Mathieu Vernier

Regulation of E2Fs and senescence by PML nuclear bodies

The tumor suppressor PML (promyelocytic leukemia protein) regulates cellular senescence and terminal differentiation, two processes that implicate a permanent exit from the cell cycle. Here, we show that the mechanism by which PML induces a permanent cell cycle exit and activates p53 and senescence involves a recruitment of E2F transcription factors bound to their promoters and the retinoblastoma (Rb) proteins to PML nuclear bodies enriched in heterochromatin proteins and protein phosphatase 1α. Blocking the functions of the Rb protein family or adding back E2Fs to PML-expressing cells can rescue their defects in E2F-dependent gene expression and cell proliferation, inhibiting the senescent phenotype. In benign prostatic hyperplasia, a neoplastic disease that displays features of senescence, PML was found to be up-regulated and forming nuclear bodies. In contrast, PML bodies were rarely visualized in prostate cancers. The newly defined PML/Rb/E2F pathway may help to distinguish benign tumors from cancers, and suggest E2F target genes as potential targets to induce senescence in human tumors.

Retinoblastoma‐independent regulation of cell proliferation and senescence by the p53–p21 axis in lamin A /C‐depleted cells

The expression of A‐type lamin is downregulated in several cancers, and lamin defects are the cause of several diseases including a form of accelerated aging. We report that depletion of lamin A/C expression in normal human cells leads to a dramatic downregulation of the Rb family of tumor suppressors and a defect in cell proliferation. Lamin A/C‐depleted cells exhibited a flat morphology and accumulated markers of cellular senescence. This senescent phenotype was accompanied by engagement of the p53 tumor suppressor and induction of the p53 target gene p21 and was prevented by small hairpin RNAs against p53, p21, or by the oncoprotein Mdm2. The expression of E2F target genes, normally required for cell cycle progression, was downregulated after lamin A/C depletion but restored after the inactivation of p53. A similar senescence response was observed in myoblasts from a patient with a lamin A mutation causing muscular dystrophy. We thus reveal a previously unnoticed mechanism of controlling cell cycle genes expression, which depends on p53 but does not require the retinoblastoma family of tumor suppressors and that can be relevant to understand the pathogenesis of laminopathies and perhaps aging.

A CDK4/6-Dependent Epigenetic Mechanism Protects Cancer Cells from PML-induced Senescence.

Promyelocytic leukemia (PML) plays a tumor suppressive role by inducing cellular senescence in response to oncogenic stress. However, tumor cell lines fail to engage in complete senescence upon PML activation. In this study, we investigated the mechanisms underlying resistance to PML-induced senescence. Here, we report that activation of the cyclin-dependent kinases CDK4 and CDK6 are essential and sufficient to impair senescence induced by PML expression. Disrupting CDK function by RNA interference or pharmacological inhibition restored senescence in tumor cells and diminished their tumorigenic potential in mouse xenograft models. Complete senescence correlated with an increase in autophagy, repression of E2F target genes, and an gene expression signature of blocked DNA methylation. Accordingly, treatment of tumor cells with inhibitors of DNA methylation reversed resistance to PML-induced senescence. Further, CDK inhibition with palbociclib promoted autophagy-dependent degradation of the DNA methyltransferase DNMT1. Lastly, we found that CDK4 interacted with and phosphorylated DNMT1 in vitro, suggesting that CDK activity is required for its stabilization. Taken together, our findings highlight a potentially valuable feature of CDK4/6 inhibitors as epigenetic modulators to facilitate activation of senescence programs in tumor cells. Cancer Res; 76(11); 3252-64. ©2016 AACR.

Mutant lamin A links prophase to a p53 independent senescence program

Expression of oncogenes or short telomeres can trigger an anticancer response known as cellular senescence activating the p53 and RB tumor suppressor pathways. This mechanism is switched off in most tumor cells by mutations in p53 and RB signaling pathways. Surprisingly, p53 disabled tumor cells could be forced into senescence by expression of a mutant allele of the nuclear envelope protein lamin A. The pro-senescence lamin A mutant contains a deletion in the sequence required for processing by the protease ZMPSTE24 leading to accumulation of farnesylated lamin A in the nuclear envelope. In addition, the serine at position 22, a target for CDK1-dependent phosphorylation, was mutated to alanine, preventing CDK1-catalyzed nuclear envelope disassembly. The accumulation of this mutant lamin A compromised prophase to prometaphase transition leading to invaginations of the nuclear lamina, nuclear fragmentation and impaired chromosome condensation. Cells exited this impaired mitosis without cytokinesis and re-replicated their DNA ultimately arresting in interphase as polyploid cells with features of cellular senescence including increased expression of inflammatory gene products and a significant reduction of tumorigenicity in vivo.

CHES1/FOXN3 regulates cell proliferation by repressing PIM2 and protein biosynthesis.

CHES1/FOXN3 inhibits cell proliferation and protein biosynthesis in tumor cell lines but not in normal fibroblasts. CHES1 directly represses the expression of the gene coding for the protein kinase PIM2, and PIM2 or eIF4E counteracts the antiproliferative effect of CHES1. The levels of CHES1 and PIM2 are inversely correlated in several human cancers.

Complete senescence: RB and PML share the task.

Cellular senescence is a program that prevents malignant transformation in part by avoiding cell cycle progression.1 The senescent cell cycle arrest is controlled by the retinoblastoma protein (RB), which negatively regulates the activity of the E2F transcription factors, leading to repression of proliferation genes.2 This event is common to senescence and quiescence, a dormant state from which cells can re-enter proliferation in response to an external signal. Thus, another layer of regulation is necessary to make the distinction between a transient and a permanent growth arrest. E2F target genes can undergo an RB-dependent heterochromatinisation process.3 More precisely, RB interacts, via a LXCXE motif, with proteins involved in chromatin remodeling, and a mutant RB lacking the LXCXE interaction domain is defective for heterochromatinization, stable repression of E2F target genes, and maintenance of senescence.4 RB cooperates with the promyelocytic leukemia protein (PML) during the establishment of senescence.5 PML is the main constituent of spherical nuclear structures known as PML bodies, in which RB/E2F complexes, along with E2F target gene promoters, can relocalize in an RB-dependent manner during senescence.5 Also, several proteins involved in heterochromatinisation have been shown to transiently localize to PML bodies prior to their integration into regions of heterochromatin embedding E2F target genes in senescence.6 Finally, PML−/− mouse fibroblasts fail to senesce in response to oncogene expression, demonstrating the importance of PML in the regulation of the process. Thus RB and PML are important players in the regulation of senescence and interact together to repress genes essential for proliferation.5 In this issue, Talluri and Dick report that the interaction between RB and PML is dependent on the LXCXE binding motif of RB, and that this interaction is critical for chromatin reorganization on promoters of cell cycle genes, leading to their stable repression and a permanent cell cycle arrest. To do so, they took advantage of an elegant mouse model carrying a mutant form of RB lacking the LXCXE interaction domain (called RB1ΔL). RB1ΔL/ΔL MEFs were infected with vectors expressing the oncogene RASV12 or PML to induce senescence. In this context, the authors observed a normal induction of early senescent events, such as an increase of β-galactosidase activity, DNA damage, and an increase in the number of PML bodies. However, the repression of E2F target genes was less efficient than in wild-type MEFs, explaining why these cells can incorporate BrdU and resume proliferation when challenged with ectopic E2F expression. Of interest, the recruitment of PML to the promoters of E2F-target genes and the marker of constitutive heterochromatin H3K9me3 were also absent in E2F target promoters in RB1ΔL/ΔL MEFs. Mechanistically, Talluri and Dick found that PML and RB only interact in the context of senescence. They also observed high molecular forms of PML in interaction with RB, suggesting that sumoylation of PML might be required. The study of Talluri and Dick sheds new light into the complexity of the senescence tumor suppressor response. Their work suggests that what we call senescence is not a single cellular state, and at least 2 types can be defined. They defined incomplete senescence (type I) as the process occurring in RB1ΔL/ΔLMEFs in response to oncogenes. These cells arrest their proliferation, are SA-β-Gal-positive, and display signs of DNA damage and an increase in the number of PML bodies. However, their cell cycle arrest is not stable, and they can resume cell proliferation. On the other hand, complete senescence (type II) is essentially a permanent process due, at least in part, to the formation of heterochromatin at E2F target genes, which requires the interaction between PML and RB via its LXCXE binding motif as defined by Talluri and Dick. Of note, Vernier et al. discovered abundant PML bodies in benign prostatic hyperplasia (BPH), which are benign prostate tumors that do not progress to cancer. In contrast, prostatic intraepithelial neoplasia (PIN), which are premalignant lesions with the potential to progress into prostate cancer, do not have PML bodies.5 These 2 lesions may represent clinical examples of complete and incomplete senescence.

Correction: Deficiency of Interleukin-15 Confers Resistance to Obesity by Diminishing Inflammation and Enhancing the Thermogenic Function of Adipose Tissues

[This corrects the article DOI: 10.1371/journal.pone.0162995.].

SOCS1 regulates senescence and ferroptosis by modulating the expression of p53 target genes

The mechanism by which p53 suppresses tumorigenesis remains poorly understood. In the context of aberrant activation of the JAK/STAT5 pathway, SOCS1 is required for p53 activation and the regulation of cellular senescence. In order to identify p53 target genes acting during the senescence response to oncogenic STAT5A, we characterized the transcriptome of STAT5A-expressing cells after SOCS1 inhibition. We identified a set of SOCS1-dependent p53 target genes that include several secreted proteins and genes regulating oxidative metabolism and ferroptosis. Exogenous SOCS1 was sufficient to regulate the expression of p53 target genes and sensitized cells to ferroptosis. This effect correlated with the ability of SOCS1 to reduce the expression of the cystine transporter SLC7A11 and the levels of glutathione. SOCS1 and SOCS1-dependent p53 target genes were induced during the senescence response to oncogenic STAT5A, RasV12 or the tumor suppressor PML. However, while SOCS1 sensitized cells to ferroptosis neither RasV12 nor STAT5A mimicked the effect. Intriguingly, PML turned cells highly resistant to ferroptosis. The results indicate different susceptibilities to ferroptosis in senescent cells depending on the trigger and suggest the possibility of killing senescent cells by inhibiting pathways that mediate ferroptosis resistance.

Abstract 3952: New advances in regulation of senescence by PML and the PML nuclear bodies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Senescence is a cellular defense mechanism activated by short telomeres, DNA damage or expression of oncogenes. The program includes the expression of high levels of the promyelocytic leukemia protein PML that form nuclear spherical bodies, known as PML-nuclear bodies (PML-NB). The expression of PML in normal fibroblasts is sufficient to induce senescence while genetic inactivation of PML inhibits the process. These results suggest that PML is a critical component of the senescence tumor suppressor mechanism. Accordingly PML is poorly expressed in malignant human tumors but highly expressed in benign tumors. We recently discovered a new mechanism of regulation of RAS-induced senescence by PML, which implicates the recruitment of the RB/E2F complex to the PML-NB via RB-PML interaction. This leads to inhibition of cell cycle and DNA repair genes, DNA damage, p53 activation and ultimately to senescence. We show now that the cyclin dependent kinase CDK4 blocks the ability of PML to regulate E2F gene expression and senescence and that CDK inhibitors potentiate the ability of PML to restore the senescence program in tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3952. doi:1538-7445.AM2012-3952