Abstract
To discover novel therapeutic targets for triple-negative breast cancer (TNBC) and cancer stem cells (CSCs), we screened long non-coding RNAs (lncRNAs) most enriched in TNBCs for high expression in CSCs defined by high Aldefluor activity and associated with worse patient outcomes. This led to the identification of non-coding RNA in the aldehyde dehydrogenase 1 A pathway (NRAD1), also known as LINC00284. Targeting NRAD1 in TNBC tumors using antisense oligonucleotides reduced cell survival, tumor growth, and the number of cells with CSC characteristics. Expression of NRAD1 is regulated by an enzyme that causes Aldefluor activity in CSCs, aldehyde dehydrogenase 1A3 (ALDH1A3) and its product retinoic acid. Cellular fractionation revealed that NRAD1 is primarily nuclear localized, which suggested a potential function in gene regulation. This was confirmed by transcriptome profiling and chromatin isolation by RNA purification, followed by sequencing (ChIRP-seq), which demonstrated that NRAD1 has enriched chromatin interactions among the genes it regulates. Gene Ontology enrichment analysis revealed that NRAD1 regulates expression of genes involved in differentiation and catabolic processes. NRAD1 also contributes to gene expression changes induced by ALDH1A3; thereby, the induction of NRAD1 is a novel mechanism through which ALDH1A3 regulates gene expression. Together, these data identify lncRNA NRAD1 as a downstream effector of ALDH1A3, and a target for TNBCs and CSCs, with functions in cell survival and regulation of gene expression.
Highlights
Triple-negative breast cancers (TNBCs) represent 15–20% of breast tumors and are associated with worse outcomes [1, 2]
To generate a shortlist of candidate long non-coding RNAs (lncRNAs) that could serve as novel functional targets for TNBCs and the cancer stem cells (CSCs) within these tumors, we identified lncRNAs that fulfilled the following prioritization strategy: (1) highly expressed in TNBC/basal-like patient tumors, (2) enriched in breast CSC populations, and (3) associated with worse patient outcomes
Our screening of lncRNAs led to the identification of TNBC/basal-like/CSC-enriched NRAD1 as a new mediator of cell survival within these tumors and cancer cells
Summary
Triple-negative breast cancers (TNBCs) represent 15–20% of breast tumors and are associated with worse outcomes [1, 2]. With the goal of identifying a novel oncogenic lncRNA that could be targeted with antisense oligonucleotides to treat TNBCs and kill CSCs within these tumors, we screened for lncRNAs that are enriched in TNBCs and CSCs and are associated with poor patient outcomes This led to the identification of a previously uncharacterized lncRNA, LINC00284, which forth shall be referred to as non-coding RNA in the aldehyde dehydrogenase 1 A pathway (NRAD1). NRAD1 is a novel downstream target of aldehyde dehydrogenase 1A3 (ALDH1A3) and the first lncRNA described to contribute to gene expression changes induced by this CSC marker and mediator of tumor progression [23,24,25,26,27,28,29,30,31] Together, these data identify the lncRNA NRAD1 as a novel oncogenic effector that is targetable with antisense oligonucleotides in the treatment of TNBC and reduction of cells with CSC characteristics
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