Xavier Deschênes-Simard

Mitochondrial dysfunction contributes to oncogene-induced senescence

ABSTRACT The expression of oncogenic ras in normal human cells quickly induces an aberrant proliferation response that later is curtailed by a cell cycle arrest known as cellular senescence. Here, we show that cells expressing oncogenic ras display an increase in the mitochondrial mass, the mitochondrial DNA, and the mitochondrial production of reactive oxygen species (ROS) prior to the senescent cell cycle arrest. By the time the cells entered senescence, dysfunctional mitochondria accumulated around the nucleus. The mitochondrial dysfunction was accompanied by oxidative DNA damage, a drop in ATP levels, and the activation of AMPK. The increase in mitochondrial mass and ROS in response to oncogenic ras depended on intact p53 and Rb tumor suppression pathways. In addition, direct interference with mitochondrial functions by inhibiting the expression of the Rieske iron sulfur protein of complex III or the use of pharmacological inhibitors of the electron transport chain and oxidative phosphorylation was sufficient to trigger senescence. Taking these results together, this work suggests that mitochondrial dysfunction is an effector pathway of oncogene-induced senescence.

ERKs in Cancer: Friends or Foes?

The extracellular signal-regulated kinase ERK1 and ERK2 (ERK1/2) cascade regulates a variety of cellular processes by phosphorylating multiple target proteins. The outcome of its activation ranges from stimulation of cell survival and proliferation to triggering tumor suppressor responses such as cell differentiation, cell senescence, and apoptosis. This pathway is intimately linked to cancer as several of its upstream activators are frequently mutated in human disease and are shown to accelerate tumorigenesis when engineered in the mouse genome. However, measurement of activated ERKs in human cancers or mouse models does not always support a role in tumorigenesis, and data consistent with a role in tumor suppression have been reported as well. The intensity of ERK signaling, negative feedback loops that regulate the pathway, and cross-talks with other signaling pathways, seem to be of primary importance in determining the final cellular outcome. Cell senescence, a putative tumor-suppression mechanism, depends on high-intensity ERK signals that trigger phosphorylation-dependent protein degradation of multiple proteins required for cell-cycle progression. This response may be circumvented during carcinogenesis by a variety of mechanisms, some of them yet to be discovered, which in essence turn ERK functions from tumor suppression to tumor promotion. The use of pharmacologic inhibitors targeting this pathway must be carefully evaluated so they are applied to cases in which ERKs are mainly oncogenic.

Metformin inhibits the senescence‐associated secretory phenotype by interfering with IKK/NF‐κB activation

We show that the antidiabetic drug metformin inhibits the expression of genes coding for multiple inflammatory cytokines seen during cellular senescence. Conditioned medium (CM) from senescent cells stimulates the growth of prostate cancer cells but treatment of senescent cells with metformin inhibited this effect. Bioinformatic analysis of genes downregulated by metformin suggests that the drug blocks the activity of the transcription factor NF‐κB. In agreement, metformin prevented the translocation of NF‐κB to the nucleus and inhibited the phosphorylation of IκB and IKKα/β, events required for activation of the NF‐κB pathway. These effects were not dependent on AMPK activation or on the context of cellular senescence, as metformin inhibited the NF‐κB pathway stimulated by lipopolysaccharide (LPS) in ampk null fibroblasts and in macrophages. Taken together, our results provide a novel mechanism for the antiaging and antineoplastic effects of metformin reported in animal models and in diabetic patients taking this drug.

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.

Cellular senescence and protein degradation: Breaking down cancer

Autophagy and the ubiquitin–proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells.

Abstract 3895: Tumor suppressor activity of the ERK/MAPK signaling: inhibition of cell reprogramming by degradation of specific proteins

In this study, we investigated the contribution of the ERK signals to tumor initiation and the differentiation state of cancer cells. Oncogenic forms of RAS are found in up to 30% of all human cancers and are established drivers of tumor initiation and maintenance. However, strong expression of these oncogenes in normal cells induces cellular senescence, a putative tumor-suppressive barrier. RAS activates several signaling pathways, such as the PI3K/AKT pathway, the RAL pathway and the classical RAF/MEK/ERK MAP Kinase pathway. We previously found that RASV12-induced senescence of primary cells is prevented by attenuation of ERK signaling. Mechanistically, strong ERK signaling promotes senescence by inducing selective proteasome-dependent protein degradation. This “Senescence-Associated Protein Degradation” (SAPD) targets proteins required for cell cycle progression, mitochondrial functions, cell migration and cell signaling. Here we show that in addition to abrogating RAS-induced senescence, a moderate ERK activity allows transformation of primary human cells stably expressing RASV12 and hTERT as well as transformation of RasV12-expressing rodent cells. Furthermore, in a Kras-driven mouse model of multistage pancreatic cancer progression, decreased p-ERK levels correlate with tumor initiation. We found that transformed cells with low p-ERK levels express markers of pluripotency and demonstrate phenotypes of tumor initiating cells, such as formation of free-floating tumor spheres, and show the expression of a gene module associated to stem cells. This depends on moderate p-ERK levels, since increasing the activity of the pathway by the pharmacological and genetic inhibition of the Dual-Specificity Phosphatases 1 and 6 (DUSP1/6) completely abrogates the stem-like cell phenotype. Our results suggest that strong ERK signals could circumvent this phenotype by promoting the degradation of key transcription factors that regulate expression of stem cell-associated genes, and this, even if the activity of the pathway is not sufficient to induce cellular senescence. Taken together, these results demonstrate a novel anti-tumor effect of strong ERK signaling and suggest that processes attenuating ERK levels and/or activity may contribute to tumor initiation and aggressiveness of oncogenic RAS-driven cancers. Therefore, we propose a model where a moderated level of activated ERK (p-ERK) in RAS-expressing cells promotes transformation and dedifferentiation whereas higher levels limit cancer initiation and maintenance by activating tumor-suppressive mechanisms like senescence and differentiation. Considering the increased resistance of tumor-initiating cells to chemotherapy and their capacity to initiate tumor development, this model suggests a need to target cancer cells with low pERK levels within a tumor. Citation Format: Xavier Deschenes-Simard, Filippos Kottakis, Frederic Lessard, Emmanuelle Saint-Germain, Veronique Bourdeau, Nabeel Bardeesy, Gerardo Ferbeyre. Tumor suppressor activity of the ERK/MAPK signaling: inhibition of cell reprogramming by degradation of specific proteins. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3895. doi:10.1158/1538-7445.AM2014-3895

Senescence-associated ribosome biogenesis defects contributes to cell cycle arrest through the Rb pathway

Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribosome biogenesis factors, the knockdown of which was also sufficient to induce senescence. Genetic analysis revealed that Rb but not p53 was required for the senescence response to altered ribosome biogenesis. Mechanistically, the ribosomal protein S14 (RPS14 or uS11) accumulates in the soluble non-ribosomal fraction of senescent cells, where it binds and inhibits CDK4 (cyclin-dependent kinase 4). Overexpression of RPS14 is sufficient to inhibit Rb phosphorylation, inducing cell cycle arrest and senescence. Here we describe a mechanism for maintaining the senescent cell cycle arrest that may be relevant for cancer therapy, as well as biomarkers to identify senescent cells.Lassard et al. demonstrate a relationship between cellular senescence and perturbed ribosome biogenesis and find that the ribosomal protein S14 is an inhibitor of CDK4, inducing an Rb-dependent cell cycle arrest.

Abstract 2246: Senescence as a result of impaired ribosome biogenesis

Senescence is a stable arrest of cell proliferation in which the cells remain viable and metabolically active but display a constitutive activation of the DNA damage response and of the tumor suppressors p53 and RB. The senescent phenotype can be induced by multiple stresses including short telomeres and oncogenes. We have shown that senescence, involves the ERK-dependent degradation of selective proteins involved in cell cycle progression and tumorigenesis. We call this process senescence associated protein degradation (SAPD) and it involves many nucleolar proteins that play a role in ribosome biogenesis. Using tritium pulse labelling we found a strong decrease of rRNA synthesis in senescent cells indicating that the degradation of nucleolar proteins is functionally relevant. Because we know how exactly the human 47S precursor rRNA is processed, it was possible to design primers on both sides of some processing sites and study their maturation by QPCR. In this way we showed defects in the processing of rRNA in senescent cells. Knocking down some of the nucleolar proteins degraded in senescence was sufficient to trigger the process indicating that a decrease in ribosome biogenesis is causal to cellular senescence. Mechanistically, the degradation of nucleolar proteins during senescence involves the ubiquitin-proteasome system suggesting that E3 ligases link the oncogenic stress that trigger senescence to nucleolar proteins degradation. We will discuss ongoing efforts to identify these enzymes. Citation Format: Frederic Lessard, Veronique Bourdeau, Xavier Deschenes-Simard, Sebastian Igelmann, Marinieve Montero, Gerardo Ferbeyre. Senescence as a result of impaired ribosome biogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2246. doi:10.1158/1538-7445.AM2014-2246

Abstract 2248: The implication of STAT3 degradation and its mitochondrial functions in cellular senescence

Cellular senescence is a stable growth arrest of the cell cycle considered to be a mechanism of tumor suppression. We have recently described a senescence associated protein degradation or SAPD process that targets many proteins required for tumor progression. The Signal transducer and activator of transcription 3 is one of the targets of SAPD and its downregualtion by RNAi is sufficient to trigger senescence. In the last decade several different functions of STAT3 have been described. Beyond its canonical role as a transcription factor, it has been shown that STAT3 is imported into mitochondria and regulate the activity of the respiratory chain. In this study we want to clarify which function of STAT3 is required to prevent senescence. To do that we have made several mutants of STAT3 affecting different domains of the protein and tested their ability to rescue the senescence response to an anti-Stat3 shRNA. We have found that phosphorylation on residue S727 of STAT3 is important to reestablish proliferation in shSTAT3 induced senescence. Further, transcriptional activity of STAT3 is not required to rescue senescence as a mutant unable to enter the nucleus or the transcriptionally death STAT3 K180A are both able to bypass shSTAT3-induced senescence. Moreover, phosphorylation on Y705 seems not to be important as Y705F STAT3 is also able to reestablish proliferation. All in all, it seems that mitochondrial function of STAT3 is required to prevent senescence since STAT3 S727 phosphorylation is linked to the mitochondrial import of STAT3. Citation Format: Sebastian Igelmann, Xavier Deschenes-Simard, Frederic Lessard, Veronique Bourdeau, Gerardo Ferbeyre. The implication of STAT3 degradation and its mitochondrial functions in cellular senescence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2248. doi:10.1158/1538-7445.AM2014-2248