Synergistic anti-proliferative activity of JQ1 and GSK2801 in triple-negative breast cancer

Abstract

Background: Triple-negative breast cancer (TNBC) constitutes 10-20% of breast cancers and is challenging to treat due to a lack of effective targeted therapies. Previous studies in TNBC cell lines showed in vitro growth inhibition when JQ1 or GSK2801 were administered alone, and enhanced activity when co-administered. Given their respective mechanisms of actions, we hypothesized the combinatorial effect could be due to the target genes affected. Hence the target genes were characterized for their expression in the TNBC cell lines to prove the combinatorial effect of JQ1 and GSK2801.

Methods: RNASeq data sets of TNBC cell lines (MDA-MB-231, HCC-1806 and SUM-159) were analyzed to identify the differentially expressed genes in single and combined treatments. The topmost downregulated genes were characterized for their downregulated expression in the TNBC cell lines treated with JQ1 and GSK2801 under different dose concentrations and combinations. The optimal lethal doses were determined by cytotoxicity assays. The inhibitory activity of the drugs was further characterized by molecular modelling studies.

Results: Global expression profiling of TNBC cell lines using RNASeq revealed different expression patterns when JQ1 and GSK2801 were co-administered. Functional enrichment analyses identified several metabolic pathways (i.e., systemic lupus erythematosus, PI3K-Akt, TNF, JAK-STAT, IL-17, MAPK, Rap1 and signaling pathways) enriched with upregulated and downregulated genes when combined JQ1 and GSK2801 treatment was administered. RNASeq identified downregulation of PTPRC, MUC19, RNA5-8S5, KCNB1, RMRP, KISS1 and TAGLN (validated by RT-qPCR) and upregulation of GPR146, SCARA5, HIST2H4A, CDRT4, AQP3, MSH5-SAPCD1, SENP3-EIF4A1, CTAGE4 and RNASEK-C17orf49 when cells received both drugs. In addition to differential gene regulation, molecular modelling predicted binding of JQ1 and GSK2801 with PTPRC, MUC19, KCNB1, TAGLN and KISS1 proteins, adding another mechanism by which JQ1 and GSK2801 could elicit changes in metabolism and proliferation.

Conclusion: JQ1-GSK2801 synergistically inhibits proliferation and results in selective gene regulation. Besides suggesting that combinatorial use could be useful therapeutics for the treatment of TNBC, the findings provide a glimpse into potential mechanisms of action for this combination therapy approach.

Keywords: Breast cancer; Differential expression analysis; Drug resistance; Expression studies; MTT assay; RNASeq.

Fig. 1

A. Schematic diagram of methods and tools. The steps/tools used to identify differentially expressed genes, along with their subsequent validation through computational and in vitro methods. B. Differential expression analysis of RNASeq data. Bar plots depicting the number of upregulated/downregulated DEGs in treated samples (JQ1, GSK2801 and JQ1&GSK2801) compared to control across different TNBC cell lines

Fig. 2

Functional enrichment analysis of DEGs identified in MDA-MB-231 cells. Metabolic pathways enriched with (A) upregulated (B) downregulated genes in JQ1 treatment. Metabolic pathways enriched with (C) upregulated (D) downregulated genes from JQ1 + GSK2801 combined treatment

Fig. 3

Functional enrichment analysis of DEGs identified in HCC-1806 cells. Metabolic pathways enriched with (A) upregulated (B) downregulated genes from JQ1 treatment. Metabolic pathways enriched with (C) upregulated (D) downregulated genes from JQ1 + GSK2801 combined treatment

Fig. 4

Functional enrichment analysis of DEGs from identified from SUM-159 cells. Metabolic pathways enriched with (A) upregulated (B) downregulated genes from JQ1 treatment. Metabolic pathways enriched with (C) upregulated genes from GSK2801 treatment. (D) upregulated and (E) downregulated genes from JQ1 + GSK2801 combined treatment

Fig. 5

A. Homology modelling of PTPRC, MUC19, KCNB1, TAGLN and KISS1 protein. The optimized conformations of five downregulated protein structures represented as cartoon models. The reactive binding domains were constructed for MUC19 and KISS1 proteins due to the lack of template availability. B. Molecular docking of JQ1 and GSK2801 against PTPRC, MUC19, KCNB1, TAGLN and KISS1 proteins. Binding mode orientation of JQ1 (Pink) and GSK2801 (Cyan) with downregulated proteins in TNBC. The ligands are shown in the binding site cavities of target proteins

Fig. 6

A. Cytotoxicity assays of JQ1 and GSK2801 against three TNBC cell lines. A. Viability curves explaining the cytotoxic effect of JQ1 and GSK2801 when treated alone on MDA-MB-231, HCC-1806 and SUM-159 TNBC cell lines. B. Viability curves explaining the cytotoxic effect of combined treatments of JQ1 and GSK2801 on MDA-MB-231, HCC-1806 and SUM-159 cell lines demonstrating the synergistic effect. Values are given as mean of three independent experiments ± SD. B. Quantification of the cell density. Bar plots showing the cell densities measured after the treatment. There is a progressive decrease in the cell density with increasing drug concertation. The cell densities are lower in the combined treatment when compared to single agent treatment

Fig. 7

Microscopic images demonstrating the decrease in cell density. With increase in the concentration of drugs (left to right) there is a progressive decrease in density of the cells which indicated a steady death of cancer cells. The cell density is lesser in the combined treatments when compared to the single agent treatment

Fig. 8

Validation of gene expression in three TNBC cell lines. A. Effect of JQ1 and GSK2801 on TAGLN and KISS1 genes in the MDA-MB-231 cells showing the downregulation in the expression both in the single and combined treatments. B. Effect of JQ1 and GSK2801 on MUC19 and KCNB1 genes in SUM-159 cells showing the downregulation in the expression. MUC19 was found to be upregulated with a higher concentration of JQ1 (250 nM). C. Effect of JQ1 and GSK2801 on MUC19, KCNB1 and KISS1 genes in HCC-1806 cells lines. MUC19 was upregulated by GSK2801 (10 µM). The increase in the JQ1 concentration in the combined treatments increased the expression of KCNB1. KISS1 was observed to be downregulated in all the single and combined treatments. Values are given as mean of three independent experiments ± SD. Statistical significance were defined at *p < 0.05 compared to DMSO control. DMSO: control, J: JQ1, G: GSK2801. JQ1 doses are in nM and GSK2801 in µM