Abstract LB-167: Off-target analysis of RNAi screens identifies microRNA seed sequence contributing to metastatic invasion

Abstract

Abstract The occurrence of metastatic events is a prominent indicator of poor survival outcomes in individuals afflicted by solid tumor cancers. The introduction of high throughput techniques for genetic knockdown or ablation, such as shRNA and CRISPR-Cas9 libraries, provides the means to effectively screen for loss of function genes that contribute to the metastatic cascade; however, screening strategies employing these methods, particularly RNAi, carry an inherent risk of false positives due to off-target effects. These off-target effects are often mediated by shRNA sequences mimicking microRNA functionality, whereby only seed sequence (base pair positions 2-8) complementarity to the 3’ UTR of transcripts is sufficient for effective targeting and downregulation. Through two separate genome-wide shRNA screens, we identified a particular shRNA that shares its seed sequence with an uncharacterized miR, miR-8087, and exhibits wide-spread, miR-like off-target effects causing increased chemotaxis and Matrigel invasiveness. A comprehensive, third generation shRNA library was utilized to screen for metastasis suppressor genes in non-aggressive breast and prostate cancer cell lines (T-47D and LNCaP; respectively) by both in vitro and in vivo methods. Enriched shRNA variants were identified by barcode sequencing of clones and one shRNA sequence in particular, targeting apolipoprotein L-II (APOL2), was consistently identified in both screens. Conversion of the shAPOL2 sequence to siRNA and subsequent transfection in LNCaP cells increased the invasive and chemotactic potential of these cells. In contrast, APOL2 knockdown with alternative siRNA sequences did not increase the invasive or migratory capability of LNCaP cells. In order to identify off-targets responsible for the invasive phenotype, the gene expression profile of LNCaP transfected with the functionally relevant siAPOL2 was compared by microarray analysis (Illumina HT-12v4) to that of cells containing APOL2-specific siRNA. The library-specific siAPOL2 results in differential downregulation of 132 gene transcripts, suggesting miR-like activity. Examination of the siRNA seed sequence reveals exact homology to miR-8087 seed sequence and many of the siAPOL2 off-targets are shared with miR-8087 gene targets as predicted by miRWalk 2.0. Currently, efforts are underway to characterize miR-8087 expression and targeting in a panel of cell lines with varying migratory potentials as well as in patient primary and metastatic prostate cancer tissue samples. The off-target analysis of RNAi screens reveals a broad acting miR sequence that contributes to the invasive potential of cells and may provide a predictive and targetable element in metastatic cancers. Citation Format: Henry G. Withers, Ryan Ransom, Irwin H. Gelman. Off-target analysis of RNAi screens identifies microRNA seed sequence contributing to metastatic invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-167.