Mechanism, Regulation and Application of miRNA pathway in cancer

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

In FY25, we extended our study beyond isomiRs and pivoted part of our efforts towards harnessing mechanistic insights we gained for potential translational studies. Despite the extensive documentation of the roles of miRNAs in tumorigenesis, the development of miRNA-based cancer treatments has lagged. This is partly due to the difficulty in identifying tumor-driver miRNA variations through profiling-based approaches. To address this challenge, we sought to focus on genetic evidence by studying recurrent mutations in miRNA biogenesis factors found in cancer patients. We reasoned that the resulting miRNA alterations likely contribute to tumor formation and/or progression. During our characterization of Drosha/Dicer mutants affecting cleavage accuracy, the unique behavior of RNase IIIb mutants caught our attention. DICER1 RNase IIIb mutations are prominent in certain pediatric cancers, including pleuropulmonary blastoma (PPB), and are hallmark mutations for DICER1 syndrome-related tumors. To elucidate the functional implications of the DICER1 RNase IIIb mutation, we generated a set of cells mimicking DICER1-associated PPB tumors using CRISPR. Our miRNA profiling with Northern blot and deep sequencing not only confirmed the expected loss of mature 5p-miRNAs in the mutant cell line but also revealed a surprising upregulation of a subset of 3p-miRNAs. The functionality of the upregulated passenger 3p-miRNAs was confirmed by reporter assays and transcriptome analyses. Analyses of patient miRNA and transcriptome datasets corroborated these findings. Thus, our study indicates that the DICER1 RNase IIIb hotspot mutation not only causes a loss-of-function (LOF) for 5p-miRNAs as previously believed, but also leads to a gain-of-function for a set of 3p-miRNAs, potentially impacting DICER1 tumor pathogenicity. In vitro and in vivo analyses implicate strand switching by AGO loading as the mechanism for the functional upregulation of passenger 3p-miRNAs, highlighting a novel mechanism of miRNA regulation through AGO strand selection. Importantly, our mechanistic understanding enables us to predict and identify functional passenger 3p-miRNAs in Dicer mutant cells, which will be tremendously useful as we extend our study beyond cultured cells to patient-relevant settings to determine downstream targets. Going forward, we will generate a novel mouse model for PPB and an isogenic cell culture-based platform to identify and validate the specific requirements for miRNAs that underlie PPB tumorigenesis. If successful, identifying miRNAs as druggable targets for PPB may provide new prospects for cancer therapy.