. 2013 Jun 4;17(6):1000-1008.

doi: 10.1016/j.cmet.2013.04.013. Epub 2013 May 23.

EGFR mutation-induced alternative splicing of Max contributes to growth of glycolytic tumors in brain cancer

Ivan Babic¹, Erik S Anderson², Kazuhiro Tanaka³, Deliang Guo⁴, Kenta Masui¹, Bing Li⁵, Shaojun Zhu⁶, Yuchao Gu⁷, Genaro R Villa⁸, David Akhavan⁹, David Nathanson⁶, Beatrice Gini¹, Sergey Mareninov¹⁰, Rui Li⁶, Carolina Espindola Camacho⁶, Siavash K Kurdistani⁵, Ascia Eskin¹¹, Stanley F Nelson¹¹, William H Yong¹⁰, Webster K Cavenee¹², Timothy F Cloughesy¹³, Heather R Christofk⁶, Douglas L Black¹⁴, Paul S Mischel¹⁵

Affiliations

¹ Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA.
² Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Molecular Biology Interdepartmental Graduate Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Neurosurgery, Kobe University, Kobe 650-0017, Japan.
⁴ Department of Radiation Oncology, James Comprehensive Cancer Center, The Ohio State University Medical Center, Columbus, OH 43210, USA.
⁵ Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁶ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁷ Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA; Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁸ Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA; Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁹ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁰ Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹¹ Department of Human Genetics, David Geffen School of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹² Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA; Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA.
¹³ Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁴ Howard Hughes Medical Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁵ Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA; Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA; Department of Pathology, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address: pmischel@ucsd.edu.

PMID: 23707073
PMCID: PMC3679227
DOI: 10.1016/j.cmet.2013.04.013

EGFR mutation-induced alternative splicing of Max contributes to growth of glycolytic tumors in brain cancer

Ivan Babic et al. Cell Metab. 2013.

. 2013 Jun 4;17(6):1000-1008.

doi: 10.1016/j.cmet.2013.04.013. Epub 2013 May 23.

Authors

Affiliations

¹ Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA.
² Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Molecular Biology Interdepartmental Graduate Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Neurosurgery, Kobe University, Kobe 650-0017, Japan.
⁴ Department of Radiation Oncology, James Comprehensive Cancer Center, The Ohio State University Medical Center, Columbus, OH 43210, USA.
⁵ Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁶ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁷ Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA; Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁸ Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA; Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁹ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; UCLA Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁰ Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹¹ Department of Human Genetics, David Geffen School of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹² Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA; Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA.
¹³ Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁴ Howard Hughes Medical Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁵ Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093, USA; Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA; Department of Pathology, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address: pmischel@ucsd.edu.

PMID: 23707073
PMCID: PMC3679227
DOI: 10.1016/j.cmet.2013.04.013

Abstract

Alternative splicing contributes to diverse aspects of cancer pathogenesis including altered cellular metabolism, but the specificity of the process or its consequences are not well understood. We characterized genome-wide alternative splicing induced by the activating EGFRvIII mutation in glioblastoma (GBM). EGFRvIII upregulates the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 splicing factor, promoting glycolytic gene expression and conferring significantly shorter survival in patients. HnRNPA1 promotes splicing of a transcript encoding the Myc-interacting partner Max, generating Delta Max, an enhancer of Myc-dependent transformation. Delta Max, but not full-length Max, rescues Myc-dependent glycolytic gene expression upon induced EGFRvIII loss, and correlates with hnRNPA1 expression and downstream Myc-dependent gene transcription in patients. Finally, Delta Max is shown to promote glioma cell proliferation in vitro and augment EGFRvIII expressing GBM growth in vivo. These results demonstrate an important role for alternative splicing in GBM and identify Delta Max as a mediator of Myc-dependent tumor cell metabolism.

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Figures

**Figure 1. EGFRvIII enhances alternative splicing, induces expression of Myc-target genes, and promotes glucose uptake in vivo**
(A) Schematic illustration of experimental design used to coordinately analyze gene expression and alternative splicing promoted by EGFRvIII *in vivo*. (B) Heat map of expression for genes in the indicated gene sets (EGF-Pathway and Myc). Gene expression in U87 and U87-EGFRvIII tumors from three mice is shown. (C) Schematic showing increased expression of Myc-regulated genes in EGFRvIII-expressing tumors. The fold upregulation of these transcripts in EGFRvIII expressing tumors is shown in green. (D) MicroPET/CT imaging comparing U87 with U87-EGFRvIII xenograft tumors (arrow). The graph represents FDG uptake averaged for three tumors (white-dotted circles) presented as percentage injected dose per gram (%ID/G) (p value by t test).

**Figure 2. EGFRvIII regulates expression of Myc and hnRNPA1 splicing factor promoting glycolysis, proliferation and shorter patient survival**
(A) Immunoblot analysis of Myc and downstream splicing factors from three U87 xenograft tumor lysates and three U87-EGFRvIII xenograft tumor lysates. Numerical values below HnRNPA1 and Myc blots (black arrows) is densitometric quantification of bands normalized to tubulin. (B) (Left) Immunoblot analysis of lysates from U373 with doxycycline-regulated EGFRvIII expression, and (right) relative mRNA transcript levels of GLUT1, GLUT3, HK2, and PDK1 upon acute loss of EGFRvIII (n=4; shown is the mean±SD; * p<0.05). (C) Immunoblot analysis of patient GBM biopsy samples having low EGFR expression or EGFR amplification and EGFRvIII expression. (D) Kaplan-Meier overall survival plots of 131 primary GBM patients stratified by survival plots for hnRNPA1 expression. The single p-value is based on the difference of the two curves, and was calculated using the log-rank test. (E) Cell proliferation of U87-EGFRvIII cells with hnRNPA1 knockdown (n=3; shown is mean±SD; ** p<0.01). (F) Uptake of fluorescein-conjugated glucose (2NBDG) after 1 hour in U87-EGFRvIII cells transfected with the indicated siRNA. Unstained are cells without 2NBDG. (G) RT-qPCR analysis for glycolytic gene expression in U87-EGFRvIII cells with hnRNPA1 knockdowns (n=3; shown is mean±SD; * p<0.05).

**Figure 3. HnRNPA1-dependent splicing of the Myc-heterodimerization partner Max results in the truncated variant Delta Max**
(A) RT-PCR for Max intron upstream of exon 5 using RNA extracted from hnRNPA1 immunoprecipitated from U87-EGFRvIII cells after UV crosslinking (CLIP) as described in Supplemental Experimental Procedures. (B) RT-PCR splicing analysis for Max exon 5 and immunoblot analysis for hnRNPA1 in neurospheres established from patient GBM biopsies. (C) RT-PCR splicing analysis for Max exon 5 in hnRNPA1 knockdown cells from Figure 2E. (D) Max immunoblot of lysates from U87-EGFRvIII cells transfected with siRNAs to hnRNPA1. (E) Immunoblot for Max in Patient GBM biopsies from Figure 2C.

**Figure 4. Delta Max promotes glycolytic gene expression and tumor growth in vitro and in vivo**
(A) Proliferation of U87 cells on glucose- or galactose-containing media after transfection with empty vector (EV), wild-type (WT) Max, or Delta Max (n=4; shown is the mean±SD; ***p<0.001). (B) Relative mRNA transcript levels of GLUT1, GLUT3, HK2, and PDK1 upon acute loss of EGFRvIII with or without Delta Max or wild-type (WT) Max overexpression (n=3; shown is the mean±SD; * p<0.05; N.S. is non-significant). (C) Xenograft tumors from U87-EGFRvIII cells with stable knockdown of Delta Max grew slower that control (Sh Scrambled) cells as measured by tumor volume (n=4; shown is the mean±SD; **p<0.01). (D) RT-qPCR analysis of glycolytic gene expression in Delta Max knockdown U87-EGFRvIII cells (n=3; shown is the mean±SD; * p<0.05). (E) Diagram illustrating EGFRvIII activation of mTOR upregulates Myc which stimulates hnRNPA1 expression and promotes splicing of Max generating Delta Max which augments Myc activity and increases aerobic glycolysis.

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References

1. Blackwood EM, Eisenman RN. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science. 1991;251(4998):1211–1217. - PubMed
1. Cancer Genome Atlas Research, N. . Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455(7216):1061–1068. - PMC - PubMed
1. Christofk HR, Vander Heiden MG, et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature. 2008;452(7184):230–233. - PubMed
1. Christofk HR, Vander Heiden MG, et al. Pyruvate kinase M2 is a phosphotyrosine-binding protein. Nature. 2008;452(7184):181–186. - PubMed
1. Clower CV, Chatterjee D, et al. The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism. Proc Natl Acad Sci U S A. 2010;107(5):1894–1899. - PMC - PubMed

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EGFR mutation-induced alternative splicing of Max contributes to growth of glycolytic tumors in brain cancer

Affiliations

EGFR mutation-induced alternative splicing of Max contributes to growth of glycolytic tumors in brain cancer

Authors

Affiliations

Abstract

Figures

References

Publication types

MeSH terms

Substances

Associated data

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Molecular Biology Databases

Research Materials

Miscellaneous