A tumor-associated splice-isoform of MAP2K7 drives dedifferentiation in MBNL1-low cancers via JNK activation

Abstract

Master splicing regulator MBNL1 shapes large transcriptomic changes that drive cellular differentiation during development. Here we demonstrate that MBNL1 is a suppressor of tumor dedifferentiation. We surveyed MBNL1 expression in matched tumor/normal pairs across The Cancer Genome Atlas and found that MBNL1 was down-regulated in several common cancers. Down-regulation of MBNL1 predicted poor overall survival in breast, lung, and stomach adenocarcinomas and increased relapse and distant metastasis in triple-negative breast cancer. Down-regulation of MBNL1 led to increased tumorigenic and stem/progenitor-like properties in vitro and in vivo. A discrete set of alternative splicing events (ASEs) are shared between MBNL1-low cancers and embryonic stem cells including a MAP2K7∆exon2 splice variant that leads to increased stem/progenitor-like properties via JNK activation. Accordingly, JNK inhibition is capable of reversing MAP2K7∆exon2-driven tumor dedifferentiation in MBNL1-low cancer cells. Our work elucidates an alternative-splicing mechanism that drives tumor dedifferentiation and identifies biomarkers that predict enhanced susceptibility to JNK inhibition.

Keywords: JNK inhibitors; MAP2K7; MBNL1; alternative splicing; tumor cell dedifferentiation.

Fig. 1.

MBNL1 is down-regulated in cancer and is a prognostic marker for survival. (A) Box plot showing median difference in variance stabilized transformed read counts (VST counts) for MBNL1. Each shaded circle (red, up-regulated; blue, down-regulated) represents a unique patient. Down-regulated indicated in red font, up-regulated by green font (log2 fold change >0.5 (abs), P value <0.05) and no change in black font. (B) The table lists number (N) of patients with higher or lower MBNL1 expression in matched tumors/normal. (C) Representative MBNL1 expression (IHC) in matched tumor and normal stomach adenocarcinoma patients in a tissue microarray (TMA). (D) Western blot showing MBNL1 expression. (E) Kaplan–Meier plots showing correlation between MBNL1 and overall survival. (F and G) Kaplan–Meier plots showing correlation between MBNL1 expression and “relapse-free survival” and “distant-metastasis-free survival.”

Fig. 2.

MBNL1 KD up-regulates CSC-specific splice isoforms and genes. (A) Wiggle plots representing an inclusion event (EXOC1) and a skip event (MAP2K7). Histograms at Right show PSI values. (B) Quantification of different classes of high-confidence ASEs. (C) Pie chart showing high-confidence ASEs that are also differentially spliced in MBNL1-low cancers. (D) Qualitative heat map showing distribution of MRTA-ASEs. (E) Venn diagram showing overlap between the SIE events of MRTA-ASEs and ESC-differential ASEs from Han et al. (12). (F) Gel electrophoresis images (Left) and quantitative bar graphs (Right) showing validation of CS-MRTA-ASEs. (G) Bar graph showing biological processes GO term enrichment upon MBNL1 knockdown. (H) Validation of up-regulation of CSC-associated genes upon MBNL1 knockdown using two independent siRNAs by qRT-PCR. Data show mean ± SD.

Fig. 3.

MBNL1 KD increases in vitro colony formation, migration, and in vivo tumor formation. (A) Western blot showing expression of MBNL1 in indicated cell lines treated with nontargeting shRNA (shNeg) or two MBNL1 shRNAs. (B and D) Bar graphs (Top) and representative images (Bottom) showing soft agar colony formation (B) and in vitro Transwell migration (D) in indicated cell lines. Data show mean ± SD. See also SI Appendix, Fig. S3. (C) X-Y graph showing mean ± SD tumor volume (mm3) over time (days) in NOD/SCID mice injected with MDA-MB-231 cells treated with shNeg control shRNA (green) vs. MBNL1 KD cells (red). (E, Left) Image of resected tumors from four replicate mice per treatment category and IHC staining with MBNL1 primary antibody (E, Right) of the excised tumors.

Fig. 4.

MBNL1 down-regulation leads to increased stem/progenitor-like properties in cancer. (A and B) Bar graph (Left) and representative images at 10× magnification (Right) showing mean (±SD) number of tumorspheres formed by cells transfected with shNeg and shMBNL1#2 in primary, secondary, and tertiary plating for indicated cell lines (n = 3). (C) X-Y graph (Top) showing mean ± SD tumor volume (mm3) over time (days) in NOD/SCID mice s.c. injected with MDA-MB-231 cells treated with shNeg control shRNA (green) vs. MBNL1 KD cells (red) at indicated dilutions. (Bottom) Representative images of excised tumors. (D) Relative expression of LGR5, CD133, OCT4, NANOG, and SOX2 in control and MBNL1 KD cells. Data show mean ± SD. (E) Scatterplot showing correlation between MBNL1 expression and stemness score in matched TCGA tumor/normal stomach and breast cancer samples. (F) Scatterplot showing correlation between MBNL1 expression and stemness score in three subtypes of breast adenocarcinoma. (G) Western blot showing MBNL1 expression in indicated cell lines grown either as monolayer or in suspension as tumor spheroids.

Fig. 5.

MBNL1 regulates MAP2K7 exon2 skipping. (A) RT-PCR validation of skipping of MAP2K7 exon2 in the stable MBNL1 KD MDA-MB-231 cell line. (B) Box plots showing median PSI values for MAP2K7 exon2 SIE in indicated cancer types using all available normal and tumor samples from TCGA. n.s. refers to not significant. (C) Scatterplots showing correlation between MBNL1 mRNA expression and MAP2K7 exon2 delta PSI values in matched tumor normal pairs in indicated cancer types. (D) Scatterplot showing correlation between stemness score and delta PSI value of MAP2K7 exon2 of matched TCGA tumor and normal cancer samples.

Fig. 6.

Low levels of MBNL1 and consequent presence of MAP2K7∆exon2 splice form is associated with increased JNK signaling and acquisition of stem/progenitor-like properties. (A) Western blot analysis of ph-JNK, JNK, ph-c-JUN, and c-JUN in MBNL1 KD and control MDA-MB-231 and HFE-145 cells in the presence or absence of serum. (B and C) Western blot analysis of ph-JNK and JNK in a panel of MBNL1-low/MAP2K7∆exon2-high and MBNL1-high/MAP2K7∆exon2-low cell lines. Note for B, all protein lysates were analyzed on the same sodium dodecyl sulfate polyacrylamide gel but image was edited to exclude MKN7 due to technical reasons. This edit is indicated by visibly separating the image. (D) MAP2K7 exon2 and GAPDH RT-PCR in HFE-145 cells transfected with control or MAP2K7 exon2 AMO. (E) Western blot showing expression of ph-JNK, JNK, ph-c-JUN, c-JUN, MAP2K7, MBNL1, and GAPDH in HFE-145 cells treated with control or MAP2K7 exon2 AMO. (F and I) qRT-PCR showing relative expression of CD133, LGR5, OCT4, NANOG, and SOX2 in HFE-145 cells transfected with control or MAP2K7 AMO and control or MAP2K7–JNK1 construct, respectively. Data show mean ± SD. (G and J) Bar graph (Top) and representative images (Bottom) showing migration (Left) and invasion (Right) in HFE-145 cells either transfected with control or MAP2K7 AMO (G) and transfected with either control or MAP2K7–JNK1 construct (J). Data show mean ± SD. (H) Western blots of ph-JNK, JNK, ph-c-JUN, and c-JUN expression in HFE-145 cells mock transfected with vehicle alone or with 0.5 µg MAP2K7–JNK1 construct. Right shows semiquantitative RT-PCR of MAP2K7 showing skipping of exon2 in HFE-145 cells transfected with MAP2K7–JNK1 construct.

Fig. 7.

JNK inhibition reverses MBNL1 KD-associated stem/progenitor-like properties. (A and B) qRT-PCR analysis showing mean (±SD) expression of CD133, LGR5, OCT4, NANOG, and SOX2 relative to GAPDH in shNeg and shMBNL1 MDA-MB-231 cells treated either with dimethyl sulfoxide or with an indicated JNK inhibitor. (C) Bar plot showing mean of effective dose (ED₅₀) of MBNL1-low/MAP2K7∆exon2-high and MBNL1-lhigh/MAP2K7∆exon2-low cell lines treated with JNK-IN-8. (D) Graphical representation of the role and function of MBNL1–MAP2K7∆exon2–JNK signaling in cancer.