptional regulation in drug-resistant cells. Furthermore, we discover that clusters of G4s (super-G4s) are associated with particularly high levels of transcriptional enhancement that surpass the effects of super-enhancers, which act as well-established regulatory sites in many cancers. Finally, we demonstrate that targeting G4s with small molecules results in significant downregulation of pathways associated with drug resistance, resulting in resensitization of resistant cells to chemotherapy agents.

Conclusions: These findings indicate that G4 structures are critical for the epigenetic regulatory networks of drug-resistant cells and represent a promising target to treat drug-tolerant ovarian cancer.

Keywords: G-quadruplexes; Ovarian cancer; Resistance; Transcription.

Fig. 1

A BG4 ChIP-qPCR with primers targeting G4 regions (MAZ, RPA3, KIF14, SPRED2) or non-G4 sites (TMCC1, IL36G) in chromatin extracted from PEO1 cells. BG4 immunoprecipitation results in an eightfold enrichment for G4 regions over non-G4 sites. B Most enriched sequence motif amongst top 1,000 G4 peaks in PEO1 and PEO4. Motif enrichment performed with MEME suite. C Genomic distribution of BG4 ChIP-seq peaks in PEO1 and PEO4 (annotated with HOMER), showing locations of all peaks and peaks unique to only PEO1 or PEO4. D Integration of ATAC, BG4-ChIP and RNA-seq data. Fold-change in expression (PEO4 relative to PEO1) of genes associated with gained ATAC peaks (that overlap OQS) or gained G4 peaks identified with BG4 ChIP-seq. Comparisons are made for ATAC and G4 peaks located at promoters, introns and intergenic regions. Statistical significance assessed by Mann–Whitney U test. Error bars show standard deviation of gene expression data collected in triplicate. FC, fold-change; ns, non-significant; ****p < 0.0001

Fig. 2

A Viability of PEO1^BRCA+ and PEO4 cells in increasing concentrations of cisplatin, treated for 72 h. Error bars are standard deviation of results collected in triplicate. B Genomic distribution of BG4 CUT&Tag peaks in PEO1^BRCA+ and PEO4, annotated with HOMER. All G4 peaks detected in PEO1/4 (PEO1/4-all) were compared to cell-type specific G4s (PEO1 only or PEO4 only). C Integration of ATAC, G4 CUT&Tag, and RNA-seq data in PEO1^BRCA+/PEO4. Fold-change in expression (PEO4 relative to PEO1) of genes associated with gained ATAC peaks (that overlap OQS) or gained G4 peaks identified with BG4 CUT&Tag. Comparisons are made for ATAC and G4 peaks located at promoters, introns, and intergenic regions. Statistical significance assessed by Mann–Whitney U test. Error bars show standard deviation of gene expression data collected in triplicate. FC, fold-change; *p < 0.05; ***p < 0.001; ****p < 0.0001

Fig. 3

A Genomic locations of enhancer peaks in PEO1^BRCA+ and PEO4, identified by H3K27ac CUT&Tag. The distribution of all peaks in either cell line is compared to peaks specific to either PEO1 or PEO4. B Fold-change in expression (PEO4 relative to PEO1) of genes associated with gained enhancers and super-enhancers. Enhancers are defined by H3K27ac peaks and super-enhancers by significant clusters of enhancers found in a 12.5 kb window, identified with the ranked ordering of super-enhancers (ROSE) algorithm [67, 68]. Genes associated with gained enhancer/super-enhancer peaks that overlap OQS are compared to genes proximal to enhancers that overlap G4 peaks identified with BG4 CUT&Tag. C Change of expression for genes associated with super-G4s regions, defined by ROSE as significant clusters of G4 CUT&Tag peaks at intronic or D intergenic regions. The expression of super-G4 linked genes is compared to genes associated with singular G4s or ATAC peaks that overlap OQS. Statistical significance assessed by Mann–Whitney U test. Error bars are standard deviation of gene expression measured across 3 biological replicates. FC, fold-change; ns, non-significant; ***p < 0.001; ****p < 0.0001

Fig. 4

A Comparison of the effects of putative G4 sequences on gene expression within three paired drug-sensitive and drug-resistant ovarian cancer cell lines: PEO1 and PEO4, PEA1 and PEA2, A2780 and cisA2780. The expression of genes associated with gained ATAC peaks was compared to genes that gained ATAC peaks also overlapping OQS (detected by G4-seq) at promoters, introns, and intergenic regions. Differences in expression between the two groups were assessed by p-value obtained from Mann–Whitney U test. Dotted line at p = 0.05. B Gene expression heat map of top 1,000 most differentially expressed genes between PEO1, PEO4, PEA1, PEA2, A2780, and cisA2780, based on previously acquired RNA-seq data. Samples clustered by average Pearson correlation. Pathway enrichment was conducted on genes uniquely expressed in PEO1/PEO4 and PEA1/PEA2 compared to A2780/cisA2780 revealing genes enriched in cell adhesion, motility and wound healing. Analysis performed in iDEP [76]

Fig. 5

A Cell viability of PEO1^BRCA+ and PEO4 in response to increasing concentrations of cisplatin with or without cotreatment with G4 ligand PDS (0.5 µM), incubated for 72 h. PDS significantly re-sensitizes PEO4 cells to cisplatin but has no effect on PEO1. Error bars are standard deviation of results collected in triplicate. B Heat map of top 1,000 most differentially expressed genes in PEO1 and PEO4 after 0, 2, and 6 h of PDS treatment (5 µM). C Number of significantly differentially expressed genes (|FC|> 1, FDR < 0.05) in PEO1 and PEO4 after 2 and 6 h of PDS (5 µM) treatment. D KEGG pathway enrichment of genes significantly downregulated in PEO1^BRCA+ and E PEO4 after 6 h PDS (5 µM) treatment. F Fold-change in gene expression for genes associated with promoter, G intronic, or H intergenic G4 peaks (identified by BG4 CUT&Tag), after treatment with PDS (5 µM) for 2 or 6 h. Expression is compared for genes proximal to G4 peaks in PEO1^BRCA+ and PEO4. Statistical significance assessed by Mann–Whitney U test. Error bars are standard deviation of gene expression data acquired in triplicate. FC, fold-change; ns, non-significant; *p < 0.05; ***p < 0.001; ****p < 0.0001

Fig. 6

Proposed mechanism of distal, G4-mediated regulation of gene expression in drug-resistant ovarian cancer cells. Clusters of G4s (super-G4s) at non-promoter sites may promote chromatin looping and recruit additional regulatory proteins, leading to the formation of membraneless condensates (depicted in grey) through liquid–liquid phase separation which in turn increase the expression of signalling proteins involved in drug responses. Incubation of cells with a G4 ligand (PDS) alters this molecular network, resulting in significant downregulation of genes associated with drug resistance and leading to a marked increase in drug sensitivity