Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

Abstract

The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Emu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Emu-Myc/+ mice. When accurate translational control is re-established in Emu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.

Figure 1. Myc-induced increases in protein synthesis regulate B lymphocyte size, division and apoptosis prior lymphomagenesis

a, Protein synthesis rates assessed by S³⁵ methionine incorporation and densitometry analysis (n=3) * p < 0.01. b, Cell size analysis (n=3) * p < 0.001 for Eμ–Myc/+ versus WT, ** p < 0.01 for Eμ–Myc/+ versus Eμ–Myc/+;L24^+/−. c, d, Cell cycle distribution and quantification of the percentage of cells in S phase (n=3). Red bar indicates S-phase, * p < 0.01, ** p < 0.05. e, Western blot analysis for cell cycle targets transcriptionally regulated by Myc f, In situ Tunnel analysis (n=3). Inserts are representative pictures of Tunnel analysis comparing Eμ–Myc/+ and Eμ–Myc/+;L24^+/− samples * p < 0.05, ** p < 0.05. a-e, all experiments were performed on freshly isolated B-lymphocytes. Error bars, s.d.

Figure 2. The ability of Myc to augment protein synthesis is necessary for its oncogenic potential

a, Representative photographs of spleens and average spleen weight (n=6 per genotype, 4 weeks of age). Error bars, s.d. b, Kaplan-Meier curves showing lymphoma-free survival (LFS) of Eμ–Myc/+ and Eμ–Myc/+;L24^+/− mice (n=30 per genotype). c, Kaplan-Meier curves showing tumor-free survival (TFS) of p53^−/− and p53^−/−;L24^+/− mice (n=25 per genotype).

Figure 3. Myc hyper-activation impairs the translational switch from cap to IRES-dependent translation control during mitosis and blocks mitotic translation of the Cdk11/p58 PITSLRE kinase

a,b, S³⁵ methionine incorporation in B-lymphocytes synchronized in S phase and mitosis. Densitometry analysis (n=3) comparing protein synthesis levels in S-phase and mitotically arrested B-lymphocytes. c, Cap-dependent activity of the Renilla luciferase reporter mRNA in asynchronous and mitotic synchronized MEFs expressing a tamoxifen inducible Myc vector (n=6 experiments performed in triplicate) *(p < 0.001) compared to WT, **(p < 0.005) compared to asynchronous values. d, HCV-IRES dependent activity of the Firefly luciferase reporter mRNA (n=6 experiments performed in triplicate) in asynchronous and mitotic synchronized MEFs ** (p < 0.001) compared to asynchronous values. e, Representative western blot of the endogenous Cdk11/p58 PITSLRE kinase in asynchronous and mitotic synchronized primary B-lymphocytes. Note that expression of Cdk11/p58 PITSLRE is only present in mitotically synchronized cells (left), and not asynchronous cells (right) via an IRES-element positioned in its 5′UTR. Densitometry analysis (n=3). f, Cdk11/p58-IRES dependent activity of Firefly luciferase reporter mRNA (n=4 experiments performed in triplicate) transfected in asynchronous and mitotic synchronized cells ** (p < 0.001) compared to asynchronous values. (c,d,f) Average steady state WT values were set to 1. The Y-axes show fold change. Error bars, s.d.

Figure 4. Aberrant translation control downstream of Myc activation underlies cytokinesis defects and genome instability

a, Myc overexpressing MEFs show increased numbers of binucleated cells (arrows). Insert illustrates dysmorphic and variably sized nuclei frequently observed in Myc overexpressing MEFs. b, Western blot of mitotic Cdk11/p58 expression showing decreased expression in MEFs expressing a Myc inducible vector. Myc+p58 cells express a retroviral Cdk11/p58 cDNA. c, The increased number of binucleated cells in Myc overexpressing cells is restored to normal level when Cdk11/p58 is reintroduced (n=4, at least 500 cells were scored per experiment) Error bars, s.d. d, Percentages of cells with normal (1 and 2) and aberrant (3) centrosome numbers in freshly isolated B-lymphocytes (n=6, at least 300 cells per experiment), *p < 0.001. Insert shows representative immunofluorescence staining with a centrosome marker. e, Comparative genomic hybridization (CGH) analysis of tumors (n=6). Error bars, s.d. f, Proposed model for how deregulations in translation control downstream of Myc activation lead to cancer initiation. Myc dependent increases in protein synthesis augment cell growth and this effect is coupled to increased cell cycle progression and a cell survival advantage. Increasing cap-dependent translation downstream of Myc-activation also gives rise to a specific molecular impairment in the modality of translation initiation employed during mitosis that leads to cytokinesis defects associated with genome instability.