An RNA editing fingerprint of cancer stem cell reprogramming

Abstract

BackgroundDeregulation of RNA editing by adenosine deaminases acting on dsRNA (ADARs) has been implicated in the progression of diverse human cancers including hematopoietic malignancies such as chronic myeloid leukemia (CML). Inflammation-associated activation of ADAR1 occurs in leukemia stem cells specifically in the advanced, often drug-resistant stage of CML known as blast crisis. However, detection of cancer stem cell-associated RNA editing by RNA sequencing in these rare cell populations can be technically challenging, costly and requires PCR validation. The objectives of this study were to validate RNA editing of a subset of cancer stem cell-associated transcripts, and to develop a quantitative RNA editing fingerprint assay for rapid detection of aberrant RNA editing in human malignancies.MethodsTo facilitate quantification of cancer stem cell-associated RNA editing in exons and intronic or 3'UTR primate-specific Alu sequences using a sensitive, cost-effective method, we established an in vitro RNA editing model and developed a sensitive RNA editing fingerprint assay that employs a site-specific quantitative PCR (RESSq-PCR) strategy. This assay was validated in a stably-transduced human leukemia cell line, lentiviral-ADAR1 transduced primary hematopoietic stem and progenitor cells, and in primary human chronic myeloid leukemia stem cells.ResultsIn lentiviral ADAR1-expressing cells, increased RNA editing of MDM2, APOBEC3D, GLI1 and AZIN1 transcripts was detected by RESSq-PCR with improved sensitivity over sequencing chromatogram analysis. This method accurately detected cancer stem cell-associated RNA editing in primary chronic myeloid leukemia samples, establishing a cancer stem cell-specific RNA editing fingerprint of leukemic transformation that will support clinical development of novel diagnostic tools to predict and prevent cancer progression.ConclusionsRNA editing quantification enables rapid detection of malignant progenitors signifying cancer progression and therapeutic resistance, and will aid future RNA editing inhibitor development efforts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0370-3) contains supplementary material, which is available to authorized users.

AZIN1 RNA editing confers cancer stemness and enhances oncogenic potential in colorectal cancer

“…To quantify AZIN1 RNA editing levels, we employed RNA editing site-specific quantitative PCR (RESSq-PCR), as described previously (14). Consistent with the outcomes of ADAR1 dysregulation, AZIN1 RNA editing levels were significantly increased in neoplastic tissues when compared with normal mucosa (P < 0.0001 in both cohorts; Figure 1B).…”

“…RNA editing of AZIN1 was analyzed using the RESSq-PCR method published previously (14). In brief, specific primers for the wild-type and edited AZIN1 sequences were designed (Supplemental Figure 1A).…”

ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis

“…Transduction efficiency was confirmed by fluorescence microscopy, nanoproteomics analysis of p- JAK2 and p -STAT5a and JAK2 qRT-PCR (Figures 2B–D and S2C). Lentiviral JAK2 transduction of human CD34 + progenitors enhanced both ADAR1 p150 and p110 transcript expression and ADAR1’s A-to-I editing activity as measured by RNA Editing Site-Specific qPCR (RESSqPCR) (Figures 2E–F) (Crews et al, 2015). While lentiviral ADAR1 transduction of BCR-ABL1 + K562 leukemia cells potentiated A-to-I RNA editing as measured by luciferase reporter activity in both the pCDH lentiviral vector and pDEST26 mammalian expression vector, this activity was further enhanced by addition of JAK2 expression (Figures S2D–F).…”

RNA rewriting, recoding, and rewiring in human disease

“…As sequencing technology, bioinformatics algorithms, and functional annotation techniques improve, the importance of A-to-I editing in the regulation of mammalian gene expression during normal tissue homeostasis and in the evolution of degenerative diseases, such as cancer, will become more apparent. However, the limitations and pitfalls intrinsically associated with high-throughput sequencing underscore the need for confirmation of specific editing sites with validation techniques such as RNA editing site-specific quantitative PCR (RESSq-PCR) [53].…”

“…Because aberrant RNA editing may be selectively targeted, it becomes of great clinical relevance to develop diagnostic and prognostic tools capable of accurately detecting aberrant RNA editing. A clinically-amenable RNA editing detection assay, such as RESSq-PCR, could prove to be useful in this regard [53]. RESSq-PCR provides a rapid method for the detection of aberrant RNA editing and has been used to quantify RNA editing in human leukemia stem cells.…”