Long Noncoding RNA URB1-Antisense RNA 1 (AS1) Suppresses Sorafenib-Induced Ferroptosis in Hepatocellular Carcinoma by Driving Ferritin Phase Separation.

Sorafenib, a first-line molecular-target drug for advanced hepatocellular carcinoma (HCC), has been shown to be a potent ferroptosis inducer in HCC. However, we found that there was a lower level of ferroptosis in sorafenib-resistant HCC samples than in sorafenib-sensitive HCC samples, suggesting that sorafenib resistance in HCC may be a result of ferroptosis suppression. Recent reports have shown that long noncoding RNAs (lncRNAs) are involved in programmed cell death (PCD), including apoptosis and ferroptosis. This study aimed to investigate the roles and underlying molecular mechanisms of lncRNAs in sorafenib-induced ferroptosis in HCC cells. Using lncRNA sequencing, we identified a ferroptosis-related lncRNA, URB1-antisense RNA 1 (AS1), which was highly expressed in sorafenib-resistant HCC samples and predicted poor survival in HCC. Furthermore, URB1-AS1 mitigates sorafenib-induced ferroptosis by inducing ferritin phase separation and reducing the cellular free iron content. Hypoxia inducible factor (HIF)-1α was identified as a key factor promoting URB1-AS1 expression in sorafenib-resistant HCC cells. Notably, we found that specifically inhibiting the expression of URB1-AS1 with N-acetylgalactosamine (GalNAc)-small interfering (si)URB1-AS1 successfully enhanced the sensitivity of HCC cells to sorafenib in an in vivo tumor model. Our study uncovered a critical role for URB1-AS1 in the repression of ferroptosis, suggesting URB1-AS1 targeting may represent a potential approach to overcome sorafenib resistance in HCC.

SPINK1-induced tumor plasticity provides a therapeutic window for chemotherapy in hepatocellular carcinoma.

Tumor lineage plasticity, considered a hallmark of cancer, denotes the phenomenon in which tumor cells co-opt developmental pathways to attain cellular plasticity, enabling them to evade targeted therapeutic interventions. However, the underlying molecular events remain largely elusive. Our recent study identified CD133/Prom1 in hepatocellular carcinoma (HCC) tumors to mark proliferative tumor-propagating cells with cancer stem cell-like properties, that follow a dedifferentiation trajectory towards a more embryonic state. Here we show SPINK1 to strongly associate with CD133 + HCC, and tumor dedifferentiation. Enhanced transcriptional activity of SPINK1 is mediated by promoter binding of ELF3, which like CD133, is found to increase following 5-FU and cisplatin treatment; while targeted depletion of CD133 will reduce both ELF3 and SPINK1. Functionally, SPINK1 overexpression promotes tumor initiation, self-renewal, and chemoresistance by driving a deregulated EGFR-ERK-CDK4/6-E2F2 signaling axis to induce dedifferentiation of HCC cells into their ancestral lineages. Depleting SPINK1 function by neutralizing antibody treatment or in vivo lentivirus-mediated Spink1 knockdown dampens HCC cancer growth and their ability to resist chemotherapy. Targeting oncofetal SPINK1 may represent a promising therapeutic option for HCC treatment.

SERPINA12 promotes the tumorigenic capacity of HCC stem cells through hyperactivation of AKT/ß-catenin signaling.

BACKGROUND AND AIMS: HCC is an aggressive disease with poor clinical outcome. Understanding the mechanisms that drive cancer stemness, which we now know is the root cause of therapy failure and tumor recurrence, is fundamental for designing improved therapeutic strategies. This study aims to identify molecular players specific to CD133 + HCC to better design drugs that can precisely interfere with cancer stem cells but not normal stem cell function. APPROACH AND RESULTS: Transcriptome profiling comparison of epithelial-specific "normal" CD133 + cells isolated from fetal and regenerating liver against "HCC" CD133 + cells isolated from proto-oncogene-driven and inflammation-associated HCC revealed preferential overexpression of SERPINA12 in HCC but not fetal and regenerating liver CD133 + cells. SERPINA12 upregulation in HCC is tightly associated with aggressive clinical and stemness features, including survival, tumor stage, cirrhosis, and stemness signatures. Enrichment of SERPINA12 in HCC is mediated by promoter binding of the well-recognized ß-catenin effector TCF7L2 to drive SERPINA12 transcriptional activity. Functional characterization identified a unique and novel role of endogenous SERPINA12 in promoting self-renewal, therapy resistance, and metastatic abilities. Mechanistically, SERPINA12 functioned through binding to GRP78, resulting in a hyperactivated AKT/GSK3ß/ß-catenin signaling cascade, forming a positive feed-forward loop. Intravenous administration of rAAV8-shSERPINA12 sensitized HCC cells to sorafenib and impeded the cancer stem cell subset in an immunocompetent HCC mouse model. CONCLUSIONS: Collectively, our findings revealed that SERPINA12 is preferentially overexpressed in epithelial HCC CD133 + cells and is a key contributor to HCC initiation and progression by driving an AKT/ß-catenin feed-forward loop.

Highly TEMPO-oxidized cellulose for removal of ionic and complexed cadmium from a complicated water system.

TEMPO-NaDCC-oxidized cellulose (TNOCS) with a large surface area and an abundance of carboxyl groups was used to remove heavy metal ions (Cd, Cu, and Pb) and their organic acid complexes [HM-OAs] (OAs, i.e., citric acid (CA) and propionic acid (PA)), and then reveal their adsorption behaviors. Taking Cd and CA as examples, the results showed that some of Cd ions were first adsorbed onto TNOCS, and then, the existence of [Cd-CA-] complexes formed a coordinated structure with preloaded Cd ions to serve as a bridge for combining TNOCS and [Cd-CA]. The maximum adsorption capacities of TNOCS for Cd and Cd-CA were 16.50 and 22.15 mg/g, respectively. Moreover, adsorption energies and molecular orbital distributions indicated that the adsorption capacity of TNOCS for [Cd-CA] was better than that for Cd alone. TNOCS can maintain greater than 90% adsorption capacity in five times regeneration experiments using EDTA, indicating that it is very efficient and stable. In addition, the electron density, deformation charge, and Mulliken charge distribution were confirmed that the electron transfer direction was from carboxyl groups to cadmium, whether it was cadmium ions or complexed cadmium.

Glucose deprivation-induced aberrant FUT1-mediated fucosylation drives cancer stemness in hepatocellular carcinoma.

Rapidly growing tumors often experience hypoxia and nutrient (e.g., glucose) deficiency because of poor vascularization. Tumor cells respond to the cytotoxic effects of such stresses by inducing molecular adaptations that promote clonal selection of a more malignant tumor-initiating cell phenotype, especially in the innermost tumor regions. Here, we report a regulatory mechanism involving fucosylation by which glucose restriction promotes cancer stemness to drive drug resistance and tumor recurrence. Using hepatocellular carcinoma (HCC) as a model, we showed that restricted glucose availability enhanced the PERK/eIF2α/ATF4 signaling axis to drive fucosyltransferase 1 (FUT1) transcription via direct binding of ATF4 to the FUT1 promoter. FUT1 overexpression is a poor prognostic indicator for HCC. FUT1 inhibition could mitigate tumor initiation, self-renewal, and drug resistance. Mechanistically, we demonstrated that CD147, ICAM-1, EGFR, and EPHA2 are glycoprotein targets of FUT1, in which such fucosylation would consequently converge on deregulated AKT/mTOR/4EBP1 signaling to drive cancer stemness. Treatment with an α-(1,2)-fucosylation inhibitor sensitized HCC tumors to sorafenib, a first-line molecularly targeted drug used for advanced HCC patients, and reduced the tumor-initiating subset. FUT1 overexpression and/or CD147, ICAM-1, EGFR, and EPHA2 fucosylation may be good prognostic markers and therapeutic targets for cancer patients.

Solution pH affects single, sequential and binary systems of sulfamethoxazole and cadmium adsorption by self-assembled cellulose: Promotion or inhibition?

A new self-assembled cellulose (SACS) containing multi-functional amine, carboxyl and hydroxyl groups was successfully obtained through etherification, cross-linking and grafting processes. Then, the adsorption of sulfamethoxazole (SMZ) and Cd(II) onto SACS at pH values of 3, 5.7 and 7.5 was systematically investigated by batch experiments of single, sequential and binary systems, characterization and density functional theory (DFT) calculations. The presence of Cd(II) decreased the adsorption of SMZ because of hydrophilic site competition, while SMZ inversely increased the adsorption of Cd(II), which was attributed to bridging and especially to electrostatic shielding effects; moreover, both the inhibitory and synergistic effects were more obvious in the binary system and at a pH of 7.5. There was a dynamic balance between the inhibitory and synergistic effects that depended on the system, pH value and concentration ratio. DFT results further indicated that SMZ- more easily coordinated with Cd(II) at sulfonyl oxygen and nitrogen sites, and the cationic bridge of Cd(II) with SMZ- mainly occurred in the sequential system. Moreover, a complexation-decomplexation-complexation balance of SMZ- and Cd(II) probably occurred in the binary system.

A new risk model comprising genes highly correlated with CD133 identifies different tumor-immune microenvironment subtypes impacting prognosis in hepatocellular carcinoma.

The existence of cancer stem cells (CSCs), marked by CD133, is the primary cause of death in hepatocellular carcinoma (HCC). Here, we generated a new risk model comprising the signatures of four genes highly correlated with CD133 (CD133(hi)) that help improve survival in HCC. Three datasets were used to identify the differential CD133(hi) genes by comparing sorted CD133+ liver CSCs and CD133- differentiated counterparts. Univariate analysis was used to screen significantly differential CD133(hi) genes associated with overall survival in the training dataset, which were used for risk model construction. High-risk patients were strongly associated with poor survival by Kaplan-Meier survival analysis in both the training and validation datasets. Clinical stratification analyses further demonstrated that the risk factors acted as independent factors and that high-risk patients were characterized by more aggressive cancer features. Functional enrichment analyses performed by gene set enrichment analysis (GSEA) and the Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed that high-risk patients showed the disturbance of immune hepatic homeostasis involving aberrant immune cells, including macrophages and T and B cells, and an abnormal inflammatory response including the IL6/Jak/STAT3 pathway and TNF signaling pathway. In conclusion, our constructed CD133(hi) gene risk model provides a resource for understanding the role of CD133+ CSCs in the progression of HCC in terms of tumor-immune interactions.

TBX2 Identified as a Potential Predictor of Bone Metastasis in Lung Adenocarcinoma via Integrated Bioinformatics Analyses and Verification of Functional Assay.

Objective: Bone metastasis from patients with advanced lung adenocarcinoma (LAC) is a very serious complication. To better understand the molecular mechanism, our current study sheds light on identification of hub genes mediating bone metastatic spread by combining bioinformatic analysis with functional verification. Methods: First, we downloaded a lung adenocarcinoma dataset (GSE76194) from Gene Expression Omnibus, analyzed differentially expressed genes (DEGs) through Limma package in R software and constructed a protein-protein interaction network. Based on that preliminary data, we further performed modular and topological analysis using Cystoscope to obtain biological connected genes. Through literature searching and performing mRNA expression analysis on the other independent public dataset (GSE10799), we finally focused on TBX2. Functional effects of TBX2 were performed in tumorigenicity assays including migration and invasion assays, cell proliferation assay, and cell cycle assay. In addition, mechanically, we found enriched pathways related to bone metastasis using Gene Set Enrichment Analysis (GSEA) and validated our results by western blot. Result: A total of 1132 significant genes were sorted initially. We selected common significant genes (log FC>2; p<0.01) from both the biological network data and microarray data. In total, 44 such genes were identified. we found TBX2, along with 10 other genes, to be reported with relevance to bone metastasis in other cancer types. Moreover, TBX2 showed significantly higher expression levels in patients that were found positive for metastasis to bone marrow compared to patients that did not exhibit this type of metastasis in the other separated cohort (GSE10799). Thus, we finally focused on TBX2. We found that TBX2 had detectable expression in LAC cell lines and silencing endogenous TBX2 expression in A549 and H1299 cell lines markedly suppressed migration and invasion, cell proliferation and arrested cell-cycle. Pathway enrichment analyses suggested that TBX2 drove LAC oncogenesis and metastasis through various pathways with epithelial mesenchymal transition (EMT) figuring prominently in the bone metastatic group, which was evidenced by western blot. Conclusion: Collectively, TBX2 plays as a potential predictor of bone metastasis from LAC, yielding a better promise view towards "driver" gene responsible for bone metastasis.

Deficiency in Embryonic Stem Cell Marker Reduced Expression 1 Activates Mitogen-Activated Protein Kinase Kinase 6-Dependent p38 Mitogen-Activated Protein Kinase Signaling to Drive Hepatocarcinogenesis.

BACKGROUND AND AIMS: Embryonic stem-cell-related transcription factors are central to the establishment and maintenance of stemness and pluripotency, and their altered expression plays key roles in tumors, including hepatocellular carcinoma (HCC), a malignancy with no effective treatment. Here, we report on the embryonic stem cell marker, reduced expression 1 (REX1; also known as zinc finger protein 42), to be selectively down-regulated in HCC tumors. APPROACH AND RESULTS: Deficiency of REX1 in HCC was attributed to a combination of hypermethylation at its promoter as well as histone modification by methylation and acetylation. Clinically, hypermethylation of REX1 was closely associated with neoplastic transition and advanced tumor stage in humans. Functionally, silencing of REX1 potentiated the tumor-initiating and metastasis potential of HCC cell lines and xenografted tumors. Lentivirus-mediated Rex1 ablation in liver of male immunocompetent mice with HCC, induced by hydrodynamic tail vein injection of proto-oncogenes, enhanced HCC development. Transcriptome profiling studies revealed REX1 deficiency in HCC cells to be enriched with genes implicated in focal adhesion and mitogen-activated protein kinase (MAPK) signaling. From this lead, we subsequently found REX1 to bind to the promoter region of mitogen-activated protein kinase kinase 6 (MKK6), thereby obstructing its transcription, resulting in altered p38 MAPK signaling. CONCLUSIONS: Our work describes a critical repressive function of REX1 in maintenance of HCC cells by regulating MKK6 binding and p38 MAPK signaling. REX1 deficiency induced enhancement of p38 MAPK signaling, leading to F-actin reorganization and activation of nuclear factor erythroid 2-related factor 2-mediated oxidative stress response, which collectively contributed to enhanced stemness and metastatic capabilities of HCC cells.

RNA-binding protein KHSRP promotes tumor growth and metastasis in non-small cell lung cancer.

BACKGROUND: KH-type splicing regulatory protein (KHSRP) plays an important role in cancer invasion, but the relevant mechanism is not well known. In the present study, we investigated the function and potential molecular mechanism of KHSRP in non-small cell lung cancer (NSCLC) metastasis and elucidated its clinical significance. METHODS: Isobaric tags for relative and absolute quantitation and the SWATH™ approach were combined with nanoliquid chromatography-tandem mass spectrometry analysis to identify metastasis-associated nucleoproteins in NSCLC. Real-time PCR and Western blot were used to screen for metastasis-associated candidate molecules. Gene knockdown and overexpression were used to investigate their functions and molecular mechanisms in lung cancer cells. Coimmunoprecipitation (Co-IP) experiments were performed to identify the interactions between candidate molecules and their interacting proteins. Gene expression and its association with multiple clinicopathologic characteristics were analyzed by immunohistochemistry (IHC) and Western blot in human lung cancer specimens. RESULTS: KHSRP was identified as a metastasis-associated candidate molecule. In NSCLC cell lines, knockdown of KHSRP significantly reduced lung cancer cell proliferation, migration, and invasion in vitro and in vivo, whereas overexpression of KHSRP did the opposite. Mechanistically, the protein heterogeneous nuclear ribonucleoprotein C (C1/C2) (HNRNPC) was identified to interact with KHSRP using Co-IP experiments. In NSCLC cell lines, overexpression of HNRNPC significantly promoted lung cancer cell proliferation, migration, and invasion in vitro and in vivo. KHSRP and HNRNPC may induce human lung cancer cell invasion and metastasis by activating the IFN-α-JAK-STAT1 signaling pathway. Drastically higher expression levels of KHSRP and HNRNPC were observed in lung cancer tissues compared to those in adjacent noncancerous tissues. Increased KHSRP and HNRNPC expression was significantly associated with advanced tumor stages and metastasis (both lymph node and distant). Kaplan-Meier survival analysis showed that patients with high KHSRP and HNRNPC expression levels were predicted to have the shortest survival times and to have a poor prognosis. CONCLUSIONS: KHSRP plays an important role in NSCLC metastasis and may serve as a potential prognostic marker and novel therapeutic target for lung cancer metastasis treatment.

Upregulation of phosphoserine phosphatase contributes to tumor progression and predicts poor prognosis in non-small cell lung cancer patients.

BACKGROUND: Growing evidence indicates that high phosphoserine phosphatase (PSPH) expression is associated with tumor prognosis in many types of cancers. However, the role of PSPH in non-small cell lung cancer (NSCLC) is unclear. The purpose of this study was to investigate the clinical significance of PSPH in NSCLC. METHODS: One hundred forty-three patients with histologically confirmed NSCLC who underwent surgery were included. Quantitative real-time PCR and Western blot were used to assess PSPH expression in paired tumor and corresponding adjacent non-tumorous tissues. The role of PSPH in invasion and cell growth was investigated in vitro. RESULTS: Compared to adjacent normal lung tissues, PSPH messenger RNA and protein levels were significantly higher in NSCLC tissues, and the PSPH expression level was positively related to clinical stage, metastasis, and recurrence. High PSPH expression was predictive of poor overall survival. A549 cells transfected with small interfering-PSPH showed inhibited cell migration, invasion, and proliferation. We further demonstrated that PSPH might promote the invasive capabilities of NSCLC cells through the AKT/AMPK signaling pathway. CONCLUSION: Our results indicate that PSPH may act as a putative oncogene in NSCLC, and may be a vital molecular marker for the metastasis and proliferation of NSCLC cells by regulating the AKT/AMPK signaling pathway.

OTUB2 stabilizes U2AF2 to promote the Warburg effect and tumorigenesis via the AKT/mTOR signaling pathway in non-small cell lung cancer.

Increasing evidence has confirmed that deubiquitinating enzymes play an important role in lung cancer progression. In the current study, we investigated the expression profile of deubiquitinating enzymes in non-small cell lung cancer (NSCLC) tissues and identified OTUB2 as an upregulated deubiquitinating enzyme. The role of OTUB2 in NSCLC is unknown. Methods: Quantitative, real-time PCR and Western blot were used to detect OTUB2 and U2AF2 expression in NSCLC tissues. The correlations between OTUB2 and U2AF2 expression and clinicopathologic features were then analyzed. We used In vitro Cell Counting Kit-8 (CCK-8) , colony formation , and trans-well invasion assays to investigate the function of OTUB2 and U2AF2 in tumorigenesis. The regulation of glycolysis by OTUB2 and U2AF2 was assessed by determining the extracellular acid ratio, glucose consumption, and lactate production. The mechanism of OTUB2 was explored through co-immunoprecipitation and mass spectrometry analyses. A xenograft model was also used to study the tumorigenesis role of OTUB2 In vivo. Results: OTUB2 expression was significantly upregulated in primary NSCLC tissues and greatly associated with metastasis, advanced tumor stages, poor survival, and recurrence. In NSCLC cell lines, OTUB2 promoted cell growth, colony formation, migration, and invasive activities. Mechanistic investigations showed that OTUB2 stimulated the Warburg effect and induced the activation of the serine/threonine kinase/mechanistic target of rapamycin kinase (AKT/mTOR) pathway in different NSCLC cells. More importantly, OTUB2 promoted NSCLC progression, which was largely dependent on the direct binding to and deubiquitination of U2AF2, at least in NSCLC cells. U2AF2 expression was also significantly upregulated in primary NSCLC tissues and dramatically associated with metastasis, advanced tumor stages, poor survival, and recurrence. Importantly, a positive correlation between the protein expression of OTUB2 and U2AF2 in NSCLC tissues was found. In vivo experiments indicated that OTUB2 promoted xenograft tumor growth of NSCLC cell. In addition, our results suggest that high expression of OTUB2, U2AF2 and PGK1 is significantly associated with worse prognosis in NSCLC patients. Conclusion: Taken together, the present study provides the first evidence that OTUB2 acts as a pivotal driver in NSCLC tumorigenesis by stabilizing U2AF2 and activating the AKT/mTOR pathway and the Warburg effect. It may serve as a new potential prognostic indicator and therapeutic target in NSCLC.

Optimal adjuvant therapy for resected hepatocellular carcinoma: a systematic review with network meta-analysis.

OBJECTIVES: Major adjuvant therapies (ATs) for resected hepatocellular carcinoma (HCC) include chemotherapy, internal radiation therapy (IRT), interferon therapy (IFNT) and immunotherapy but the optimum regimen remains inconclusive. We aim to compare these therapies in terms of patient survival and recurrence rates. METHODS: We searched PubMed, EMBASE and Cochrane library databases for randomized trials comparing the above four therapies until 31 March 2014. We estimated the HRs for survival and ORs for overall recurrence among different therapies. Toxic effects were also evaluated. RESULTS: Fourteen eligible articles were included. IFNT improved 5-year survival greatly (HR 1.81, 95% CI 1.01-3.81, P = 0.034), whereas chemotherapy (HR 0.33, 95% CI 0.03-2.02), IRT (HR 0.31, 95% CI 0.02-3.33) and immunotherapy (HR 0.73, 95% CI 0.05-9.12) all provided a poorer survival outcome after 1-year. Similarly, for 5-year survival rates, although differing, IRT did not provide a significant improvement in survival (HR 1.38, 95% CI 0.34-5.19) compared with IFNT. Chemotherapy (HR 0.49, 95% CI 0.18-1.14) and immunotherapy (HR 0.56, 95% CI 0.17-1.59) did not appear to provide benefit over IFNT. Chemotherapy was ranked the worst in overall recurrence (OR 0.99, 95% CI 0.18-5.38) and most likely to cause toxic effects. CONCLUSIONS: IFNT was the most efficacious AT regimen both for short and long term survivals. Immunotherapy and IFNT were the most two effective in preventing overall relapse for resected HCC.