SMYD2 regulates lymph node metastasis derived from intrahepatic cholangiocellular carcinoma through CCR7-dependent...

Intrahepatic cholangiocellular carcinoma (ICC) has poor prognosis, especially with lymph node (LN) metastasis (LNM). Lysine methyltransferase SET and MYND domain-containing 2 (SMYD2) has critical roles and is reportedly associated with poor prognosis in various cancers, but the role of SMYD2 in ICC is unclear. Here, we examined the role of SMYD2 in the progression of LNM of ICC. We immunohistochemically analyzed SMYD2 expression using resected specimens first in primary ICC and then in metastatic LNs derived from ICC. For functional analyses, we then performed cell proliferation assay following SMYD2 inhibition using two ICC cell lines such as HuCCT-1 and HuH28. To explore the detailed mechanisms of LNM, immunocytochemistry and western blotting related to epithelial-mesenchymal transition (EMT) were first performed. Moreover, we performed clinically immunohistochemical staining of C-C motif chemokine receptor 7 (CCR7) in metastatic LNs, immunofluorescence staining and western blotting for functional analyses to evaluate the relationship between SMYD2 and CCR7. Finally, p65 of nuclear factor-kappa B (NF-κB) and phospho-p65 (Ser536) status were checked as the regulator of CCR7. Protein expression levels of SMYD2 in the primary ICC lesion were not associated with any clinicopathological characteristics or prognosis. However, SMYD2 was expressed in metastatic LNs of all cases and LNM was strongly associated with poor disease-free and overall survival also as previously reported. This study therefore aimed to clarify the molecular-biological roles for LNM by SMYD2. SMYD2 knockdown significantly inhibited ICC cell proliferation. According to subsequent EMT-related research, SMYD2 knockdown did not induce morphological changes or cadherin switching. Next, chemotaxis assay revealed that SMYD2 knockdown reduced chemotaxis in HuCCT-1 from metastatic ascites but not in HuH28 from the primary lesion. CCR7 expression in metastatic LNs was immunologically confirmed in all cases and had a positive correlation with SMYD2 expression. Decreased expression of CCR7 was observed in SMYD2-suppressed HuCCT-1 cells by immunofluorescence staining and western blotting simultaneously with the decreasing phosphorylation of p65^S536. SMYD2 could be demonstrated to express in all metastatic LNs derived from ICC and may regulate this metastatic mechanism through CCR7-dependent chemotaxis rather than EMT via the SMYD2-phospho-p65^S536-CCR7 pathway.