Cellular stiffness regulates metastasis of hepatocellular carcinoma through JNK

Abstract

Distant metastases rather than the primary tumor from which these malignant lesions were initially caused are responsible for more than 90% of human mortality from carcinomas. Hepatocellular carcinoma (HCC), a primary type of liver cancer, has become one of the most prevalent cancer and one of the leading causes of cancer deaths around the whole world. Most HCC cases are distributed in Asia, more than 50% of all cases occur in China alone. Although great advance has been achieved, it remains quite challenging in the prognosis and effective treatment of HCC, due to the nature of chemoresistance, recurrence, and metastasis. Accumulating evidence suggests that HCC contained a subset of cancer cells with the ability to form tumors and give rise to multiple tumor cells, which is called cancer stem cells (CSCs). CSCs are highly invasive and able to spread to other organs and generate secondary tumors. These cells are resistant to chemotherapy and radiotherapy and speculated to play a key role in cancer metastasis and relapse. Therefore, CSCs have been proposed to drive tumor progression and metastasis. To develop effective therapeutic strategies against HCC metastasis, understanding the mechanisms underlying high metastatic ability of HCC is essential and necessary. It is known that significant alterations in cellular cytoskeleton could induce changes in cell mechanics, which are associated with tumor progression. Compared to cells of normal tissue, cancer cells, especially CSCs exhibit lower cellular stiffness in many types of cancer, which is correlated with malignancy and poor clinical outcomes. However, it remains largely elusive whether cellular stiffness could regulate metastatic potential and the underlying mechanism. In this study, we aimed to dissect the regulatory effect of cellular stiffness on HCC metastasis. For this purpose, we employed different HCC cell lines and firstly confirm the correlation between their cellular stiffness and metastatic ability. Different methods were used to change cellular stiffness, including activators, inhibitors, plasmids and small interfering RNAs (siRNA)s targeting several molecules that directly related to cytoskeleton. Upon the confirmation of the correlation between cellular stiffness and metastatic ability in HCC cell lines and the efficiency of methods to change cellular stiffness, we examined the functional effect of cellular stiffness on metastasis. We found decreasing cellular stiffness could enhance metastatic ability of HCC cells and in contrast metastasis was impaired when increasing cellular stiffness. These phenomena were proved both in 2D migration and 3D invasion condition. Not only in HCC cells, we also further confirmed the findings in CSCs obtained from 3D soft-fibrin method, metastatic potential was obviously decreased when stiffening CSCs. Our results confirmed the regulatory roles of cancer cell stiffness in HCC metastasis. To better understand the mechanism underlying the finding that cellular stiffness could regulate metastatic ability, we analysed several related signalling pathways through qPCR and found JNK might play an important role. It is known that JNK functions as activated state by phosphorylation at the site of T183 and Y185, so we examined the change of phosphorylated JNK at protein level by western blotting assay and found phosphorylated JNK was reduced after decreasing cellular stiffness. On the contrary, phosphorylated JNK was augmented when increasing cellular stiffness. Except for the alterations at protein level, we performed rescue experiments to prove it at the functional level. Increasing phosphorylation of JNK through activator Anisomycin could decrease metastasis after soften HCC cells and inhibitor SP600125 that decreases phosphorylation of JNK could conversely increase metastasis. In conclusion, our study proved the regulatory role of cellular stiffness on metastatic ability through JNK pathway. The outcomes of this study may provide new evidence to support the opinion that cancer may be not only a genetic disease but also related to mechanics. The causal role of low mechanical stiffness in promoting metastasis may identify cellular stiffness as a mechanical marker as a therapeutic target for liver cancer metastasis treatment, which will facilitate the development of novel strategies against cancer cell stiffness and eventually benefit patients with liver cancer.