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Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression
Roberto Ferrarese, … , Maria S. Carro, Markus Bredel
Roberto Ferrarese, … , Maria S. Carro, Markus Bredel
Published May 27, 2014
Citation Information: J Clin Invest. 2014;124(7):2861-2876. https://doi.org/10.1172/JCI68836.
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Research Article Oncology Article has an altmetric score of 21

Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression

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Abstract

Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones.

Authors

Roberto Ferrarese, Griffith R. Harsh IV, Ajay K. Yadav, Eva Bug, Daniel Maticzka, Wilfried Reichardt, Stephen M. Dombrowski, Tyler E. Miller, Anie P. Masilamani, Fangping Dai, Hyunsoo Kim, Michael Hadler, Denise M. Scholtens, Irene L.Y. Yu, Jürgen Beck, Vinodh Srinivasasainagendra, Fabrizio Costa, Nicoleta Baxan, Dietmar Pfeifer, Dominik von Elverfeldt, Rolf Backofen, Astrid Weyerbrock, Christine W. Duarte, Xiaolin He, Marco Prinz, James P. Chandler, Hannes Vogel, Arnab Chakravarti, Jeremy N. Rich, Maria S. Carro, Markus Bredel

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Figure 2

PTBP1 mediates exon 6 skipping in ANXA7 pre-RNA.

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PTBP1 mediates exon 6 skipping in ANXA7 pre-RNA.
(A) PTBP1 mRNA expressi...
(A) PTBP1 mRNA expression in normal brain, glioblastoma, and glioblastoma-derived lines by qRT-PCR. n = 8 (normal brain); n = 6 (glioblastoma tumors); n = 13 (glioblastoma-derived lines). Error bars represent the mean ± SD. (B) PTBP1, ANXA7-I1, and ANXA7-I2 protein abundance upon PTBP1 silencing in SNB19, LN229, and BTSC23 lines, the latter being run on a different gel (spliced blot is indicated by a thin black line). (C–F) qRT-PCR analysis showing changes in the ANXA7-I1/ANXA7-I2 mRNA ratio upon PTBP1 silencing in SNB19 (C), LN229 (D), and BTSC23 (E) lines (high endogenous PTBP1 expression) and upon PTBP1 overexpression in BTSC145 cells (low endogenous PTBP1 expression) (F). n = 3 independent experiments for all samples. Error bars represent the mean ± SD. (G and H) ANXA7 RNA IP with PTBP1 antibody in MB003 line. PTBP1 protein precipitation upon RNA IP (G). ANXA7 amplification from the anti-PTBP1 coprecipitated RNA fraction (H). (I) Splicing reporter (ET) assay after cloning ANXA7 genomic region from exon 5 to exon 7 showing reexpression of ANXA7-I1 in SNB19 shPTBP1-ET-ANXA7 cells. (J) Representation of the cloned ANXA7 minigene with predicted PTBP1-binding sites and RIP primer location. Heatmap based on PTB CLIP-seq data modeling showing the influence of individual nucleotide mutations 1–7 (superimposed); scores shown are from dark blue (positive) to white (negative); green represents midrange. Blue bars show PTB affinity to the original sequence (top) and the reduced PTB affinity of all 7 mutations (bottom). (K) Changes in ANXA7-I1/ANXA7-I2 mRNA ratio by qRT-PCR after mutagenesis in the cloned ANXA7 minigene. P values refer to each mutation versus wild-type. n = 3 independent experiments for all samples. Error bars represent the mean ± SD.

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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