Dual-specificity Phosphatase 6 Expression Research Articles

DUSP6, a tumor suppressor, is involved in differentiation and apoptosis in esophageal squamous cell carcinoma

Dual-specificity phosphatase 6 (DUSP6), a specific negative feedback regulator of phosphorylated extracellular signal-regulated kinase, was found to play an important role in numerous types of solid tumors as a tumor suppressor. In this study, 64.2% (61/95) of esophageal squamous cell carcinoma (ESCC) specimens studied exhibited reduced DUSP6 protein expression, compared with 91% (81/89) of normal esophageal specimens that displayed moderate or strong DUSP6 protein expression in tissue microarray analysis. In total, 36.8% (7/19) of the tumor biopsies displayed at least two-fold downregulation of DUSP6 compared with their paired normal counterparts, by qPCR. Significant loss of DUSP6 was observed in EC9706 and KYSE150 ESCC cell lines by immunoblotting assay. Low DUSP6 protein expression was significantly associated with pathological grade in ESCC by immunohistochemistry (P<0.05). Treatment with 5-aza-2′-deoxycytidine restored DUSP6 expression in the two ESCC cell lines, and the expression varied according to the drug concentration. Methylation-specific PCR analysis showed methylation-specific products in the two ESCC cell lines. We observed significant differences in the early and total apoptotic proportion between the control and experimental groups of the two ESCC cell lines and their transfectants (P<0.001) by annexin/propidium iodide assay. The presence of cleaved PARP product, a marker of caspase-mediated apoptosis, expressed in the two pCMV-DUSP6 transfectants in marked contrast to the parental and pCMV-transfected EC9706 and KYSE150 cells, was observed by immunoblotting. Overall, our results support the role of DUSP6 as a novel candidate tumor suppressor gene in ESCC, which may be a potential prognostic marker for ESCC.

Haloperidol induces demethylation and expression of the dual specificity phosphatase 6 gene in MIA PaCa-2 human pancreatic cancer cells

AimsThe effects of antipsychotics on various gene expressions through change in DNA methylation have been reported. Dual-specificity phosphatase 6 (DUSP6) is an extracellular signal regulated kinase 1/2 (ERK1/2)-selective phosphatase, and its expression can be suppressed by intronic methylation. Antipsychotic agent haloperidol affects ERK1/2 activity and could induce changes in DNA methylation as well as histone acetylation. In this study, we examined the effects of haloperidol on DUSP6 expression related to DNA methylation changes. Main methodsThe effects of haloperidol and 5-azacytidine, a demethylating agent, on expression and methylation of DUSP6 were quantitatively measured in MIA PaCa-2 human pancreatic carcinoma cells, in which DUSP6 expression is suppressed due to intronic hypermethylation. The growth rate of MIA PaCa-2 cells was also examined after treatment with haloperidol or 5-azacytidine. Key findingsHaloperidol increased DUSP6 expression in a concentration-dependent manner and inhibited MIA PaCa-2 cell proliferation; effects were comparable to those of 5-azacytidine. However, haloperidol did not induce DUSP6 expression in PANC-1 cells, another pancreatic cancer cell line without transcriptional suppression of DUSP6. Pyrosequencing methylation analysis confirmed the intronic hypermethylation of DUSP6 in MIA PaCa-2 and revealed that haloperidol and 5-azcytidine induced demethylation of CpG sequences in this region. SignificanceHaloperidol induced DUSP6 expression related to intronic demethylation and inhibited MIA PaCa-2 cell proliferation, which suggests demethylating activity and anti-cancer effects of haloperidol. These findings suggest the possible involvement of epigenetic regulatory mechanisms in the action mechanism of haloperidol.

Dual specificity phosphatase 6 as a predictor of invasiveness in papillary thyroid cancer

The genetic mutations causing the constitutive activation of MEK/ERK have been regarded as an initiating factor in papillary thyroid carcinoma (PTC). The ERK-specific dual specificity phosphatase 6 (DUSP6) is part of the ERK-dependent transcriptional output. Therefore, the coordinated regulation of the activities of ERK kinases and DUSP6 may need to be reestablished to make new balances in PTC. To investigate the role of DUSP6 in the regulation of ERK1/2 (MAPK3/1)-dependent transcription, 42 benign neoplasms and 167 PTCs were retrospectively analyzed by immunohistochemistry with dideoxy sequencing to detect BRAF(V600E) mutation. The expressions of total ERK1/2, DUSP6, c-Fos (FOS), c-Myc (MYC), cyclin D1, and PCNA were markedly increased in PTC compared with those in benign neoplasms. However, phospho-ERK1/2 was detected in only eight (4.8%) cases out of 167 PTC samples. Unexpectedly, the staining intensity and nuclear localization of ERK1/2 were not affected by the presence or absence of the BRAF(V600E) mutation. However, the expressions of c-Fos and PCNA were elevated in BRAF(V600E)-positive PTC compared with those in BRAF(V600E)-negative PTC. Interestingly, the higher staining intensities of DUSP6 were associated with the level of total ERK1/2 expression (P=0.04) and with high-risk biological features such as age (P=0.05), tumor size (P=0.01), and extrathyroidal extension (linear by linear association, P=0.02). In addition, DUSP6 silencing significantly decreased the cell viability and migration rate of FRO cells. The coordinated upregulation of total ERK1/2 and its phosphatase, DUSP6, is related to bare detection of phospho-ERK1/2 in PTC regardless of BRAF(V)(600E) mutation status. A link between DUSP6 expression and high-risk features of PTC suggested that DUSP6 is an important independent factor affecting the signaling pathways in established PTC.

Differential expression of DUSP6 with expression of ERK and Ki-67 in non-small cell lung carcinoma

Dual specificity phosphatase 6 (DUSP6) is a member of the MAP kinase phophatase family. DUSP6 inactivates extracellular signal-regulated kinase (ERK), belonging to the MAP kinase family, and can act in tumor suppressive pathways. The aim of this study was to investigate associations of DUSP6 expression with expression of ERK and Ki-67 and with clinicopathological parameters in lung adenocarcinoma and squamous cell carcinoma. A total of 102 squamous cell carcinomas and 66 adenocarcinomas were studied using immunohistochemistry for DUSP6, ERK1/2, and Ki-67. In 66 adenocarcinomas, high DUSP6 expression was positively correlated with ERK1/2 expression. High DUSP6 expression was correlated with lower histological grade and lower Ki-67 index in the adenocarcinomas. In 102 squamous cell carcinomas, high DUSP6 expression was correlated with lower ERK expression, with greater smoking pack-years, but not with the Ki-67 index. These results indicate that DUSP6 acts as a negative feedback regulator of ERK in adenocarcinoma progression, but that DUSP6 does not play a role in the downregulation of ERK in squamous cell carcinoma. The differential expression of DUSP6 correlated with Ki-67 index, suggesting that DUSP6 plays an important role in cancer resistance in different subtypes of non-small cell lung carcinoma.

Tumor suppressor dual‐specificity phosphatase 6 (DUSP6) impairs cell invasion and epithelial‐mesenchymal transition (EMT)‐associated phenotype

Suppressive effects of DUSP6 in tumorigenesis and EMT-associated properties were observed. Dual-specificity phosphatase (DUSP6) is a MAP kinase phosphatase (MKP) negatively regulating the activity of ERK, one of the major molecular switches in the MAPK signaling cascade propagating the signaling responses during malignancies. The impact of DUSP6 in EMT and its contribution to tumor dissemination has not yet been characterized. Due to differences in tumor microenvironments affecting cell signaling during cancer progression, DUSP6 may play varying roles in tumor development. We sought to examine the potential role of DUSP6-mediated tumorigenesis and EMT-associated properties in two aerodigestive tract cancers, namely, esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). Significant loss of DUSP6 was observed in 100% of 11 ESCC cell lines and 71% of seven NPC cell lines. DUSP6 expression was down-regulated in 40% of 30 ESCC tumor tissues and 75% of 20 NPC tumor tissues compared to their respective normal counterparts. Suppressive effects of DUSP6 in tumor formation and cancer cell mobility are seen in in vivo tumorigenicity assay and in vitro colony formation, three-dimensional Matrigel culture, cell migration and invasion chamber tests. Notably, overexpression of DUSP6 impairs EMT-associated properties. Furthermore, tissue microarray analysis reveals a clinical association of DUSP6 expression with better patient survival. Taken together, our study provides a novel insight into understanding the functional impact of DUSP6 in tumorigenesis and metastasis of ESCC and NPC.

Dual specificity phosphatase 6 (DUSP6) is an ETS-regulated negative feedback mediator of oncogenic ERK signaling in lung cancer cells

Mitogen-activated protein kinase (MAPK) pathway signaling plays an important role in the majority of non-small-cell lung cancers (NSCLCs). In a prior microarray analysis of epidermal growth factor receptor (EGFR) inhibition in NSCLC cell lines, we noted that several dual specificity phosphatases (DUSPs) were among the most highly and immediately regulated genes. DUSPs act as natural terminators of MAPK signal transduction and therefore, we hypothesized a tumor suppressive role via feedback mechanisms. In the current study, we focus on the assessment of DUSP6, a cytoplasmic DUSP with high specificity for extracellular signal-regulated kinase (ERK). We demonstrate that DUSP6 expression tracks in tandem with ERK inhibition and that regulation of DUSP6 is mediated at the promoter level by ETS1, a well-known nuclear target of activated ERK. Small interfering RNA knockdown in DUSP6-high H441 lung cancer cells significantly increased ERK activation and cellular proliferation, whereas plasmid-driven overexpression in DUSP6-low H1975 lung cancer cells significantly reduced ERK activation and cellular proliferation and promoted apoptosis. Also, DUSP6 overexpression synergized with EGFR inhibitor treatment in EGFR-mutant HCC827 cells. Our results indicate that DUSP6 expression is regulated by ERK signaling and that DUSP6 exerts antitumor effects via negative feedback regulation, pointing to an important feedback loop in NSCLC. Further studies assessing the tumor suppressive role of DUSP6 and strategies aimed at modulation of its activity are warranted.

Identification of an ES cell pluripotent state-specific DUSP6 enhancer

To identify genes with pluripotent state-specific expression in embryonic stem (ES) cells, we compared gene expression profiles between undifferentiated and differentiated mouse ES cells using DNA microarrays. Among the numerous genes identified, we focused on dual specificity phosphatase 6 (DUSP6), which had previously been shown to be expressed in undifferentiated human ES cells. We have identified and characterized a regulatory enhancer that we have termed PEDRE that controls pluripotent state-specific expression of DUSP6. This 82-base pair enhancer overlaps with, but is distinct from, a recently identified regulatory element that is regulated by the FGF-ERK pathway. The sequence of PEDRE is 100% identical between mouse and human DUSP6, suggesting that the molecular basis of DUSP6 gene expression in undifferentiated state of ES cells is highly conserved during evolution.

Cisplatin‐mediated activation of extracellular signal‐regulated kinases 1/2 (ERK1/2) by inhibition of ERK1/2 phosphatases

The mechanism(s) underlying neurodegeneration-associated activation of ERK1/2 remain poorly understood. We report that in cultured rat cortical neurons, whose basal ERK1/2 phosphorylation required NMDA receptors (NMDAR), the neurotoxic DNA intercalating drug cisplatin increased ERK1/2 phosphorylation via NMDAR despite reducing their activity. The rate of ERK1/2 dephosphorylation was lowered by cisplatin. Cisplatin-treated neurons showed general transcription inhibition likely accounting for the reduced expression of the ERK1/2-selective phosphatases including the dual specificity phosphatase-6 (DUSP6) and the DUSP3 activator vaccinia-related kinase-3 (VRK3). Hence, cisplatin effects on ERK1/2 may be due to the deficient ERK1/2 inhibition by the transcription-regulated phosphatases. Indeed, the transcription inhibitor actinomycin D reduced expression of DUSP6 and VRK3 while inducing the NMDAR-dependent activation of ERK1/2 and the impairment of ERK1/2 dephosphorylation. Thus, cisplatin-mediated transcriptional inhibition of ERK1/2 phosphatases contributed to delayed and long lasting accumulation of phospho-ERK1/2 that was driven by the basal NMDAR activity. Our results provide the first direct evidence for transcriptionally-regulated inactivation of neuronal ERK1/2. Its disruption likely contributes to neurodegeneration-associated activation of ERK1/2.