Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.

Metastasis is one of the main causes of poor prognosis for hepatocellular carcinoma (HCC), which has been linked to cell-death resistance. Autophagy is an important survival mechanism under conditions of cell stress. We hypothesized that autophagy may play a role in HCC metastasis due to its prosurvival effect. Highly metastatic HCC cell lines with stable autophagy inhibition were established via lentivirus-mediated silencing of BECN1 and ATG5 genes. Mouse models of pulmonary metastasis were then developed using the cells with or without autophagy inhibition. The analysis of lung metastasis by histopathological examination and small animal imaging showed that autophagy inhibition significantly decreased the incidence of pulmonary metastases in vivo. Further invasion, migration, detachment, lung colonization, and epithelial-mesenchymal transition (EMT) assays indicated that autophagy inhibition did not affect cell invasiveness, migration or EMT but attenuated the anoikis-resistance and lung colonization of HCC cells. Investigation of the molecular mechanisms underlying showed that the autophagy-inhibition-mediated anoikis-resistance attenuation was associated with the regulation of apoptotic signaling. As autophagy inhibition was shown to be able to suppress HCC metastasis, an autophagy-based HCC tissue-specific target therapy system (AFP-Cre/LoxP-shRNA) was constructed. In vitro and in vivo analyses showed that the system was able to efficiently inhibit autophagy of HCC cells and tissue in a tissue-specific manner. Further in vivo metastasis assay showed that intratumoral administration of the system could significantly suppress lung metastasis. Together, our findings suggest that autophagy may be involved in HCC metastasis through facilitating anoikis resistance and lung colonization of HCC cells. Autophagy-based HCC tissue-specific target therapy may be a new strategy for the management of HCC metastasis.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide because of rapid progression and high incidence of metastasis or recurrence. Accumulating evidence shows that CD73-expressing tumor cell is implicated in development of several types of cancer. However, the role of CD73 in HCC cell has not been systematically investigated and its underlying mechanism remains elusive. CD73 expression in HCC cell was determined by RT-PCR, Western blot, and immunohistochemistry staining. Clinical significance of CD73 was evaluated by Cox regression analysis. Cell counting kit-8 and colony formation assays were used for proliferation evaluation. Transwell assays were used for motility evaluations. Co-immunoprecipitation, cytosolic and plasma membrane fractionation separation, and ELISA were applied for evaluating membrane localization of P110β and its catalytic activity. NOD/SCID/γc(null) (NOG) mice model was used to investigate the in vivo functions of CD73. In the present study, we demonstrate that CD73 was crucial for epithelial-mesenchymal transition (EMT), progression and metastasis in HCC. CD73 expression is increased in HCC cells and correlated with aggressive clinicopathological characteristics. Clinically, CD73 is identified as an independent poor prognostic indicator for both time to recurrence and overall survival. CD73 knockdown dramatically inhibits HCC cells proliferation, migration, invasion, and EMT in vitro and hinders tumor growth and metastasis in vivo. Opposite results could be observed when CD73 is overexpressed. Mechanistically, adenosine produced by CD73 binds to adenosine A2A receptor (A2AR) and activates Rap1, which recruits P110β to the plasma membrane and triggers PIP3 production, thereby promoting AKT phosphorylation in HCC cells. Notably, a combination of anti-CD73 and anti-A2AR achieves synergistic depression effects on HCC growth and metastasis than single agent alone. CD73 promotes progression and metastasis through activating PI3K/AKT signaling, indicating a novel prognostic biomarker for HCC. Our data demonstrate the importance of CD73 in HCC in addition to its immunosuppressive functions and revealed that co-targeting CD73 and A2AR strategy may be a promising novel therapeutic strategy for future HCC management.

Background: Hepatocellular carcinoma (HCC) is a typical malignancy in a background of chronic inflammation. Th17 cells (a major source of IL-17) constitute crucial components of infiltrating inflammatory/immune cells in HCC and can amplify inflammatory response via binding to interleukin-17 receptor (IL-17R). Thus, we investigated the expression and clinical significance of IL-17 and IL-17 receptor family cytokines in HCC. Methods: The expression and prognostic value of IL-17 and IL-17R (A-E) were examined in 300 HCC patients after resection. Six Th17 associated cytokines in serum (n = 111) were quantified using enzyme-linked immunosorbent assays. Phenotypic features of IL-17⁺ CD4⁺ T cells were determined by flow cytometry analysis. Results: High expression of intratumoral IL-17 and IL1-7RE were significantly associated with poorer survival (p = 0.016 and <0.001, respectively) and increased recurrence (both P < 0.001) of HCC patients. Moreover, intratumoral IL-17, individually or synergistically with IL-17RE, could predict HCC early recurrence and late recurrence. Also, peritumoral IL-17RE showed the prognostic ability in HCC (P < 0.001 for OS/TTR). Furthermore, expression levels of Th17 associated cytokines including IL-6, -22, -17R and TNF-α were increased in serum of HCC patients compared to haemangioma patients. Importantly, activated human hepatic stellate cells induced in vitro expansion of IL-17⁺ CD4⁺ T cells. Conclusions: High expression of IL-17 and IL-17RE were promising predictors for poor outcome of HCC patients. The protumor power of IL-17 producing CD4⁺ T cells was probably involved in the crosstalk with different types of inflammatory/immune cells in HCC.

Oct4 and Nanog are key regulatory genes that maintain the pluripotency and self-renewal properties of embryonic stem cells. We previously reported that the two stemness markers were tightly associated with cancer progression and poor outcomes of hepatocellular carcinoma. In this study, we demonstrate that coexpression of Oct4/Nanog modulates activation of signal transducer and activator of transcription 3 (Stat3), an oncogenic transcription factor that is activated in many human malignancies including hepatocellular carcinoma (HCC), as well as the expression of Snail, a key regulator implicated in epithelial-mesenchymal transition and tumor metastasis. Oct4 and Nanog were ectopic expressed in MHCC97-L cell lines via lentiviral gene transfection. The stemness characteristics including self-renewal, proliferation, chemoresistance, and tumorigenicity were assessed. The effect of coexpression of Oct4 and Nanog on epithelial-mesenchymal transition change, and the underlying molecular signaling was investigated. Ectopic coexpression of Oct4 and Nanog empowered MHCC97-L cells with cancer stem cell (CSC) properties, including self-renewal, extensive proliferation, drug resistance, and high tumorigenic capacity. Significantly, Oct4 and Nanog encouraged epithelial-mesenchymal transition change contributing to tumor migration, invasion/metastasis in vitro and in vivo. Following molecular mechanism investigation indicated Oct4/Nanog-regulated epithelial-mesenchymal transition change through Stat3-dependent Snail activation. Moreover, silencing Stat3 abrogates Oct4/Nanog-mediated epithelial-mesenchymal transition (EMT) change and invasion/metastasis in HCC. We delineate Oct4 and Nanog initiate stem cell characteristics in hepatocellular carcinoma and promote epithelial-mesenchymal transition through activation of Stat3/Snail signaling. Our findings propose Stat3/Snail pathway as a novel therapeutic target for the treatment of progression and metastasis of HCC with CSC-like signatures and epithelial-mesenchymal transition phenotype.

Cancer-associated fibroblasts (CAFs) are commonly acquired activated extracellular matrix (ECM)-producing myofibroblasts, a phenotypes with multiple roles in hepatic fibrogenesis and carcinogenesis via crosstalk with cohabitating stromal/cancer cells. Here, we discovered a mechanism whereby CAF-derived cytokines enhance hepatocellular carcinoma (HCC) progression and metastasis by activating the circRNA-miRNA-mRNA axis in tumor cells. CAFs secreted significantly higher levels of CXCL11 than normal fibroblasts (NFs), and CXCL11 also had comparatively higher expressions in HCC tissues, particularly in metastatic tissues, than para-carcinoma tissues. Both CAF-derived and experimentally introduced CXCL11 promoted HCC cell migration. Likewise, CAFs promoted tumor migration in orthotopic models, as shown by an increased number of tumor nodules, whereas CXCL11 silencing triggered a decrease of it. CXCL11 stimulation upregulated circUBAP2 expression, which was significantly higher in HCC tissues than para-carcinoma tissues. Silencing circUBAP2 reversed the effects of CXCL11 on the expression of IL-1β/IL-17 and HCC cell migration. Further downstream, the IFIT1 and IFIT3 levels were significantly upregulated in HCC cells upon CXCL11 stimulation, but downregulated upon circUBAP2 silencing. IFIT1 or IFIT3 silencing reduced the expression of IL-17 and IL-1β, and attenuated the migration capability of HCC cells. Herein, circUBAP2 counteracted miR-4756-mediated inhibition on IFIT1/3 via sponging miR-4756. miR-4756 inhibition reversed the effects induced by circUBAP2 silencing on the IL-17 and IL-1β levels and HCC cell migration. In orthotopic models, miR-4756 inhibition also reversed the effects on metastatic progression induced by silencing circUBAP2.