Dissecting the signaling pathways associated with the oncogenic activity of MLK3 P252H mutation

Sérgia Velho,Raquel Seruca,Ana Pinto,Elia Stupka,Danilo Licastro,Maria José Oliveira,Filipa Sousa

doi:10.1186/1471-2407-14-182

Abstract

BackgroundMLK3 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.MethodsIn 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.ResultsOur 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.ConclusionThe 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.

Highlights

Mixed-lineage kinase 3 (MLK3) gene mutations were described to occur in about 20% of microsatellite unstable gastrointestinal cancers and to harbor oncogenic activity
MLK3 P252H mutation affects fundamental colorectal cancer-associated pathways In order to assess the effect of a tumorigenic MLK3 mutant on a genome-wide level, we performed microarray-based
Corroborating our results, a recent study using both in vivo and in vitro approaches showed that MLK3 signaling is important in intestinal mucosal healing and epithelial cell motility [24], implicating MLK3 signaling in the maintenance of intestinal epithelial homeostasis

Summary

Introduction

MLK3 gene mutations were described to occur in about 20% of microsatellite unstable gastrointestinal cancers and to harbor oncogenic activity. The MLK3 signaling activation was associated with an increase in the migratory and invasive capacity of tumor cells in gastric [9], breast [10,11,12], lung [13] and ovarian [14] cancers. All together, these observations implicate MLK3 as a cancerrelated gene until recently, nothing was known about MLK3 gene deregulation in primary cancer tissues

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Conclusion