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

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

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 1

Lineage-specific splicing of a brain-enriched exon in ANXA7 abrogates EGFR regulation.

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Lineage-specific splicing of a brain-enriched exon in ANXA7 abrogates EG...
(A) Schematic representation of the ANXA7 exon structure to highlight the alternative splicing between exons 5 and 7 generating the ANXA7-I2 variant. (B and C) Expression of brain-specific ANXA7-I1 mRNA and splice variant ANXA7-I2 mRNA (shown as a ratio) in normal brain (NB), normal human astrocytes, neural precursor cells (NPCs), A2B5-positive and -negative glial precursor cells (GPCs), glioblastoma (GBM) tumors, and GBM-derived cell cultures measured by qRT-PCR (B) and corresponding box plot summary (C) showing the smallest and largest observations (upper and lower whiskers, respectively), the interquartile range (box), and the median (black line); data points more than 1.5 times the interquartile range lower than the first quartile or 1.5 times the interquartile range higher than the third quartile were considered to be outliers; P value by an unpaired t test. (D) ANXA7-I1 and ANXA7-I2 protein expression in NB and glioblastoma-derived cell cultures. (E) EGFR pathway activation upon EGF ligand stimulation for various time periods in SNB19 glioblastoma cells transduced with lentivirus to express empty vector (EV), ANXA7-I2, or ANXA7-I1 (both His-tagged). Lanes were run on the same gel but were noncontiguous (indicated by thin black lines). (F) EGFR endosomal marker (EEA1, M6PR) coimmunostaining of EGF-stimulated SNB19-EV, SNB19-ANXA7-I2, and SNB19-ANXA7-I1 cells; nuclei were counterstained with DAPI. Scale bars: 50 μm; 10 μm (insets). (G) Time-dependent EGFR protein abundance and His-tagged ANXA7-I2 and ANXA7-I1 in EGF-stimulated SNB19-EV, SNB19-ANXA7-I2, and SNB19-ANXA7-I1 cells treated with cycloheximide (CHX) to halt protein synthesis. (H) Quantification of EGFR.