Naoya Miyashita

Achaete-scute family bHLH transcription factor 1 (ASCL1) is a master transcription factor involved in neuroendocrine differentiation. ASCL1 is expressed in approximately 10% of lung adenocarcinomas (LUAD) and exerts tumor-promoting effects. Here, we explored miRNA profiles in ASCL1-positive LUADs and identified several miRNAs closely associated with ASCL1 expression, including miR-375, miR-95–3p/miR-95–5p, miR-124–3p, and members of the miR-17∼92 family. Similar to small cell lung cancer, Yes1 associated transcriptional regulator (YAP1), a representative miR-375 target gene, was suppressed in ASCL1-positive LUADs. ASCL1 knockdown followed by miRNA profiling in a cell culture model further revealed that ASCL1 positively regulates miR-124–3p and members of the miR-17∼92 family. Integrative transcriptomic analyses identified ZFP36 ring finger protein like 1 (ZFP36L1) as a target gene of miR-124–3p, and IHC studies demonstrated that ASCL1-positive LUADs are associated with low ZFP36L1 protein levels. Cell culture studies showed that ectopic ZFP36L1 expression inhibits cell proliferation, survival, and cell-cycle progression. Moreover, ZFP36L1 negatively regulated several genes including E2F transcription factor 1 (E2F1) and snail family transcriptional repressor 1 (SNAI1). In conclusion, our study revealed that suppression of ZFP36L1 via ASCL1-regulated miR-124–3p could modulate gene expression, providing evidence that ASCL1-mediated regulation of miRNAs shapes molecular features of ASCL1-positive LUADs. Implications: Our study revealed unique miRNA profiles of ASCL1-positive LUADs and identified ASCL1-regulated miRNAs with functional relevance.

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor with dismal prognosis. Recently, molecular subtypes of SCLC have been defined by the expression status of ASCL1, NEUROD1, YAP1, and POU2F3 transcription regulators. ASCL1 is essential for neuroendocrine differentiation and is expressed in the majority of SCLC. Although previous studies investigated ASCL1 target genes in SCLC cells, ASCL1‐mediated regulation of miRNAs and its relationship to molecular subtypes remain poorly explored. Here, we performed genome‐wide profiling of chromatin modifications (H3K27me3, H3K4me3, and H3K27ac) by CUT&Tag assay and ASCL1 knockdown followed by RNA sequencing and miRNA array analyses in SCLC cells. ASCL1 could preferentially regulate genes associated with super‐enhancers (SEs) defined by enrichment of H3K27ac marking. Moreover, ASCL1 positively regulated several SE‐associated miRNAs, such as miR‐7, miR‐375, miR‐200b‐3p, and miR‐429, leading to repression of their targets, whereas ASCL1 suppressed miR‐455‐3p, an abundant miRNA in other molecular subtypes. We further elucidated unique patterns of SE‐associated miRNAs in different SCLC molecular subtypes, highlighting subtype‐specific miRNA networks with functional relevance. Notably, we found apparent de‐repression of common target genes of different miRNAs following ASCL1 knockdown, suggesting combinatorial action of multiple miRNAs underlying molecular heterogeneity of SCLC (e.g., co‐targeting of YAP1 by miR‐9 and miR‐375). Our comprehensive analyses provide novel insights into SCLC pathogenesis and a clue to understanding subtype‐dependent phenotypic differences.