Mitogen-Activated Protein Kinase Kinase Kinase 4 Deficiency in Intrahepatic Cholangiocarcinoma Leads to Invasive Growth and Epithelial-Mesenchymal Transition

The molecular pathogenesis of intrahepatic cholangiocarcinoma (iCCA) is poorly understood, and its incidence continues to increase worldwide. Deficiency of mitogen-activated protein kinase kinase kinase 4 (MAP3K4) has been reported to induce the epithelial-mesenchymal transition (EMT) process of placental and embryonic development, yet its role in human cancer remains unknown. MAP3K4 has somatic mutation in iCCA so we sequenced all exons of MAP3K4 in 124 iCCA patients. We identified nine somatic mutations in 10 (8.06%) patients, especially in those with lymph node metastasis and intrahepatic metastasis. We also showed that messenger RNA and protein levels of MAP3K4 were significantly reduced in iCCA versus paired nontumor tissues. Furthermore, knockdown of MAP3K4 in cholangiocarcinoma cells markedly enhanced cell proliferation and invasiveness in vitro and tumor progression in vivo, accompanied by a typical EMT process. In contrast, overexpression of MAP3K4 in cholangiocarcinoma cells obviously reversed EMT and inhibited cell invasion. Mechanistically, MAP3K4 functioned as a negative regulator of EMT in iCCA by antagonizing the activity of the p38/nuclear factor kappa B/snail pathway. We found that the tumor-inhibitory effect of MAP3K4 was abolished by inactivating mutations. Clinically, a tissue microarray study containing 322 iCCA samples from patients revealed that low MAP3K4 expression in iCCA positively correlated with aggressive tumor characteristics, such as vascular invasion and intrahepatic or lymph node metastases, and was independently associated with poor survival and increased recurrence after curative surgery. Conclusions: MAP3K4, significantly down-regulated, frequently mutated, and potently regulating the EMT process in iCCA, was a putative tumor suppressor of iCCA.