Abstract 3619: EP4 promoted cell migration via mitochondrial biogenesis in oral cancer cells
Authors: Ishikawa Soichiro , Umemura Masanari , Nakakaji Rina , Nagasako Akane , Nagao Kagemichi , Mizuno Yuto , Suzuki Fumina , Osawa Kohei , Kioi Mitomu , Mitsudo Kenji , Ishikawa Yoshihiro 
Published: April 2023

Introduction: Lymph node metastasis caused by migration of oral cancer cells is an important prognostic factor. We previously reported that EP4, a prostaglandin E2 (PGE2) receptor, regulates the cell migration in oral cancer cells via Ca2+ signaling. However, how Ca2+ signaling regulates cell migration remains unclear. The intracellular Ca2+ regulates a variety of signaling pathways, either directly or by forming complexes with proteins such as calmodulin (CaM), and then phosphorylates calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Furthermore, Ca2+ signaling is directly or indirectly involved in cancer cell proliferation and migration. Therefore, we hypothesized that EP4 regulates the cell migration via CaMKK2 and its downstream signaling in oral cancer.

Materials and Methods: Human gingival fibroblasts, HGnF and Human tongue squamous cell carcinoma cell lines, HSC-3 were used. EP4 agonist (ONO-AE1-437) and CaMKK2 inhibitor (STO-609) were used. HSC-3 cells were transduced with CaMKK2 shRNA, and scramble control shRNA using lentivirus. The protein expressions were evaluated by western blotting. The intracellular calcium concentrations were measured using Fura-2, the Fluorescent Ca2+ Indicator. The cell migration ability was evaluated by scratch assay. Immunocytochemistry was performed to evaluate the lamellipodium. The mRNA transcriptions of the mitochondrial associated genes were evaluated by real-time qPCR. The intracellular adenosine triphosphate (ATP) production and the reactive oxygen species (ROS) production were also evaluated by luciferase activity assay and DCFH-DA staining.

Results: EP4 protein expression of HSC-3 cells was higher than that of HGnF cells (n=4, p&lt;0.001). EP4 agonist rapidly increased the intracellular calcium in HSC-3 cells but not in HGnF cells (n=4, p&lt;0.001). EP4 agonist also promoted the cell migration and increased the lamellipodia formation in HSC-3 cells (n=3, p&lt;0.001). Furthermore, EP4 agonist promoted the phosphorylation of CaMKK2 and AMP-activated protein kinase (AMPK) (n=4, p&lt;0.001). In contrast, CaMKK2 inhibitor and CaMKK2 knockdown suppressed EP4-stimulated cell migration and AMPK phosphorylation (n=4, p&lt;0.001). Furthermore, EP4 agonist increased the expression of the transcriptional coactivator PGC1-α, mtDNA and mitochondrial transcription factor A (TFAM) (n=4, p&lt;0.01). Furthermore, EP4 agonist increased the intracellular ATP production and the ROS production (n=6, p&lt;0.01). Taken together, EP4 regulated the mitochondrial biogenesis via CaMKK2 and AMPK, and promoted the cell migration in oral cancer.

Conclusion: EP4/CaMKK2/AMPK pathway is a novel signal transduction in oral cancer and may be a new target for oral cancer therapy.

Citation Format: Soichiro Ishikawa, Masanari Umemura, Rina Nakakaji, Akane Nagasako, Kagemichi Nagao, Yuto Mizuno, Fumina Suzuki, Kohei Osawa, Mitomu Kioi, Kenji Mitsudo, Yoshihiro Ishikawa. EP4 promoted cell migration via mitochondrial biogenesis in oral cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3619.