HCC is a highly metastatic tumor that is associated with high recurrence rates and low survival rates (Siegel et al., 2018). The molecular mechanisms underlying the suppression of invasion and migration in HCC have been extensively studied to discover new therapeutic targets and predictive markers. The EMT is an important step in tumor migration and invasion, and it is characterized by the loss of the epithelial properties of cells such as adhesion and the expression of the epithelial marker E-cadherin, and the acquisition of mesenchymal properties, such as increased cell motility and the upregulation of the mesenchymal markers N-cadherin and vimentin (Lamouille, Xu, & Derynck, 2014).
The extracellular matrix (ECM) is an important tissue barrier for tumor metastasis. The migration and invasion of malignant tumors are often accompanied by changes in the expression of the ECM and its cell surface receptors (Lu, Takai, Weaver, & Werb, 2011). The degradation of the ECM by MMPs is one of the key aspects of tumor cell metastasis. Many malignant tumors are associated with increased secretion and activity of MMPs (Shay, Lynch, & Fingleton, 2015).
Mortalin, a member of the Hsp70 family, plays an important carcinogenic role in cancer cells through a variety of mechanisms. In addition, mortalin is considered as a target for many kinds of cancer therapy (Ando et al., 2014; Jin et al., 2016; Starenki, Hong, Lloyd, & Park, 2015; Wadhwa et al., 2006; Yang, Li, Jiang, Zuo, & Liu, 2013). It is important to find specific mortalin inhibitors for the treatment of tumors. Current studies have shown that mortalin has good surface properties and can make small molecules dock with high affinity and specificity. Wadhwa et al. (Wadhwa et al., 2016) used molecular docking software Autodock to analyze that caffeic acid phenylethyl ester (CAPE) could dock with mortalin and destroy the interaction of mortalin-p53 complex, resulting in nuclear ectopia of mortalin and activation of p53 anti-cancer function, and inhibited the growth of cancer cells through p53-GAD45a-p21 pathway. Over-inhibition of matrix metalloproteinase inhibits cell invasion and metastasis. At the same time, CAPE can increase the sensitivity of anticancer drugs by targeting mortalin in many ways (Yun et al., 2017). CAPE and Sal B in this study belong to caffeic acid derivatives, so we speculate whether Sal B also has the same targeting effect, targeting mortalin to play its anti-cancer role. We also found that another specific inhibitor of mortalin, MKT-077, binds to mortalin in this region, and MKT-077 inhibits its interaction with p53 without affecting mortalin expression, thus activating the anti-cancer function of p53 (Grover et al., 2012; Wadhwa et al., 2000). As shown in Fig. 6, we found that Sal B can significantly combine with mortalin protein. Therefore, we consider that Sal B can be used as a new inhibitor of mortalin protein. So how does Sal B affect mortalin expression? It has been reported that ubiquitin-like protein UBXN2A promotes the carboxyl end of mortalin's HSP70 interacting protein (CHIP) dependent ubiquitination. Subsequently, it was found that UNXN2A increased mortalin proteasome degradation. Subcellular regionalization experiments showed that induction of UNXN2A reduced mortalin and its partner HSP60 levels. Upregulation of UNXN2A by small molecules of veratridine (VTD) can decrease mortalin level in cancer cells. Consistent with the results in vitro, UNXN2A+/- mice showed increased mortalin selectivity in colon tissues. Recombinant UNXN2A can enhance the degradation of mortalin proteasome in mouse colon tissue (Sane et al., 2018). Our results also proved that Sal B degrades mortalin protein through ubiquitination.
More and more studies have been done on mortalin and migration and invasion of tumors recently. It has been proved that mortalin can promote migration and invasion of tumors, including breast cancer, liver cancer, cholangiocarcinoma, etc (Q. Kang et al., 2017; Na et al., 2016; Yi et al., 2008). However, the mechanism of its effect on the migration and invasion of tumors is still unclear. Our current experiments confirm that mortalin affects the migration and invasion of HCC through EMT and matrix metalloproteinase pathways. RECK and STAT3 are also involved as upstream signaling molecules. RECK is down-regulated in many tumors, and the mechanism of this down-regulation is multifactorial and tumor-specific. One of the common targets of inhibition is the Sp1 site in the promoter sequence of RECK. Early studies have speculated that the oncogene Ras promotes the phosphorylation or other modification of Sp1/Sp3 factor by activating extracellular signal-regulated kinase (ERK) pathway, increases the affinity of Sp1 site on RECK promoter, and reduces the expression of RECK (Oh et al., 2001; Sasahara, Takahashi, & Noda, 1999). Other studies hypothesized that the interaction between histone deacetylase and Sp1 might help to inhibit RECK transcription (Liu, Chang, Chiang, & Hung, 2003). However, our study showed that Sal B could up-regulate the expression of RECK and down-regulate the expression of mortalin, and mortalin could affect the protein level of RECK. This indicated that mortalin could affect RECK not through the transcriptional regulation of Sp1, but through other ways.
Overactivation of STAT3 is essential for metastasis. RECK regulates STAT3 activation, cytokine signaling and induction of vascular endothelial growth factor and uPA by forming complex with cell surface receptors (Walsh et al., 2015). MMP9 is secreted paracrine in cancer cells with high STAT3 signal transduction, and STAT3 can regulate MMP9 (Orgaz et al., 2014). In addition, STAT3 blocked MMP2 expression and MMP9 promoter activity by using chromatin immunoprecipitation of anti-STAT3 antibody (Zhang et al., 2015). We suggest that Sal B induces the up-regulation of RECK, which leads to the down-regulation of p-STAT3Y705 and Ac-STAT3K685, and inhibits the ability of MMPs to destroy ECM, thus inhibiting the migration and invasion of HCC (Fig. 6). Yuki et al. showed that RECK was up-regulated after EMT in non-malignant epithelial cells, but in cancer-derived cell lines, the down-regulation of E-cadherin and up-regulation of RECK were not related. Our current studies suggest that Sal B regulates EMT markers and STAT3/MMPs signal transduction in HCC by up-regulating RECK. All changes in EMT-related molecular markers knocked down by RECK were based on Sal B treatment. Whether RECK can affect EMT in HCC cells without Sal B remains to be further confirmed.
Na et al. (Na et al., 2016)showed that JAK-STAT signaling was involved in mortalin-induced migration and invasion of breast cancer through DNA microarray analysis. However, the relationship between mortalin and STAT signaling pathway is still lack of experimental evidence. It is the first time that Sal B can significantly reduce the levels of p-STAT3 and Ac-STAT3, mortalin can regulate the levels of p-STAT3 and Ac-STAT3, and high expression of mortalin can reverse the down-regulation of p-STAT3 and Ac-STAT3 by Sal B. However, further mechanism research is still needed.