Original ArticlesLow-level shear stress promotes migration of liver cancer stem cells via the FAK-ERK1/2 signalling pathway
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.
Section snippets
Cell culture
The human hepatoma cell line, MHCC97H, was bought from the Liver Cancer Institute, Zhongshan Hospital, Fudan University (Shanghai, China), and maintained as monolayer cultures in high-glucose DMEM supplemented with 10% foetal bovine serum (FBS, HyClone, Logan, UT, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL), in a 37 °C, 5% CO2 incubator. Liver cancer stem cells (LCSCs) were enriched and identified from MHCC97H in our lab [2]. Cancer stem cell related proteins, including CD133,
Shear stress regulates LCSC migration
Transwell assays were performed to evaluate the migration of LCSCs exposed to 2 dyne/cm2 shear stress. As shown in Fig. 1A and B, shear stress significantly promoted migration. Next, MTT assays were performed to exclude the possibility that cell proliferation interfered with the migration results. These results showed that none of the treatments significantly affected cell quantities (Fig. 1C), indicating that shear stress indeed promotes LCSC migration.
Shear stress activates FAK and ERK1/2 phosphorylation in LCSCs
The FAK-ERK1/2 cascade is the primary
Discussion
Cell migration not only plays major roles in physiological events, such as embryo implantation, embryogenesis, morphogenesis, neurogenesis, angiogenesis, wound healing and inflammation [[29], [30], [31]], but it is also implicated in the pathophysiology of many diseases such as cancer. Indeed, metastatic behaviour, which varies widely between cancers, defines the malignancy of tumours, and metastasis is one of the main causes of cancer-related mortality [32]; additionally, metastases are
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (11272365, 11772073, 31700810 and 11532004), the Exchange Program of the National Natural Science Foundation of China and Japan Society for the Promotion of Science (11511140092).
Conflicts of interest statement
The authors have declared that no competing interest exists.
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