Frédéric Lessard

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.

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.

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

PO-117 Role of nfe2l3 in colon cancer by regulating cancer cells proliferation

Introduction Dysfunctional transcriptional and signalling networks play a fundamental role in colorectal cancer (CRC), one of the most common and fatal malignancies worldwide. Different molecular CRC subtypes have been identified, and understanding of the underlying pathogenesis of CRC formation is crucial for predicting prognosis and treatment response. Constitutive NF-κB activation is a hallmark of colon tumour development. In this study, we aimed to unravel the cellular network governing NFE2L3 regulation and function. We report that NFE2L3 acts as a central player in a newly identified NF-kB signalling pathway that controls colon cancer cell growth. Material and methods We analysed the level of NFE2L3 in colon cancer samples extracted from patients. We compared by qPCR and immunohistochemistry the level NFE2L3 expression in normal and tumour tissues. We performed a knockdown of NFE2L3 in different colon cancer cell lines. We characterised the phenotype associated to its depletion in vitro and in vivo. Then, we used a CHIP sequencing analysis to identify the targets of NFE2L3 in our models and we validated by qPCR the direct link between NFE2L3 expression and targets discovered. Finally, by using a strategy of immunoprecipitation associated to mass spectrometry analysis, we identified potential proteins implicated in the pathway of NFE2L3 in colon cancer. Results and discussions Firstly, we observed that the expression of NFE2L3 in colon cancer samples is significantly higher compared to in normal colon tissues. Moreover, the expression of this protein directly correlated to the survival of patients and could be used as a prognostic marker in colon adenocarcinoma. After that, we depleted the expression of NFE2L3 and we observed that the proliferation of colon cancer cells was slow down. Then, we identified NF-kB pathway as a key regulator of NFE2L3 expression by regulating promoter region. Finally, we demonstrated that NFE2L3 controls the cell cycle in cancer cells by modulating the expression of negative regulators of proliferation. Conclusion Taken together, based on our observations, we propose the existence of a novel oncogenic pathway, comprising the NF–kB, NFE2L3 and cell cycle regulators, that controls cancer cell growth. Our study establishes a key role for the NFE2L3 transcription factor that regulates cell cycle progression in colon cancer cells.

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

Abstract A09: Downregulation of PI3K signaling by high ERK activity prevents transformation by oncogenic RAS

In this study, we investigated the contribution of strong ERK signals to tumor suppression. 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, low 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 progression, decreased p-ERK levels correlate with tumor initiation. Taken together, these observations suggest that overactivated ERK may serve as a barrier for oncogenic transformation of cells acquiring activating RAS mutations. We found that the antitumor effects of aberrant ERK signaling include the inhibition of the phosphoinositide-3-kinase/AKT (PI3K/AKT) signaling pathway. Consequently, transformed RasV12-expressing cells with reduced ERK signaling show hyperactivation of the PI3K/AKT pathway, GSK3b inhibition, c-MYC stabilization and expression of stem cell-associated genes. In these cells, the hyperactivation of the PI3K/AKT pathway is essential in order to maintain their transformed phenotype since pharmacological inhibition of the pathway abrogates their tumorigenicity. Collectively, 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 malignant transformation of oncogenic RAS-expressing cells. Therefore, we propose a model where a moderated level of activated ERK (p-ERK) in RAS-expressing cells promotes transformation whereas higher levels limit cancer initiation by activating tumor-supressive mechanisms. This model suggests caution in clinical use of inhibitors of the ERK-MAPK pathway. Although complete pathway shut-down may counter tumorigenesis, partial reductions in ERK signaling may be deleterious by allowing a bypass of tumor-suppressive mechanisms in other premalignant lesions or in cells with strong ERK activity. Citation Format: Xavier Deschenes-Simard, Filippos Kottakis, Frederic Lessard, Nabeel Bardeesy, Gerardo Ferbeyre. Downregulation of PI3K signaling by high ERK activity prevents transformation by oncogenic RAS. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A09.