Antitumorigenic potential of STAT3 alternative splicing modulation

Abstract

Signal transducer and activator of transcription 3 (STAT3) plays a central role in the activation of multiple oncogenic pathways. Splicing variant STAT3β uses an alternative acceptor site within exon 23 that leads to a truncated isoform lacking the C-terminal transactivation domain. Depending on the context, STAT3β can act as a dominant-negative regulator of transcription and promote apoptosis. We show that modified antisense oligonucleotides targeted to a splicing enhancer that regulates STAT3 exon 23 alternative splicing specifically promote a shift of expression from STAT3α to STAT3β. Induction of endogenous STAT3β leads to apoptosis and cell-cycle arrest in cell lines with persistent STAT3 tyrosine phosphorylation compared with total STAT3 knockdown obtained by forced splicing-dependent nonsense-mediated decay (FSD-NMD). Comparison of the molecular effects of splicing redirection to STAT3 knockdown reveals a unique STAT3β signature, with a down-regulation of specific targets (including lens epithelium-derived growth factor, p300/CBP-associated factor, CyclinC, peroxisomal biogenesis factor 1, and STAT1β) distinct from canonical STAT3 targets typically associated with total STAT3 knockdown. Furthermore, similar in vivo redirection of STAT3 alternative splicing leads to tumor regression in a xenograft cancer model, demonstrating how pharmacological manipulation of a single key splicing event can manifest powerful antitumorigenic properties and validating endogenous splicing reprogramming as an effective cancer therapeutic approach.

Novel role of microRNA146b in promoting mammary alveolar progenitor cell maintenance

“…More recent studies have demonstrated that STAT3b may activate a specific set of genes and possess non-redundant functions to STAT3a. Zammarchi et al used morpholino oligomers to redirect endogenous STAT3 alternative splicing from STAT3a to STAT3b (Zammarchi et al, 2011). This study showed that inducing the STAT3b isoform in MDA-MB-435 cells caused cell death and tumor regression in the xenograft models.…”

“…The same study showed that STAT3 canonical target genes were not affected by the switch from STAT3a to STAT3b. Rather, a unique expression signature seems to be associated with the physiological a-to-b splicing shift (Zammarchi et al, 2011). In mammary gland development, it has become clear that STAT3 regulates a complex series of processes involving alveolar epithelial cell death during involution by maintaining the balance of inflammatory and anti-inflammatory signaling (Pensa et al, 2009).…”

“…Since STAT3 transcription factors were reported to play key roles in mammary epithelial cell apoptosis at the onset of involution, we explored the role of miR146b in regulating STAT3 isoforms. STAT3 has two main isoforms: the full-length STAT3a and the truncated STAT3b, by alternative mRNA splicing of exon 23 (Huang et al, 2007;Zammarchi et al, 2011). In STAT3b, the 55-amino acid C-terminal acidic transactivation domain of STAT3a is replaced by seven unique amino acid residues (Zammarchi et al, 2011).…”

“…STAT3 has two main isoforms: the full-length STAT3a and the truncated STAT3b, by alternative mRNA splicing of exon 23 (Huang et al, 2007;Zammarchi et al, 2011). In STAT3b, the 55-amino acid C-terminal acidic transactivation domain of STAT3a is replaced by seven unique amino acid residues (Zammarchi et al, 2011). An important factor in STAT3 functional heterogeneity may be the existence of these two alternatively spliced isoforms.…”

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

“…STAT3 is of particular interest because of its pleiotropic and uncertain functionality, with conflicting reports on whether it acts as an oncoprotein or tumor suppressor in cancer (reviewed in reference 33 ). Differences in STAT3 α and β splice variant function had been characterized previously 34,35 , and our protocol facilitated a knock-down/re-expression analysis that suggests a need for an optimal ratio of S and ΔS transcripts 19 .…”