Circular RMST cooperates with lineage-driving transcription factors to govern neuroendocrine transdifferentiation
Circular RNA (circRNA) is a class of noncoding RNA with regulatory potentials. It is unexplored how they may be functionally involved in the transdifferentiation of prostate and lung adenocarcinoma to neuroendocrine prostate cancer (NEPC) and small cell lung cancer (SCLC). Here we identified an exceptionally abundant circRNA circRMST predominantly expressed in NEPC and SCLC and conserved between humans and mice. Using shRNA, siRNA, CRISPR-Cas13 and Cas9, we consistently showed that circRMST is essential for tumour growth and for the expression of ASCL1, a master regulator of neuroendocrine fate. Genetic knockout of Rmst in NEPC genetic engineered mouse models led to a prevention of neuroendocrine transdifferentiation and sustained tumours in adenocarcinoma state. Mechanistically, circRMST physically interacts with lineage transcription factors NKX2-1 and SOX2. Loss of circRMST induces NKX2-1 protein degradation through autophagy-lysosomal pathway and alters the genomic binding of SOX2, which collectively leads to the loss of ASCL1 transcription. Overall design: To investigate function of circRMST, we designed two shRNAs and performed stable knockdown experiments in SHP77 small cell lung cancer cell line. shGFP was used as the negative control. Two biological replicates for each shRNA were performed. We identified that circRMST physically interacts with SOX2 and NKX2-1. To understand the interactions and the downstream target genes, we performed siRNA knockdown of SOX2 and NKX2-1 with two independent siRNAs each in SHP77. Two biological replicates were performed. In addition, we performed ChIP-seq of SOX2 and NKX2-1 in SHP77 SCLC-A cells with and without circRMST shRNAs to investigate how the cistromes for the two transcription factors are altered without circRMST. Two biological replicates were performed for each transcription factor.