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

• Published in: International journal of oral science 2018-10, Vol.10 (4), p.30-10, Article 30
• Main Authors: Noi, Masaharu, Mukaisho, Ken-Ichi, Yoshida, Saori, Murakami, Shoko, Koshinuma, Shinya, Adachi, Takeshi, Machida, Yoshisato, Yamori, Masashi, Nakayama, Takahisa, Yamamoto, Gaku, Sugihara, Hiroyuki
• Format: Article
• Language: English
• Subjects: Actin; AKT protein; Animal research; Cancer; Carcinoma in Situ - metabolism; Carcinoma in Situ - pathology; Carcinoma, Squamous Cell - metabolism; Carcinoma, Squamous Cell - pathology; Cell culture; Cell Culture Techniques - methods; Cell migration; Cell Movement; Cell Proliferation; Cytology; Cytoskeletal Proteins - metabolism; Dentistry; Extracellular signal-regulated kinase; Extracellular Signal-Regulated MAP Kinases - metabolism; Ezrin; Humans; Invasiveness; Kinases; Medicine; Morphology; Neoplasm Invasiveness - pathology; Oral and Maxillofacial Surgery; Oral cancer; Orthopedics; Phosphorylation; Podosomes - pathology; Proto-Oncogene Proteins c-akt - metabolism; Silica; Silicon Dioxide; siRNA; Squamous cell carcinoma; Surgical Orthopedics; Tongue; Tongue Neoplasms - metabolism; Tongue Neoplasms - pathology; Transfection; Tumor cell lines; Tumor Cells, Cultured; Tumors
• ISSN: 1674-2818; 2049-3169
• DOI: 10.1038/s41368-018-0033-y

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 Cellbed TM with a structure resembling the loose connective tissue morphology in a novel 3D culture system. We confirmed that the 3D system using Cellbed TM 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. Oncology: ERK contribution to tongue cancer development Extracellular signal-regulated kinases (ERKs) are enzymes that are involved in a variety of cell functions, and one in vitro study suggests that ERKs play a role in tongue cancer development by increasing cancer cell proliferation and migration. Using a novel 3-D cell culture system called Cellbed to mimic cancer cell morphology, a team headed by Ken-ichi Mukaisho at Shiga University of Medical Science, Japan found that ERKs activate cortactin (a protein located in the cell cytoplasm) and contribute to the formation of invadopodia (invasive cell protrusions associated with cancer cells) in tongue cancer cells and tumor devel