Loss of MKK4 expression in ovarian cancer: a potential role for the epithelial to mesenchymal transition Shamima Yeasmin, Kentaro Nakayama, Mohammed Tanjimur Rahman, Munmun Rahman, Masako Ishikawa, Atsuko Katagiri, Kouji Iida, Naomi Nakayama, and Kohji Miyazaki Department of Obstetrics and Gynecology, Shimane University School of Medicine, Izumo, Shimane 6938501, Japan In the current study, we investigated the mechanism relating downregulation of mitogen-activated protein kinase kinase 4 (MKK4) expression to development of ovarian cancer. Over-expression of the MKK4 gene in TOV-21 G cells, a line with homozygous deletion of MKK4, resulted in morphologic changes in which cells growing in a scattered, fibroblast-like pattern formed tightly packed colonies. Based on a wound healing assay and a Matrigel invasion assay, we determined that both motility and invasiveness of MKK4-transfected TOV-21G cells were significantly reduced compared to control vector- transfected cells. To confirm that MKK4 expression related to tumor invasion resulted from an epithelial to mesenchymal transition (EMT)-like morphological change, we used 2 independent but complementary approaches. MKK4 gene knockdown in MDAH 2774 cells over-expressing MKK4 increased invasion activity. Additionally, engineered expression of MKK4 in SKOV3 cells, a line with low endogenous MKK4 expression, produced a phenotype similar to that of TOVG-21G. Interestingly, we found that MKK4 upregulation caused downregulation of phosphorylated NF-jB and Twist, as well as upregulation of E- cadherin, in TOVG-21G and SKOV3 cells. Reciprocal results were obtained in MDAH 2774 cells with MKK4 knockdown. Our results suggest that MKK4 downregulation causes increased phosphorylation NF-jB. This promotes Twist over-expression, resulting in E-cadherin downregulation that induces EMT in ovarian cancer. Ovarian cancer is the most lethal gynecological malignancy in the world. 1 As there is no method of early detection, the majority of patients present with peritoneal dissemination and distant metastasis at the time of diagnosis. With dissemi- nated disease, treatment is often unsuccessful and overall sur- vival is low. The identification of an invasion-related mole- cule associated with early and rapid spread of ovarian cancer is the current focus of many investigators. Recently, inactiva- tion or downregulation of metastasis suppressor genes has been associated with ovarian cancer progression. Mitogen- activated protein kinase kinase 4 (MKK4), a member of the stress-activated protein kinase signaling cascade, has been identified as a metastasis-suppressor gene. 2 Progressive loss of expression occurs in prostate, 3 pancreatic 4 and ovarian cancers. 5 The consequences of downregulation of MKK4 could include development of a more aggressive phenotype, one prone to invasion and metastasis that is more difficult to optimally debulk and is more chemoresistant. 6 The role of MKK4 in cancer is incompletely studied 4–6 and further stud- ies will be required to clarify the function. Recently, we iden- tified homozygous deletion of MKK4, suggesting a possible mechanism for the downregulation of MKK4 in ovarian can- cer. 7 Cells with a homozygous deletion of MKK4 are an ideal model to analyze its function using gene transfection meth- ods. In the current study, we successfully identified an ovar- ian cancer cell line carrying a homozygous MKK4 deletion. We utilized this line in a gene transfection assay in order to clearly demonstrate the involvement of MKK4 in tumor growth and invasion both in vitro and in vivo. Invasion and metastasis are the biologic hallmarks of malignancy. The molecular mechanism responsible for inva- sion and metastasis is a key area to investigate. A number of molecules related to tumor invasion and spread in ovarian cancer have been reported and include the following: Twist, 8 E-cadherin, 9 NF-jB 10 and Snail. 11 However, the molecular changes associated with acquisition of metastatic ability in ovarian cancer progression are poorly understood. Recently, the epithelial to mesenchymal transition (EMT) has been described as an important mechanism promoting invasion and causing metastasis of cancer. 12 The EMT is basically an embryonic trait through which cells adopt a phenotype more amenable to migration and invasion. 13 The phenotypic change after forced expression of MKK4 in our present model raised the possibility that the molecular mechanism by which MKK4 suppressed metastasis involved the EMT. In light of this, we also measured expression levels of other invasion-related molecules promoting EMT changes in MKK4 deleted cells. Our findings are the first evidence link- ing EMT to the metastasis suppressive function of MKK4. Key words: MKK4, EMT, ovarian cancer, twist, NF-jB, E-cadherin Additional Supporting Information may be found in the online version of this article. DOI: 10.1002/ijc.25332 History: Received 22 Jul 2009; Accepted 25 Feb 2010; Online 22 Mar 2010 Correspondence to: Kentaro Nakayama, Shimane University School of Medicine, Enyacho 89-1, Izumo, Shimane, Japan 6938501, Tel.: 81-853-20-2268, Fax: þ81-853-20-2264, E-mail: kn88@med. shimane-u.ac.jp Cancer Genetics Int. J. Cancer: 128, 94–104 (2011) V C 2010 UICC International Journal of Cancer IJC