Abstract
Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype characterised by a poor outcome when compared to other breast cancers. The lack of safe and targeted therapy for TNBC promotes the need for the development of novel and efficacious anticancer agents. Our laboratory developed RL71, a second-generation curcumin analogue, which demonstrated potent anticancer activity. However, the hydrophobic nature of RL71 limited its bioavailability and impaired its clinical application. The encapsulation of RL71 into a copoly (styrene-maleic acid) micelles (SMA-RL71) improved the anticancer effect of RL71 in vivo. In the current study, we examined the gene expression profile in response to RL71 and SMA-RL71, respectively, in MDA-MB-231, MDA-MB-468 and HS578t TNBC cell lines using total RNA sequencing. Differential expression (DE) analysis was conducted using empirical analysis of digital gene expression data in R (edgeR). P values reported by edgeR were used to calculate false discovery rates (FDR) for each gene by the method of Benjamini and Hochberg. Fold-changes were reported as the log (base 2) of normalized count samples. Genes with a FDR <0.05 and at least a 2 fold change were considered to be differentially expressed between groups with and without drug treatment. In the RNA sequencing study, we identified 1546 upregulated genes and 453 downregulated genes in response to RL71, and 1210 upregulated and 396 downregulated genes in response to SMA-RL71 in MDA-MB-231 cells. In HS578t cells, a total of 5,925 genes were differentially expressed with 3,459 upregulated and 2,466 downregulated in response to RL71, while a total of 5,786 genes were affected by SMA-RL71, 3,263 of which were upregulated and 2,523 were downregulated. In MDA-MB-468 cells, a total of 536 genes and 525 genes were identified to be differentially expressed in response to RL71 and SMA-RL71, respectively. Furthermore, network analysis of differentially expressed genes (DEGs) found that the treatment of RL71 or SMA-RL71 significantly affect a set of genes associated with PI3K-Akt, MAPK, JAK-STAT, WNT, p53 signalling pathways. Some PI3K-Akt signalling pathway-linked genes were downregulated (PRKCA, IKBKB, IKBKG, and Akt) by RL71 and SMA-RL71, and a series of genes related to the serine/threonine protein phosphatase (PP2A), such as PPP2R1A, PPP2R3B and PPP2R2A, were steadily upregulated by RL71 and SMA-RL71 in MDA-MB-231 or/and HS578t cells. MEK1/2, MEKK4, MKK4, GADD45B, DUSP2 and MNK2 represent the major key regulators in MAPK signalling pathway, and these genes showed differential expression in response to RL71 and SMA-RL71 in MDA-MB-231 or/and HS578t cells. Some JAK-STAT pathway related genes, such as STAT3, STAT5A, STAT5B, PTPN11, PIAS4, and SOCS2, differentially expressed on addition of RL71 and SMA-RL71 in MDA-MB-231 or/and HS578t cells. A number of genes that regulates the WNT signalling pathway (e.g. WNT5A, WNT3, FZD7, Pontin 52, STBM, JUK3 and DVL1) were up- or down-regulated following the treatment of RL71 and SMA-RL71 in MDA-MB-231 or/and HS578t cells. In addition, a set of p53 signalling pathway linked genes (GADD45B, cyclin G2, and p21) were upregulated on addition of RL71 and SMA-RL71 in both MDA-MB-231 and HS578t cells. Therefore, multiple cancer-related molecular signalling pathways were identified to be modulated by RL71 and SMA-RL71. To support the genetic changes, a few proteins were analysis by Western blotting. For instance, the expression of JAK2 and JAK3 were reduced by RL71 and SMA-RL71. The expression of STAT3 was not affected by RL71 and SMA-RL71, but the pSTAT3 were significantly downregulated following the treatment of RL71 and SMA-RL71 in HS578t cells. Moreover, a panel of six genes (INPP4B, GNL1, CALD1, AQP3, MICB, and DNAH14) were differentially expressed across all the cell lines, suggesting these genes might provide future targets in the regulation of TNBCs.