JNK Dependency of Intercellular Junction Development in YD-10B cells

Objectives: Recently we reported that the development of intercellular junction (ICJ) of normal human oral keratinocytes is under the control of adhesion strength of cells to the substrates and the activity of c-Jun N-terminal kinase (JNK) (J Dent Res, in press). The present study explored if this mechanism contributes to the disruption of ICJ of the oral carcinoma cells which is a crucial pre-requisite for metastasis of cancer cells.
Methods: YD-10B cells, an oral carcinoma cell line established from human oral mucosa, were cultured on dishes pre-coated with various amounts of fibronectin (FN) to vary the adhesion strengths to substrates. JNK activity was inhibited or enhanced by pharmacological drugs or lentiviral transfection methods. Expression of proteins was analyzed by Western blotting analysis or immunocytochemistry.
Results: YD-10B cells transited to mesenchymal-type cells (MTC) on substrates pre-coated with more than 0.05 μg/cm² FN, whereas normal oral keratinocyte, HOK-16B cells, did on substrates more than 0.5 μg/cm² FN. E-cadherin and non-phosphorylated β-catenin (S33, S37, S41) (NPB) disappeared from plasma membrane of YD-10B on transition to MTC, which was accompanied with an increase in pJNK level. Then, JNK inhibition prevented YD-10B cells from disintegration and loss of E-cadherin and NPB from the plasma membrane despite the culture on substrates pre-coated with more than the amount of FN disrupting ICJ. JNK inhibition increased NPB, but decreased phosphorylated β-catenin (S33, S37, S41) (PB) in Western blotting analysis. On contrary, JNK activation of YD-10B cells disrupted ICJ of cells with loss of E-cadherin and NPB from plasma despite the culture on the substrates pre-coated with FN maintaining ICJ. JNK activation decreased NPB, but increased PB in the Western blotting analysis.
Conclusions: These results indicate that threshold of adhesion strength disrupting ICJ through activation of JNK that phosphorylates β-catenin at cell membrane is much lowered in YD-10B oral carcinoma cells.