RAF1–MEK/ERK pathway‐dependent ARL4C expression promotes ameloblastoma cell proliferation and osteoclast formation

Abstract

Ameloblastoma is an odontogenic neoplasm characterized by slow intraosseous growth with progressive jaw resorption. Recent reports have revealed that ameloblastoma harbours an oncogenic BRAFV600E mutation with mitogenactivated protein kinase (MAPK) pathway activation and described cases of ameloblastoma harbouring a BRAFV600E mutation in which patients were successfully treated with a BRAF inhibitor. Therefore, the MAPK pathway may be involved in the development of ameloblastoma; however, the precise mechanism by which it induces ameloblastoma is unclear. The expression of ADP-ribosylation factor (ARF)-like 4c (ARL4C), induced by a combination of the EGF-MAPK pathway and Wnt/β-catenin signalling, has been shown to induce epithelial morphogenesis. It was also reported that the overexpression of ARL4C, due to alterations in the EGF/RAS-MAPK pathway and Wnt/β-catenin signalling, promotes tumourigenesis. However, the roles of ARL4C in ameloblastoma are unknown. We investigated the involvement of ARL4C in the development of ameloblastoma. In immunohistochemical analyses of tissue specimens obtained from 38 ameloblastoma patients, ARL4C was hardly detected in non-tumour regions but tumours frequently showed strong expression of ARL4C, along with the expression of both BRAFV600E and RAF1 (also known as C-RAF). Loss-of-function experiments using inhibitors or siRNAs revealed that ARL4C elevation depended on the RAF1-MEK/ERK pathway in ameloblastoma cells. It was also shown that the RAF1-ARL4C and BRAFV600E-MEK/ ERK pathways promoted cell proliferation independently. ARL4C-depleted tumour cells (generated by knockdown or knockout) exhibited decreased proliferation and migration capabilities. Finally, when ameloblastoma cells were co-cultured with mouse bone marrow cells and primary osteoblasts, ameloblastoma cells induced osteoclast formation. ARL4C elevation in ameloblastoma further promoted its formation capabilities through the increased RANKL expression of mouse bone marrow cells and/or primary osteoblasts. These results suggest that the RAF1-MEK/ ERK-ARL4C axis, which may function in cooperation with the BRAFV600E-MEK/ERK pathway, promotes ameloblastoma development.

The role of Wnt, ARL4C, and Sema3A in developmental process and disease pathogenesis

“…A recent report shows that ARL4C is also expressed in ameloblastoma, which shows slow intraosseous growth and progressive bone resorption of the jaw 3 . An immunohistochemical analysis showed that ARL4C expression was detected in 28/38 (73.7%) tumor cases 126 . ARL4C was strongly expressed in the cellular cytoplasm of tumor cells and occasionally localized in the nucleus of tumor cells (Figure 6a).…”

“…As ARL4C harbors the characteristics to be localized in nucleus and/or cell membrane, 101,102,105,106 it is required to investigate the effect of its localization on tumor formation in the future. Because ARL4C expression is hardly detected in non‐tumor regions, ARL4C might be a highly specific marker for above malignant tumors as well as benign tumors 108,114,120,126 …”

“…These results suggest that the expression of ARL4C might depend on the RAF1‐MEK/ERK pathway in ameloblastoma cells (Figure 6b). 126 …”

“…Loss‐of‐function experiments using a clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated protein 9 system or siRNAs revealed that the expression of ARL4C is involved in cellular growth and migration of AM‐1 ameloblastoma cells 126 …”

BML‐111 inhibits osteoclast differentiation by suppressing the MAPK and NF‐κB pathways, alleviating deterioration of the knee joints in a CIA rat model

“…In this study, we verified that BML-111 inhibits MAPK consists of three subfamilies, ERK, JNK, and p38, and can be activated by the binding of RANKL to its receptor RANK. Activated ERK enters the nucleus and modulates the transcription of many proteins, such as C-FOS, and thus drives the commitment of osteoclast precursors to become large multinucleated mature osteoclasts (Fujii et al, 2022). Phospho-JNK induces expression of osteoclastogenesis-related genes through activation of transcription factor AP-1 and MMP, thus directing osteoclast differentiation, fusion, activation, and survival (Lorenzo, 2017).…”