Sébastien Cagnol

ERK and cell death: Mechanisms of ERK‐induced cell death – apoptosis, autophagy and senescence

The Ras/Raf/extracellular signal‐regulated kinase (ERK) signaling pathway plays a crucial role in almost all cell functions and therefore requires exquisite control of its spatiotemporal activity. Depending on the cell type and stimulus, ERK activity will mediate different antiproliferative events, such as apoptosis, autophagy and senescence in vitro and in vivo. ERK activity can promote either intrinsic or extrinsic apoptotic pathways by induction of mitochondrial cytochrome c release or caspase‐8 activation, permanent cell cycle arrest or autophagic vacuolization. These unusual effects require sustained ERK activity in specific subcellular compartments and could depend on the presence of reactive oxygen species. We will summarize the mechanisms involved in Ras/Raf/ERK antiproliferative functions.

Inhibition of glycogen synthase kinase-3 activity triggers an apoptotic response in pancreatic cancer cells through JNK-dependent mechanisms.

Recent evidences suggest that the activity of glycogen synthase kinase-3 (GSK3) contributes to the tumorigenic potential of pancreatic cancer cells through modulation of cell proliferation and survival. However, further investigations are needed to identify GSK3-dependent mechanisms involved in the control of pancreatic cancer cell proliferation and survival. This study was undertaken to provide further support for a role of GSK3 in pancreatic cancer cell growth as well as to identify new cellular and molecular mechanisms involved. Herein, we demonstrate that prolonged inhibition of GSK3 triggers an apoptotic response only in human pancreatic cancer cells but not in human non-transformed pancreatic epithelial cells. We show that prolonged inhibition of GSK3 activity increases Bim messenger RNA and protein expressions. Moreover, we provide evidence that activation of the c-jun N-terminal kinase (JNK) pathway is necessary for the GSK3 inhibition-mediated increase in Bim expression and apoptotic response. Finally, we demonstrate that concomitant inhibition of GSK3 potentiates the death ligand-induced apoptotic response in pancreatic cancer cells but not in non-transformed pancreatic epithelial cells and that this effect also requires JNK activity. Considering that different approaches leading to stimulation of death receptor signaling are under clinical trials for treatment of unresectable or metastatic pancreatic cancer, inhibition of GSK3 could represent an attractive new avenue to improve their effectiveness.

Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis

Cathepsin B is a cysteine proteinase that primarily functions as an endopeptidase within endolysosomal compartments in normal cells. However, during tumoral expansion, the regulation of cathepsin B can be altered at multiple levels, thereby resulting in its overexpression and export outside of the cell. This may suggest a possible role of cathepsin B in alterations leading to cancer progression. The aim of this study was to determine the contribution of intracellular and extracellular cathepsin B in growth, tumorigenesis, and invasion of colorectal cancer (CRC) cells. Results show that mRNA and activated levels of cathepsin B were both increased in human adenomas and in CRCs of all stages. Treatment of CRC cells with the highly selective and non‐permeant cathepsin B inhibitor Ca074 revealed that extracellular cathepsin B actively contributed to the invasiveness of human CRC cells while not essential for their growth in soft agar. Cathepsin B silencing by RNAi in human CRC cells inhibited their growth in soft agar, as well as their invasion capacity, tumoral expansion, and metastatic spread in immunodeficient mice. Higher levels of the cell cycle inhibitor p27Kip1 were observed in cathepsin B‐deficient tumors as well as an increase in cyclin B1. Finally, cathepsin B colocalized with p27Kip1 within the lysosomes and efficiently degraded the inhibitor. In conclusion, the present data demonstrate that cathepsin B is a significant factor in colorectal tumor development, invasion, and metastatic spreading and may, therefore, represent a potential pharmacological target for colorectal tumor therapy.

The serine protease inhibitor serpinE2 is a novel target of ERK signaling involved in human colorectal tumorigenesis

BackgroundAmong the most harmful of all genetic abnormalities that appear in colorectal cancer (CRC) development are mutations of KRAS and its downstream effector BRAF as they result in abnormal extracellular signal-related kinase (ERK) signaling. In a previous report, we had shown that expression of a constitutive active mutant of MEK1 (caMEK) in normal rat intestinal epithelial cells (IECs) induced morphological transformation associated with epithelial to mesenchymal transition, growth in soft agar, invasion and metastases in nude mice. Results from microarrays comparing control to caMEK-expressing IECs identified the gene encoding for serpinE2, a serine protease inhibitor, as a potential target of activated MEK1.Results1- RT-PCR and western blot analyses confirmed the strong up-regulation of serpinE2 expression and secretion by IECs expressing oncogenic MEK, Ras or BRAF. 2- Interestingly, serpinE2 mRNA and protein were also markedly enhanced in human CRC cells exhibiting mutation in KRAS and BRAF. 3- RNAi directed against serpinE2 in caMEK-transformed rat IECs or in human CRC cell lines HCT116 and LoVo markedly decreased foci formation, anchorage-independent growth in soft agarose, cell migration and tumor formation in nude mice. 4- Treatment of CRC cell lines with U0126 markedly reduced serpinE2 mRNA levels, indicating that expression of serpinE2 is likely dependent of ERK activity. 5- Finally, Q-PCR analyses demonstrated that mRNA levels of serpinE2 were markedly increased in human adenomas in comparison to healthy adjacent tissues and in colorectal tumors, regardless of tumor stage and grade.ConclusionsOur data indicate that serpinE2 is up-regulated by oncogenic activation of Ras, BRAF and MEK1 and contributes to pro-neoplastic actions of ERK signaling in intestinal epithelial cells. Hence, serpinE2 may be a potential therapeutic target for colorectal cancer treatment.

Epithelial Tyrosine Phosphatase SHP-2 Protects against Intestinal Inflammation in Mice

ABSTRACT Polymorphisms of PTPN11 encoding SHP-2 are biomarkers for ulcerative colitis (UC) susceptibility. However, their functional relevance is unknown. We thus investigated the role of epithelial SHP-2 in the control of intestinal homeostasis. Mice with an intestinal epithelial cell-specific SHP-2 deletion (SHP-2IEC-KO mice) were generated. Control and SHP-2IEC-KO mice were monitored for clinical symptoms and sacrificed for histological staining and Western blot analyses. Cytokines and chemokines, as well as intestinal permeability, were quantified. SHP-2 mRNA expression was evaluated in control and UC patients. SHP-2IEC-KO mice showed growth retardation compared to control littermates and rapidly developed severe colitis. Colon architecture was markedly altered with infiltration of immune cells, crypt abscesses, neutrophil accumulation, and reduced goblet cell numbers. Decreased expression of claudins was associated with enhanced intestinal permeability in mutant SHP-2IEC-KO mice. Inflammatory transcription factors Stat3 and NF-κB were hyperactivated early in the mutant colonic epithelium. Levels of several epithelial chemokines and cytokines were markedly enhanced in SHP-2IEC-KO mice. Of note, antibiotic treatment remarkably impaired the development of colitis in SHP-2IEC-KO mice. Finally, SHP-2 mRNA levels were significantly reduced in intestinal biopsy specimens from UC patients. Our results establish intestinal epithelial SHP-2 as a critical determinant for prevention of gut inflammation.

ERK-associated changes in E2F4 phosphorylation, localization and transcriptional activity during mitogenic stimulation in human intestinal epithelial crypt cells

BackgroundThe transcription factor E2F4 controls proliferation of normal and cancerous intestinal epithelial cells. E2F4 localization in normal human intestinal epithelial cells (HIEC) is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, the intracellular signaling mechanisms regulating such E2F4 localization remain unknown.ResultsTreatment of quiescent HIEC with serum induced ERK1/2 activation, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition while inhibition of MEK/ERK signaling by U0126 prevented these events. Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition. Furthermore, Akt and GSK3β phosphorylation levels were markedly enhanced in serum- or LPA-stimulated HIEC but not by EGF. Importantly, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition were all observed in response to EGF when GSK3 activity was concomitantly inhibited by SB216763. Finally, E2F4 was found to be overexpressed, phosphorylated and nuclear localized in epithelial cells from human colorectal adenomas exhibiting mutations in APC and KRAS or BRAF genes, known to deregulate GSK3/β-catenin and MEK/ERK signaling, respectively.ConclusionsThe present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC. This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.

Dual-specificity phosphatase 6 deletion protects the colonic epithelium against inflammation and promotes both proliferation and tumorigenesis: BEAUDRY et al.

The Ras/mitogen‐activated protein kinase (MAPK) pathway controls fundamental cellular processes such as proliferation, differentiation, and apoptosis. The dual‐specificity phosphatase 6 (DUSP6) regulates cytoplasmic MAPK signaling by dephosphorylating and inactivating extracellular signal‐regulated kinase (ERK1/2) MAPK. To determine the role of DUSP6 in the maintenance of intestinal homeostasis, we characterized the intestinal epithelial phenotype of Dusp6 knockout (KO) mice under normal, oncogenic, and proinflammatory conditions. Our results show that loss of Dusp6 increased crypt depth and epithelial cell proliferation without altering colonic architecture. Crypt regeneration capacity was also enhanced, as revealed by ex vivo Dusp6 KO organoid cultures. Additionally, loss of Dusp6 induced goblet cell expansion without affecting enteroendocrine and absorptive cell differentiation. Our data also demonstrate that Dusp6 KO mice were protected from acute dextran sulfate sodium‐induced colitis, as opposed to wild‐type mice. In addition, Dusp6 gene deletion markedly enhanced tumor load in Apc Min/+ mice. Decreased DUSP6 expression by RNA interference in HT29 colorectal cancer cells enhanced ERK1/2 activation levels and promoted both anchorage‐independent growth in soft agar as well as invasion through Matrigel. Finally, DUSP6 mRNA expression in human colorectal tumors was decreased in advanced stage tumors compared with paired normal tissues. These results demonstrate that DUSP6 phosphatase, by controlling ERK1/2 activation, regulates colonic inflammatory responses, and protects the intestinal epithelium against oncogenic stress.

Abstract A05: The control of nuclear ERK activity by the DUSP phosphatases in colorectal cancer

The RAS/RAF/MEK/ERK pathway is suspected to be strongly implicated in malignant transformation of colorectal epithelial cells since KRAS and BRAF genes are mutated in up to 60% of human colorectal cancers (CRCs). Despite intensive efforts made in the understanding of KRAS and BRAF functions in colorectal carcinogenesis, little is known about the oncogenic regulation of ERK activity in CRC cells, especially by the ERK-specific nuclear DUSP phosphatases. We have recently demonstrated in CRC cells that ERK1/2 activity is confined to the cytoplasm because of a pervanadate-dependent phosphatase activity. Accordingly, DUSP4 mRNA was found to be highly expressed, in a MEK-dependent manner, in CRC cells. Thus, DUSP4 and other nuclear DUSPs may function as part of a negative feedback mechanism in the control of the duration and magnitude of nuclear ERK activation during colorectal carcinogenesis. We thus speculate that the modification of nuclear ERK activity might strongly impact on the oncogenic properties of CRC. Methods: Expression of DUSPs was analyzed by PCR in CRC cells and qPCR in human colorectal tumors from different stages. To elucidate the role of nuclear DUSPs, RNA interference was used to specifically silence their expression in human HT29 CRC cell line, a cell line exhibiting BRafV600E mutation. Inducible expression of a DUSP4 inactivated mutant (DUSP4C284S) was done in a model of IEC-6 cells that express an inducible form of BRaf oncogene (BRafV600E:ER). Results: 1- DUSP2, DUSP4 and DUSP5 mRNAs were found to be highly expressed, in a MEK-dependent manner, in CRC cells. 2- DUSP4 and DUSP5 mRNA levels were markedly increased in colorectal tumors in comparison to healthy matched adjacent tissues. 3- Silencing of DUSP 2, 4 or 5 in HT29 cells increased ERK phosphorylation levels and markedly reduced colony formation in soft agar. 4- Furthermore, expression of DUSP4C284S in BRafV600E-expressing IEC-6 cells increased ERK phosphorylation into the nucleus and markedly reduced colony formation in soft agar. Conclusion: We are currently searching by which mechanisms sustained nuclear ERK activity reduces colorectal tumor cell growth. Therefore, targeting nuclear DUSP activity could become a new strategy to treat CRC. Citation Format: Sebastien Cagnol, Nathalie Rivard. The control of nuclear ERK activity by the DUSP phosphatases in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A05. doi: 10.1158/1557-3125.RASONC14-A05