Matrix stiffness enhances viability, migration, invasion and invadopodia formation of oral cancer cells via PI3K/AKT pathway in vitro

Abstract

Background: Oral cancer (OC) is one of the major types of cancer and the most common cause of cancer-related mortality in Asia. In recent years, matrix stiffness in the tumor microenvironment has been found to play an important role in regulating tumor cell behavior. However, the regulatory mechanisms associated with matrix stiffness in OC cells remain unclear.

Methods: In this study, polyacrylamide gels with different stiffness were prepared to simulate low versus high matrix stiffness environments in tumor tissues by adjusting the acrylamide and cross-linker concentrations. Subsequently, the effects of different stiffness on OC cell survival, migration, invasion and invadopodia formation were explored based on cell counting kit-8 (CCK-8), Transwell and confocal microscopy. Meanwhile, the levels of markers relevant to phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), apoptosis (BAX and BCL2) as well as metastasis (Cadherin-1, CDH1; Cadherin-2, and CDH2) were calculated via western blotting and real-time quantitative PCR.

Results: According to the results, high matrix stiffness was seen to contribute to the increased number of migrated and invaded cells as well as the enhanced viability of OC cells, along with the aggravated invadopodia formation and the up-regulation in CDH2 and BCL2 levels yet the down-regulation in CDH1 and BAX levels. Elevated PI3K/AKT phosphorylation levels were also seen in high matrix stiffness-mediated OC cells, and the intervention using LY294002 could visibly overturned the effects of high matrix stiffness on the cell migration, invasion and invadopodia formation of OC cells.

Conclusions: This study reveals that matrix stiffness may enhance the invasiveness and anti-apoptotic ability of OC cells by activating the PI3K/AKT pathway, which provides a new idea for exploring the microenvironmental regulation of tumor mechanics and targeted intervention strategies.

Keywords: Invadopodia; Matrix stiffness; Metastasis; Oral cancer; PI3K/AKT pathway.

Fig. 1

High matrix stiffness enhanced the migration and invasion of oral cancer cells in vitro. A Indicated processes showing the preparation of polyacrylamide gels. B Proportion of polyacrylamide gels at different stiffness degrees. C–E Transwell migration/invasion assay determining the migration and invasion of oral cancer cells at 48 h. F CCK-8 assay demonstrating the viability of oral cancer cells at different timepoints. The statistical analyses were implemented with Student’s t test or two-way analysis of variance (ANOVA). ^**p < 0.01; ^****p < 0.0001

Fig. 2

High matrix stiffness promoted the invadopodia formation in oral cancer cells in vitro. A Visualization on invadopodia formation in oral cancer cells with different mechanical forces via confocal microscopy. B Quantified number of invadopodia in oral cancer cells based on the results. All statistical analyses were implemented with Student’s t test. ^**p < 0.01

Fig. 3

High matrix stiffness activated PI3K/AKT pathway in oral cancer cells in vitro. A Oral cancer cells were exposed to different mechanical forces and the phosphorylation levels of PI3K and AKT were determined based on western blotting assay. B, C Phosphorylation levels of PI3K (B) and AKT (C) in accordance with western blotting assay. All statistical analyses were implemented with Student’s t test. ^***p < 0.001

Fig. 4

Modulation of high matrix stiffness on metastasis- and apoptosis-related proteins in oral cancer cells in vitro. A Oral cancer cells were exposed to different mechanical forces and the protein levels of metastasis- and apoptosis-related markers in oral cancer cells were accordingly gauged using western blotting assay. B–E Levels of apoptosis-related markers [BAX (B) and BCL2 (C)] and metastasis-related markers [CDH1 (D) and CDH2 (E)] based on western blotting assay. All statistical analyses were implemented with Student’s t test. ^*p < 0.05; ^**p < 0.01

Fig. 5

Inhibition of PI3K/AKT diminished the number of migrated and invaded cells as well as the invadopodia formation in oral cancer cells in vitro. A–C Transwell migration/invasion assay determining the migration and invasion of oral cancer cells with high mechanical forces and PI3K inhibitor LY294002 at 48 h. D, E Visualization on invadopodia formation in oral cancer cells with high mechanical forces and PI3K inhibitor LY294002 via confocal microscopy and the quantified number of invadopodia in oral cancer cells. All statistical analyses were implemented with Student’s t test. ^**p < 0.01

Fig. 6

Metastasis- and apoptosis-related markers in OC cells exposed to high matrix stiffness in vitro. A–D Levels of apoptosis-related markers [BAX (A) and BCL2 (B)] and metastasis-related markers [CDH1 (C) and CDH2 (D)] in oral cancer cells with high mechanical forces and PI3K inhibitor LY294002, quantified via RT-qPCR. All statistical analyses were implemented with Student’s t test. ^***p < 0.001; ^****p < 0.0001