SUMO-modified nuclear cyclin D1 bypasses Ras-induced senescence

Abstract

Oncogene-induced senescence represents a key tumor suppressive mechanism. Here, we show that Ras oncogene-induced senescence can be mediated by the recently identified haploinsufficient tumor suppressor apoptosis-stimulating protein of p53 (ASPP) 2 through a novel and p53/p19 Arf /p21 waf1/cip1 -independent pathway. ASPP2 suppresses Ras-induced small ubiquitin-like modifier (SUMO)-modified nuclear cyclin D1 and inhibits retinoblastoma protein (Rb) phosphorylation. The lysine residue, K33, of cyclin D1 is a key site for this newly identified regulation. In agreement with the fact that its nuclear localization is required for its oncogenic activity, we show that nuclear cyclin D1 is far more potent than wild-type (WT) cyclin D1 in bypassing Ras-induced senescence. Thus, this study identifies SUMO modification as a positive regulator of nuclear cyclin D1, and reveals a new way by which cell cycle entry and senescence are regulated. Cell Death and Differentiation (2011) 18, 304-314; doi:10.1038/cdd.2010; published online 27 August 2010Cellular senescence is an irreversible cell cycle arrest and one of the major cellular processes that suppress tumor growth. Cyclin-dependent kinases (CDKs) are among the main regulators of cellular senescence, predominantly achieved through their phosphorylation of the retinoblastoma protein (Rb), preventing it from binding and inhibiting the E2F family of transcription factors that are involved in cell cycle progression. Deregulation of CDK pathways is common in the development of human cancers. Amplification and overexpression of cyclin D1, a regulatory subunit of CDK4/CDK6, has been observed in around 50% of human breast and esophageal cancers. 1,2 Interestingly, however, elevation of cyclin D1 alone is not sufficient to induce tumor growth in vivo, 3,4 partly owing to the fact that the cyclin D1/CDK4 or cyclin D1/CDK6 complex needs to locate in the nucleus to phosphorylate Rb during S-phase to promote cell proliferation. 5,6 Nuclear export, coupled with ubiquitin-dependent destruction of cyclin D1 in the cytoplasm during S-phase, is one of the best known mechanisms of cyclin D1/CDK4 kinase activity control. Hence, nuclear accumulation of cyclin D1 may be a key mechanism by which cyclin D1 exerts its oncogenic effects. In agreement with this, overexpression of mutant cyclin D1 (D1T286A), defective in phosphorylation-mediated nuclear export and subsequent proteolysis, induces cell transformation in vitro and triggers B-cell lymphoma in vivo. 6,7 Furthermore, transgenic mice overexpressing the same mutant, driven by mouse mammary tumor virus (MMTV) promoter (MMTV-D1T286A), developed mammary adenocarcinoma with a shorter latency relative to mice overexpressing wild-type (WT) cyclin D1 (MMTV-D1). 8 These observations support the notion that the subcellular localization of cyclin D1 is critical in controlling its tumorigenicity. Identifying the molecular mechanisms that regulate the nuclear localization of cyclin D1 is, therefore, vital for our better understanding of tumor development....

Autophagic activity dictates the cellular response to oncogenic RAS

“…High levels of nuclear cyclin D1/CDK4 kinase activity are essential for cells to phosphorylate retinoblastoma protein (Rb) and to bypass cell cycle arrest and cellular senescence. We observed recently that ASPP2 uses a p53/ p19 Arf /p21 waf1 independent pathway to mediate RAS-induced senescence by inhibiting RAS-induced nuclear accumulation of small ubiquitin-like modifier (SUMO)-modified cyclin D1 (15). Because ASPP2 deficiency enhances RAS-induced autophagy and elevated autophagy is sufficient to bypass RAS-induced senescence, it is possible that elevated autophagic activity may use the same pathway as that of ASPP2 deficiency to bypass RASinduced senescence.…”

“…For autophagy activity assays, measurement of the degradation of long-lived proteins, GFP-LC3 immunofluorescence, and LC3II/I Western blotting were performed. Analysis of senescence in cell culture was performed using a senescence β-gal staining kit (Cell Signaling) or BrdU incorporation experiments (15). To quantify SA-β-gal-or BrdU-positive readings, at least 200 cells were counted in random fields in each of the duplicated wells.…”

“…Oncogenic RAS induces senescence in primary fibroblasts. We showed recently that ASPP2 deficiency bypasses oncogenic RAS-induced senescence independent of p53 (15). To identify the region of ASPP2 that mediates RAS-induced senescence, two ASPP2 mutants were generated: ASPP2(1-360) and ASPP2(123-1,128).…”

“…Interestingly, an NMR study showed that the N-terminal ASPP2, a known activator of p53, shares high structure similarity with LC3 and ATG12 (14). Additionally, we showed recently that ASPP2 deficiency can bypass oncogenic RAS-induced senescence independent of p53 (15). ASPP2 was first identified as a p53 binding protein, with subsequent studies showing that it enhances p53-induced apoptosis in vitro (16) and p53-mediated tumor suppression in vivo (17).…”

SUMO1‐conjugation is altered during normal aging but not by increased amyloid burden

“…Consistent with this initial finding, our statistical analysis revealed a small group of candidate SUMO1 targets whose SUMOylation in vivo appeared to be altered during aging, but no SUMO1 candidate was identified to be altered during increased amyloid burden. Interestingly, these age‐related proteins are components of cellular signaling pathways with roles mainly in RNA processing, but also in protein folding and Ras signaling, many of which are known to play a role during aging (Balchin, Hayer‐Hartl & Hartl, 2016; Liu, Cali & Lee, 2017; Longo, 2004; Wang et al., 2011). Most of the nuclear proteins identified as age‐related SUMO1 candidate proteins are involved in the regulation of gene expression, chromatin remodeling, RNA processing, and protein folding.…”