Discovery and Characterization of Non-Coding RNAs with Therapeutic and Diagnostic Potential in Prostate Cancer

Abstract

Prostate cancer is the most common malignancy and the second leading cause of death in American men. Primary prostate cancer is often hormone-dependent and relies on signaling through the androgen receptor (AR); therefore, the majority of patients are responsive to front-line treatment with androgen-deprivation therapy. However, some cases progress to an incurable stage of the disease known as castration-resistant prostate cancer (CRPC), which still relies on AR signaling.

Substantial efforts have been undertaken to study sustained AR signaling in CRPC. The mechanisms identified include AR amplifications, AR splice variants, alterations in cofactor recruitment, and ligand-independent activation via crosstalk with signal transduction pathways. However, few studies have investigated the roles of non-coding RNAs (ncRNAs) in AR signaling axis. Noncoding RNAs are a class of transcripts with diverse and largely uncharacterized biological functions. Through crosstalk with chromatin, DNA, RNA, and proteins, ncRNAs function via chromatin remodeling as well as transcriptional and post-transcriptional regulation.

To discover long non-coding RNAs (lncRNAs) with therapeutic or diagnostic potential in prostate cancer, we have performed a comprehensive RNA-seq profile investigation of cancer-associated, AR-regulated lncRNAs from prostate cancer cell lines and patient tissue samples. Through this analysis, we identified ARLNC1 (AR-regulated long noncoding RNA 1), an important lncRNA that is significantly enriched in prostate lineage and associates with prostate cancer progression.

ARLNC1 is directly induced by the AR protein and modulates AR signaling. ARLNC1 knockdown suppresses AR expression, AR-responsive genes, and prostate cancer growth in vitro and in vivo. Further dissection of ARLNC1 cellular mechanism showed that the lncRNA stabilized the AR transcript via RNA-RNA interaction. This positive feedback loop, where AR transcriptionally regulates ARLNC1 and ARLNC1 regulates its master regulator (AR) post-transcriptionally, suggests that lncRNAs can serve as novel regulatory nodes on AR signaling pathway.

Because modulation of ARLNC1 expression levels resulted in a striking proliferation phenotype, it is hypothesized that ARLNC1 inhibition could be used therapeutically for the treatment of prostate cancer. Supporting this hypothesis, antisense oligonucleotides (ASOs) targeting ARLNC1 inhibited prostate cancer cell growth in vitro and in vivo. The fact that ARLNC1 acts upstream of AR signaling presents the possibility that targeting ARLNC1 may afford an additional option to patients that have de novo or acquired resistance to therapies targeting AR itself.

In addition, we evaluated the clinical relevance of ARLNC1 as a diagnostic biomarker. Among the prostate samples, ARLNC1 expression was significantly higher in localized and metastatic prostate cancers than in benign tissues, as assessed by RNA-seq and RNA in situ hybridization. Moreover, there exists an association of ARLNC1 levels with accentuated AR signaling and luminal epithelial differentiation in patient tumors, both of which are important clinical considerations for anti-androgen treatment.

Finally, we characterized expression patterns of another type of non-coding RNA, circular RNA (circRNA). A set of circRNAs were identified as candidate biomarkers for prostate cancer. They have higher stability compared to linear transcripts. We further validated the existence of a novel class of circular transcripts, termed read-through circRNAs, that involve exons originating from different genes. Additionally, circRNAs can be detected in patient urine samples and may be developed into non-invasive biomarkers.

Taken together, this work defines essential roles for ARLNC1 in prostate cancer progression, uncovers novel aspects of AR signaling and lncRNA biology, and underscores the clinical potential of non-coding RNAs as diagnostic markers or therapeutic targets in prostate cancer.