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The MAP3K ZAK, a Novel Modulator of ERK-Dependent Migration, Is Upregulated in Colorectal Cancer

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The MAP3K ZAK, a Novel Modulator of ERK-Dependent Migration, Is Upregulated in Colorectal Cancer Oncogene (2016) 35, 3190–3200 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc ORIGINAL ARTICLE The MAP3K ZAK, a novel modulator of ERK-dependent migration, is upregulated in colorectal cancer C Rey1, B Faustin2, I Mahouche1, R Ruggieri3, C Brulard1, F Ichas4, I Soubeyran5, L Lartigue1,6 and F De Giorgi4,6 Often described as a mediator of cell cycle arrest or as a pro-apoptotic factor in stressful conditions, the MAP3K ZAK (Sterile alpha motif and leucine zipper-containing kinase) has also been proven to positively regulate epidermal growth factor receptor (EGFR) and WNT signaling pathways, cancer cell proliferation and cellular neoplastic transformation. Here, we show that both isoforms of ZAK, ZAK-α and ZAK-β are key factors in cancer cell migration. While ZAK depletion reduced cell motility of HeLa and HCT116 cells, its overexpression triggered the activation of all three mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK) and p38, as well as an increase in cell motion. On the contrary, the kinase-dead mutants, ZAK-α K45M and ZAK-β K45M, were not able to provoke such events, and instead exerted a dominant-negative effect on MAPK activation and cell migration. Pharmacological inhibition of ZAK by nilotinib, preventing ZAK-autophosphorylation and thereby auto-activation, led to the same results. Activated by epidermal growth factor (EGF), we further showed that ZAK constitutes an essential element of the EGF/ERK-dependent cell migration pathway. Using public transcriptomic databases and tissue microarrays, we finally established that, as strong factors of the EGFR signaling pathway, ZAK-α and/or ZAK-β transcripts and protein(s) are frequently upregulated in colorectal adenoma and carcinoma patients. Notably, gene set enrichment analysis disclosed a significant correlation between ZAK+ colorectal premalignant lesions and gene sets belonging to the MAPK/ERK and motility-related signaling pathways of the reactome database, strongly suggesting that ZAK induces such pro-tumoral reaction cascades in human cancers. Oncogene (2016) 35, 3190–3200; doi:10.1038/onc.2015.379; published online 2 November 2015 INTRODUCTION constitutes the cellular basis for invasion and metastatic The mitogen-activated protein kinase (MAPK) signaling cascade dissemination of cancer cells, it remains of crucial importance to consists of a three-step activation of protein kinases that permit better understand the ERK-migrating signaling cascade. integration and transmission of numerous extracellular signals to ZAK (sterile alpha motif and leucine zipper-containing kinase) control cell response to environmental stress, growth factors and belongs to a subfamily of MAP3Ks referred to as mixed-lineage inflammatory cytokines. These signals thereby regulate vital cell kinases (MLKs). Two differential splice products of ZAK have been 8 9 functions such as cell growth, proliferation, migration, differentia- described, namely ZAK-α (also called MLTK-α or MRK-α ) and 8 9 10 tion and death.1,2 In such pathways, signals basically transit from ZAK-β (also known as MLTK-β, MRK-β and MLK-7). Identical upstream MAP3Ks to MAP2Ks that, in turn, activate the four in the N-terminus (amino-acid, aa 1–331), ZAK-α and ZAK-β classical downstream MAPKs: ERK (extracellular signal-regulated sequences completely diverge in the C-terminal region: the kinase 1/2), p38, c-JUN N-terminal kinase (JNK) or ERK5.3 Each of longest isoform ZAK-α (800 aa) has a sterile-α motif domain in these modules has shown some specificity: ERK is generally this region, which is not present in ZAK-β (455 aa).10,11 ZAK is responsive to mitogenic and differentiation stimuli, and JNK activated by phosphorylation, but until now, only one real and p38 are responsive to external and genotoxic stresses.2 upstream and/or scaffold partner has been identified for ZAK-α, Nonetheless, a high degree of redundancy and interconnections the protein kinase N α.12 Protein kinase N α enzymatic activity is exist inside and between these signaling pathways, revealing that stimulated by fatty acids (such as arachidonic acid) and Rho even more complicated regulatory mechanisms regulate cell fate GTPases.13 Interestingly, a recent report has also shown that ZAK-β and behavior. Alternative spliced variants, external factors and the activation downstream of lysophosphatidic acid and Rho C leads countless substrates of the various MAPKs add to this complexity.3 to an ERK- and p38-dependent cell-invasive process.14 This has been widely reported for ERK, a kinase for which 4200 ZAK intracellular function has been widely investigated in substrates have been indexed.4 Thus, in addition to its role in cell cardiomyocytes, where the kinase seems to have a prominent role proliferation, ERK has been reported to be involved in cell motility in cardiac cell hypertrophy.10,15,16 In cancer, however, its role has through focal adhesion kinase (FAK), calpain or myosin light chain just begun to be investigated. Although ZAK has been shown – kinase (MLCK)5,6 and more recently via the phosphatase STYX to have antitumoral properties,11,16 18 recent evidence supports (serine/threonine/tyrosine-interacting protein) and the transcrip- pro-oncogenic functions. Cho et al.19 reported that increasing tion factor Slug.7 Considering that directional cell migration ZAK-α expression level in JB6 Cl41 skin epidermal cells promotes 1Laboratoire de Validation et Identification de Nouvelles Cibles en Oncologie (VINCO), INSERM U916, Institut Bergonié, Université Victor Segalen Bordeaux 2, Bordeaux, France; 2CNRS – CIRID, Bordeaux Cedex, France; 3Laboratory of Cell Signaling, The Feinstein Institute for Medical Research, Manhasset, NY, USA; 4Fluofarma, 2 rue R. Escarpit, Pessac, France and 5Département de Biopathologie, Institut Bergonié, Bordeaux, France. Correspondence: Dr L lartigue, Laboratoire de Validation et Identification de Nouvelles Cibles en Oncologie (VINCO), INSERM U916, Institut Bergonié, Université Victor Segalen Bordeaux 2, 229 cours de l’Argonne, Bordeaux 33 076, France. E-mail: [email protected] or [email protected] 6These authors contributed equally to this work. Received 4 April 2015; revised 30 August 2015; accepted 4 September 2015; published online 2 November 2015 The MAP3K ZAK controls ERK-dependent cell migration C Rey et al 3191 their neoplastic transformation post-epidermal growth factor p38 and phospho-JNK in comparison with control cells in both (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA or PMA) conditions (Figures 2a and b), without altering total MAPKs treatment, as well as tumor formation in nude mice. More expression (Figure 2c). Interestingly, ZAK was also able to activate recently, it has also been identified as a gene preferentially ERK in the absence of serum (Figures 2b and c). In contrast to native overexpressed in gastric, breast, bladder and colorectal cancers proteins, ZAK-α and ZAK-β kinase-dead mutants’ overexpression (CRCs)20 and one of the splice gene candidates through which did not trigger any MAPK activation (Figure 2d and Supplementary PRPF6 would favor colorectal tumor growth.21 In that respect, ZAK Figure 5a). They instead behaved like dominant-negative mutants, figured among the eight genes found to be able to drive both decreasing the endogenous level of phospho-p38, phospho-JNK proliferation and Wnt signaling pathway activation in CRC cells.22 and phospho-ERK proteins. This effect not only highlighted the At last, a computational approach investigating a directed protein ability of endogenous ZAK to regulate these MAPKs, but certainly interaction network recently revealed the kinase as a strong also explained why the cells carrying these mutants migrate more positive modulator of the epidermal growth factor receptor (EGFR) slowly than their WT counterparts (Figure 1h and Supplementary signaling cascade,23 strengthening the possibility that ZAK Figure 3c). It is also to be noted that WT ZAK-α and ZAK-β constitutes a key factor of this pro-oncogenic pathway. overexpression resulted in the appearance of two bands on western In this study, we evaluated the role of ZAK-α and ZAK-β in blot membranes incubated with anti-ZAK antibody (Figure 2d, cancer progression, and its interconnection with the EGFR pathway. arrows columns 2 and 3). As shown in Figure 2d and Our results show that both ZAK isoforms regulate cancer cell Supplementary Figure 4, the upper band either disappeared after migration in a kinase-dependent manner. Most importantly, a mutation of ZAK-α and ZAK-β kinase domain (Figure 2d, columns we showed that ZAK regulates endogenous ERK activity through 4 and 5) or a phosphatase treatment (Supplementary Figure 4), which it exerts its pro-migratory effects. Public transcriptomic which presumably indicates that overexpressing ZAK results in its database, as well as tissue microarrays (TMA) analyses, further autophosphorylation and, in turn, its activation. These results were revealed ZAK-α and/or ZAK-β transcripts and proteins as being confirmed by confocal microscopy using HCT116, HT-29 and HeLa frequently upregulated in colorectal adenoma and carcinoma (CRC). cells (Figures 2e and f and Supplementary Figures 5b and c), Interestingly, when compared with the rest of the cohort by gene implying that ZAK is a central regulator of MAPK signaling in set enrichment analysis, ZAK-overexpressing patients presented a cancer cells. co-activation of the MAPK/ERK and motility-related signaling pathways, supporting a role of ZAK in activating these pro- Pharmacological inhibition of ZAK prevents its tumoral cascades in vivo. autophosphorylation and ability to induce MAPK activation and cell migration in HCT116 cells RESULTS It has previously been shown that nilotinib (Tasigna), a second- generation compound targeting BCR–ABL in chronic myeloid ZAK controls cancer cell motility 24–26 leukemia, binds ZAK with great affinity (K 8–10 nM) in vitro. α β d In order to clarify ZAK- and - intracellular activity in cancer cells, Nilotinib may thus be a pharmacological tool able to inhibit ZAK.
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