Search

The mammalian target of rapamycin (mTOR) pathway is frequently deregulated and has critical roles in cancer progression. mTOR inhibitor has been widely used in several kinds of cancers and is strongly recommended in patients with hepatocellular carcinoma (HCC) after liver transplantation (LT). However, the poor response to mTOR inhibitors due to resistance remains a challenge. Hypoxia-associated resistance limits the therapeutic efficacy of targeted drugs. The present study established models of HCC clinical samples and cell lines resistance to mTOR inhibitor sirolimus and screened out E2F7 as a candidate gene induced by hypoxia and promoting sirolimus resistance. E2F7 suppressed mTOR complex 1 via directly binding to the promoter of the TSC1 gene and stabilizes hypoxia-inducible factor-1α activating its downstream genes, which are responsible for E2F7-dependent mTOR inhibitor resistance. Clinically, low E2F7 expression could be an effective biomarker for recommending patients with HCC for anti-mTOR-based therapies after LT. Targeting E2F7 synergistically inhibited HCC growth with sirolimus in vivo. E2F7 is a promising target to reverse mTOR inhibition resistance. Collectively, our study points to a role for E2F7 in promoting mTOR inhibitor resistance in HCC and emphasizes its potential clinical significance in patients with HCC after LT.

Background The use of the immunosuppressive agent sirolimus (SRL) following liver transplantation (LT) in patients with hepatocellular carcinoma (HCC) is controversial. Sirolimus is a typical mammalian target of rapamycin (mTOR) inhibitor, and tuberous sclerosis 1-tuberous sclerosis 2 complex (TSC1/TSC2) is an important negative effector in the mTOR pathway. In this study, we investigated the effect of SRL-based immunosuppression on the prognosis of LT recipients with HCC beyond the Milan criteria based on TSC1/2 expression and explored the effect of TSC1 on HCC in vitro and in vivo. Methods We retrospectively analyzed 120 HCC patients who underwent LT in our hospital between January 1, 2015 and December 30, 2018. All patients had HCC beyond the Milan criteria and were divided into the SRL group (n = 50) and non-SRL group (n = 70). TSC1/2 expression levels in paraffin-embedded tissues were determined by immunohistochemistry (IHC) and then analyzed as subgroups. Overall survival (OS) and disease-free survival (DFS) were analyzed using the Kaplan-Meier method. TSC1 expression was silenced in Huh-7 and Bel-7402 cell lines for further cell function experiments. Results 88.3% of patients were HBV LT recipients. The SRL group exhibited better DFS and OS compared to the non-SRL group (P = 0.02, P = 0.003). Subgroup (TSC1-based or TSC2-based) analyses revealed that patients with low TSC1 or TSC2 expression benefited from sirolimus (DFS: P = 0.046, OS: P = 0.006 for TSC1; DFS: P = 0.05, OS: P = 0.003 for TSC2) compared with patients with high expression. TSC1 knockdown in Huh-7 and Bel-7402 HCC cell lines activated the mTORC1 pathway and enhanced cell proliferation, migration and sensitivity to SRL in vitro and in vivo. Conclusion TSC1/2 expression could be used to predict the prognosis of patients with HCC beyond the Milan criteria who underwent SRL-based immunosuppression following LT. TSC1 knockdown promoted HCC malignancy and enhanced sensitivity to SRL.

Background Numerous studies have revealed that gamma delta (γδ) T cell infiltration plays a crucial regulatory role in hepatocellular carcinoma (HCC) development. Nonetheless, a comprehensive analysis of γδ T cell infiltration in prognosis evaluation and therapeutic prediction remains unclear. Methods Multi-omic data on HCC patients were obtained from public databases. The CIBERSORT algorithm was applied to decipher the tumor immune microenvironment (TIME) of HCC. Weighted gene co-expression network analysis (WGCNA) was performed to determine significant modules with γδ T cell-specific genes. Kaplan-Meier survival curves and receiver operating characteristic analyses were used to validate prognostic capability. Additionally, the potential role of RFESD inhibition by si-RFESD in vitro was investigated using EdU and CCK-8 assays. Results A total of 16,421 genes from 746 HCC samples (616 cancer and 130 normal) were identified based on three distinct cohorts. Using WGCNA, candidate modules (brown) with 1755 significant corresponding genes were extracted as γδ T cell-specific genes. Next, a novel risk signature consisting of 11 hub genes was constructed using multiple bioinformatic analyses, which presented great prognosis prediction reliability. The risk score exhibited a significant correlation with ICI and chemotherapeutic targets. HCC samples with different risks experienced diverse signalling pathway activities. The possible interaction of risk score with tumor mutation burden (TMB) was further analyzed. Subsequently, the potential functions of the RFESD gene were explored in HCC, and knockdown of RFESD inhibited cell proliferation in HCC cells. Finally, a robust prognostic risk-clinical nomogram was developed and validated to quantify clinical outcomes. Conclusions Collectively, comprehensive analyses focusing on γδ T cell patterns will provide insights into prognosis prediction, the mechanisms of immune infiltration, and advanced therapy strategies in HCC.

The downstaging of hepatocellular carcinoma (HCC) has been confirmed to benefit liver transplantation (LT) patients whose tumors are beyond the transplantation criteria. Milan criteria (MC), a tumor size and number-based assessment, is currently used as the endpoint in these patients. However, many studies believe that tumor biological behavior should be added to the evaluation criteria for downstaging efficacy. Hence, this study aimed to explore the feasibility of Hangzhou criteria (HC), which introduced tumor grading and alpha-fetoprotein in addition to tumor size and number, as an endpoint of downstaging.We performed a multicenter and retrospective study of 206 patients accepted locoregional therapy (LRT) as downstaging/bridge treatment prior to LT in three centers of China.Recipients were divided into four groups: failed downstaging to the HC (group A, n = 46), successful downstaging to the HC (group B, n = 30), remained within the HC all the time (group C, n = 113), and tumor progressed (group D, n = 17). The 3-year HCC recurrence probabilities of groups B and C were not significantly different (10.3% vs. 11.6%, P = 0.87). The HCC recurrent rate was significantly higher in group A (52.3%) compared with that in group B/C (P 0.05). Seven patients (7/76, 9.2%) whose tumor exceeded the the HC were successfully downstaged to the MC, and 39.5% (30/76) to the the HC. In group B, 23 patients remained beyond the MC and their survivals were as well as those of patients within the MC.Compared to the MC, HC downstaging criteria can give more HCC patients access to LT and furthermore, the outcome of these patients is the same as those matching MC downstaging criteria. Hangzhou downstaging criteria therefore is applicable in clinical practice.

Background Ubiquitin-specific protease 22 (USP22) is described as a key subunit of the Spt-Ada-Gcn5 acetyl transferase complex, which plays an important role in the prognosis and resistance to chemotherapy drugs in hepatocellular carcinoma (HCC). Silent information regulator 1 (SIRT1) is a member of the sirtuin family that is deubiquitinated by USP22. However, it is still unknown whether USP22 and SIRT1 co-expression is associated with disease progression and 5-Fluorouracil (5-FU) resistance in HCC. Methods 141 patients who received hepatectomy at our hospital from January 2010 to December 2014 were enrolled in this study. The expression of USP22 and SIRT1 was detected by immunohistochemical staining. Clinicopathological features, including age, gender, tumor number, tumor size, tumor differentiation, tumor stage, alpha-fetoprotein and microscopic vascular invasion, were assessed. Further experiments confirmed the role of SIRT1 in 5-FU drug resistance in vivo. Results Immunohistochemical staining showed that the high expression of USP22 and SIRT1 was frequently observed in HCC tissues relative to normal liver tissues. Overexpression of USP22 is associated with microscopic vascular invasion (MVI). Further analysis showed that the co-expression of USP22 and SIRT1 was more effective in predicting the prognosis of HCC. The SIRT1 inhibitor EX-527 dramatically inhibited the expression of Cyclin B1 and resistance-associated protein 3 (MRP3) to reduce 5-FU drug resistance in vivo. Conclusion These findings suggest that the co-expression of USP22 and SIRT1 is significantly associated with unfavorable HCC progression. The inhibition of SIRT1 in vivo could be valuable in improving 5-FU drug sensitivity and inhibiting tumor cell proliferation and inducing apoptosis.

The incidence of portal vein tumor thrombus (PVTT) has been reported to be as high as approximately 10%-40% in patients with hepatocellular carcinoma (HCC). The long-term prognosis of deceased donor liver transplantation (DDLT) in HCC patients with PVTT remains unknown.Data of 961 HCC patients who underwent DDLT between 2015 and 2018 in six centers were analyzed. Based on the Milan criteria (MC) and Cheng's classification of PVTT, the patients were divided into 4 groups: within MC, beyond MC without PVTT, type 1 PVTT, and type 2 PVTT groups.489 (50.9%) were within the MC, 296 (30.8%) beyond the MC but without PVTT, 83 (8.6%) type 1 PVTT, and 93 (9.7%) type 2 PVTT. Kaplan-Meier analysis showed that type 1 or 2 PVTT patients with alpha-fetoprotein (AFP) ≤ 100 ng/mL had overall survival (OS) similar to that of patients within the MC (P = 0.957), and superior OS (P = 0.003 and 0.009) and recurrence-free survival (RFS) (P = 0.038 and 0.001) than those of patients beyond the MC and PVTT patients with AFP 100 ng/mL. Multivariable Cox-regression analysis identified type 1 and 2 PVTT to be independent risk factor for RFS [hazard ratio (HR) 1.523 95% confidence interval (CI) 1.162-1.997, P = 0.002], but not for OS (HR 1.283, 95%CI 0.922-1.786, P = 0.139).HCC patients with type 1 or 2 PVTT may be acceptable candidates for DDLT. To achieve better outcomes, preoperative AFP levels should be seriously considered when selecting patients with PVTT for DDLT.

Mammalian target of rapamycin (mTOR) inhibitor as an attractive drug target with promising antitumor effects has been widely investigated. High quality clinical trial has been conducted in liver transplant (LT) recipients in Western countries. However, the pertinent studies in Eastern world are paucity. Therefore, we designed a clinical trial to test whether sirolimus can improve recurrence-free survival (RFS) in hepatocellular carcinoma (HCC) patients beyond the Milan criteria after LT. This is an open-labeled, single-arm, prospective, multicenter, and real-world study aiming to evaluate the clinical outcomes of early switch to sirolimus-based regimens in HCC patients after LT. Patients with a histologically proven HCC and beyond the Milan criteria will be enrolled. The initial immunosuppressant regimens are center-specific for the first 4-6 weeks. The following regimens integrated sirolimus into the regimens as a combination therapy with reduced calcineurin inhibitors based on the condition of patients and centers. The study is planned for 4 years in total with a 2-year enrollment period and a 2-year follow-up. We predict that sirolimus conversion regimen will provide survival benefits for patients particular in the key indicator RFS as well as better quality of life. If the trial is conducted successfully, we will have a continued monitoring over a longer follow-up time to estimate indicator of overall survival. We hope that the outcome will provide better evidence for clinical decision-making and revising treatment guidelines based on Chinese population data. Trial register: Trial registered at http://www.chictr.org.cn: ChiCTR2100042869.

Liver transplantation (LT), one of the radical methods of treating liver cancer, has brought new hope for the treatment of unresectable liver cancer. Currently, patients who meet transplant criteria can achieve a favorable prognosis, but those who exceed transplant criteria tend not to have very satisfactory outcomes. For patients whose tumor burden exceeds the transplant criteria, downstaging treatment is a promising method to reduce tumor burden to within the transplant criteria that may lead to good posttransplant survival. Multiple treatments, such as transcatheter arterial chemoembolization (TACE), transarterial radioembolization (TARE), percutaneous ethanol injection (PEI), and radiofrequency ablation (RFA), have been used as downstaging treatments. However, there are still some issues that limit the effectiveness of downstaging treatments, such as the inclusion criteria for downstaging, which the choice of downstaging treatment method, and the endpoint of downstaging, all of which are worthy of further discussion. Based on the published literature, this review discusses these issues.

Drug treatments for hepatocellular carcinoma (HCC) often fail because of multidrug resistance (MDR). The mechanisms of MDR are complex but cancer stem cells (CSCs), which are able to self-renew and differentiate, have recently been shown to be involved. The deubiquitinating enzyme ubiquitin-specific protease 22 (USP22) is a marker for CSCs. This study aimed to elucidate the role of USP22 in MDR of HCC and the underlying mechanisms. Using in vitro and in vivo assays, we found that modified USP22 levels were responsible for the altered drug-resistant phenotype of BEL7402 and BEL/FU cells. Downregulation of USP22 dramatically inhibited the expression of ABCC1 (encoding MRP1) but weakly influenced ABCB1 (encoding P-glycoprotein). Sirtuin 1 (SIRT1) was reported previously as a functional mediator of USP22 that could promote HCC cell proliferation and enhance resistance to chemotherapy. In this study, USP22 directly interacted with SIRT1 and positively regulated SIRT1 protein expression. Regulation of the expression of both USP22 and SIRT1 markedly affected the AKT pathway and MRP1 expression. Inhibition of the AKT pathway by its specific inhibitor LY294002 resulted in downregulation of MRP1. USP22 and MRP1 expression was detected in 168 clinical HCC samples by immunohistochemical staining, and a firm relationship between USP22 and MRP1 was identified. Together, these results indicate that USP22 could promote the MDR in HCC cells by activating the SIRT1/AKT/MRP1 pathway. USP22 might be a potential target, through which the MDR of HCC in clinical setting could be reversed.

ObjectiveWe aimed to elucidate the mutual regulation mechanism of ubiquitin-specific protease 22 (USP22) and hypoxia inducible factor-1α (HIF1α), and the mechanism they promote the stemness of hepatocellular carcinoma (HCC) cells under hypoxic conditions.DesignCell counting, migration, self-renewal ability, chemoresistance and expression of stemness genes were established to detect the stemness of HCC cells. Immunoprecipitation, ubiquitination assay and chromatin immunoprecipitation assay were used to elucidate the mutual regulation mechanism of USP22 and HIF1α. HCC patient samples and The Cancer Genome Atlas data were used to demonstrate the clinical significance. In vivo USP22-targeting experiment was performed in mice bearing HCC.ResultsUSP22 promotes hypoxia-induced HCC stemness and glycolysis by deubiquitinating and stabilising HIF1α. As direct target genes of HIF1α, USP22 and TP53 can be transcriptionally upregulated by HIF1α under hypoxic conditions. In TP53 wild-type HCC cells, HIF1α induced TP53-mediated inhibition of HIF1α-induced USP22 upregulation. In TP53-mutant HCC cells, USP22 and HIF1α formed a positive feedback loop and promote the stemness of HCC. HCC patients with a loss-of-function mutation at TP53 and high USP22 and/or HIF1α expression tend to have a worse prognosis. The USP22-targeting lipopolyplexes caused high tumour inhibition and high sorafenib sensitivity in mice bearing HCC.ConclusionUSP22 promotes hypoxia-induced HCC stemness by a HIF1α/USP22 positive feedback loop on TP53 inactivation. USP22 is a promising target for the HCC therapy.

Hepatocellular carcinoma (HCC) is one of the most common cancers and a leading cause of death worldwide. Due to latent liver disease, late diagnosis, and nonresponse to systemic treatments, surgical resection and/or biopsy specimens are still generally considered as the gold standard by clinicians for clinical decision-making until now. Since the conventional tissue biopsy is invasive and contains small tissue samples, it is unable to represent tumor heterogeneity or monitor dynamic tumor progression. Therefore, it is imperative to find a new less invasive or noninvasive diagnostic strategy to detect HCC at an early stage and to monitor HCC recurrence. Over the past years, a new diagnostic concept known as “liquid biopsy” has emerged with substantial attention. Liquid biopsy is noninvasive and allows repeated analyses to monitor tumor recurrence, metastasis or treatment responses in real time. With the advanced development of new molecular techniques, HCC circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) detection have achieved interesting and encouraging results. In this review, we focus on the clinical applications of CTCs and ctDNA as key components of liquid biopsy in HCC patients.

Apoptotic liver cancer cells have important roles in liver tumorigenesis and liver cancer progression. Recent studies have shown that δ-opioid receptors are highly expressed in human liver and liver cancer cells. The present study aimed to investigate the role of activated δ-opioid receptors on human liver cancer cell apoptosis and its interrelation with the mitochondria and the protein kinase C/extracellular-signal-regulated kinase (PKC/ERK) signaling pathway. H2O2 was used to induce apoptosis in human liver cancer cells. During apoptosis, mitochondrial transmembrane potentials were observed to decrease, cytochrome c expression was found to increase and B cell lymphoma 2 (Bcl‑2) expression decreased. These findings suggested that H2O2-induced apoptosis was mediated through the mitochondrial pathway. Of note, activated δ-opioid receptors were observed to inhibit H2O2-induced apoptosis in human liver cancer cells. Following δ-opioid receptor activa- tion, the number of apoptotic liver cancer cells decreased, mitochondrial transmembrane potentials were restored, cytoplasmic cytochrome c and Bcl-2-associated X protein expression decreased and Bcl-2 expression increased. These data suggested that δ-opioid receptor activation inhibited mitochondria-mediated apoptosis. In addition, activation of δ-opioid receptors was observed to increase the expression of PKC and ERK in human liver cancer cells. Furthermore, upon inhibition of the PKC/ERK signaling pathway, the protective effect associated with the δ-opioid receptor on liver cancer cell apoptosis was inhibited, which was not asso- ciated with the status of δ-opioid receptor activation. These findings suggested that the PKC/ERK signaling pathway has an important role in δ-opioid receptor-mediated inhibition of apoptosis in human liver cancer cells.

The multi‑kinase inhibitor sorafenib is the only drug for which randomized control trials have shown improved patient survival in advanced hepatocellular carcinoma (HCC). However, life expectancy is extended in these cases by only a few months. The anti‑type II diabetes agent metformin was used in this study in an effort to find a more efficient approach to HCC treatment. Sorafenib effectively reversed the activation status of mTORC2 induced by metformin and enhanced the suppression of the mTORC1 and MAPK pathway by metformin in HCC cells, which may be responsible for reduced proliferation upon combined treatment. The metformin and sorafenib combination led to increased impaired proliferation and tumor inhibition of HCC in vitro and in vivo compared to single agent, which was partially bridged by disrupting the mTORC1/mTORC2 feedback loop. Metformin and sorafenib cooperated to promote apoptosis and autophagy in HCC cells. Pharmacological inhibition of autophagy sensitized HCC cells to metformin and sorefenib‑induced apoptotic cell death. Therefore, the anti‑autophagy treatment should be considered in metformin and sorafenib-based treatments in HCC cells.

Hepatocellular carcinoma (HCC) is a highly malignant tumor that can evolve rapidly to acquire resistance to conventional chemotherapies. Arsenic trioxide (ATO) is a traditional Asian medicine, and a phase II study has shown that treatment with ATO alone was not effective against HCC. Bcl2 is an antiapoptotic protein that regulates chemotherapy in HCC. Metformin is reported to decrease Bcl2 expression, and the purpose of this study was to verify whether metformin could potentiate the anti-HCC efficacy of ATO in vitro. In the present study, we used metformin and ATO alone or in combination and then tested proliferation, apoptosis, and Bcl2 level of HCC cells. The results showed that metformin enhanced both the proliferation-inhibiting and apoptosis-inducing effects of ATO on HCC cell lines HepG2 and BEL7402. Furthermore, this activity proceeded via a mechanism involving metformin-induced downregulation of Bcl2. A combination of ATO and metformin is therefore a potentially promising approach for HCC therapy.