Low-level shear stress promotes migration of liver cancer stem cells via the FAK-ERK1/2 signalling pathway

Highlights

• Low-level shear stress promotes migration of liver cancer stem cells (LCSCs).

• Shear stress enhances the phosphorylation levels of FAK and ERK1/2 in LCSCs.

• Shear stress leads to reduction of organized F-actin and decreases cell stiffness.

• Shear stress induces LCSC migration via altering cell stiffness by FAK-ERK1/2 pathway.

Abstract

Cancer stem cells (CSCs) are a small subpopulation of tumour cells that have been proposed to be responsible for cancer initiation, chemotherapy resistance and cancer recurrence. Shear stress activated cellular signalling is involved in cellular migration, proliferation and differentiation. However, little is known about the effects of shear stress on the migration of liver cancer stem cells (LCSCs). Here, we studied the effects of shear stress that are generated from a parallel plated flow chamber system, on LCSC migration and the activation of focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2), using transwell assay and western blot, respectively. We found that 2 dyne/cm² shear stress loading for 6 h promotes LCSC migration and activation of the FAK and ERK1/2 signalling pathways, whereas treatment with the FAK phosphorylation inhibitor PF573228 or the ERK1/2 phosphorylation inhibitor PD98059 suppressed the shear stress-promoted migration, indicating the involvement of FAK and ERK1/2 activation in shear stress-induced LCSC migration. Additionally, atomic force microscopy (AFM) analysis showed that shear stress lowers LCSC stiffness via the FAK and ERK1/2 pathways, suggesting that the mechanism by which shear stress promotes LCSC migration might partially be responsible for the decrease in cell stiffness. Further experiments focused on the role of the actin cytoskeleton, demonstrating that the F-actin filaments in LCSCs are less well-defined after shear stress treatment, providing an explanation for the reduction in cell stiffness and the promotion of cell migration. Overall, our study demonstrates that shear stress promotes LCSC migration through the activation of the FAK-ERK1/2 signalling pathways, which further results in a reduction of organized actin and softer cell bodies.

Introduction

The ability to metastasize is the defining character of a cancer and the leading cause for resultant mortality. It is hypothesized that a rare subset of cancer cells, often operationally referred to as “cancer stem cells” (CSCs), are responsible for sustaining cancer metastasis and recurrence [1]. They are generally more resistant to chemotherapeutics compared to their fully differentiated tumour cell counterparts [2]. Liver cancer is one of the most common cancers worldwide and has a high mortality rate. Accumulating evidences have demonstrated the existence of CSCs in liver cancer tissue and some hepatoma cell lines, these stem cells are called liver cancer stem cells (LCSCs) [[3], [4], [5]]. Currently, LCSCs are considered an important targeting subset for the successful treatment of liver cancer. Therapies that can eradicate these cells may eventually lead to cancer cures [6]. To better understand the characteristics of CSCs and eradicate them, how chemical signals guide the physiological processes of CSCs has recently been extensively studied [7,8]. However, accumulating evidence has also shown that cancer cells in the tumour microenvironment are exposed to multiple forces including shear stress, hydrostatic pressure, tension, mechanical compression and the rigidity of the extracellular matrix, which in turn regulate the migration, invasion and proliferation of cancer cells [[9], [10], [11]].

Shear stress is a relevant mechanical signal for liver physiology. In and around normal or tumour tissues, shear stress alters the local mechanical microenvironment and regulates intracellular signalling pathways. Previous studies have reported that, under physiological conditions in solid tumours, interstitial fluid velocities in tumours vary between 0.1 and 2 μm/s, and shear stress varies on the order of 0.01 Pa–0.2 Pa (0.1–2 dyne/cm²) [12]. 2 dyn/cm² shear stress markedly upregulates matrix metalloproteinase-12 (MMP-12) expression and promotes chondrosarcoma cell invasion [13]. Further work has focused on the effects of shear stress on the motility of cancer cells, such as renal carcinoma cell line SN12L1 [14] and chondrosarcoma cell line SW1353 [15]. Yu et al. suggested that HepG2 migration capability is increased after lower shear stress loading [16]. Furthermore, the integrin-mediated FAK-Rho GTPase signalling pathway is found to promote migration and metastasis in HepG2 cells under 1.4 dyn/cm² shear stress. Shah et al. proposed that the CXCR4/CSCL12 and MEK/ERK pathways are involved in the promotion of liver cancer cell migration in response to interstitial flow stimuli [17]. Multiple studies have demonstrated that shear stress, as a type of force loading, plays significant roles in regulating cancer cells and other cell types. Gojova et al. showed that treatment with 1.9 Pa shear stress significantly increases the migration capability of bovine aortic ECs [18]. Our previous studies have also report that 0.2 Pa shear stress promotes the migration of human mesenchymal stem cells [19,20]. However, there has been little research focused on the migration of cancer stem cells from a biomechanical perspective, which has great potential for promoting further research on CSCs, providing new insight on the mechanics of liver cancer occurrence, and most importantly, providing an experimental basis for clinical strategies targeting LCSCs. Therefore, the molecular mechanisms behind the migration of CSC under shear stress are worth investigating.

Focal adhesion kinase (FAK) plays vital roles in the formation of focal adhesion sites responsible for mechanotransduction [21]. Moreover, FAK has been found to regulate cellular invasion and migration. Reduced cell motility and enhanced focal adhesion contact are observed in FAK deficient cells [22], whereas the invasiveness of human cancer cells requires elevated FAK expression [23]. FAK has many downstream signals, the best known of which are extracellular signal-regulated kinases 1 and 2 (ERK1/2), two force-activated protein kinases that are activated by shear stress and cyclic stretching [21,24]. It has also been shown that cell motility can be regulated via ERK1/2 activation [25,26].

This study aimed to investigate the effects of low-level shear stress on the migration of liver cancer stem cells (LCSCs) and to identify the possible signalling pathway involved in this process. To provide a more in-depth understanding, the changes in cell stiffness and F-actin stimulated by shear stress were also explored.