Bortezomib in previously treated phosphatase and tension homology-deficient patients with advanced intrahepatic cholangiocarcinoma: An open-label, prospective and single-centre phase II trial.

INTRODUCTION: Intrahepatic cholangiocarcinoma (ICC) is characterized by a dismal prognosis with limited therapeutic alternatives. To explore phosphatase and tension homolog (PTEN) as a biomarker for proteasome inhibition in ICC, we conducted a phase II trial to assess the second-line efficacy of bortezomib in PTEN-deficient advanced ICC patients. METHODS: A total of 130 patients with advanced ICC in our centre were screened by PTEN immunohistochemical staining between 1 July 2017, and 31 December 2021, and 16 patients were ultimately enrolled and treated with single-agent bortezomib 1.3 mg/m2 on days 1, 4, 8 and 11 of a 21-day cycle. The primary endpoint was the objective response rate (ORR) according to Response Evaluation Criteria in Solid Tumors v1.1. RESULTS: The median follow-up was 6.55 months (95% confidence interval [CI]: 0.7-19.9 months). Among the 16 enrolled patients, the ORR was 18.75% (3/16) and the disease control rate was 43.75% (7/16). The median progress-free survival was 2.95 months (95% CI: 2.1-5.1 months) and the median overall survival (mOS) was 7.2 months (95% CI: 0.7-21.6 months) in the intent-to-treat-patients. Treatment-related adverse events of any grade were reported in 16 patients, with thrombopenia being the most common toxicity. Patients with PTEN staining scores of 0 were more likely to benefit from bortezomib than those with staining scores > 0. CONCLUSIONS: Bortezomib yielded an encouraging objective response and a favourable OS as a second-line agent in PTEN-deficient ICC patients. Our findings suggest bortezomib as a promising therapeutic option for patients with PTEN-deficient ICC. HIGHLIGHTS: There is a limited strategy for the second-line option of intrahepatic cholangiocarcinoma (ICC). This investigator-initiated phase 2 study evaluated bortezomib in ICC patients with phosphatase and tension homology deficiency. The overall response rate was 18.75% and the overall survival was 7.2 months in the intent-to-treat cohort. These results justify further developing bortezomib in ICC patients with PTEN deficiency.

Arsenic trioxide augments immunogenic cell death and induces cGAS-STING-IFN pathway activation in hepatocellular carcinoma.

The treatment of hepatocellular carcinoma (HCC) is particularly challenging due to the inherent tumoral heterogeneity and easy resistance towards chemotherapy and immunotherapy. Arsenic trioxide (ATO) has emerged as a cytotoxic agent effective for treating solid tumors, including advanced HCC. However, its effectiveness in HCC treatment remains limited, and the underlying mechanisms are still uncertain. Therefore, this study aimed to characterize the effects and mechanisms of ATO in HCC. By evaluating the susceptibilities of human and murine HCC cell lines to ATO treatment, we discovered that HCC cells exhibited a range of sensitivity to ATO treatment, highlighting their inherent heterogeneity. A gene signature comprising 265 genes was identified to distinguish ATO-sensitive from ATO-insensitive cells. According to this signature, HCC patients have also been classified and exhibited differential features of ATO response. Our results showed that ATO treatment induced reactive oxygen species (ROS) accumulation and the activation of multiple cell death modalities, including necroptosis and ferroptosis, in ATO-sensitive HCC cells. Meanwhile, elevated tumoral immunogenicity was also observed in ATO-sensitive HCC cells. Similar effects were not observed in ATO-insensitive cells. We reported that ATO treatment induced mitochondrial injury and mtDNA release into the cytoplasm in ATO-sensitive HCC tumors. This subsequently activated the cGAS-STING-IFN axis, facilitating CD8+ T cell infiltration and activation. However, we found that the IFN pathway also induced tumoral PD-L1 expression, potentially antagonizing ATO-mediated immune attack. Additional anti-PD1 therapy promoted the anti-tumor response of ATO in ATO-sensitive HCC tumors. In summary, our data indicate that heterogeneous ATO responses exist in HCC tumors, and ATO treatment significantly induces immunogenic cell death (ICD) and activates the tumor-derived mtDNA-STING-IFN axis. These findings may offer a new perspective on the clinical treatment of HCC and warrant further study.

N6-methyladenosine modification promotes hepatocarcinogenesis through circ-CDYL-enriched and EpCAM-positive liver tumor-initiating exosomes.

CircRNAs play multiple roles in a variety of cellular processes. We found that Circ-CDYL is highly enriched in early HCC plasma exosomes. Moreover, EpCAM+ HCC cells and exosomes had significant Circ-CDYL levels. We postulated that Circ-CDYL-enriched and EpCAM-positive exosomes would function as liver tumor-initiating exosomes (LTi-Exos). As predicted, intercellular transfer of LTi-Exos activates the HDGF-PI3K-AKT-mTOR and HIF1AN-NOTCH2 axes in recipient cells, promoting malignancy. Upstream, we found that the N6-methyladenosine (m6A) modification of Circ-CDYL exerted its action in HCC cells through a dual mechanism. First, it stimulated back-splicing processes via YTHDC1 to promote Circ-CDYL biogenesis. Second, it facilitates the active sorting of Circ-CDYL into exosomes via hnRNPA2/B1. Clinically, the combination of LTi-Exos and plasma alpha-fetoprotein (AFP) provides a promising early diagnostic biomarker for HCC with an AUC of 0.896. This study highlights the effect and mechanism by which m6A modification promotes hepatocarcinogenesis via modulation of the tumor microenvironment by LTi-Exos.

URI alleviates tyrosine kinase inhibitors-induced ferroptosis by reprogramming lipid metabolism in p53 wild-type liver cancers.

The clinical benefit of tyrosine kinase inhibitors (TKIs)-based systemic therapy for advanced hepatocellular carcinoma (HCC) is limited due to drug resistance. Here, we uncover that lipid metabolism reprogramming mediated by unconventional prefoldin RPB5 interactor (URI) endows HCC with resistance to TKIs-induced ferroptosis. Mechanistically, URI directly interacts with TRIM28 and promotes p53 ubiquitination and degradation in a TRIM28-MDM2 dependent manner. Importantly, p53 binds to the promoter of stearoyl-CoA desaturase 1 (SCD1) and represses its transcription. High expression of URI is correlated with high level of SCD1 and their synergetic expression predicts poor prognosis and TKIs resistance in HCC. The combination of SCD1 inhibitor aramchol and deuterated sorafenib derivative donafenib displays promising anti-tumor effects in p53-wild type HCC patient-derived organoids and xenografted tumors. This combination therapy has potential clinical benefits for the patients with advanced HCC who have wild-type p53 and high levels of URI/SCD1.

PTEN deficiency facilitates gemcitabine efficacy in cancer by modulating the phosphorylation of PP2Ac and DCK.

Gemcitabine is a nucleoside analog that has been successfully used in the treatment of multiple cancers. However, intrinsic or acquired resistance reduces the chemotherapeutic potential of gemcitabine. Here, we revealed a previously unappreciated mechanism by which phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, dominates the decision-making process that is central to the regulation of gemcitabine efficacy in cholangiocarcinoma (CCA). By investigating a gemcitabine-treated CCA cohort, we found that PTEN deficiency was correlated with the improved efficacy of gemcitabine-based chemotherapy. Using cell-based drug sensitivity assays, cell line-derived xenograft, and patient-derived xenograft models, we further confirmed that PTEN deficiency or genetic-engineering down-regulation of PTEN facilitated gemcitabine efficacy both in vitro and in vivo. Mechanistically, PTEN directly binds to and dephosphorylates the C terminus of the catalytic subunit of protein phosphatase 2A (PP2Ac) to increase its enzymatic activity, which further dephosphorylates deoxycytidine kinase (DCK) at Ser74 to diminish gemcitabine efficacy. Therefore, PTEN deficiency and high phosphorylation of DCK predict a better response to gemcitabine-based chemotherapy in CCA. We speculate that the combination of PP2A inhibitor and gemcitabine in PTEN-positive tumors could avoid the resistance of gemcitabine, which would benefit a large population of patients with cancer receiving gemcitabine or other nucleoside analogs.

Orthogonally Engineered Albumin with Attenuated Macrophage Phagocytosis for the Targeted Visualization and Phototherapy of Liver Cancer.

The five-year survival rate of hepatocellular carcinoma (HCC) remains unsatisfactory. This reflects, in part, the paucity of effective methods that allow the target-specific diagnosis and therapy of HCC. Here, we report a strategy based on engineered human serum albumin (HSA) that permits the HCC-targeted delivery of diagnostic and therapeutic agents. Covalent cysteine conjugation combined with the exploitation of host-guest chemistry was used to effect the orthogonal functionalization of HSA with two functionally independent peptides. One of these peptides targets glypican-3 (GPC-3), an HCC-specific biomarker, while the second reduces macrophage phagocytosis through immune-checkpoint stimulation. This orthogonally engineered HSA proved effective for the GPC-3-targeted delivery of near-infrared fluorescent and phototherapeutic agents, thus permitting target-specific optical visualization and photodynamic ablation of HCC in vivo. This study thus offers new insights into specificity-enhanced fluorescence-guided surgery and phototherapy of HCC through the orthogonal engineering of biocompatible proteins.

Tertiary lymphoid structures predict the prognosis and immunotherapy response of cholangiocarcinoma.

Introduction: Cholangiocarcinoma (CCA) is a malignant tumor of the biliary epithelium with a poor prognosis. The lack of biomarkers to predict therapeutic response and prognosis is one of the major challenges for CCA treatment. Tertiary lymphoid structures (TLS) provide a local and pivotal microenvironment for tumor immune responses. The prognostic value and clinical relevance of TLS in CCA remain unclear. We aimed to explore the characteristics and clinical significance of TLS in CCA. Methods: We investigated the prognostic value and clinical relevance of TLS in CCA using a surgery cohort containing 471 CCA patients (cohort 1) and an immunotherapy cohort containing 100 CCA patients (cohort 2). Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were used to evaluate the maturity of TLS. Multiplex IHC (mIHC) was employed to characterize the composition of TLS. Results: Different maturity of TLS were observed in CCA tissue sections. Strong staining of the four-gene signature including PAX5, TCL1A, TNFRSF13C, and CD79A were found in TLS regions. A high density of intra-tumoral TLS (T-score high) were significantly correlated with longer overall survival (OS) both in CCA cohort 1 (p = 0.002) and cohort 2 (p = 0.01), whereas a high density of peri-tumoral TLS (P-score high) were associated with shorter OS in these two cohorts (p = 0.003 and p = 0.03, respectively). Conclusion: The established four-gene signature efficiently identified the TLS in CCA tissues. The abundance and spatial distribution of TLS were significantly correlated with the prognosis and immune checkpoint inhibitors (ICIs) immunotherapy response of CCA patients. The presence of intra-tumoral TLS are positive prognostic factors for CCA, which provide a theoretical basis for the future diagnosis and treatment of CCA.

Development and validation of prognostic dynamic nomograms for hepatitis B Virus-related hepatocellular carcinoma with microvascular invasion after curative resection.

Background and Aim: The prediction models of postoperative survival for hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) with microvascular invasion (MVI) have not been well established. The study objective was the development of nomograms to predict disease recurrence and overall survival (OS) in these patients. Methods: Data were obtained from 1046 HBV-related MVI-positive HCC patients who had undergone curative resection from January 2014 to December 2017. The study was approved by the Eastern Hepatobiliary Surgery Hospital and Jinling Hospital ethics committee, and patients provided informed consent for the use of their data. Nomograms for recurrence and OS were created by Cox regression model in the training cohort (n=530). The modes were verified in an internal validation cohort (n= 265) and an external validation cohort (n= 251). Results: The nomograms of recurrence and OS based on preoperative serological indicators (HBV-DNA, neutrophil-lymphocyte ratio, a-fetoprotein), tumor clinicopathologic features (diameter, number), surgical margin and postoperative adjuvant TACE achieved high C-indexes of 0.722 (95% confidence interval [CI], 0.711-0.732) and 0.759 (95% CI, 0.747-0.771) in the training cohort, respectively, which were significantly higher than conventional HCC staging systems (BCLC, CNLC, HKLC).The nomograms were validated in the internal validation cohort (0.747 for recurrence, 0.758 for OS) and external validation cohort(0.719 for recurrence, 0.714 for OS) had well-fitted calibration curves. Our nomograms accurately stratified patients with HBV-HCC with MVI into low-, intermediate- and high-risk groups of postsurgical recurrence and mortality. Prediction models for recurrence-free survival (https://baishileiehbh.shinyapps.io/HBV-MVI-HCC-RFS/) and OS (https://baishileiehbh.shinyapps.io/HBV-MVI-HCC-OS/) were constructed. Conclusions: The two nomograms showed good predictive performance and accurately distinguished different recurrence and OS by the nomograms scores for HBV-HCC patients with MVI after resection.

PTEN Deficiency Facilitates Exosome Secretion and Metastasis in Cholangiocarcinoma by Impairing TFEB-mediated Lysosome Biogenesis.

BACKGROUND & AIMS: In eukaryotes, the ubiquitin-proteasome system and the autophagy-lysosome pathway are essential for maintaining cellular proteostasis and associated with cancer progression. Our previous studies have demonstrated that phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, limits proteasome abundance and determines chemosensitivity to proteasome inhibitors in cholangiocarcinoma (CCA). However, whether PTEN regulates the lysosome pathway remains unclear. METHODS: We tested the effects of PTEN on lysosome biogenesis and exosome secretion using loss- and gain-of-function strategies in CCA cell lines. Using in vitro dephosphorylation assays, we explored the regulatory mechanism between PTEN and the key regulator of lysosome biogenesis, transcription factor EB (TFEB). Using the migration assays, invasion assays, and trans-splenic liver metastasis mouse models, we evaluated the function of PTEN deficiency, TFEB-mediated lysosome biogenesis, and exosome secretion on tumor metastasis. Moreover, we investigated the clinical significance of PTEN expression and exosome secretion by retrospective analysis. RESULTS: PTEN facilitated lysosome biogenesis and acidification through its protein phosphatase activity to dephosphorylate TFEB at Ser211. Notably, PTEN deficiency increased exosome secretion by reducing lysosome-mediated degradation of multi-vesicular bodies, which further facilitated the proliferation and invasion of CCA. TFEB agonist curcumin analog C1 restrained the metastatic phenotype caused by PTEN deficiency in mouse models, and we highlighted the correlation between PTEN deficiency and exosome secretion in clinical cohorts. CONCLUSIONS: In CCA, PTEN deficiency impairs lysosome biogenesis to facilitate exosome secretion and cancer metastasis in a TFEB phosphorylation-dependent manner.

The therapeutic effect of KSP inhibitors in preclinical models of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an epithelial malignancy with a dismal prognosis owing to limited treatment options. Here, we identified several compound candidates against CCA using a high-throughput drug screen with approved or emerging oncology drugs, among which kinesin spindle protein (KSP) inhibitors showed potent cytotoxic effects on CCA cells. Treatment with KSP inhibitors SB743921 and ARRY520 caused significant tumor suppression in CCA xenograft models in vivo. Mechanistically, KSP inhibitors led to the formation of abnormal monopolar spindles, which further resulted in the mitotic arrest and cell death of CCA cells both in vivo and in vitro. KEGG pathway analysis of transcriptional data confirmed this finding. Moreover, our clinical data as well as the TCGA database showed KIF11 expression was abundant in most CCA tumor specimens and associated with poor outcomes of CCA patients. Our results demonstrate that the therapeutic regimen of KSP inhibitors could be a promising treatment strategy in CCA.

Metabolic Reprogramming and Its Relationship to Survival in Hepatocellular Carcinoma.

Hepatocarcinogenesis is frequently accompanied by substantial metabolic reprogramming to maximize the growth and proliferation of cancer cells. In this study, we carried out a comprehensive study of metabolomics and lipidomics profiles combined with gene expression analysis to characterize the metabolic reprogramming in hepatocellular carcinoma (HCC). Compared with adjacent noncancerous liver tissue, the enhanced aerobic glycolysis and de novo lipogenesis (DNL) and the repressed urea cycle were underscored in HCC tissue. Furthermore, multiscale embedded correlation analysis was performed to construct differential correlation networks and reveal pathologically relevant molecule modules. The obtained hub nodes were further screened according to the maximum biochemical diversity and the least intraclass correlation. Finally, a panel of ornithine, FFA 18:1, PC O-32:1 and TG (18:1_17:1_18:2) was generated to achieve the prognostic risk stratification of HCC patients (p < 0.001 by log-rank test). Altogether, our findings suggest that the metabolic dysfunctions of HCC detected via metabolomics and lipidomics would contribute to a better understanding of clinical relevance of hepatic metabolic reprogramming and provide potential sources for the identification of therapeutic targets and the discovery of biomarkers.

Metabolic Reprogramming and Risk Stratification of Hepatocellular Carcinoma Studied by Using Gas Chromatography-Mass Spectrometry-Based Metabolomics.

Hepatocellular carcinoma (HCC) displays a high degree of metabolic and phenotypic heterogeneity and has dismal prognosis in most patients. Here, a gas chromatography-mass spectrometry (GC-MS)-based nontargeted metabolomics method was applied to analyze the metabolic profiling of 130 pairs of hepatocellular tumor tissues and matched adjacent noncancerous tissues from HCC patients. A total of 81 differential metabolites were identified by paired nonparametric test with false discovery rate correction to compare tumor tissues with adjacent noncancerous tissues. Results demonstrated that the metabolic reprogramming of HCC was mainly characterized by highly active glycolysis, enhanced fatty acid metabolism and inhibited tricarboxylic acid cycle, which satisfied the energy and biomass demands for tumor initiation and progression, meanwhile reducing apoptosis by counteracting oxidative stress. Risk stratification was performed based on the differential metabolites between tumor and adjacent noncancerous tissues by using nonnegative matrix factorization clustering. Three metabolic clusters displaying different characteristics were identified, and the cluster with higher levels of free fatty acids (FFAs) in tumors showed a worse prognosis. Finally, a metabolite classifier composed of six FFAs was further verified in a dependent sample set to have potential to define the patients with poor prognosis. Together, our results offered insights into the molecular pathological characteristics of HCC.

An RNA-RNA crosstalk network involving HMGB1 and RICTOR facilitates hepatocellular carcinoma tumorigenesis by promoting glutamine metabolism and impedes immunotherapy by PD-L1+ exosomes activity.

Hepatocellular carcinoma (HCC) is the global leading cause of cancer-related deaths due to the deficiency of targets for precision therapy. A new modality of epigenetic regulation has emerged involving RNA-RNA crosstalk networks where two or more competing endogenous RNAs (ceRNAs) bind to the same microRNAs. However, the contribution of such mechanisms in HCC has not been well studied. Herein, potential HMGB1-driven RNA-RNA crosstalk networks were evaluated at different HCC stages, identifying the mTORC2 component RICTOR as a potential HMGB1 ceRNA in HBV+ early stage HCC. Indeed, elevated HMGB1 mRNA was found to promote the expression of RICTOR mRNA through competitively binding with the miR-200 family, especially miR-429. Functional assays employing overexpression or interference strategies demonstrated that the HMGB1 and RICTOR 3'untranslated regions (UTR) epigenetically promoted the malignant proliferation, self-renewal, and tumorigenesis in HCC cells. Intriguingly, interference against HMGB1 and RICTOR in HCC cells promoted a stronger anti-PD-L1 immunotherapy response, which appeared to associate with the production of PD-L1+ exosomes. Mechanistically, the HMGB1-driven RNA-RNA crosstalk network facilitated HCC cell glutamine metabolism via dual mechanisms, activating a positive feedback loop involving mTORC2-AKT-C-MYC to upregulate glutamine synthetase (GS) expression, and inducing mTORC1 signaling to derepress SIRT4 on glutamate dehydrogenase (GDH). Meanwhile, this crosstalk network could impede the efficacy of immunotherapy through mTORC1-P70S6K dependent PD-L1 production and PD-L1+ exosomes activity. In conclusion, our study highlights the non-coding regulatory role of HMGB1 with implications for RNA-based therapeutic targeting together with a prediction of anti-PD-L1 immunotherapy in HCC.

Prognosis prediction of hepatocellular carcinoma after surgical resection based on serum metabolic profiling from gas chromatography-mass spectrometry.

Comprehensive prognostic risk prediction of hepatocellular carcinoma (HCC) after surgical treatment is particularly important for guiding clinical decision-making and improving postoperative survival. Hence, we aimed to build prognostic models based on serum metabolomics data, and assess the prognostic risk of HCC within 5 years after surgical resection. A pseudotargeted gas chromatography-mass spectrometry (GC-MS)-based metabolomics method was applied to analyze serum profiling of 78 HCC patients. Important metabolic features with discriminant ability were identified by a novel network-based metabolic feature selection method based on combinational significance index (N-CSI). Subsequently, phenylalanine and galactose were further identified to be relevant with mortality by the Cox regression analysis, while galactose and tyrosine were associated with recurrence and metastasis. Two models to predict risk of mortality (risk score of overall survival, RSOS) and risk of recurrence and metastasis (risk score of disease-free survival, RSDFS) were generated based on two panels of metabolites, respectively, which present favorable ability to predict prognosis of HCC, especially when combined with clinical staging system. The performance of models was further validated in an external independent cohort from 91 HCC patients. This study demonstrated that metabolomics is a powerful tool for risk screening of HCC prognosis.

PTEN deficiency facilitates the therapeutic vulnerability to proteasome inhibitor bortezomib in gallbladder cancer.

Gallbladder cancer (GBC) is an aggressive malignancy of biliary tract with poor prognosis. Although several studies have shown the frequency of relevant genetic alterations, there are few genetic models or translational studies that really benefit for GBC treatment in the era of precision medicine. By targeted sequencing and immunohistochemistry staining, we identified that phosphate and tension homology deleted on chromosome ten (PTEN) was frequently altered in GBC specimens, and loss of PTEN expression was independently correlated with poor survival outcomes. Further drug screening assays revealed proteasome inhibitor bortezomib as a promising agent for GBC treatment, and knockdown of PTEN increased bortezomib efficacy both in vivo and in vitro. Therapeutic evaluation of patient derived xenografts (PDXs) strongly supported the utilization of bortezomib in PTEN deficient GBC. Mechanically, functional PTEN inhibited ARE-dependent transcriptional activity, the same machinery regulating the transcription of proteasome subunits, thus PTEN suppressed proteasome activity and bortezomib sensitivity. Through siRNA screening, we identified the ARE-related transcriptional suppressor BACH1 involved in PTEN-mediated proteasome inhibition and regulated by PTEN-AKT1 axis. In summary, our study indicates that proteasome activity represents a prime therapeutic target in PTEN-deficient GBC tumors, which is worthy of further clinical validation.

PTEN status determines chemosensitivity to proteasome inhibition in cholangiocarcinoma.

Patient-derived xenografts (PDXs) and PDX-derived cells (PDCs) are useful in preclinical research. We performed a drug screening assay using PDCs and identified proteasome inhibitors as promising drugs for cholangiocarcinoma (CCA) treatment. Furthermore, we determined that phosphate and tensin homology deleted on chromosome ten (PTEN) deficiency promotes protein synthesis and proteasome subunit expression and proteolytic activity, creating a dependency on the proteasome for cancer cell growth and survival. Thus, targeting the proteasome machinery with the inhibitor bortezomib inhibited the proliferation and survival of CCA cells lacking functional PTEN. Therapeutic evaluation of PDXs, autochthonous mouse models, and patients confirmed this dependency on the proteasome. Mechanistically, we found that PTEN promoted the nuclear translocation of FOXO1, resulting in the increased expression of BACH1 and MAFF BACH1 and MAFF are transcriptional regulators that recognize the antioxidant response element, which is present in genes encoding proteasome subunits. PTEN induced the accumulation and nuclear translocation of these proteins, which directly repressed the transcription of genes encoding proteasome subunits. We revealed that the PTEN-proteasome axis is a potential target for therapy in PTEN-deficient CCA and other PTEN-deficient cancers.

Liquid Chromatography-Mass Spectrometry-Based Nontargeted Metabolomics Predicts Prognosis of Hepatocellular Carcinoma after Curative Resection.

Assessment and prediction of prognostic risk in patients with hepatocellular carcinoma (HCC) would greatly benefit the optimal treatment selection. Here, we aimed to identify the critical metabolites associated with the outcomes and develop a risk score to assess the prognosis of HCC patients after curative resection. A total of 78 serum samples of HCC patients were analyzed by liquid chromatography-mass spectrometry to characterize the metabolic profiling. A novel network-based feature selection method (NFSM) was developed to define the critical metabolites with the most discriminant capacity to outcomes. The metabolites defined by NFSM was further reduced by Cox regression analysis to generate a prognostic metabolite panel-phenylalanine and choline. Furthermore, univariate and multivariate Cox regression analyses were applied to combine the metabolite panel with the presence of satellite nodes to generate a global prognostic index (GPI) score for overall survival assessment. Compared with the current clinical classification systems, including the Barcelona-clinic liver cancer stage, tumor-node-metastasis stage, and albumin-bilirubin grade, the GPI score presented comparable performance, according to the time-dependent receiver operating characteristic curves and was validated in an independent cohort, which suggested that metabolomics could serve as a helpful tool to stratify the HCC prognostic risk after operation.

The Bcr-Abl inhibitor GNF-7 inhibits necroptosis and ameliorates acute kidney injury by targeting RIPK1 and RIPK3 kinases.

Necroptosis is a form of programmed, caspase-independent cell death that is involved in various pathologic disorders such as ischemia/reperfusion injury, acute kidney injury and inflammatory bowel diseases. Identification of necroptosis inhibitors has great therapeutic potential for the treatment of necroptosis-associated diseases. In this study, we identified that the Bcr-Abl inhibitor GNF-7 was a potent inhibitor of necroptosis. GNF-7 inhibited necroptosis in both human and mouse cells, while not protecting cells from apoptosis. Drug affinity responsive target stability assay (DARTS) demonstrated that it binded with RIPK1 and RIPK3. GNF-7 inhibited RIPK1 and RIPK3 kinase activities and thus disrupted RIPK1-RIPK3 necrosome complex formation. In vivo, GNF-7 ameliorated both cisplatin- and ischemia/reperfusion-induced AKI. Orally administration of GNF-7 attenuated renal cell necrosis and reduced pro-inflammatory responses in mouse models of AKI. Taken together, our study shows that GNF-7 is a novel necroptosis inhibitor and has great potential for the treatment of acute renal inflammatory disorders by targeting both RIPK1 and RIPK3 kinases.

RPB5-Mediating Protein Promotes Cholangiocarcinoma Tumorigenesis and Drug Resistance by Competing With NRF2 for KEAP1 Binding.

BACKGROUND AND AIMS: Cancer cell survival depends on the balance between reactive oxygen species production and scavenging, which is regulated primarily by NRF2 during tumorigenesis. Here, we demonstrate that deletion of RBP5-mediating protein (RMP) in an autonomous mouse model of intrahepatic cholangiocarcinoma (ICC) delays tumor progression. APPROACH AND RESULTS: RMP-overexpressing tumor cells exhibited enhanced tolerance to oxidative stress and apoptosis. Mechanistically, RMP competes with NRF2 for binding to the Kelch domain of KEAP1 (Kelch-like ECH-associated protein 1) through the E**E motif, leading to decreased NRF2 degradation via ubiquitination, thus increasing NRF2 nuclear translocation and downstream transactivation of antioxidant genes. This RMP-KEAP1-NRF2 axis promotes ICC tumorigenesis, metastasis, and drug resistance. Consistent with these findings, the RMP level in human ICC is positively correlated with the protein level of NRF2 and is associated with poor prognosis. CONCLUSION: These findings reveal that RMP is involved in the oxidative stress defense program and could be exploited for targeted cancer therapies.

Six2 is negatively correlated with prognosis and facilitates epithelial-mesenchymal transition via TGF-ß/Smad signal pathway in hepatocellular carcinoma.

BACKGROUND: Increasing evidence indicates that Six2 contributes to tumorigenesis in various tumor including hepatocellular carcinoma (HCC). This study aimed to determine the role of Six2 in HCC and to elucidate the association of Six2 with clinical pathological characteristics. METHODS: The expressions of Six2 in HCC tumor, para-tumor tissue and portal vein tumor thrombus (PVTT) were detected by tissue microarray technique, immunohistochemistry, real-time RT-PCR and Western blotting. Chi-square and Kaplan-Meier analysis were used to analyze the correlation between Six2 expression and prognosis of HCC patients. Lentivirus mediated Six2 knockdown, spheroid formation assay, proliferation assay and subcutaneous tumor implantation were performed to determine the function of Six2. RESULTS: In 274 HCC samples, Six2 was strongly expressed. Kaplan-Meier analysis revealed that high expression of Six2 was correlated with a shorter overall survival (OS) and disease-free survival (DFS). Moreover, Six2 expression was associated with sex, alpha-fetoprotein, tumor size and portal vein invasion. Six2 was highly expressed in PVTT. Six2 knockdown inhibited HCC cell lines proliferation, migration, and self-renewal in vitro and in vivo. In addition, low-expression of Six2 weakened TGF-ß induced Smad4 activation and epithelial-mesenchymal transition in HCC cell lines. CONCLUSIONS: Elevated Six2 expression in HCC tumor patients was associated with negative prognosis. Upregulated Six2 promoted tumor growth and facilitated HCC metastasis via TGF-ß/Smad signal pathway.