ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system

Masaharu Noi,Ken-Ichi Mukaisho,Yoshisato Machida,Saori Yoshida,Takeshi Adachi,Masashi Yamori,Shoko Murakami,Shinya Koshinuma,Gaku Yamamoto,Takahisa Nakayama,Hiroyuki Sugihara

doi:10.1038/s41368-018-0033-y

Abstract

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase (ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet CellbedTM with a structure resembling the loose connective tissue morphology in a novel 3D culture system. We confirmed that the 3D system using CellbedTM accurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3D-cultured tongue cancer cell lines than in 2D cultures. Typically, under conventional 2D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells. However, in the 3D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3D culture systems using Cellbed™ are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

Highlights

Oral cancer ranks 15th worldwide in both morbidity and mortality.[1,2] In Japan, the number of patients with oral cancer has been increasing each year; oral cancer develops most frequently in the tongue.[3]
We found that extracellular signal-related kinase (ERK) and ezrin were significantly overexpressed in invasive squamous cell carcinoma (SCC) compared to carcinoma in situ (CIS)
It has been reported that AKT is associated with the progression of tongue cancer, AKT staining showed no significant difference in the degree of protein expression between CIS and SCC samples in our study

Summary

Introduction

Oral cancer ranks 15th worldwide in both morbidity and mortality.[1,2] In Japan, the number of patients with oral cancer has been increasing each year; oral cancer develops most frequently in the tongue.[3]. We previously reported that ezrin contributes to the development of tongue cancer, suggesting its usefulness as a novel therapeutic target.[4] To screen for additional treatment targets, we first evaluated the possible contributions of extracellular signal-related kinase (ERK) and AKT to the development of tongue cancer by immunohistochemical analyses. It has been reported that AKT is associated with the progression of tongue cancer, AKT staining showed no significant difference in the degree of protein expression between CIS and SCC samples in our study.

Methods

Results

Conclusion