THE CYTOSKELETAL ALTERATION MODULATES CELL INVASIVENESS OF OSCC CELLS THROUGH RHOA-YAP SIGNALING IN STROMAL fiBROBLASTS

Abstract

Objectives Cancer-associated fibroblasts (CAFs) are most abundant stromal cells among tumor microenvironment that participate in carcinogenesis. This study aimed to investigate the mechanism of cytoskeletal alteration of CAFs and its role in carcinogenesis of oral squamous cell carcinoma (OSCC). Findings We first evaluated if immortalized normal fibroblasts(hTERT-hNOFs) can be substituted for CAFs. hTERT- hNOFs co-cultured with OSCC cells showedmyofibroblastic and senescent phenotypeslike CAFs.Next, we observed the cytoskeletal alteration in hTERT-hNOFs co-cultured with OSCC cells, including enlarged cellular size, distinct F-actin assembly (stress fibers). To further understand the mechanisms, we identified the expression of RhoGT- Pase gene family. Among them, RhoA was significantly increased. These results were confirmed by RhoA-ROCK inhibitor(Y27632). In spite of fibroblasts grown with OSCC cells, Y27632 reduced cell size and stress fibers. Furthermore,YAP distribution, as a downstream transcriptional factor of RhoA, was examined. YAP was mainly localized at nucleus in hTERT-hNOF co-cultured with OSCC cells, unlike hTERT-hNOFs co-cultured with HEK(human normal epidermal keratinocyte). To further verify if RhoA and cytoskeletal change modulate YAP distribution, Actin polymerization inhibitor(Lat.A) and Y27632 were used. As results, the inhibitors interrupted nuclear YAP localization, suggesting that YAP can be regulated by RhoA-induced cytoskeletal alteration. Lastly, we examined if nuclear YAP localization of fibroblasts exacerbates OSCC progression. YAPS127A mutant fibroblasts, maintained in nuclear YAP, were generated. As results, YAPS127A showed cytoskeletal rearrangement, such as increased gel contractility and matrix stiffness, and thereby enhanced the invasiveness of OSCC cells. Conclusions The alteration of tumor microenvironment, such as cytoskeletal change and matrix remodeling via RhoA-YAP in CAFs, modulates OSCC progression. These understandings will provide the novel approaches for CAFs-based OSCC therapy.