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Critical tumor suppression pathways in brain tumors have yet to be fully defined. Along with mutational analyses, genome-wide epigenetic investigations may reveal novel suppressor elements. Using differential methylation hybridization, we identified a CpG-rich region of the promoter of the dual-specificity mitogen-activated protein kinase phosphatase-2 gene (DUSP4/MKP-2) that is hypermethylated in gliomas. In 83 astrocytic gliomas and 5 glioma cell lines examined, hypermethylation of the MKP-2 promoter was found to occur relatively more frequently in diffuse or anaplastic astrocytomas and secondary glioblastomas relative to primary glioblastomas. MKP-2 hypermethylation was associated with mutations in TP53 and IDH1, exclusive of EGFR amplification, and with prolonged survival of patients with primary glioblastoma. Expression analysis established that promoter hypermethylation correlated with reduced expression of MKP-2 mRNA and protein. Consistent with a regulatory role, reversing promoter hypermethylation by treating cells with 5-aza-2'-deoxycytidine increased MKP-2 mRNA levels. Furthermore, we found that glioblastoma cell growth was inhibited by overexpression of exogenous MKP-2. Our findings reveal MKP-2 as a common epigenetically silenced gene in glioma, the inactivation of which may play a significant role in glioma development.

The extracellular signal-regulated kinase (Erk) is one of the downstream effectors of the Ras pathway whose activation is essential for the proliferation and survival of cancer cells. Erk activation is negatively regulated by mitogen-activated protein kinase (MAPK) phosphatases (MKP), which are generally up-regulated by Erk activation, thus forming a feedback loop for regulation of Erk activity. In searching for early alterations in the Ras pathway in epidermal carcinogenesis, we identified MKP4, a cytosolic MKP with specificity to not only Erk, but also, to a lesser extent, c-jun-NH(2)-kinase and p38. MKP4 is down-regulated at initiation and lost at malignant conversion in a clonal model of epidermal carcinogenesis that lacks Ras mutation. The loss of MKP4 was associated with squamous cell carcinoma (SCC) but not benign papilloma clonal lineages and with independently induced SCC relative to benign tumors in mouse skin. Reconstitution of MKP4 expression in malignant tumor cells leads to cell death and tumor suppression. Unlike Erk inhibition that blocks cell cycle entry, MKP4 reconstitution resulted in G(2)-M associated cell death and microtubule disruption. Thus, microtubule disruption by MKP4 provides a novel mechanism for tumor suppression by a cytosolic MKP and implies a novel therapeutic strategy through combined MAPK inhibitions that mimic the function of MKP4.