Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma
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
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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Research Article Cell biology Hepatology

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 8

Intersection of gene-expression profiling and chromatin binding identifies E2F1 and E2F3B targets.

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Intersection of gene-expression profiling and chromatin binding identifi...
(A) Heat map of Affymetrix microarray data showing differentially expressed genes in 3a1KI/1KI liver tumors when compared with normal liver samples from E2f+/+ and 3a–/– age-matched controls. Differentially expressed genes are defined as having a fold change of 1.5 or more (P ≤ 0.05) relative to 3a–/– samples. (B) Heat map of Affymetrix microarray data showing differentially expressed genes in 3a3bKI/3bKI liver tumors when compared with normal liver samples from E2f+/+ and 3a–/– age-matched controls. Differentially expressed genes are defined as having a fold change of 1.5 or more (P ≤ 0.05) relative to 3a–/– samples. (C) Venn diagram illustrating the overlap of E2F1-specific promoter peaks with upregulated or downregulated genes in 3a1KI/1KI liver tumors identified in A. (D) Venn diagram illustrating the overlap of E2F3B-specific promoter peaks with upregulated or downregulated genes in 3a3bKI/3bKI liver tumors identified in B. (E) Sequence tag-density heat map showing the distribution of E2F1, E2F3A, and E2F3B binding to targets identified in C and D (overlapping groups). (F) ChIP-qPCR validation using E2F1, E2F3, or IgG antibodies in SNU-449 and PLC/PRF5 HCC-derived cells. Occupancy of E2Fs on selected target promoters is shown. A nonpromoter region of TUBA4A (TUBA4A neg) was used as a negative control. Primers were designed to amplify ChIP-seq–identified peak regions.