Frédérick A. Mallette

RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites

In response to DNA damage, cells initiate complex signalling cascades leading to growth arrest and DNA repair. The recruitment of 53BP1 to damaged sites requires the activation of the ubiquitination cascade controlled by the E3 ubiquitin ligases RNF8 and RNF168, and methylation of histone H4 on lysine 20. However, molecular events that regulate the accessibility of methylated histones, to allow the recruitment of 53BP1 to DNA breaks, are unclear. Here, we show that like 53BP1, the JMJD2A (also known as KDM4A) tandem tudor domain binds dimethylated histone H4K20; however, JMJD2A is degraded by the proteasome following the DNA damage in an RNF8‐dependent manner. We demonstrate that JMJD2A is ubiquitinated by RNF8 and RNF168. Moreover, ectopic expression of JMJD2A abrogates 53BP1 recruitment to DNA damage sites, indicating a role in antagonizing 53BP1 for methylated histone marks. The combined knockdown of JMJD2A and JMJD2B significantly rescued the ability of RNF8‐ and RNF168‐deficient cells to form 53BP1 foci. We propose that the RNF8‐dependent degradation of JMJD2A regulates DNA repair by controlling the recruitment of 53BP1 at DNA damage sites.

The DNA Damage Signaling Pathway Connects Oncogenic Stress to Cellular Senescence

The mechanisms of tumor suppression must be linked to the oncogenic threats that may affect a normal cell. An important cancer causing mechanism is the accidental activation of genes that stimulate cell proliferation (oncogenes) by a variety of endogenous or environmental mutagens. This event has been experimentally modelled by enforcing the expression of oncogenes in primary cells. The astonishing outcome of these manipulations is that oncogenes trigger antiproliferative responses preventing progression to malignant transformation. These responses bring to an end proliferation due to cell death or a permanent cell cycle arrest called senescence. Here we review evidence indicating that oncogene induced senescence (OIS) involves activation of p53 via the DNA damage response (DDR). These results imply mechanisms of DNA damage in cells expressing oncogenes, that may be secondary to reactive oxygen species and/or some form of “oncogenic stress” that affect normal DNA replication. Interestingly, DNA damage signals persist in cells that escape from senescence. The implications of these signals for tumorigenesis are also discussed. Given that DNA damage signals have now been observed in cells treated with any stimuli known to induce senescence, the process can be redefined as a metabolically viable but permanent cell cycle arrest with persistent DNA damage signaling.

Human fibroblasts require the Rb family of tumor suppressors, but not p53, for PML-induced senescence.

Cellular senescence is a permanent cell cycle arrest that can be triggered by a variety of stresses including short telomeres and activated oncogenes. Promyelocytic leukemia protein (PML) is a central component of the senescence response, and is able to trigger the process when overexpressed in human diploid fibroblasts (HDFs). Senescence induced by PML in HDFs is characterized by a modest increase in p53 levels and activity, the accumulation of hypophosphorylated Rb and a reduced expression of E2F-dependent genes. To dissect the p53 and Rb family requirements for PML-induced senescence, we used the oncoproteins E6 and E7 from human papillomavirus type 16. We found that the coexpression of E6 and E7 inhibited the growth arrest and senescence induced by PML. In addition, these viral oncoproteins blocked the formation of PML bodies and excluded both p53 and Rb from PML bodies. Expression of dominant-negative p53 alone failed to block PML-induced senescence and expression of E6 only delayed the process. On the other hand, expression of E7 was sufficient to block PML-induced senescence, while an E7 mutant unable to bind Rb did not. Together, these data indicate that PML-induced senescence engages the Rb tumor-suppressor pathway predominantly.

SOCS1 links cytokine signaling to p53 and senescence.

SOCS1 is lost in many human tumors, but its tumor suppression activities are not well understood. We report that SOCS1 is required for transcriptional activity, DNA binding, and serine 15 phosphorylation of p53 in the context of STAT5 signaling. In agreement, inactivation of SOCS1 disabled p53-dependent senescence in response to oncogenic STAT5A and radiation-induced apoptosis in T cells. In addition, SOCS1 was sufficient to induce p53-dependent senescence in fibroblasts. The mechanism of activation of p53 by SOCS1 involved a direct interaction between the SH2 domain of SOCS1 and the N-terminal transactivation domain of p53, while the C-terminal domain of SOCS1 containing the SOCS Box mediated interaction with the DNA damage-regulated kinases ATM/ATR. Also, SOCS1 colocalized with ATM at DNA damage foci induced by oncogenic STAT5A. Collectively, these results add another component to the p53 and DNA damage networks and reveal a mechanism by which SOCS1 functions as a tumor suppressor.

Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation

Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence.

JMJD2A promotes cellular transformation by blocking cellular senescence through transcriptional repression of the tumor suppressor CHD5.

Senescence is a cellular response preventing tumorigenesis. The Ras oncogene is frequently activated or mutated in human cancers, but Ras activation is insufficient to transform primary cells. In a search for cooperating oncogenes, we identify the lysine demethylase JMJD2A/KDM4A. We show that JMJD2A functions as a negative regulator of Ras-induced senescence and collaborates with oncogenic Ras to promote cellular transformation by negatively regulating the p53 pathway. We find CHD5, a known tumor suppressor regulating p53 activity, as a target of JMJD2A. The expression of JMJD2A inhibits Ras-mediated CHD5 induction leading to a reduced activity of the p53 pathway. In addition, we show that JMJD2A is overexpressed in mouse and human lung cancers. Depletion of JMJD2A in the human lung cancer cell line A549 bearing an activated K-Ras allele triggers senescence. We propose that JMJD2A is an oncogene that represents a target for Ras-expressing tumors.

Ablation of PRMT6 reveals a role as a negative transcriptional regulator of the p53 tumor suppressor

Arginine methylation of histones is a well-known regulator of gene expression. Protein arginine methyltransferase 6 (PRMT6) has been shown to function as a transcriptional repressor by methylating the histone H3 arginine 2 [H3R2(me2a)] repressive mark; however, few targets are known. To define the physiological role of PRMT6 and to identify its targets, we generated PRMT6−/− mouse embryo fibroblasts (MEFs). We observed that early passage PRMT6−/− MEFs had growth defects and exhibited the hallmarks of cellular senescence. PRMT6−/− MEFs displayed high transcriptional levels of p53 and its targets, p21 and PML. Generation of PRMT6−/−; p53−/− MEFs prevented the premature senescence, suggesting that the induction of senescence is p53-dependent. Using chromatin immunoprecipitation assays, we observed an enrichment of PRMT6 and H3R2(me2a) within the upstream region of Trp53. The PRMT6 association and the H3R2(me2a) mark were lost in PRMT6−/− MEFs and an increase in the H3K4(me3) activator mark was observed. Our findings define a new regulator of p53 transcriptional regulation and define a role for PRMT6 and arginine methylation in cellular senescence.

Myc Down-regulation as a Mechanism to Activate the Rb Pathway in STAT5A-induced Senescence

Senescence is a general antiproliferative program that avoids the expansion of cells bearing oncogenic mutations. We found that constitutively active STAT5A (ca-STAT5A) can induce a p53- and Rb-dependent cellular senescence response. However, ca-STAT5A did not induce p21 and p16INK4a, which are responsible for inhibiting cyclin-dependent protein kinases and engaging the Rb pathway during the senescence response to oncogenic ras. Intriguingly, ca-STAT5A led to a down-regulation of Myc and Myc targets, including CDK4, a negative regulator of Rb. The down-regulation of Myc was in part proteasome-dependent and correlated with its localization to promyelocytic leukemia bodies, which were found to be highly abundant during STAT5-induced senescence. Introduction of CDK4 or Myc bypassed STAT5A-induced senescence in cells in which p53 was also inactivated. These results uncover a novel mechanism to engage the Rb pathway in oncogene-induced senescence and indicate the existence of oncogene-specific pathways that regulate senescence.

Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer.

BackgroundUrodele amphibians like the axolotl are unique among vertebrates in their ability to regenerate and their resistance to develop cancers. It is unknown whether these traits are linked at the molecular level.ResultsBlocking p53 signaling in axolotls using the p53 inhibitor, pifithrin-α, inhibited limb regeneration and the expression of p53 target genes such as Mdm2 and Gadd45, suggesting a link between tumor suppression and regeneration. To understand this relationship we cloned the p53 gene from axolotl. When comparing its sequence with p53 from other organisms, and more specifically human we observed multiple amino acids changes found in human tumors. Phylogenetic analysis of p53 protein sequences from various species is in general agreement with standard vertebrate phylogeny; however, both mice-like rodents and teleost fishes are fast evolving. This leads to long branch attraction resulting in an artefactual basal emergence of these groups in the phylogenetic tree. It is tempting to assume a correlation between certain life style traits (e.g. lifespan) and the evolutionary rate of the corresponding p53 sequences. Functional assays of the axolotl p53 in human or axolotl cells using p53 promoter reporters demonstrated a temperature sensitivity (ts), which was further confirmed by performing colony assays at 37°C. In addition, axolotl p53 was capable of efficient transactivation at the Hmd2 promoter but has moderate activity at the p21 promoter. Endogenous axolotl p53 was activated following UV irradiation (100 j/m2) or treatment with an alkylating agent as measured using serine 15 phosphorylation and the expression of the endogenous p53 target Gadd45.ConclusionUrodele p53 may play a role in regeneration and has evolved to contain multiple amino acid changes predicted to render the human protein defective in tumor suppression. Some of these mutations were probably selected to maintain p53 activity at low temperature. However, other significant changes in the axolotl proteins may play more subtle roles on p53 functions, including DNA binding and promoter specificity and could represent useful adaptations to ensure p53 activity and tumor suppression in animals able to regenerate or subject to large variations in oxygen levels or temperature.

The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and Is Disrupted in Cancer

Background: The relevance of ASXL2 to the function of the histone H2A deubiquitinase BAP1 remains unknown. Results: ASXL2 promotes the assembly by BAP1 of a composite ubiquitin-binding interface (CUBI) required for DUB activity and coordination of cell proliferation. Conclusion: Cancer-associated mutations of BAP1 disrupt BAP1-ASXL2 interaction and function. Significance: We provide novel insights into BAP1 tumor suppressor function. The deubiquitinase (DUB) and tumor suppressor BAP1 catalyzes ubiquitin removal from histone H2A Lys-119 and coordinates cell proliferation, but how BAP1 partners modulate its function remains poorly understood. Here, we report that BAP1 forms two mutually exclusive complexes with the transcriptional regulators ASXL1 and ASXL2, which are necessary for maintaining proper protein levels of this DUB. Conversely, BAP1 is essential for maintaining ASXL2, but not ASXL1, protein stability. Notably, cancer-associated loss of BAP1 expression results in ASXL2 destabilization and hence loss of its function. ASXL1 and ASXL2 use their ASXM domains to interact with the C-terminal domain (CTD) of BAP1, and these interactions are required for ubiquitin binding and H2A deubiquitination. The deubiquitination-promoting effect of ASXM requires intramolecular interactions between catalytic and non-catalytic domains of BAP1, which generate a composite ubiquitin-binding interface (CUBI). Notably, the CUBI engages multiple interactions with ubiquitin involving (i) the ubiquitin carboxyl hydrolase catalytic domain of BAP1, which interacts with the hydrophobic patch of ubiquitin, and (ii) the CTD domain, which interacts with a charged patch of ubiquitin. Significantly, we identified cancer-associated mutations of BAP1 that disrupt the CUBI and notably an in-frame deletion in the CTD that inhibits its interaction with ASXL1/2 and DUB activity and deregulates cell proliferation. Moreover, we demonstrated that BAP1 interaction with ASXL2 regulates cell senescence and that ASXL2 cancer-associated mutations disrupt BAP1 DUB activity. Thus, inactivation of the BAP1/ASXL2 axis might contribute to cancer development.