Here, we report a cell-intrinsic mechanism by which oncogenic RAS promotes senescence while predisposing cells to senescence bypass by allowing for secondary hits. We show that oncogenic RAS inactivates the BRCA1 DNA repair complex by dissociating BRCA1 from chromatin. This event precedes senescence-associated cell cycle exit and coincides with the accumulation of DNA damage. Downregulation of BRIP1, a physiological partner of BRCA1 in the DNA repair pathway, triggers BRCA1 chromatin dissociation. Conversely, ectopic BRIP1 rescues BRCA1 chromatin dissociation and suppresses RAS-induced senescence and the DNA damage response. Significantly, cells undergoing senescence do not exhibit a BRCA1-dependent DNA repair response when exposed to DNA damage. Overall, our study provides a molecular basis by which oncogenic RAS promotes senescence. Because DNA damage has the potential to produce additional "hits" that promote senescence bypass, our findings may also suggest one way a small minority of cells might bypass senescence and contribute to cancer development.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.
"Diminished sumoylation of chromatinassociated substrates in this fraction is therefore most likely to be masked by the presence of the PML NBs and other insoluble SUMO susbtrates. However, in agreement with previous reports (Tu et al. 2011), a reduction in BRCA1 is seen in the nuclear pellet fraction of the senescent cells (Fig. 7C). Thus, although global sumoylation increases in senescent cells, the ChIP-seq experiments reveal a general loss of sumoylated chromatin-associated proteins in the senescent state. "
[Show abstract][Hide abstract] ABSTRACT: Despite numerous studies on specific sumoylated transcriptional regulators, the global role of SUMO on chromatin in relation to transcription regulation remains largely unknown. Here, we determined the genome-wide localisation of SUMO1 and SUMO2/3, as well as of UBC9 (encoded by UBE2I) and PIASY (encoded by PIAS4), two markers for active sumoylation, along with Pol II and histone marks in proliferating versus senescent human fibroblasts together with gene expression profiling. We found that, whereas SUMO alone is widely distributed over the genome with strong association at active promoters, active sumoylation occurs most prominently at promoters of histone and protein biogenesis genes, as well as Pol I rRNAs and Pol III tRNAs. Remarkably, these four classes of genes are up-regulated by inhibition of sumoylation, indicating that SUMO normally acts to restrain their expression. In line with this finding, sumoylation-deficient cells show increase in both cell size and global protein levels. Strikingly, we found that, in senescent cells, the SUMO machinery is selectively retained at histone and tRNA gene clusters, whereas it is massively released from all other unique chromatin regions. These data, which reveal the highly dynamic nature of the SUMO landscape, suggest that maintenance of a repressive environment at histone and tRNA loci is a hallmark of the senescent state. The approach taken in our study thus permitted the identification of a common biological output and uncovered hitherto unknown functions for active sumoylation at chromatin as a key mechanism that, in dynamically marking chromatin by a simple modifier, orchestrates concerted transcriptional regulation of a network of genes essential for cell growth and proliferation.
"Immunofluorescence, BrdU Labeling, and SA-b-Gal Staining Immunofluorescence staining and BrdU labeling were performed as described previously using antibodies described above (Tu et al., 2011; Zhang et al., 2005, 2007a, 2007b). SA-b-gal staining was performed as previously described (Dimri et al., 1995). "
[Show abstract][Hide abstract] ABSTRACT: Oncogene-induced senescence is characterized by a stable cell growth arrest, thus providing a tumor suppression mechanism. However, the underlying mechanisms for this phenomenon remain unknown. Here, we show that a decrease in deoxyribonucleotide triphosphate (dNTP) levels underlies oncogene-induced stable senescence-associated cell growth arrest. The decrease in dNTP levels is caused by oncogene-induced repression of ribonucleotide reductase subunit M2 (RRM2), a rate-limiting protein in dNTP synthesis. This precedes the senescence-associated cell-cycle exit and coincides with the DNA damage response. Consistently, RRM2 downregulation is both necessary and sufficient for senescence. Strikingly, suppression of nucleotide metabolism by RRM2 repression is also necessary for maintenance of the stable senescence-associated cell growth arrest. Furthermore, RRM2 repression correlates with senescence status in benign nevi and melanoma, and its knockdown drives senescence of melanoma cells. These data reveal the molecular basis whereby the stable growth arrest of oncogene-induced senescence is established and maintained through suppression of nucleotide metabolism.
"The human breast cancer cell line MCF7, which is BRCA1 and 53BP1 proficient, was depleted of BRCA1 via lentiviral transduction with shRNAs (Fig. 1 A and Fig. S1 A). As previously shown in human fibroblasts (Tu et al., 2011), depletion of BRCA1 in MCF7 cells induces growth arrest (Fig. 1 B). BRCA1-deficient cells did not show differences in the levels of 53BP1 or CTSL proteins immediately after growth arrest (Fig. 1 C). "
[Show abstract][Hide abstract] ABSTRACT: Loss of 53BP1 rescues BRCA1 deficiency and is associated with BRCA1-deficient and triple-negative breast cancers (TNBC) and with resistance to genotoxic drugs. The mechanisms responsible for decreased 53BP1 transcript and protein levels in tumors remain unknown. Here, we demonstrate that BRCA1 loss activates cathepsin L (CTSL)-mediated degradation of 53BP1. Activation of this pathway rescued homologous recombination repair and allowed BRCA1-deficient cells to bypass growth arrest. Importantly, depletion or inhibition of CTSL with vitamin D or specific inhibitors stabilized 53BP1 and increased genomic instability in response to radiation and poly(adenosine diphosphate-ribose) polymerase inhibitors, compromising proliferation. Analysis of human breast tumors identified nuclear CTSL as a positive biomarker for TNBC, which correlated inversely with 53BP1. Importantly, nuclear levels of CTSL, vitamin D receptor, and 53BP1 emerged as a novel triple biomarker signature for stratification of patients with BRCA1-mutated tumors and TNBC, with potential predictive value for drug response. We identify here a novel pathway with prospective relevance for diagnosis and customization of breast cancer therapy.
Full-text · Article · Jan 2013 · The Journal of Cell Biology