Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma
Lindsey N. Kent, … , James M. Pipas, Gustavo Leone
Lindsey N. Kent, … , James M. Pipas, Gustavo Leone
Published January 30, 2017
Citation Information: J Clin Invest. 2017;127(3):830-842. https://doi.org/10.1172/JCI87583.
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Research Article Cell biology Hepatology Article has an altmetric score of 2

Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma

Abstract

Disruption of the retinoblastoma (RB) tumor suppressor pathway, either through genetic mutation of upstream regulatory components or mutation of RB1 itself, is believed to be a required event in cancer. However, genetic alterations in the RB-regulated E2F family of transcription factors are infrequent, casting doubt on a direct role for E2Fs in driving cancer. In this work, a mutation analysis of human cancer revealed subtle but impactful copy number gains in E2F1 and E2F3 in hepatocellular carcinoma (HCC). Using a series of loss- and gain-of-function alleles to dial E2F transcriptional output, we have shown that copy number gains in E2f1 or E2f3b resulted in dosage-dependent spontaneous HCC in mice without the involvement of additional organs. Conversely, germ-line loss of E2f1 or E2f3b, but not E2f3a, protected mice against HCC. Combinatorial mapping of chromatin occupancy and transcriptome profiling identified an E2F1- and E2F3B-driven transcriptional program that was associated with development and progression of HCC. These findings demonstrate a direct and cell-autonomous role for E2F activators in human cancer.

Authors

Lindsey N. Kent, Sooin Bae, Shih-Yin Tsai, Xing Tang, Arunima Srivastava, Christopher Koivisto, Chelsea K. Martin, Elisa Ridolfi, Grace C. Miller, Sarah M. Zorko, Emilia Plevris, Yannis Hadjiyannis, Miguel Perez, Eric Nolan, Raleigh Kladney, Bart Westendorp, Alain de Bruin, Soledad Fernandez, Thomas J. Rosol, Kamal S. Pohar, James M. Pipas, Gustavo Leone

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Figure 3

Copy number increases in E2f1 or E2f3b result in HCC.

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Copy number increases in E2f1 or E2f3b result in HCC. (A) Diagram of the...
(A) Diagram of the murine E2f3 locus and E2f knockin alleles where the coding region of E2f1 or exon 1 of E2f3b has been inserted into the first exon of E2f3a, resulting in the loss of E2f3a and expression of E2f1 (3a1KI) or E2f3b (3a3bKI) driven by the E2f3a promoter. (B) Immunoblot of 1- and 12-month-old liver extracts from 3a1KI/1KI mice. The blot was probed with an E2F1-specific antibody. Liver from 1–/– mice was used to validate the antibody. Asterisks indicate nonspecific bands, and tubulin was used as a loading control. (C) Representative pictures of livers from 12- to 18-month-old mice (top), sections stained with H&E (middle), or probed with Ki-67–specific antibodies (bottom). Areas of HCC are outlined by dotted lines. T, tumor; N, normal liver. Scale bars: 1 cm (top); 100 μm (middle and bottom). (D) Box plots showing the liver/body weight of male mice from C (top): *P = 0.012, E2f3a1KI/1KI vs. E2f+/+; †P = 0.043, E2f3a3bKI/3bKI vs. E2f+/+, Wilcoxon method with Bonferroni’s correction for multiple tests. Histopathological classification (bottom): ‡P = 0.002, E2f3a1KI/+ vs. E2f+/+; §P < 0.001, E2f3a1KI/1KI vs. E2f+/+; P = 0.032, E2f3a3bKI/+ vs. E2f+/+; #P = 0.02, E2f3a3bKI/3bKI vs. E2f+/+, Fisher’s exact tests with Bonferroni’s correction. (E) Box plots showing the liver/body weight of female mice from C (top): *P = 0.002, E2f3a1KI/+ vs. E2f+/+; †P = 0.001, E2f3a1KI/1KI vs. E2f+/+; ‡P = 0.015, E2f3a3bKI/+ vs. E2f+/+; §P = 0.005, E2f3a3bKI/3bKI vs. E2f+/+, Wilcoxon method with Bonferroni’s correction. Histopathological classification (bottom): P < 0.001, E2f3a1KI/+ vs. E2f+/+ liver; #P < 0.001, E2f3a1KI/1KI vs. E2f+/+ liver; **P = 0.014, E2f3a3bKI/+ vs. E2f+/+ liver; ††P < 0.001, E2f3a3bKI/3bKI vs. E2f+/+ liver, Fisher’s exact tests with Bonferroni’s correction. (F) Box plots showing the liver/body weight (top) or bar graph showing histopathological classification (bottom) of DEN-treated E2f+/+ and 3a1KI/1KI male mice at 9 months of age.