Cellular senescence and protein degradation: breaking down cancer

Abstract

Autophagy and the ubiquitin-proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells.

Keywords: E3 ligases; ERK kinases; Ras oncogene; SASP; ubiquitin.

Figure 1. Oncogenic Ras increases overall protein degradation, but does not increase proteasome activity. (A) Normal human fibroblasts (IMR90; from ATCC) cultured in Dulbecco modified Eagle medium (DMEM; Wisent) and expressing oncogenic Ras (R) or an empty pWZL vector (V), 10 d after retroviral infection. Total protein extracts after a pulse with 0.5 µCi [35S]-methionine for 2 h, followed by a treatment with 75 µg/mL cycloheximide (CHX; Sigma-Aldrich) for the indicated times. (B) Bands were quantitated using Image Lab 4.0 (M = slope). An immunoblot for α-tubulin (1:5000; clone B-5–1-2, T6074, Sigma-Aldrich) was used for normalization. (C) Immunoblots for HRas (1:250; clone F235, Sc-29, Santa Cruz), α-tubulin and mono-polyubiquitylated conjugates (1:1000; clone FK2, BML-PW8810, Enzo Life sciences). Protein extracts from IMR90 cells as in (A), but treated with DMSO or 20 µM MG132 (Sigma-Aldrich) for 18 h. (D) Fibroblasts as in (A) were treated with 500 nM of the proteasome activity probe Me4BodipyFL-Ahx3Leu3VS (Boston Biochem, I-190) for 1 h. Total protein extracts were subjected to SDS-PAGE, and fluorescence was analyzed on a ChemiDoc^™ MP System (Bio-Rad). Multiple catalytic subunits are visible (β1, 2, and 5).

Figure 2. Senescence-associated phenotypes likely regulated by SAPD targets. Normal human fibroblasts, 10 d after infection with H-RasV12, were treated 18 h with DMSO (control) or 20 µM MG132 (Sigma-Aldrich). Then, cells were harvested, and protein extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for phosphoproteomics. Almost 3000 phosphopeptides from 1018 proteins were enriched. A FatiGO single enrichment analysis with the Babelomics 4.3 platform was perform in order to identify Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome terms significantly enriched. The terms related to a senescence phenotype and their associated peptides (398 proteins) were grouped into the indicated phenotypes.

Figure 3. The proteins corresponding to the genes downregulated by RB1 in Ras-induced senescent fibroblasts are also unstable. Unbiased Gene Set Enrichment Analysis (GSEA) of the proteomic data as in Figure 2. The gene set CHICAS_RB1_TARGETS_SENESCENT (Systematic name: M2125) was the second most significant result among the proteins stabilized by MG132 in Ras-induced senescent cells. The normalized enrichment score (NES), the nominal P value determined by an empirical phenotype-based permutation test procedure and the false discovery rate (FDR; Q value) are shown.

Figure 4. Modulation of protein stability for proteins regulated by phosphorylation-driven ubiquitination and proteasome-dependent degradation. (A) Under normal conditions, competition between the activity of kinases vs. phosphatases (PPases) and E3 ubiquitin ligases vs. deubiquitinases (DUBs) ensures the maintenance of appropriate levels of a specific protein. The turnover of this protein can be increased by (B) increasing the activity of its kinases; (C) increasing the activity of its E3 ubiquitin ligases; (D) both (B and C); (E) decreasing the activity of its PPases; (F) decreasing the activity of its DUBs; (G) both (E and F). Of note, different combinations of (B) to (G) can be involved. Also, a similar scenario can be applied for SUMO-dependent ubiquitination; kinases and PPases can be substituted by SUMO E3 ligases and deSUMOylase. Ub, ubiquitin; P, phosphorylation.

Figure 5. The balance of oncogenic vs. tumor suppressor E3 ubiquitin ligases. The activities of oncogenic vs. tumor suppressor E3 ligases are in equilibrium to maintain cells in a normal state. Tipping the balance in one direction or the other can be critical for determining whether a cell under oncogenic stress will undergo tumor suppression or neoplastic transformation.

Figure 6. Theoretical purpose of oncogene-induced senescence and contribution of protein degradation. Increasing evidence suggests that the destiny of senescent cells in many organs is clearance by the immune system. This implies a central role for the cytokine production characteristic of the SASP in the recruitment of immune cells (in red). Specific protein degradation (SAPD) may contribute directly and/or indirectly to the initial cell cycle arrest, but may also cooperate with macroautophagy to produce antigenic peptides and to support the SASP. Proteolysis may redistribute cellular energy to the SASP and may supply nutrient building blocks for biosynthetic reactions.