E2F7 promotes mammalian target of rapamycin inhibitor resistance in hepatocellular carcinoma after liver transplantation.

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.

Efficacy and safety of sirolimus early conversion protocol in liver transplant patients with hepatocellular carcinoma: A single-arm, multicenter, prospective study.

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.

USP22 promotes hypoxia-induced hepatocellular carcinoma stemness by a HIF1α/USP22 positive feedback loop upon TP53 inactivation.

OBJECTIVE: We 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. DESIGN: Cell 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. RESULTS: USP22 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. CONCLUSION: USP22 promotes hypoxia-induced HCC stemness by a HIF1α/USP22 positive feedback loop on TP53 inactivation. USP22 is a promising target for the HCC therapy.

Ubiquitin-Specific Protease 22/Silent Information Regulator 1 Axis Plays a Pivotal Role in the Prognosis and 5-Fluorouracil Resistance in Hepatocellular Carcinoma.

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.

Hangzhou criteria as downstaging criteria in hepatocellular carcinoma before liver transplantation: A multicenter study from China.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

MCM family in HCC: MCM6 indicates adverse tumor features and poor outcomes and promotes S/G2 cell cycle progression.

BACKGROUND: Minichromosome Maintenance family (MCMs), as replication licensing factors, is involved in the pathogenesis of tumors. Here, we investigated the expression of MCMs and their values in hepatocellular carcinoma (HCC). METHODS: MCMs were analyzed in 105 samples including normal livers (n = 15), cirrhotic livers (n = 40), HCC (n = 50) using quantitative polymerase chain reaction (qPCR) (Cohort 1). Significantly up-regulated MCMs were verified in 102 HCC and matched peritumoral livers using PCR (Cohort 2), and the correlations with clinical features and outcomes were determined. In addition, the focused MCMs were analyzed in parallel immunohistochemistry of 345 samples on spectrum of hepatocarcinogenesis (Cohort 3) and queried for the potential specific role in cell cycle. RESULTS: MCM2-7, MCM8 and MCM10 was significantly up-regulated in HCC in Cohort 1. In Cohort 2, overexpression of MCM2-7, MCM8 and MCM10 was verified and significantly correlated with each other. Elevated MCM2, MCM6 and MCM7 were associated with adverse tumor features and poorer outcomes. In Cohort 3, MCM6 exhibited superior HCC diagnostic performance compared with MCM2 and MCM7 (AUC: 0.896 vs. 0.675 and 0.771, P < 0.01). Additionally, MCM6 other than MCM2 and MCM7 independently predicted poorer survival in 175 HCC patients. Furthermore, knockdown of MCM6 caused a delay in S/G2-phase progression as evidenced by down-regulation of CDK2, CDK4, CyclinA, CyclinB1, CyclinD1, and CyclinE in HCC cells. CONCLUSIONS: We analyze MCMs mRNA and protein levels in tissue samples during hepatocarcinogenesis. MCM6 is identified as a driver of S/G2 cell cycle progression and a potential diagnostic and prognostic marker in HCC.

Fibrinogen and D-dimer levels elevate in advanced hepatocellular carcinoma: High pretreatment fibrinogen levels predict poor outcomes.

AIM: Plasma fibrinogen and D-dimer have been reported to predict survival in several types of malignancies. The aim of this study is to investigate their predictive value in patients with hepatocellular carcinoma (HCC). METHODS: We retrospectively analyzed plasma fibrinogen and D-dimer levels from 252 subjects: control (n = 20), hepatitis (n = 20), cirrhosis (n = 20), and HCC (n = 192) subjects. The clinical involvement and prognostic value of fibrinogen and D-dimer was analyzed in HCC subjects. To confirm the effects of tumor on hypercoagulability and fibrinolysis, fibrinogen and D-dimer levels were measured in nude mice following HCC inoculation. RESULTS: Fibrinogen decreased and D-dimer increased in cirrhosis subjects relative to other groups. In HCC subjects, elevated fibrinogen and D-dimer levels were significantly associated with adverse tumor features (increased size, stage, and grade) and systemic inflammation. Patients with HCC with either elevated fibrinogen or D-dimer levels had significantly higher 3-year tumor recurrence rates (65% vs. 41%, P < 0.001 for fibrinogen; 67% vs. 40%, P = 0.011 for D-dimer) and significantly lower 3-year overall survival rates (57% vs. 79%, P < 0.001 for fibrinogen; 56% vs. 80%, P = 0.001 for D-dimer). After multivariate analysis, elevated fibrinogen levels remained an independent predictor of poor prognosis in HCC patients. Finally, elevated levels of fibrinogen and D-dimer were confirmed in nude mice following tumor inoculation. CONCLUSION: The fibrinogen and D-dimer levels, elevating after carcinogenesis, may serve as simple but effective predictors of adverse tumor profiles and outcomes in HCC.

Predictive value of tumor markers in patients with recurrent hepatocellular carcinoma in different vascular invasion pattern.

BACKGROUND: Four tumor markers for hepatocellular carcinoma (HCC), alpha-fetoprotein (AFP), glypican-3 (GPC3), vascular endothelial growth factor (VEGF) and des-gamma-carboxy prothrombin (DCP), are closely associated with tumor invasion and patient's survival. This study estimated the predictability of preoperative tumor marker levels along with pathological parameters on HCC recurrence after hepatectomy. METHODS: A total of 140 patients with HCC who underwent hepatectomy between January 2012 and August 2012 were enrolled. The demographics, clinical and follow-up data were collected and analyzed. The patients were divided into two groups: patients with macroscopic vascular invasion (MaVI+) and those without MaVI (MaVI-). The predictive value of tumor markers and clinical parameters were evaluated by univariate and multivariate analysis. RESULTS: In all patients, tumor size (>8 cm) and MaVI were closely related to HCC recurrence after hepatectomy. For MaVI+ patients, VEGF (>900 pg/mL) was a significant predictor for recurrence (RR=2.421; 95% CI: 1.272-4.606; P=0.007). The 1- and 2-year tumor-free survival rates for MaVI+ patients with VEGF ≤900 pg/mL versus for those with VEGF >900 pg/mL were 51.5% and 17.6% versus 19.0% and 4.8% (P<0.001). For MaVI- patients, DCP >445 mAu/mL and tumor size >8 cm were two independent risk factors for tumor recurrence (RR=2.307, 95% CI: 1.132-4.703, P=0.021; RR=3.150, 95% CI: 1.392-7.127, P=0.006; respectively). The 1- and 2-year tumor-free survival rates for the patients with DCP ≤445 mAu/mL and those with DCP >445 mAu/mL were 90.4% and 70.7% versus 73.2% and 50.5% respectively (P=0.048). The 1- and 2-year tumor-free survival rates for the patients with tumor size ≤8 cm and >8 cm were 83.2% and 62.1% versus 50.0% and 30.0%, respectively (P=0.003). CONCLUSIONS: The MaVI+ patients with VEGF ≤900 pg/mL had a relatively high tumor-free survival than those with VEGF >900 pg/mL. In the MaVI- patients, DCP >445 mAu/mL and tumor size >8 cm were predictive factors for postoperative recurrence.

Potent suppression of neuroendocrine tumors and gastrointestinal cancers by CDH17CAR T cells without toxicity to normal tissues.

Gastrointestinal cancers (GICs) and neuroendocrine tumors (NETs) are often refractory to therapy after metastasis. Adoptive cell therapy using chimeric antigen receptor (CAR) T cells, though remarkably efficacious for treating leukemia, is yet to be developed for solid tumors such as GICs and NETs. Here we isolated a llama-derived nanobody, VHH1, and found that it bound cell surface adhesion protein CDH17 upregulated in GICs and NETs. VHH1-CAR T cells (CDH17CARTs) killed both human and mouse tumor cells in a CDH17-dependent manner. CDH17CARTs eradicated CDH17-expressing NETs and gastric, pancreatic and colorectal cancers in either tumor xenograft or autochthonous mouse models. Notably, CDH17CARTs do not attack normal intestinal epithelial cells, which also express CDH17, to cause toxicity, likely because CDH17 is localized only at the tight junction between normal intestinal epithelial cells. Thus, CDH17 represents a class of previously unappreciated tumor-associated antigens that is 'masked' in healthy tissues from attack by CAR T cells for developing safer cancer immunotherapy.

Sirolimus-based immunosuppression improves the prognosis of liver Transplantation Recipients with low TSC1/2 expression in hepatocellular carcinoma beyond the Milan Criteria.

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.

Self-Activated Cascade-Responsive Sorafenib and USP22 shRNA Co-Delivery System for Synergetic Hepatocellular Carcinoma Therapy.

Resistance to sorafenib severely hinders its effectiveness against hepatocellular carcinoma (HCC). Cancer stemness is closely connected with resistance to sorafenib. Methods for reversing the cancer stemness remains one of the largest concerns in research and the lack of such methods obstructs current HCC therapeutics. Ubiquitin-specific protease 22 (USP22) is reported to play a pivotal role in HCC stemness and multidrug resistance (MDR). Herein, a galactose-decorated lipopolyplex (Gal-SLP) is developed as an HCC-targeting self-activated cascade-responsive nanoplatform to co-delivery sorafenib and USP22 shRNA (shUSP22) for synergetic HCC therapy. Sorafenib, entrapped in the Gal-SLPs, induced a reactive oxygen species (ROS) cascade and triggered rapid shUSP22 release. Thus, Gal-SLPs dramatically suppressed the expression of USP22. The downregulation of USP22 suppresses multidrug resistance-associated protein 1 (MRP1) to induce intracellular sorafenib accumulation and hampers glycolysis of HCC cells. As a result, Gal-SLPs efficiently inhibit the viability, proliferation, and colony formation of HCC cells. A sorafenib-insensitive patient-derived xenograft (PDX) model is established and adopted to evaluate in vivo antitumor effect of Gal-SLPs. Gal-SLPs exhibit potent antitumor efficiency and biosafety. Therefore, Gal-SLPs are expected to have great potential in the clinical treatment of HCC.

Sirolimus-based immunosuppression improves outcomes in liver transplantation recipients with hepatocellular carcinoma beyond the Hangzhou criteria.

BACKGROUND: The administration of calcineurin inhibitors (CNIs) posttransplant has been implicated as an independent risk factor for the recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT). The new immunosuppressive agent sirolimus (SRL) acts as a primary immunosuppressant or antitumor agent. In this study we investigated the effect of sirolimus-based immunosuppression compared to CNIs (non-SRL) on the outcomes of LT candidates with HCC. METHODS: We retrospectively analyzed 204 HCC patients who underwent LT in our hospital between January 2, 2014 and December 10, 2017. The median of the follow-up duration of patients was 24.5 months. The patients were divided into a sirolimus (SRL) group (76 patients) and a non-sirolimus (non-SRL) group (128 patients). Patients exceeding the LT criteria were analyzed as subgroups. Disease-free survival (DFS) and overall survival (OS) after tumor recurrence were compared using the Kaplan-Meier method. Univariate and multivariate Cox analyses were used to compare OS between the SRL and non-SRL groups. RESULTS: The SRL group achieved better OS compared to the non-SRL group, while there was no significant difference in DFS. Subgroup (Milan criteria-based or Hangzhou criteria-based) analyses revealed that patients exceeding, rather than meeting, the Milan or Hangzhou criteria benefited from SRL (exceeding the Milan criteria: P=0.002; exceeding the Hangzhou criteria: P<0.001). There was no significant difference in OS between the SRL group and the non-SRL group that met the Milan or Hangzhou criteria. CONCLUSIONS: SRL can improve survival outcomes in LT patients with HCC exceeding the Hangzhou criteria.

Enucleation versus Anatomic Resection for Giant Hepatic Hemangioma: A Meta-Analysis.

BACKGROUND: Hepatic hemangiomas are the most common benign liver tumors, and the management of giant hepatic hemangioma (GHH) is still in controversial. The aim of this meta-analysis was to compare the postoperative outcomes of enucleation versus anatomic resection for GHH. METHODS: PubMed, Embase, Web of Science, and the Cochrane Library were searched from January 1988 to December 2015 to identify studies comparing the outcomes of enucleation versus anatomic resection for GHH. Finally, we performed this meta-analysis using the Review Manager 5.3 software, and the results were presented as risk ratio (RR) or mean difference (MD) with corresponding 95% confidence interval (CI). The major limitation is that all data were derived from nonrandomized studies, and we cannot exclude potential selection bias. RESULTS: Nine studies involving 1,185 patients were included. The results showed that there was a lower incidence of complications (RR = 0.66, 95% CI 0.52 to 0.84, I2 = 0%, p = 0.0007); no incidents of death occurred among the 9 included trials. Blood loss (MD = -419.07 mL, 95% CI -575.04 to -263.09, I2 = 83%, p < 0.00001), duration of surgery (MD = -23.5 min, 95% CI -45.28 to -1.74, I2 = 0%, p = 0.03), and length of hospital stay (MD = -1.59 days, 95% CI -3.06 to -0.13, I2 = 0%, p = 0.03) were much lower in the enucleation group. CONCLUSIONS: GHH can be removed safely by either enucleation or anatomic resection. Enucleation can preserve more hepatic parenchyma and reduce postoperative complications, which is why it should be the preferred surgical procedure for suitable lesions.

Metformin ameliorates arsenic trioxide hepatotoxicity via inhibiting mitochondrial complex I.

Arsenic trioxide (ATO) is a well-accepted chemotherapy agent in managing promyelocytic leukemia. ATO often causes severe health hazards such as hepatotoxicity, dermatosis, neurotoxicity, nephrotoxicity and cardiotoxicity. The production of reactive oxygen species, (ROS) play a significant role in ATO-induced hepatotoxicity. The oral hypoglycemic drug, metformin, is considered to be a potential novel agent for chemoprevention in the treatment of cancer. Moreover, metformin has also been shown to have hepatoprotective effects. In the present study, we demonstrated that metformin protected normal hepatocytes from ATO-induced apoptotic cell death in vitro and in vivo. Gene expression screening revealed that glucose metabolism might be related to the metformin-induced protective effect on ATO-treated AML12 cells. The metformin-promoted or induced glycolysis was not responsible for the protection of AML12 cells from ATO-induced apoptotic cell death. Instead, metformin increased the intracellular NADH/NAD+ ratio by inhibiting mitochondrial respiratory chain complex I, further decreasing the intracellular ROS induced by ATO. Treatment with low glucose or rotenone, a mitochondrial respiratory chain complex I inhibitor, also protected AML12 cells from ATO-induced apoptotic cell death. We show for the first time that metformin protects the hepatocyte from ATO by regulating the mitochondrial function. With its properties of chemoprevention, chemosensitization and the amelioration of liver damage, metformin has great prospects for clinical application other than type 2 diabetes mellitus (T2DM).

USP22 mediates the multidrug resistance of hepatocellular carcinoma via the SIRT1/AKT/MRP1 signaling pathway.

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.

Metformin potentiates the effect of arsenic trioxide suppressing intrahepatic cholangiocarcinoma: roles of p38 MAPK, ERK3, and mTORC1.

BACKGROUND: Arsenic trioxide (ATO) is commonly used in the treatment of acute promyelocytic leukemia (APL), but does not benefit patients with solid tumors. When combined with other agents or radiation, ATO showed treatment benefits with manageable toxicity. Previously, we reported that metformin amplified the inhibitory effect of ATO on intrahepatic cholangiocarcinoma (ICC) cells more significantly than other agents. Here, we investigated the chemotherapeutic sensitization effect of metformin in ATO-based treatment in ICC in vitro and in vivo and explored the underlying mechanisms. METHODS: ICC cell lines (CCLP-1, RBE, and HCCC-9810) were treated with metformin and/or ATO; the anti-proliferation effect was evaluated by cell viability, cell apoptosis, cell cycle, and intracellular-reactive oxygen species (ROS) assays. The in vivo efficacy was determined in nude mice with CCLP-1 xenografts. The active status of AMPK/p38 MAPK and mTORC1 pathways was detected by western blot. In addition, an antibody array was used screening more than 200 molecules clustered in 12 cancer-related pathways in CCLP-1 cells treated with metformin and/or ATO. Methods of genetic modulation and pharmacology were further used to demonstrate the relationship of the molecule. Seventy-three tumor samples from ICC patients were used to detect the expression of ERK3 by immunohistochemistry. The correlation between ERK3 and the clinical information of ICC patients were further analyzed. RESULTS: Metformin and ATO synergistically inhibited proliferation of ICC cells by promoting cell apoptosis, inducing G0/G1 cell cycle arrest, and increasing intracellular ROS. Combined treatment with metformin and ATO efficiently reduced ICC growth in an ICC xenograft model. Mechanistically, the antibody array revealed that ERK3 exhibited the highest variation in CCLP-1 cells after treatment with metformin and ATO. Results of western blot confirm that metformin and ATO cooperated to inhibit mTORC1, activate AMP-activated protein kinase (AMPK), and upregulate ERK3. Metformin abrogated the activation of p38 MAPK induced by ATO, and this activity was partially dependent on AMPK activation. Inactivation of p38 MAPK by SB203580 or specific short interfering RNA (siRNA) promoted the inactivation of mTORC1 in ICC cells treated with metformin and ATO. Activation of p38 MAPK may be responsible for resistance to ATO in ICC. The relationship between p38 MAPK and ERK3 was not defined by our findings. Finally, AMPK is a newfound positive regulator of ERK3. Overexpression of EKR3 in ICC cells inhibited cell proliferation through inactivation of mTORC1. ERK3 expression is associated with a better prognosis in ICC patients. CONCLUSIONS: Metformin sensitizes arsenic trioxide to suppress intrahepatic cholangiocarcinoma via the regulation of AMPK/p38 MAPK-ERK3/mTORC1 pathways. ERK3 is a newfound potential prognostic predictor and a tumor suppressor in ICC.

A genome-wide assessment of rare copy number variants in colorectal cancer.

Colorectal cancer (CRC) is a complex disease with an estimated heritability of approximately 35%. However, known CRC-related common single nucleotide polymorphisms (SNPs) can only explain ~0.65% of the heritability. This "missing heritability" may be explained partially by rare copy number variants (CNVs). In this study, we performed a genome-wide scan using Illumina Human-Omni Express BeadChip, 694 sporadic CRC cases and 1641 controls were eventually included in our analysis after quality control. The global burden analysis revealed a 1.53-fold excess of rare CNVs in CRC cases compared with controls (P < 1 × 10(-6)), and the difference being more pronounced for genic rare CNVs and CNVs overlapped with coding regions (1.65-fold and 1.84-fold, respectively, both P < 1 × 10(-6)). Interestingly, both the cases in the lowest and middle tertile of age carried a higher burden of rare CNVs comparing to the highest tertile. Furthermore, 639 CNV-disrupted genes exclusive to CRC cases were found to be significantly enriched in gene ontology (GO) terms concerning nucleosome assembly and olfactory receptor activity. Our study was the first to evaluate the burden of rare CNVs in sporadic CRC and suggested that rare CNVs contributed to the missing heritability of CRC.