MST4 negatively regulates the EMT, invasion and metastasis of HCC cells by inactivating PI3K/AKT/Snail1 axis.

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has a poor prognosis due to the high incidence of invasion and metastasis-related progression. However, the underlying mechanism remains elusive, and valuable biomarkers for predicting invasion, metastasis, and poor prognosis of HCC patients are still lacking. Methods: Immunohistochemistry (IHC) was performed on HCC tissues (n = 325), and the correlations between MST4 expression of the clinical HCC tissues, the clinicopathologic features, and survival were further evaluated. The effects of MST4 on HCC cell migratory and invasive properties in vitro were evaluated by Transwell and Boyden assays. The intrahepatic metastasis mouse model was established to evaluate the HCC metastasis in vivo. The PI3K inhibitor, LY294002, and a specific siRNA against Snail1 were used to investigate the roles of PI3K/AKT pathway and Snail1 in MST4-regulated EMT, migration, and invasion of HCC cells, respectively. Results: In this study, by comprehensively analyzing our clinical data, we discovered that low MST4 expression is highly associated with the advanced progression of HCC and serves as a prognostic biomarker for HCC patients of clinical-stage III-IV. Functional studies indicate that MST4 inactivation induces epithelial-to-mesenchymal transition (EMT) of HCC cells, promotes their migratory and invasive potential in vitro, and facilitates their intrahepatic metastasis in vivo, whereas MST4 overexpression exhibits the opposite phenotypes. Mechanistically, MST4 inactivation elevates the expression and nuclear translocation of Snail1, a key EMT transcription factor (EMT-TF), through the PI3K/AKT signaling pathway, thus inducing the EMT phenotype of HCC cells, and enhancing their invasive and metastatic potential. Moreover, a negative correlation between MST4 and p-AKT, Snail1, and Ki67 and a positive correlation between MST4 and E-cadherin were determined in clinical HCC samples. Conclusions: Our findings indicate that MST4 suppresses EMT, invasion, and metastasis of HCC cells by modulating the PI3K/AKT/Snail1 axis, suggesting that MST4 may be a potential prognostic biomarker for aggressive and metastatic HCC.

MST4 inhibits human hepatocellular carcinoma cell proliferation and induces cell cycle arrest via suppression of PI3K/AKT pathway.

Objective: MST4 has exhibited functions in regulating cell polarity, Golgi apparatus, cell migration, and cancer. Mechanistically, it affects the activity of p-ERK, Hippo-YAP pathway and autophagy. The aim of this study is to further examine the functions of MST4 in hepatocellular carcinoma (HCC) and the underlying mechanism. Methods: The expression level of MST4 in HCC and noncancer adjacent liver tissues was determined by qRT-PCR and immunohistochemistry staining. Wild-type MST4 (MST4) and a dominant-negative mutant of MST4 (dnMST4) were overexpressed in HCC cell lines, respectively. CCK-8 assay, EdU incorporation assay, and soft agar assay were used to determine cell proliferation in vitro. The xenograft mouse model was employed to determine HCC cell growth in vivo. Cell cycle analysis was performed by PI staining and flow cytometry. The expression of key members in PI3K/AKT pathway was detected by Western blot analysis. Results: In our study, we reported new evidence that MST4 was frequently down-regulated in HCC tissues. Gain-of-function and loss-of-function experiments demonstrated that MST4 negatively regulated in vitro HCC cell proliferation. Additionally, MST4 overexpression suppressed Bel-7404 cell tumor growth in nude mice. Further experiments revealed that the growth-inhibitory effect of MST4 overexpression was partly due to a G1-phase cell cycle arrest. Importantly, mechanistic investigations suggested that dnMST4 significantly elevated the phosphorylation levels of key members of PI3K/AKT pathway, and the selective PI3K inhibitor LY294002 can reverse the proliferation-promoting effect of dnMST4. Conclusions: Overall, our results provide a new insight into the clinical significance, functions and molecular mechanism of MST4 in HCC, suggesting that MST4 might have a potential therapeutic value in the HCC clinical treatment.

miR-19 regulates the expression of interferon-induced genes and MHC class I genes in human cancer cells.

MicroRNA-19 (miR-19) is identified as the key oncogenic component of the miR-17-92 cluster. When we explored the functions of the dysregulated miR-19 in lung cancer, microarray-based data unexpectedly demonstrated that some immune and inflammatory response genes (i.e., IL32, IFI6 and IFIT1) were generally down-regulated by miR-19 overexpression in A549 cells, which prompted us to fully investigate whether the miR-19 family (i.e., miR-19a and miR-19b-1) was implicated in regulating the expression of immune and inflammatory response genes in cancer cells. In the present study, we observed that miR-19a or miR-19b-1 overexpression by miRNA mimics in the A549, HCC827 and CNE2 cells significantly downregulated the expression of interferon (IFN)-regulated genes (i.e., IRF7, IFI6, IFIT1, IFITM1, IFI27 and IFI44L). Furthermore, the ectopic miR-19a or miR-19b-1 expression in the A549, HCC827, CNE2 and HONE1 cells led to a general downward trend in the expression profile of major histocompatibility complex (MHC) class I genes (such as HLA-B, HLA-E, HLA-F or HLA-G); conversely, miR-19a or miR-19b-1 inhibition by the miRNA inhibitor upregulated the aforementioned MHC Class I gene expression, suggesting that miR-19a or miR-19b-1 negatively modulates MHC Class I gene expression. The miR-19a or miR-19b-1 mimics reduced the expression of interleukin (IL)-related genes (i.e., IL1B, IL11RA and IL6) in the A549, HCC827, CNE2 or HONE1 cells. The ectopic expression of miR-19a or miR-19b-1 downregulated IL32 expression in the A549 and HCC827 cells and upregulated IL32 expression in CNE2 and HONE1 cells. In addition, enforced miR-19a or miR-19b-1 expression suppressed IL-6 production by lung cancer and nasopharyngeal carcinoma (NPC) cells. Taken together, these findings demonstrate, for the first time, that miR-19 can modulate the expression of IFN-induced genes and MHC class I genes in human cancer cells, suggesting a novel role of miR-19 in linking inflammation and cancer, which remains to be fully characterized.

Recognition and killing of cancer stem-like cell population in hepatocellular carcinoma cells by cytokine-induced killer cells via NKG2d-ligands recognition.

There is an urgent need for more potent and safer approaches to eradicate cancer stem cells (CSCs) for curing cancer. In this study, we investigate cancer-killing activity (CKA) of cytokine-induced killer (CIK) cells against CSCs of hepatocellular carcinoma (HCC). To visualize CSCs in vitro by fluorescence imaging, and image and quantify CSCs in tumor xenograft-bearing mice by bioluminescence imaging, HCC cells were engineered with CSC detector vector encoding GFP and luciferase controlled by Nanog promoter. We found that CIK cells have a strong CKA in vitro against putative CSCs of HCC, as shown by tumorsphere formation and time-lapse imaging. Additionally, time-lapse recording firstly revealed that putative CSCs were attacked simultaneously by many CIK cells and finally eradicated by CIK cells, indicating the necessity of achieving sufficient effector-to-target ratios. We firstly illustrated that anti-NKG2D antibody blocking partially but significantly inhibited CKA of CIK cells against putative CSCs. More importantly, intravenous infusion of CIK cells remarkably delayed tumor growth in mice with a significant decrease in putative CSC number monitored by bioluminescence imaging. Taken together, these findings demonstrate CKA of CIK cells against putative CSCs of HCC, at least in part, by NKG2D-ligands recognition.

Cytokine-induced killer cells efficiently kill stem-like cancer cells of nasopharyngeal carcinoma via the NKG2D-ligands recognition.

Cancer stem cells (CSCs) are considered to be the root cause for cancer treatment failure. Thus, there remains an urgent need for more potent and safer therapies against CSCs for curing cancer. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against putative CSCs of nasopharyngeal carcinoma (NPC) was fully evaluated in vitro and in vivo. To visualize putative CSCs in vitro by fluorescence imaging, and image and quantify putative CSCs in tumor xenograft-bearing mice by in vivo bioluminescence imaging, NPC cells were engineered with CSC detector vector encoding GFP and luciferase (Luc) under control of Nanog promoter. Our study reported in vitro intense tumor-killing activity of CIK cells against putative CSCs of NPC, as revealed by percentage analysis of side population cells, tumorsphere formation assay and Nanog-promoter-GFP-Luc reporter gene strategy plus time-lapse recording. Additionally, time-lapse imaging firstly illustrated that GFP-labeled or PKH26-labeled putative CSCs or tumorspheres were usually attacked simultaneously by many CIK cells and finally killed by CIK cells, suggesting the necessity of achieving sufficient effector-to-target ratios. We firstly confirmed that NKG2D blockade by anti-NKG2D antibody significantly but partially abrogated CIK cell-mediated cytolysis against putative CSCs. More importantly, intravenous infusion of CIK cells significantly delayed tumor growth in NOD/SCID mice, accompanied by a remarkable reduction in putative CSC number monitored by whole-body bioluminescence imaging. Taken together, our findings suggest that CIK cells demonstrate the intense tumor-killing activity against putative CSCs of NPC, at least in part, by NKG2D-ligands recognition. These results indicate that CIK cell-based therapeutic strategy against CSCs presents a promising and safe approach for cancer treatment.