LncRNA DSCAM-AS1 interacts with YBX1 to promote cancer progression by forming a positive feedback loop that activates FOXA1 transcription network

Abstract

Rationale: The forkhead box A1 (FOXA1) is a crucial transcription factor in initiation and development of breast, lung and prostate cancer. Previous studies about the FOXA1 transcriptional network were mainly focused on protein-coding genes. Its regulatory network of long non-coding RNAs (lncRNAs) and their role in FOXA1 oncogenic activity remains unknown. Methods: The Cancer Genome Atlas (TCGA) data, RNA-seq and ChIP-seq data were used to analyze FOXA1 regulated lncRNAs. RT-qPCR was used to detect the expression of DSCAM-AS1, RT-qPCR and Western blotting were used to determine the expression of FOXA1, estrogen receptor α (ERα) and Y box binding protein 1 (YBX1). RNA pull-down and RIP-qPCR were employed to investigate the interaction between DSCAM-AS1 and YBX1. The effect of DSCAM-AS1 on malignant phenotypes was examined through in vitro and in vivo assays. Results: In this study, we conducted a global analysis of FOXA1 regulated lncRNAs. For detailed analysis, we chose lncRNA DSCAM-AS1, which is specifically expressed in lung adenocarcinoma, breast and prostate cancer. The expression level of DSCAM-AS1 is regulated by two super-enhancers (SEs) driven by FOXA1. High expression levels of DSCAM-AS1 was associated with poor prognosis. Knockout experiments showed DSCAM-AS1 was essential for the growth of xenograft tumors. Moreover, we demonstrated DSCAM-AS1 can regulate the expression of the master transcriptional factor FOXA1. In breast cancer, DSCAM-AS1 was also found to regulate ERα. Mechanistically, DSCAM-AS1 interacts with YBX1 and influences the recruitment of YBX1 in the promoter regions of FOXA1 and ERα. Conclusion: Our study demonstrated that lncRNA DSCAM-AS1 was transcriptionally activated by super-enhancers driven by FOXA1 and exhibited lineage-specific expression pattern. DSCAM-AS1 can promote cancer progression by interacting with YBX1 and regulating expression of FOXA1 and ERα.

Keywords: ERα; FOXA1; breast cancer; lncRNAs; lung adenocarcinoma; super-enhancer.

Figure 1

Identification of lineage specific, FOXA1-regulated lncRNAs. (A) Expression levels of FOXA1 in normal and tumor samples of The Cancer Genome Atlas. Each point represents one tissue sample. (B) Expression levels of FOXA1 in the Cancer Cell Line Encyclopedia project of cancer cell lines. Each point represents one cell line. (C) Venn diagram showing the screening of FOXA1-specific lncRNAs. Aberrantly overexpressed lncRNAs were analyzed in BRCA, PRAD and LUAD using the TCGA database. A total of 71 lncRNAs were highly expressed in these three cancer types (fold change [FC] ≥3, P <0.05). These lncRNAs were then overlapped with 214 FOXA1 induced lncRNAs in the T47D cell line (FC≥ 2, P <0.05) identified by RNA-seq. Five lncRNAs intersections were then analyzed for potential association in the promoter region with FOXA1 using ChIP-seq data in ENCODE datasets. (D) Expression levels of DSCAM-AS1 in The Atlas of Noncoding RNAs in Cancer project of normal and tumor samples. Each point represents one tissue sample. (E) Expression levels of DSCAM-AS1 in cancer cell lines of the Cancer Cell Line Encyclopedia project. Each point represents one cell line. (F-G) The correlation between FOXA1 and DSCAM-AS1 expression levels in BRCA (F) and LUAD (G) samples from TANRIC dataset.

Figure 2

DSCAM-AS1 is positively and directly regulated by FOXA1 driven super enhancers. (A) ChIP-seq profiles of H3K27ac, FOXA1 and ERα at representative super-enhancer-associated gene loci in lung adenocarcinoma and breast cancer cell lines. The predicted SEs are depicted as black bars. The y-axis represents reads per million (rpm) of ChIP-seq. The location of ChIP-qPCR primers is indicated with a short black line. The FOXA1 and ERα binding motif are shown on the bottom. (B) The heat map of Hi-C interactions generated using 3D Genome Browser (http://promoter.bx.psu.edu/hi-c/index.html). The SEs and transcriptional directions are indicated below the heat map. (C) The expression levels of DSCAM-AS1, c-Myc and GAPDH were analyzed by RT-qPCR after treating with different concentrations of JQ1 in MCF7 cells. (D) Indicated cell lines of BRCA, LUAD and PRAD were transfected with siNC and siFOXA1 pool, the expression level of DSCAM-AS1 were detected by RT-qPCR. (E-F) ChIP-qPCR of H3K27ac using different primers in MCF7 (E) and NCI-H1437 (F) cell lines, the locations of primers were indicated in (A). (G-H) ChIP-qPCR showed enrichment of FOXA1 in MCF7 (G) and NCI-H1437 cells (H). The position of qPCR is indicated in (A).

Figure 3

DSCAM-AS1 plays oncogenic roles through regulation of FOXA1 and ERα expression. (A, B) MTT (A) and colony formation assay (B) showed a significant reduction of cell growth after knocking down of DSCAM-AS1 with three individual siRNAs respectively. (C) Schematic diagram showed knocking out of DSCAM-AS1 using CRISPR/Cas9. (D) Decrease of colony formation after knocking out of DSCAM-AS1 in MCF7 and NCI-H1437 cells. (E-F) The mRNA (E) and protein (F) levels of FOXA1 after silencing of DSCAM-AS1. (G) qPCR showing the mRNA level of FOXA1 after knocking out of DSCAM-AS1 in MCF7 and NCI-H1437 cells. (H-K) Protein level of ERα after silencing DSCAM-AS1 using siRNAs in MCF7 (H) and T47D (J) cells. The mRNA level of ERα in MCF7 (I) and T47D (K) is also shown.

Figure 4

DSCAM-AS1 interacts with YBX1. (A) Silver staining of DSCAM-AS1-associated proteins after RNA pull-down using tRNA scaffold to a Streptavidin aptamer (tRSA) system; the empty tRSA vector is used as control. The band indicated in the red box is excised for mass spectrum analysis. (B) Mass spectrum results of the excised band. (C) Western blotting assay of proteins after RNA pull-down using YBX1 antibody. (D-E) RIP-qPCR showed enrichment of DSCAM-AS1 after immunoprecipitation of YBX1 in MCF7 (D) and NCI-H1437 (E). (F) qPCR showed the knocking down efficiency of YBX1 siRNAs. (G-H) MTT assay (G) and colony formation assay (H) showed the change of cell growth after knocking down YBX1.

Figure 5

DSCAM-AS1 enhances the recruitment of YBX1 in the promoter regions of FOXA1 and ERα to promote their expression. (A-C) Western blotting showed the protein level of YBX1, FOXA1 and ERα after knocking down YBX1 in MCF7 (A), T47D (B) and NCI-H1437 (C). (D-F) qPCR showed the RNA level of YBX1, FOXA1 and ERα after knocking down YBX1 in MCF7 (D), T47D (E) and NCI-H1437 (F). (G, I) ChIP-qPCR showed enrichment of YBX1 at the promoter of FOXA1 in MCF7 (G) and NCI-H1437 (I) cell lines. (H) ChIP-qPCR showed enrichment of YBX1 at the promoter of ERα. (J, L) ChIP-qPCR results showed YBX1 recruitment change at the promoter of FOXA1 after silencing of DSCAM-AS1 in MCF7 (J) and NCI-H1437 (L) cell lines. (K) ChIP-qPCR indicated YBX1 recruitment change at the promoter of ERα after knocking down DSCAM-AS1 in NCI-H1437 cells.

Figure 6

DSCAM-AS1 is a potential biomarker and therapeutic target. (A, C) Kaplan-Meier plots showed the association between DSCAM-AS1 expression levels and overall survival in ER+ breast cancer (A) and lung adenocarcinoma (C). (B, D) Kaplan-Meier plots indicating the association between DSCAM-AS1 expression levels and relapse-free survival in ER+ breast cancer (B) and lung adenocarcinoma (D). (E) Growth curve of nude mice inoculated with wild type (KO-NC) or KO-DSCAM-AS1 MCF7 cells. (F) Representative image of tumors at the end point. (G) Proposed working model showed DSCAM-AS1 could cooperate with YBX1 to promote the expression of FOXA1 and ERα.