3′UTR shortening and EGF signaling: implications for breast cancer

Abstract

Alternative polyadenylation (APA) plays a role in gene expression regulation generally by shortening of 3'UTRs (untranslated regions) upon proliferative signals and relieving microRNA-mediated repression. Owing to high proliferative indices of triple negative breast cancers (TNBCs), we hypothesized APA to cause 3'UTR length changes in this aggressive subgroup of breast cancers. Our probe-based meta-analysis approach identified 3'UTR length alterations where the significant majority was shortening events (∼70%, 113 of 165) of mostly proliferation-related transcripts in 520 TNBC patients compared with controls. Representative shortening events were further investigated for their microRNA binding potentials by computational predictions and dual-luciferase assay. In silico-predicted 3'UTR shortening events were experimentally confirmed in patient and cell line samples. To begin addressing the underlying mechanisms, we found CSTF2 (cleavage stimulation factor 2), a major regulator of 3'UTR shortening to be up-regulated in response to epidermal growth factor (EGF). EGF treatment also resulted with further shortening of the 3'UTRs. To investigate the contribution of CSTF2 and 3'UTR length alterations to the proliferative phenotype, we showed pharmacological inhibition of the EGF pathway to lead to a reduction in CSTF2 levels. Accordingly, RNAi-induced silencing of CSTF2 decreased the proliferative rate of cancer cells. Therefore, our computational and experimental approach revealed a pattern of 3'UTR length changes in TNBC patients and a potential link between APA and EGF signaling. Overall, detection of 3'UTR length alterations of various genes may help the discovery of new cancer-related genes, which may have been overlooked in conventional microarray gene expression analyses.

Alternative Polyadenylation: Another Foe in Cancer

“…In addition, a set of the 3 0 UTR shortening events were correlated with relapse free survival times, which also supports APA based proto-oncogene activation (62). The use of such meta-analysis tools of gene expression data is very beneficial for detecting APA-generated isoforms that may have been overlooked in conventional analysis.…”

“…0 UTR shortening of all tested genes in breast cancer cells (61,62). While E2F family of transcription factors has been implicated in CSTF2 upregulation, further studies are needed to fully uncover the mechanisms causing upregulation of CSTF2 as part of cancer-associated signaling pathways (63).…”

“…Moreover, dependence on poly(A) site annotation databases such as the PolyA_DB can be somewhat limiting for identification of novel poly(A) sites; however, it is possible to identify candidate novel poly(A) sites by seeking break sites where probe set means differ between each other. For such probe-based analysis of microarray data, PLATA (50), APADetect (62), and ERI (102) are dependent on the PolyA_DB database for poly(A) signal position information. An important advantage of APADetect is that it can readily be used as a meta-analysis tool capable of working with multiple Affymetrix datasets from both human and mouse platforms.…”

Alternative polyadenylation and RNA-binding proteins

“…For example, decreased CFIm25 expression correlated with 3′-UTR shortening events in glioblastoma patients (Masamha et al 2014). Furthermore, a combinatorial approach of computation and experimentation revealed a pattern of 3′-UTR length changes in breast cancer patients/cells and a potential link between APA, CSTF2, and EGF (epidermal growth factor) signaling (Akman et al 2015). Apart from the core proteins, numerous RBPs have been implicated in regulating APA events by either competing with or enhancing the binding of the polyadenylation machinery proteins to their target sites (Fig.…”

The Butterfly Effect of RNA Alterations on Transcriptomic Equilibrium

“…APA-mediated 3 UTR shortening has been described in multiple cancers [63][64][65][66][67][68][69][70][71][72][73][74][75]. NUDT21, a key regulator of APA, is frequently downregulated in several cancers [66,67,69,70,76].…”