Mixed lineage kinase 3 gene mutations in mismatch repair deficient gastrointestinal tumours

IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, 4200-465 Porto, Portugal.
Human Molecular Genetics (Impact Factor: 6.39). 12/2009; 19(4):697-706. DOI: 10.1093/hmg/ddp536
Source: PubMed


Mixed lineage kinase 3 (MLK3) is a serine/threonine kinase, regulating MAPkinase signalling, in which cancer-associated mutations have never been reported. In this study, 174 primary gastrointestinal cancers (48 hereditary and 126 sporadic forms) and 7 colorectal cancer cell lines were screened for MLK3 mutations. MLK3 mutations were significantly associated with MSI phenotype in primary tumours (P = 0.0005), occurring in 21% of the MSI carcinomas. Most MLK3 somatic mutations identified were of the missense type (62.5%) and more than 80% of them affected evolutionarily conserved residues. A predictive 3D model points to the functional relevance of MLK3 missense mutations, which cluster in the kinase domain. Further, the model shows that most of the altered residues in the kinase domain probably affect MLK3 scaffold properties, instead of its kinase activity. MLK3 missense mutations showed transforming capacity in vitro and cells expressing the mutant gene were able to develop locally invasive tumours, when subcutaneously injected in nude mice. Interestingly, in primary tumours, MLK3 mutations occurred in KRAS and/or BRAF wild-type carcinomas, although not being mutually exclusive genetic events. In conclusion, we have demonstrated for the first time the presence of MLK3 mutations in cancer and its association to mismatch repair deficiency. Further, we demonstrated that MLK3 missense mutations found in MSI gastrointestinal carcinomas are functionally relevant.

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    • "Our group reported the occurrence of MLK3 mutations in microsatellite unstable (MSI) gastrointestinal tumors (both sporadic and hereditary forms) in a frequency of about 20%. Using in vitro transforming assays, we demonstrated that several MLK3 mutations affecting different domains of the protein had transforming potential when compared to cells expressing the wild-type and the kinase-dead forms of the protein [15]. These results were further supported by in vivo studies in which one of the two most transforming mutations (P252H – located in the kinase domain) was found to be tumorigenic and to give rise to highly invasive tumors when subcutaneously injected in nude mice. "
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    ABSTRACT: MLK3 gene mutations were described to occur in about 20% of microsatellite unstable gastrointestinal cancers and to harbor oncogenic activity. In particular, mutation P252H, located in the kinase domain, was found to have a strong transforming potential, and to promote the growth of highly invasive tumors when subcutaneously injected in nude mice. Nevertheless, the molecular mechanism underlying the oncogenic activity of P252H mutant remained elusive. In this work, we performed Illumina Whole Genome arrays on three biological replicas of human HEK293 cells stably transfected with the wild-type MLK3, the P252H mutation and with the empty vector (Mock) in order to identify the putative signaling pathways associated with P252H mutation. Our microarray results showed that mutant MLK3 deregulates several important colorectal cancer- associated signaling pathways such as WNT, MAPK, NOTCH, TGF-beta and p53, helping to narrow down the number of potential MLK3 targets responsible for its oncogenic effects. A more detailed analysis of the alterations affecting the WNT signaling pathway revealed a down-regulation of molecules involved in the canonical pathway, such as DVL2, LEF1, CCND1 and c-Myc, and an up-regulation of DKK, a well-known negative regulator of canonical WNT signaling, in MLK3 mutant cells. Additionally, FZD6 and FZD10 genes, known to act as negative regulators of the canonical WNT signaling cascade and as positive regulators of the planar cell polarity (PCP) pathway, a non-canonic WNT pathway, were found to be up-regulated in P252H cells. The results provide an overall view of the expression profile associated with mutant MLK3, and they support the functional role of mutant MLK3 by showing a deregulation of several signaling pathways known to play important roles in the development and progression of colorectal cancer. The results also suggest that mutant MLK3 may be a novel modulator of WNT signaling, and pinpoint the activation of PCP pathway as a possible mechanism underlying the invasive potential of MLK3 mutant cells.
    BMC Cancer 03/2014; 14(1):182. DOI:10.1186/1471-2407-14-182 · 3.36 Impact Factor
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    • "Merlin inhibits MLK3 activation by directly interfering with MLK3 binding to Cdc42, a major MLK3 activator; thus providing a critical link between previous reports describing merlin suppression of Rac1 or MAPK activity (Chadee et al., 2006; Morrison et al., 2007). MLK3 is required for cell proliferation in normal and neoplastic cells, and a number of studies have demonstrated that exogenous expression of members of the MLK family is sufficient to promote neoplastic transformation (Cho et al., 2004; Hartkamp et al., 1999; Velho et al., 2010). However, deregulated MLK3 kinase activity in tumor-derived cells has not been previously reported. "
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    ABSTRACT: The Neurofibromatosis-2 (NF2) tumor suppressor merlin negatively regulates cell proliferation in numerous cell types. We have previously shown that the NF2 protein (merlin/schwannomin) associates with mixed lineage kinase 3 (MLK3), a mitogen-activated protein kinase (MAPK) kinase kinase that is required for the proliferation of normal and neoplastic cells. In this study, we show that merlin inhibits MLK3 activity, as well as the activation of its downstream effectors, B-Raf, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). The ability of merlin to regulate MLK3 activity requires a direct association between MLK3 and residues in the C-terminal region of merlin. Merlin integrates Rho GTPase family signaling with MAPK activity by inhibiting the binding between MLK3 and its upstream activator, Cdc42. Furthermore, we demonstrate that MLK3 is required for merlin-mediated suppression of cell proliferation and invasion. Collectively, these results establish merlin as a potent inhibitor of MLK3, ERK and JNK activation in cancer, and provide a mechanistic link between deregulated MAPK and Rho GTPase signaling in NF2 growth control.
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