Autophagy-mediated degradation of nuclear envelope proteins during oncogene-induced senescence.

Cellular senescence is a largely irreversible form of cell cycle arrest triggered by various types of damage and stress, including oncogene expression (termed oncogene-induced senescence or OIS). We and others have previously demonstrated that OIS occurs in human benign lesions, acting as a potent tumor suppressor mechanism. Numerous phenotypic changes occur during OIS, both in the cytoplasm and in the nucleus. These include the activation of autophagy, a catabolic process operating in the cytoplasm and downregulation of lamin B1, a component of the nuclear lamina. However, it is unknown whether these changes relate to each other. We discovered that cells entering BRAF(V600E)- or H-RAS(G12V)-induced senescence downregulate not only lamin B1 but also lamin A, as well as several other nuclear envelope (NE) proteins, resulting in an altered NE morphology. Depletion of LMNB1 or LMNA/C was sufficient to recapitulate some OIS features, including cell cycle exit and downregulation of NE proteins. We further found that the global loss of NE proteins is a consequence of their degradation by the autophagy machinery, which occurs concomitantly with autophagy induction and increased lysosomal content and activity. Our study therefore reveals a previously unknown connection between autophagy and the disruption of NE integrity during OIS.

Functional identification of LRF as an oncogene that bypasses RASV12-induced senescence via upregulation of CYCLIN E.

Mutant RAS (RAS(V12)) is known to transform most immortal cells but to induce premature senescence in primary cells. RAS(V12)-induced senescence in murine cells depends on the induction of the ARF/p53 and the retinoblastoma (Rb) family tumor suppressor pathways. We and others have shown previously that oncogene-induced senescence in vitro can be used as a tool to identify new cancer-related genes. In addition, we have shown that oncogene-induced senescence corresponds to an in vivo tumor suppressive mechanism. Therefore, we extended our search for novel genes that bypass of RAS(V12)-induced senescence, with the help of a previously designed unbiased functional screen with cDNA expression libraries. In this screen, we expected to find new mediators feeding into the p53 or Rb pathways or novel signaling factors. We report here the identification of leukemia/lymphoma related factor (Lrf) encoding a transcription factor with a BTB/POZ domain and Krüppel-like zinc fingers. This gene was previously identified as a potential oncogene that is overexpressed in human cancer. We find that LRF enhances E2F-dependent transcription and that it synergizes with RAS(V12) in activating E2F. Indeed, LRF-mediated bypass of RAS(V12)-induced senescence is accompanied by the induction of several E2F-target genes, including Cyclin E, Cyclin A and p107. Unexpectedly, LRF exerted this activity independent of several critical senescence inducers, such as p19(ARF), p21(CIP) and p16(INK4A). We show that CYCLIN E is necessary for LRF-mediated bypass, suggesting that it corresponds to a critical mediator of LRF-driven oncogenic transformation. Thus, LRF bypasses RAS(V12)-induced senescence in a CYCLIN E-dependent manner, which conceivably contributes to its role in cancer.

Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network.

Oncogene-induced cellular senescence (OIS) is emerging as a potent cancer-protective response to oncogenic events, serving to eliminate early neoplastic cells from the proliferative pool. Using combined genetic and bioinformatic analysis, we find that OIS is linked specifically to the activation of an inflammatory transcriptome. Induced genes included the pleiotropic cytokine interleukin-6 (IL-6), which upon secretion by senescent cells acted mitogenically in a paracrine fashion. Unexpectedly, IL-6 was also required for the execution of OIS, but in a cell-autonomous mode. Its depletion caused the inflammatory network to collapse and abolished senescence entry and maintenance. Furthermore, we demonstrate that the transcription factor C/EBPbeta cooperates with IL-6 to amplify the activation of the inflammatory network, including IL-8. In human colon adenomas, IL-8 specifically colocalized with arrested, p16(INK4A)-positive epithelium. We propose a model in which the context-dependent cytostatic and promitogenic functions of specific interleukins contribute to connect senescence with an inflammatory phenotype and cancer.

Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB.

Metastasis is a major factor in the malignancy of cancers, and is often responsible for the failure of cancer treatment. Anoikis (apoptosis resulting from loss of cell-matrix interactions) has been suggested to act as a physiological barrier to metastasis; resistance to anoikis may allow survival of cancer cells during systemic circulation, thereby facilitating secondary tumour formation in distant organs. In an attempt to identify metastasis-associated oncogenes, we designed an unbiased, genome-wide functional screen solely on the basis of anoikis suppression. Here, we report the identification of TrkB, a neurotrophic tyrosine kinase receptor, as a potent and specific suppressor of caspase-associated anoikis of non-malignant epithelial cells. By activating the phosphatidylinositol-3-OH kinase/protein kinase B pathway, TrkB induced the formation of large cellular aggregates that survive and proliferate in suspension. In mice, these cells formed rapidly growing tumours that infiltrated lymphatics and blood vessels to colonize distant organs. Consistent with the ability of TrkB to suppress anoikis, metastases--whether small vessel infiltrates or large tumour nodules--contained very few apoptotic cells. These observations demonstrate the potent oncogenic effects of TrkB and uncover a specific pro-survival function that may contribute to its metastatic capacity, providing a possible explanation for the aggressive nature of human tumours that overexpress TrkB.

E2F transcriptional repressor complexes are critical downstream targets of p19(ARF)/p53-induced proliferative arrest.

The p16(INK4a)/pRB/E2F and p19(ARF)/p53 tumor suppressor pathways are disrupted in most human cancers. Both p19(ARF) and p53 are required for the induction of senescence in primary mouse embryonic fibroblasts (MEFs), but little is known about their downstream targets. Disruption of E2F-mediated transcriptional repression in MEFs caused a general increase in the expression of E2F target genes, including p19ARF. We detected no contribution of E2F-mediated transactivation in this setting, indicating that a predominant role of endogenous E2F in asynchronously growing primary MEFs is to repress its target genes. Moreover, relief of transcriptional repression by E2F rendered MEFs resistant to senescence induced by either p19(ARF), p53, or RAS(V12). Thus, E2F transcriptional repressor complexes are critical downstream targets of antiproliferative p19(ARF)/p53 signaling.

A functional screen identifies hDRIL1 as an oncogene that rescues RAS-induced senescence.

Primary fibroblasts respond to activated H-RAS(V12) by undergoing premature arrest, which resembles replicative senescence. This irreversible 'fail-safe mechanism' requires p19(ARF), p53 and the Retinoblastoma (Rb) family: upon their disruption, RAS(V12)-expressing cells fail to undergo senescence and continue to proliferate. Similarly, co-expression of oncogenes such as c-MYC or E1A rescues RAS(V12)-induced senescence. To identify novel genes that allow escape from RAS(V12)-induced senescence, we designed an unbiased, retroviral complementary DNA library screen. We report on the identification of DRIL1, the human orthologue of the mouse Bright and Drosophila dead ringer transcriptional regulators. DRIL1 renders primary murine fibroblasts unresponsive to RAS(V12)-induced anti-proliferative signalling by p19(ARF)/p53/p21(CIP1), as well as by p16(INK4a). In this way, DRIL1 not only rescues RAS(V12)-induced senescence but also causes these fibroblasts to become highly oncogenic. Furthermore, DRIL1 immortalizes mouse fibroblasts, in the presence of high levels of p16(INK4a). Immortalization by DRIL1, whose product binds the pRB-controlled transcription factor E2F1 (ref. 8), is correlated with induction of E2F1 activity. Correspondingly, DRIL1 induces the E2F1 target Cyclin E1, overexpression of which is sufficient to trigger escape from senescence. Thus, DRIL1 disrupts cellular protection against RAS(V12)-induced proliferation downstream of the p19(ARF)/p53 pathway.