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Angiogenesis plays an important role in the occurrence and development of tumors. Registered tyrosine kinase inhibitors targeting vascular endothelial growth factor reduce angiogenesis. Apatinib, a tyrosine kinase inhibitor, can specifically inhibit vascular endothelial growth factor receptor 2, showing encouraging anti-tumor effects in a variety of tumors including advanced hepatocellular carcinoma (HCC). This article intends to review the clinical research and application prospects of apatinib in the field of HCC.

Most hepatocellular carcinoma (HCC) patients are diagnosed at an advanced stage; however, the effect of systemic therapy on advanced HCC remains undetermined. Therefore, new treatment targets must be identified. We analyzed Gene Expression Omnibus datasets from two HCC patient cohorts and found that NT5DC2 was associated with vascular invasion and poor survival. In two hepatoma cell lines, NT5DC2 overexpression promoted HCC cell proliferation and clone formation in vitro and promoted tumor growth in vivo. Coimmunoprecipitation assays and liquid chromatography with tandem mass spectrometry analysis revealed that NT5DC2 bound directly to epidermal growth factor receptor (EGFR). NT5DC2 upregulated EGFR expression by downregulating EGFR ubiquitination and preventing its degradation via the ubiquitin-proteasome pathway but did not upregulate its transcription. EGFR upregulation activated downstream signal transduction, which played a critical role in the protumor effects of NT5DC2. Erlotinib, a small-molecule inhibitor of EGFR, blocked the effect of NT5DC2 in promoting HCC cell proliferation. In a cohort of 79 patients who underwent curative resection for HCC, NT5DC2 expression in the tumors was associated with larger tumors and microvascular invasion. NT5DC2 expression was also independently associated with recurrence-free survival. The present study demonstrated for the first time that NT5DC2 promotes tumor cell proliferation in HCC and may serve as a potential molecular target for treating HCC. EGFR blockage could be used to treat selected patients with NT5DC2 upregulation.

Oxidative stress and hypoxia are two opposite microenvironments involved in HCC metastasis. Thioredoxin (TXN) and hypoxia-inducible factor 2α (HIF-2α) are typical proteins involved in these two different microenvironments, respectively. How these two factors interact to influence the fate on tumor cells remains unknown. Hypoxia facilitated HCC cells withstood oxidative stress and eventually promoted HCC cells metastasis, in which TXN and HIF-2α were mostly involved. Upregulation of TXN/HIF-2α correlated with poor HCC prognosis and promoted HCC metastasis both in vitro and in vivo. Epithelial-mesenchymal transition (EMT) process was involved in TXN/HIF-2α-enhanced invasiveness of HCC cells. Additionally, the stability and activity of HIF-2α were precisely regulated by TXN via SUMOylation and acetylation, which contributed to HCC metastasis. Our data revealed that the redox protein TXN and HIF-2α are both associated with HCC metastasis, and the fine regulation of TXN on HIF-2α contributes essentially during the process of metastasis. Our study provides new insight into the interaction mechanism between hypoxia and oxidative stress and implies potential therapeutic benefits by targeting both TXN and HIF-2α in the treatment of HCC metastasis.

Anti-angiogenic drugs (AADs) are currently the main choice for systemic treatment of hepatocellular carcinoma, but treatment-related proteinuria can affect the routine use of AADs, which in turn affects the overall efficacy, its prevention is a clinical aspiration. At present, most clinicians give angiotensin-converting enzyme inhibitors (ACEIs) to alleviate proteinuria according to diabetic nephropathy guidelines or expert recommendations. However, its efficacy and whether it promotes cancer are controversial. Our clinical work has found that the use of ACEIs has not effectively relieved proteinuria, and some cases even have obvious tumor progression. Here we confirmed that in different tumor-bearing mouse models, ACEIs not only did not delay the appearance of proteinuria or reduce the degree of proteinuria caused by AADs, but also reduced the anti-cancer efficacy of AADs, and the reduction of anticancer efficacy has nothing to do with the change of VEGF signaling pathway, Our study show the combination of ACEIs and AADs aggravates the production of kidney-derived erythropoietin(EPO). In turn, EPO compromises the anti-angiogenic effects of AADs and decreases antitumor activity. This study presents treatment of proteinuria caused by AADs with ACEIs is useless and plays a role in drug resistance. Our purpose is to contribute to the rational management of side effects of AADs and to develop relevant guidelines.

Background: Currently, most clinicians prescribe angiotensin-converting enzyme inhibitors (ACEIs) to alleviate proteinuria caused by anti-angiogenic drugs (AADs) according to diabetic nephropathy guidelines or expert recommendations. However, the efficacy ACEIs and potential cancer promoting effect are controversial. This study aimed at confirming whether ACEIs are beneficial in anti-angiogenesis therapy for hepatocellular carcinoma. Methods: In clinic, we observed the impact of ACEIs on AADs during the treatment of hepatocellular carcinoma. Next, we established different tumor-bearing mouse models to confirm that whether ACEIs have any effect on the anti-cancer efficacy and proteinuria side effects of AADs. Further, we confirmed the relevant mechanism in vivo. Findings: Our clinical data have shown that some hepatocellular carcinoma (HCC) patients experienced tumor progression by ACEIs administration for the treatment of hypertension or proteinuria caused by AADs. We have confirmed that in different tumor-bearing mouse models, ACEIs did not delay the appearance of proteinuria or alleviate proteinuria caused by AADs but compromised the anticancer efficacy of AADs. This effect is unrelated to a change in the VEGF signaling pathway. Our data showed that the combination of ACEIs and AADs flared the production of kidney-derived erythropoietin (EPO). In turn, EPO compromises the anti-angiogenic effects of AADs and decreases antitumor activity. Interpretation: For the treatment of proteinuria caused by AADs, ACEIs have no efficacy but promote drug resistance. Kidney-derived EPO is mainly responsible for ACEIs induced anti-angiogenesis resistance. Funding Statement: This study was supported by the National Natural Science Foundation of China (No. 81672884) and the National Science and Technology Major Project of China (No. 2017ZX10203207-004-005). Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: All human studies were approved by the Clinical Research Ethics Committee of Tianjin Medical University Cancer Institute. All animal experiments were carried out in accordance with a protocol approved by the ethics committee of the Institutional Animal Care of Tianjin Medical University Cancer Institute and Hospital.

Interleukin‑6 (IL‑6) is involved in various biological responses, including tumor progression, metastasis and chemoresistance. However, the role and molecular mechanism of IL‑6 in the treatment of sorafenib in liver cancer remain unclear. In the present study, through western blot analysis, Transwell assay, flow cytometric assay, ELISA analysis and immunohistochemistry it was revealed that sorafenib promoted metastasis and induced epithelial‑mesenchymal transition (EMT) in liver cancer cells in vitro and in vivo, and significantly increased IL‑6 expression. Endogenous or exogenous IL‑6 affected metastasis and EMT progression in liver cancer cells through Janus kinase 2/signal transducer and activator of transcription 3 (STAT3) signaling. Knocked out IL‑6 markedly attenuated the pro‑metastasis effect of sorafenib and increased the susceptibility of liver cancer cells to it. In conclusion, the present results indicated that IL‑6/STAT3 signaling may be a novel therapeutic strategy for liver cancer.

Metastasis is a serious risk that may occur during the treatment of hepatocellular carcinoma (HCC), preventing many patients from being surgical candidates and contributing to poor prognosis. Hypoxia has been proved an important factor of metastasis through the epithelial–mesenchymal transition (EMT) pathway. Acetyl‐CoA synthase 2 (ACSS2) provides an acetyl group for the acetylation of hypoxia‐inducible factor (HIF)‐2α, and this epigenetic modification affects the activity of HIF‐2α and the subsequent EMT process. Here, we showed that ACSS2 expression was negatively correlated with HCC malignancy. Knockdown of ACSS2 increased the invasion and migration ability of HCC cells and promoted EMT without increasing the total protein level of HIF‐2α, even in hypoxic conditions. The immunoprecipitation assay revealed downregulated acetylation levels of HIF‐2α after ACSS2 knockdown in hypoxic conditions, which resulted in enhanced HIF‐2α activity. Finally, decreased expression of ACSS2 was found to be related to advanced stage and poor overall survival and disease‐free survival rates in a cohort of patients with HCC. In conclusion, ACSS2 plays an important role in the acetylation process of HIF‐2α, which effectively modifies the activity of HIF‐2α under hypoxic conditions and greatly impacts on the prognosis of patients with HCC.

We previously found that a low dose of sorafenib had a prometastatic effect on hepatocellular carcinoma (HCC), which was caused by downregulation of TIP30 expression. More recently, metformin has been shown to have potential as a preventive and therapeutic agent for different cancers, including HCC. This study evaluated whether the combination of sorafenib and metformin is sufficient to revert the expression of TIP30, thereby simultaneously reducing lung metastasis and improving survival. Our data show that the combination of sorafenib and metformin inhibits proliferation and invasion in vitro, prolongs median survival, and reduces lung metastasis of HCC in vivo. This effect is closely associated with the upregulation of TIP30, partly through activating AMP-activated protein kinase. Thioredoxin, a prometastasis factor, is negatively regulated by TIP30 and plays an essential role during the process of HCC metastasis. Overall, our results suggest that metformin might be a potent enhancer for the treatment of HCC by using sorafenib.

Platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) is a well-known marker of endothelial cells and a key factor for adhesion and accumulation of platelets. CD31 plays roles in cell proliferation, apoptosis, migration, and cellular immunity. CD31 is also expressed on tumor cells, such as breast cancer cells and non-Hodgkin's lymphomas, and contributes to tumor cell invasion. Here, our experiments show that CD31 promotes metastasis by inducing the epithelial-mesenchymal transition in hepatocellular carcinoma by up-regulating integrin β1 via the FAK/Akt signaling pathway.

Background High frequency of recurrence is the major cause of the poor outcomes for patients with hepatocellular carcinoma (HCC). microRNA (miR)-182-5p emerged as a high-priority miRNA in HCC and was found to be related to HCC metastasis. Whether the expression of miR-182-5p in tumor tissue correlated with early recurrence in HCC patients underwent curative surgery was unknown. Methods Real-time PCR (RT-PCR) and in situ hybridization (ISH) were conducted to assess the expression of miR-182-5p in HCC cells and tissues. Cell Counting Kit-8 (CCK-8), transwell assays were performed to detected cells proliferation and migration ability. Flow cytometry assays were used to detect cell apoptosis rate, and xenograft model was employed to study miR-182-5p in HCC growth and lung metastasis. The target of miR-182-5p was validated with a dual-luciferase reporter assay and western blotting. Immunohistochemistry, immumoblotting, and immunoprecipitation were performed to test relative protein expression. Results We showed that high expression of miR-182-5p in tumor tissues correlated with poor prognosis as well as early recurrence in HCC patients underwent curative surgery. miR-182-5p enhanced motility and invasive ability of HCC cells both in vitro and in vivo. miR-182-5p directly targets 3′-UTR of FOXO3a and repressed FOXO3a expression, activating AKT/FOXO3a pathway to promote HCC proliferation. Notably, miR-182-5p activated Wnt/β-catenin signaling by inhibiting the degradation of β-catenin and enhancing the interaction between β-catenin and TCF4 which was mediated by repressed FOXO3a. Conclusions Consistently, miR-182-5p can be a potential predictor of early recurrence for HCC patients underwent curative surgery, and FOXO3a plays a key mediator in miR-182-5p induced HCC progression.

Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key molecule of glucose metabolism in cytoplasm, has been found in various tumors. Emerging evidence has suggested that PFKFB3 is also located in the nucleus and apparent in regulatory functions other than glycolysis. In this study, we found that PFKFB3 expression is associated with hepatocellular carcinoma (HCC) growth and located mainly in the nucleus of tumor cells. PFKFB3 overexpression was associated with large tumor size (p = 0.04) and poor survival of patients with HCC (p = 0.027). Knockdown of PFKFB3 inhibited HCC growth, not only by reducing glucose consumption but also by damaging the DNA repair function, leading to G2/M phase arrest and apoptosis. In animal studies, overexpression of PFKFB3 is associated with increased tumor growth. Mechanistically, PFKFB3 silencing decreased AKT phosphorylation and reduced the expression of ERCC1, which is an important DNA repair protein. Moreover, PFK15, a selective PFKFB3 inhibitor, significantly inhibited tumor growth in a xenograft model of human HCC. PFKFB3 is a potential novel target in the treatment of HCC.

Our previous studies demonstrated that traditional Chinese herbal medicine 'Songyou Yin' inhibited the growth and invasion of hepatocellular carcinoma (HCC) cells, and altered epithelial‑mesenchymal transition (EMT) markers in oxaliplatin‑treated HCC tissues and cell lines. In the present study, we aimed to explore whether astragaloside IV (AS-IV), a component of 'Songyou Yin', can affect the growth and invasion of HCC cells and the underlying mechanism involved. Human HCC cell lines Huh7 and MHCC97-H, with low and high metastatic potential, respectively, were treated with increasing doses of AS-IV. The Cell Counting Kit-8 (CCK-8), plate clone formation, Transwell, wound healing and immunofluorescence assays were used to investigate the effects of AS-IV on HCC cell proliferation, migration and invasion. The protein expression levels were analyzed by western blotting and immunofluorescence assay. The CCK-8 and plate clone formation assays showed that AS-IV had little effect on the proliferation of HCC cells in vitro. However, the Transwell and wound healing assays demonstrated that AS-IV inhibited the migration and invasion of HCC cells in a dose-dependent manner and the morphology of HCC cells was altered from spindle into oval shaped in the AS-IV pretreated groups. The upregulation of E-cadherin and downregulation of N-cadherin, vimentin, α-SMA and Slug were also observed in the AS-IV pretreated groups. Additionally, AS-IV treatment resulted in a profound decrease in the phosphorylated forms of Akt and GSK-3β, which in turn inhibited the expression of β-catenin. Thus, we conclude that AS-IV attenuates the invasive and migratory abilities of HCC cells through the inhibition of EMT by targeting the Akt/GSK-3β/β-catenin pathway.

Background Sorafenib is recognized as a standard treatment for advanced hepatocellular carcinoma (HCC). However, many patients have to adopt dose reduction or terminate the use of sorafenib because of side effects. In addition, a large number of patients are resistant to sorafenib. Thus, it is essential to investigate the underlying mechanisms of the resistance to sorafenib and seek potential strategy to enhance its efficacy. Methods The protein expression of hypoxia-inducible factors (HIF)-2α, 30-kDa HIV Tat-interacting protein (TIP30), E-cadherin, N-cadherin, and pAMPK was detected by Western blot. Cell viability assays were performed to study the influence of metformin and sorafenib on cell proliferation. Annexin V-FITC apoptosis assays were used to detect the influence of metformin and sorafenib on cell apoptosis. The relationship between HIF-2α and TIP30 was studied using gene silencing approach and chromatin immunoprecipitation assay. To investigate the effect of metformin and sorafenib on postoperative recurrence and lung metastasis of HCC in tumor-bearing mice, the mice were orally treated either with metformin or sorafenib once a day for continuous 37 days after the operation to remove the lobe where the tumor was implanted. CD31, Ki67, and TUNEL were examined by immunohistochemistry. Results Our study demonstrated that metformin synergized with sorafenib reduced HIF-2α expression as examined by Western blot. Gene silencing approach indicated TIP30 was upregulated after knocking-down of HIF-2α and chromatin immunoprecipitation assay revealed that HIF-2α could bind to TIP30 promoter under hypoxic condition. Cell Counting Kit-8 (CCK8) cell viability assay and Annexin V-FITC apoptosis assay showed that metformin in combination with sorafenib suppressed cell proliferation and promoted cell apoptosis. Besides, combined therapy suppressed epithelial-mesenchymal transition (EMT) process both in vitro and in vivo. Moreover, metformin in combination with sorafenib significantly minimized postoperative recurrence and lung metastasis of HCC in orthotopic mouse model. Combined therapy inhibited CD31 and Ki67 expression but promoted TUNEL expression. Conclusions Metformin may potentially enhance the effect of sorafenib to inhibit HCC recurrence and metastasis after liver resection by regulating the expression of HIF-2α and TIP30. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0253-6) contains supplementary material, which is available to authorized users.

The original article [1] contains an error in Fig. 5a whereby the Western blot bands representing CyclinD1 have mistakenly been duplicated over the Western blot bands intended to represent SGK.

M2-polarized (alternatively activated) macrophages play an important role in the progression of hepatocellular carcinoma (HCC). Allograft inflammatory factor 1 (AIF1) is overexpressed in M2-polarized macrophages. This study explored the role of AIF1 in tumor-associated macrophages in HCC. Macrophages were stimulated with colony-stimulating factor 1 (CSF1) to characterize the regulatory pathway of AIF1 in macrophages. The chromatin immunoprecipitation and luciferase reporter gene assay were conducted to examine transcription factors associated with AIF1 expression. AIF1 was down or upregulated, and the effects on tumor progression were evaluated by using in vitro and in vivo co-culture systems. A cytokine array was performed to screen the downstream functional components of AIF1. Tumor tissue from 206 patients with HCC were used to explore the clinical significance of AIF1. AIF1 induced a M2-like phenotype of macrophages. By facilitating the binding of c-Jun to the promoter of AIF1, CSF1 secreted from hepatoma cells increased AIF1 expression through the CSF1R-MEK1/2-Erk1/2-c-Jun axis. AIF1 expressed in macrophages promoted the migration of hepatoma cells in co-culture system of RAW264.7 and Hepa1-6 and tumor growth in an animal model. The cytokine array showed that CXCL16 was increased in RAW264.7 cells with overexpressed AIF1, leading to enhanced tumor cell migration. In human HCC tissue, AIF1-positive macrophages in the adjacent microenvironment was associated with microvascular invasion and advanced TNM stages and with patients' overall and disease-free survival (p = 0.002 for both). AIF1 expression in macrophages plays a pivotal role in the interaction between macrophages and hepatoma cells.