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.

Fig. 1.

Modulation of STAT3 alternative splicing. (A) Alternative 3′ splice site use in exon 23 generates STAT3α or STAT3β, where the TAD (green) is substituted by a unique 7-aa tail (red). DBD, DNA binding domain. (B) STAT3 exon 23. Deletion mutants within the 50-nt α-specific region are indicated (Upper), as are the positions of the morpholinos (red lines) (Lower). The putative mapped ESEs are depicted in green. (C and D) RT-PCR analysis of STAT3 α/β levels in HeLa cells transfected with the 9-nt (ΔA–ΔG) or 3-nt (ΔA1–ΔB3) deletion mutants. (E) RT-PCR and Western blot analysis of STAT3 α/β levels in MDA-MB-435s cells treated with increasing concentrations of ST1, ST2, and ST3 for 4 d. NT, untreated. (F) RT-PCR analysis of STAT3α/β levels in MDA-MB-435s cells pretreated with either control (INV) or ST2 for 4 d and then grown in fresh media for up to 9 d.

Fig. 2.

Effect of STAT3 alternative splicing modulation in vitro. (A) Knockdown of STAT3 by FSD-NMD. Morpholinos ST6 and ST7 cause skipping of exon 6, leading to a frameshift and a PTC, ultimately causing RNA degradation by NMD. (B) RT-PCR and WB analysis of total STAT3α/β levels in MDA-MB-435s cells treated with 16 μM ST6, ST7, or INV for 4 d. α-Tubulin was the loading control for WBs. (C) RT-PCR and WB analysis of STAT3 α/β levels in MDA-MB-453, MDA-MB-435s, and MDA-MB-468 cells treated with 16 μM INV, ST2, or ST6 for 4 d. β-Actin was used as control for RT-PCR. (D) Cell-death quantification (represented as fold change relative to control treatment) determined by trypan blue exclusion for treatments in C. The average of at least three independent experiments is represented, each in triplicate. P values were calculated by the stratified χ² test; bars represent SE of the rate. See Figs. S7 and S8 for effects on cell cycle and apoptosis.

Fig. 3.

Effect of STAT3 alternative splicing modulation in vivo. (A) Following s.c. injection of 10 × 10⁶ MDA-MB-435s cells, tumors of ∼100 mm³ were treated IT with 0.12 mg of Vivo-Morpholino (INV, ST2, and ST6) twice a week for 3 wk (arrows), and tumor volumes were measured. (B) Representative image of mice in each treatment group at time of sacrifice, with H&E staining of tumor sections underneath. (C and D) RT-PCR analysis of STAT3 exon 23 and exon 6 levels in three tumors for each treatment group. GAPDH was used as control. Primers recognize both human and mouse genes. (E) Epitope location of STAT3 antibodies used for IHC staining. (F–I) Representative images of tumor sections from the four different treatment groups following IHC using P-Y705, P-S727, and total STAT3 antibodies (F–H) or H&E staining.

Fig. 4.

STAT3β switch modulates specific target genes. (A) MDA-MB-435s cells treated with 16 μM ST2, ST6, or INV for 4 d were analyzed by qPCR. Results are represented as a comparison between ST2 and INV (blue) or between ST6 and INV treatments (red), expressed as −ddC(t) values after normalization to hypoxanthine phosphoribosyl transferase (HPRT). Data represent the average of at least three independent experiments. (B) IL8 quantification by ELISA on conditioned media from MDA-MB-435s cells treated as in A (n = 4). P values were calculated by two-tailed Student's t test. To validate STAT3β targets identified by microarray analysis, cDNAs from A were analyzed as above by qPCR (C) and lysates were analyzed by WB, with α-tubulin as control (D). (E) Quantitative PCR analysis of STAT3β target genes from tumor RNAs from xenograft experiments in which tumors were injected IT twice in a week with vehicle (C) or ST2s. Results are represented as a comparison between ST2 and control, expressed as −ddC(t) values after normalization to β-2-microglobulin. Data are from at least three independent experiments. Bars represent SD. (Inset) RT-PCR analysis of STAT3 levels in the same RNAs.

Fig. 5.

Knockdown and overexpression of STAT3β target genes. (A–E) (Left) MDA-MB-435s cells were treated for 72 h with siRNA against GFP or LEDGF (A), PEX1 (B), PCAF (C), IL8 (D), or CyclinC (E). Knockdown was verified by WB (A–C, with α-tubulin as control) or by qPCR analysis (D and E), displayed as −dC(t) values after normalization to HPRT. (Right) Cell-death quantification was determined by trypan blue exclusion for MDA-MB-435s cells treated as described. Data are displayed as fold change of treated samples (orange) relative to control (green). Data are from three independent experiments (each in triplicate). P values were calculated by the stratified χ² test; bars represent SE of the rate. (F) (Left) Lysates from MDA-MB-435s cells transiently transfected with LEDGF or empty vector were immunoblotted using an antibody to LEDGF. (Right) Transfected cells were concurrently treated with 16 μM INV or ST2 for 4 d. Cell death was quantified as above. (G–J) (Left) Stable MDA-MB-435s clones overexpressing STAT1β (G), PCAF (H), IL8 (I), CyclinC (J), or selected for the EV were treated with 16 μM INV or ST2 for 4 d. Overexpression levels were determined by WB (G and H, with α-tubulin as control) or qPCR analysis (I and J), and displayed as −dC(t) values normalized to HPRT. (Right) Cell death was quantified as above.