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.

Estrogen receptor (ER) plays important roles in cell growth, development and tumorigenesis. Although ER-regulated genes have been extensively investigated, little is known about roles of ER-regulated lncRNAs in breast cancer. Here, we conducted genome-wide study of ER-regulated lncRNAs by using RNA-seq, ChIP-seq and TCGA data. A total of identified 114 ER-regulated lncRNAs were identified, many of them were overexpressed in ER+ breast cancer and co-expressed with some key regulators. Silencing one of most prominent lncRNA, AP000439.3, resulted in inhibition of cell cycle progression and proliferation. Further study revealed AP000439.3 can regulate expression of CCND1 through enhancing estrogen receptor induction of CCND1. This finding revealed lncRNAs may serve as important effectors of ER in regulation of gene expression and cell phenotype in breast cancer.

Background Long noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown. Results We found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3ʹ-end. Consequently, the secondary structures were changed, the RNA–protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3ʹ-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs. Conclusions Our study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study. Graphic Abstract

Lineage-specific expression of genes plays important roles in tumorigenesis. It has been suggested that lncRNAs tend to be more lineage-specifically expressed than protein-coding genes. However, the regulation and role of lineage-specific lncRNAs remains largely unknown. In this study, we reported a lncRNA DSCAM-AS1 that specifically expressed in lung adenocarcinoma and luminal breast cancer. The expression of DSCAM-AS1 was regulated by two super enhancer clusters driven by Foxa1. Foxa1 can directly bind to promoter of DSCAM-AS1. In breast cancer, DSCAM-AS1 was also transcriptionally regulated by ERα, which formed complex with Foxa1. Higher expression of DSCAM-AS1 was associated with poor poor prognosis in both lung adenocarcinoma and luminal breast cancer patients. Silencing or knock-out of DSCAM-AS1 significantly reduce the growth of lung adenocarcinoma and luminal breast cancer cells. We observed silencing or knock-out of DSCAM-AS1 could reduce the expression of Foxa1 in lung adenocarcinoma and breast cancer cells. We also found a significant decrease of ERα expression after silencing or knock-out of DSCAM-AS1 in luminal breast cancer cells. Mechanistically, we found DSCAM-AS1 could interact with YBX1 and enhance recruitment of YBX1 at the promoter region of Foxa1 and ERα. DSCAM-AS1 could form a positive feedback loop with Foxa1 and ERα. Our findings may provide another regulatory loop: Foxa1 and ERα drive expression of super enhancer associated lncRNA DSCAM-AS1 and form a positive feedback loop with it, which play important roles in maintaining cancer cell identity and function. Citation Format: Yin Zhang, Yong-xin Huang, Dan-lan Wang, Hai-yan Yan, Yin Dong. A lineage-specific lncRNA driven by Foxa1 associated super enhancer promotes tumor growth through interacting YBX1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3702.