Abstract P6-05-01: A novel cleaved cytoplasmic lncRNA LacRNA interacts with PHB2 and suppresses breast cancer metastasis via repressing MYC targets

Abstract

Abstract Long noncoding RNAs (lncRNAs) have been implicated in breast cancer metastases through largely unknown mechanisms. In this study, we used microarray analysis to compare lncRNAs expression levels between matched pairs of breast lymph node metastatic tissues and primary tumors. We discovered that lncRNA LINC00478 was substantially downregulated in the metastatic tumor samples. Interestingly, we found that LINC00478 could be cleaved by RNase to simultaneously generates the mature 5' ends of cytoplasmic RNA and 3' ends of nuclear RNA by polyadenylation. We named 5' ends 791-nt RNA as LacRNA (LINC00478-assciated cytoplasmic RNA). Over expression of full-length LINC00478 and LacRNA, but not LINC00478 3' RNA, significantly inhibited breast cancer proliferation, invasion and metastasis in vitro and in vivo.We used CRISPR-dCas9 complex to mediate efficient transcriptional activation of LacRNA at endogenous genomic loci followed by RNA-seq analyses. Gene set enrichment analysis (GSEA) showed that the MYC pathway/targets were prominent gene sets negatively enriched in LacRNA-activated cells. Further study revealed that LacRNA exerted its tumor suppressive activity by directly binding with prohibitin2(PHB2) to enhance its protein stability, which promoted PHB2 competing with MYC for transcriptionally suppressing the MYC target genes (e.g., CDC20, CDC45, CCNA2 and MAD2L1). Mechanistically, LacRNA inhibits breast cancer invasion and metastasis by interacting with PHB2 through LacRNA's 1-300nt region. In addition, taking advantage of CRISPR system to knock-out and activate the expression of LacRNA, as well as rescue experiment, we uncovered the positive correlation between LacRNA and PHB2 and their role in suppressing MYC target genes and cancer metastasis. At the same time, LacRNA can attenuated the MYC induced activation of MYC targets through binding with PHB2, indicating that LacRNA plays a central role in the suppression of MYC target genes. We further explored the role of LacRNA in inhibiting lung metastasis by implanting LacRNA-activated LM2 cells into the mammary fat pads of NOD-SCID mice. Luciferase imaging and histological analysis were used to detect lung metastasis and found that LacRNA significantly suppressed lung metastasis. Immunohistochemistry were used to detect the expression of PHB2 and MYC targets in both orthotopic tumors and lung metastasis and verified their correlation in vivo. Extensive analyses of clinical data indicated that LacRNA level was substantially downregulated in metastases tumors accompanied by enrichment of MYC targets. The robustness value of LacRNA expression was further verified in two independent patient cohorts, including 530 invasive breast cancer tumors in Fudan University Shanghai Cancer Center (FUSCC) and 819 breast patients' data from TCGA. High LacRNA expression level had a significantly better clinical outcome in both cohorts and represented an independent prognostic predictor for DFS (HR=0.48, P=0.006, multivariate analysis) and OS (HR=0.32, P=0.009, multivariate analysis) in FUSCC cohort. Collectively, LacRNA functions as a tumor suppressor lncRNA that inhibits breast cancer invasion-metastasis cascade. Citation Format: Guo R, Su Y-H, Xue J-y, Si J, Chi Y-y, Wu J. A novel cleaved cytoplasmic lncRNA LacRNA interacts with PHB2 and suppresses breast cancer metastasis via repressing MYC targets [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-05-01.