6416

MAP2K4

JNKK|JNKK1|MAPKK4|MEK4|MKK4|PRKMK4|SAPKK-1|SAPKK1|SEK1|SERK1|SKK1;mitogen-activated protein kinase kinase 4;MAP2K4;mitogen-activated protein kinase kinase 4

JNK-activated kinase 1|JNK-activating kinase 1|MAP kinase kinase 4|MAPK/ERK kinase 4|MAPKK 4|MEK 4|SAPK/ERK kinase 1|c-Jun N-terminal kinase kinase 1|dual specificity mitogen-activated protein kinase kinase 4|stress-activated protein kinase kinase 1

17p12

protein-coding

The dual Ser/Thr kinase MKK4 and its downstream targets JNK and p38 regulate critical cellular functions during embryogenesis and development. MKK4 has been identified as a putative tumor-suppressor gene in human solid tumors of breast, prostate and pancreas. To clarify the mechanisms underlying the transforming potential of molecular defects targeting MKK4, we have generated totipotent embryonic stem (ES) cells expressing the dominant-negative mutant DN-MKK4(Ala), S257A/T261A. Stably transfected DN-MKK4-ES cells exhibit a transformed fibroblast-like morphology, reduced proliferation rate, were no more submitted to cell contact inhibition, were growing in soft agar, and were much more tumorigenic than parental ES cells in athymic nude mice. These phenotypic changes: (i) are consistent with the protection of DN-MKK4-transfected ES cells from spontaneous, cell density-dependent, and stress-induced apoptosis (DAPI staining and poly (ADP-ribose) polymerase (PARP) cleavage) and (ii) correlated with alterations in JNK, p38, and Erk-1/-2 MAPK/SAPK signaling. Taken together, our data provide a new mechanism linking the MKK4 signaling pathways to cancer progression and identify MKK4 as a tumor-suppressor gene implicated in several transforming functions.

CYLD is a tumor suppressor that is mutated in familial cylindromatosis, an autosomal dominant predisposition to multiple tumors of the skin appendages. Recent studies suggest that transfected CYLD has deubiquitinating enzyme activity and inhibits the activation of transcription factor NF-kappaB. However, the role of endogenous CYLD in regulating cell signaling remains poorly defined. Here we report a critical role for CYLD in negatively regulating the c-Jun NH(2)-terminal kinase (JNK). CYLD knockdown by RNA interference results in hyper-activation of JNK by diverse immune stimuli, including tumor necrosis factor-alpha, interleukin-1, lipopolysaccharide, and an agonistic anti-CD40 antibody. The JNK-inhibitory function of CYLD appears to be specific for immune receptors because the CYLD knockdown has no significant effect on stress-induced JNK activation. Consistently, CYLD negatively regulates the activation of MKK7, an upstream kinase known to mediate JNK activation by immune stimuli. We further demonstrate that CYLD also negatively regulates IkappaB kinase, although this function of CYLD is seen in a receptor-dependent manner. These findings identify the JNK signaling pathway as a major downstream target of CYLD and suggest a receptor-dependent role of CYLD in regulating the IkappaB kinase pathway.

Our previous studies indicate that loss of MKK4 expression is associated with the progression of ovarian cancer. However, direct evidence that MKK4 inhibits the malignant phenotype of ovarian cancer cells is limited. In the current study, we investigated the mechanism relating loss of MKK4 expression to the development of ovarian cancer. Using cell growth and anchorage-independent assays, we determined that both the growth and colony-forming ability of MKK4-transfected TOV-21G cells, a line with a homozygous deletion of MKK4, were significantly reduced compared to control vector-transfected cells. Overexpression of the MKK4 gene in TOV-21G cells resulted in reduced proliferative activity and increased apoptosis. To confirm that MKK4 expression related to tumor suppress function, we used two independent but complementary approaches. MKK4 gene knockdown in OVK18#2 and MDAH2774 cells, which overexpressed MKK4, increased proliferation activity. Additionally, the engineered expression of MKK4 in SKOV3 cells, a line with low endogenous MKK4 expression, produced a phenotype similar to that of TOV-21G. Similar results were produced in tumor xenografts in nude mice. These results indicated that MKK4 acts as a tumor suppressor and may represent an important therapeutic target for the treatment of ovarian cancer.

BACKGROUND: MAP2K4 is a putative tumor and metastasis suppressor gene frequently found to be deleted in various cancer types. We aimed to conduct a comprehensive analysis of this gene to assess its involvement in ovarian cancer. METHODS: We screened for mutations in MAP2K4 using High Resolution Melt analysis of 149 primary ovarian tumors and methylation at the promoter using Methylation-Specific Single-Stranded Conformation Polymorphism analysis of 39 tumors. We also considered the clinical impact of changes in MAP2K4 using publicly available expression and copy number array data. Finally, we used siRNA to measure the effect of reducing MAP2K4 expression in cell lines. RESULTS: In addition to 4 previously detected homozygous deletions, we identified a homozygous 16 bp truncating deletion and a heterozygous 4 bp deletion, each in one ovarian tumor. No promoter methylation was detected. The frequency of MAP2K4 homozygous inactivation was 5.6% overall, and 9.8% in high-grade serous cases. Hemizygous deletion of MAP2K4 was observed in 38% of samples. There were significant correlations of copy number and expression in three microarray data sets. There was a significant correlation between MAP2K4 expression and overall survival in one expression array data set, but this was not confirmed in an independent set. Treatment of JAM and HOSE6.3 cell lines with MAP2K4 siRNA showed some reduction in proliferation. CONCLUSIONS: MAP2K4 is targeted by genetic inactivation in ovarian cancer and restricted to high grade serous and endometrioid carcinomas in our cohort.

MAP2K4 encodes a dual-specificity kinase (mitogen-activated protein kinase kinase 4, or MKK4) that is mutated in a variety of human malignancies, but the biochemical properties of the mutant kinases and their roles in tumorigenesis have not been fully elucidated. Here we showed that 8 out of 11 cancer-associated MAP2K4 mutations reduce MKK4 protein stability or impair its kinase activity. On the basis of findings from bioinformatic studies on human cancer cell lines with homozygous MAP2K4 loss, we posited that MKK4 functions as a tumor suppressor in lung adenocarcinomas that develop in mice owing to expression of mutant Kras and Tp53. Conditional Map2k4 inactivation in the bronchial epithelium of mice had no discernible effect alone but increased the multiplicity and accelerated the growth of incipient lung neoplasias induced by oncogenic Kras. MKK4 suppressed the invasion and metastasis of Kras-Tp53-mutant lung adenocarcinoma cells. MKK4 deficiency increased peroxisomal proliferator-activated receptor gamma2 (PPARgamma2) expression through noncanonical MKK4 substrates, and PPARgamma2 enhanced tumor cell invasion. We conclude that Map2k4 functions as a tumor suppressor in lung adenocarcinoma and inhibits tumor cell invasion by decreasing PPARgamma2 levels.

Mitogen-activated protein kinases function in signal transduction pathways that are involved in controlling key cellular processes in many organisms. A mammalian member of this kinase family, MKK4/JNKK1/SEK1, has been reported to link upstream MEKK1 to downstream stress-activated protein kinase/JNK1 and p38 mitogen-activated protein kinase. This mitogen-activated protein kinase pathway has been implicated in the signal transduction of cytokine- and stress-induced apoptosis in a variety of cell types. Here, we report that two human tumor cell lines, derived from pancreatic carcinoma and lung carcinoma, harbor homozygous deletions that eliminate coding portions of the MKK4 locus at 17p, located approximately 10 cM centromeric of p53. In addition, in a set of 88 human cancer cell lines prescreened for loss of heterozygosity, we detected two nonsense and three missense sequence variants of MKK4 in cancer cell lines derived from human pancreatic, breast, colon, and testis cells. In vitro biochemical assays revealed that, when stimulated by MEKK1, four of the five altered MKK4 proteins lacked the ability to phosphorylate stress-activated protein kinase. Thus, the incidence of coding mutations of MKK4 in the set of cell lines is 6 of 213 (approximately 3%). These findings suggest that MKK4 may function as a suppressor of tumorigenesis or metastasis in certain types of cells.

Mitogen-activated protein kinase (MAPK) kinase 4 (MKK4) is a component of a stress and cytokine-induced signal transduction pathway involving MAPK proteins. The MKK4 protein has been implicated in activation of JNK1 and p38 MAPK on phosphorylation by conserved kinase pathways. A recent report on the deletion and mutation of the MKK4 gene in human pancreatic, lung, breast, testicle, and colorectal cancer cell lines suggests an additional role for MKK4 in tumor suppression. Both the gene function and the infrequency of mutations might be considered atypical for many human tumor suppressor genes, and constitutional DNA was not previously available to determine whether the reported sequence variants had preceded tumor development. Here, we report that homozygous deletions are detected in 2 of 92 pancreatic adenocarcinomas (2%), 1 of 16 biliary adenocarcinomas (6%), and 1 of 22 breast carcinomas (when combined with reported sequence alterations, 3 of 22 or 14%). In addition, in a panel of 45 pancreatic carcinomas prescreened for loss of heterozygosity, one somatic missense mutation of MKK4 is observed and confirmed in the primary tumor (2%). Mapping of the homozygous deletions further indicated MKK4 to lie at the target of deletion. The finding of a somatic missense mutation in the absence of any other nucleotide polymorphisms or silent nucleotide changes continues to favor MKK4 as a mutationally targeted tumor suppressor gene. Coexistent mutations of other tumor suppressor genes in MKK4-deficient tumors suggest that MKK4 may participate in a tumor suppressive signaling pathway distinct from DPC4, p16, p53, and BRCA2.