Combined Hepatocellular-Cholangiocarcinoma: Biology, Diagnosis, and Management.

Background: Combined hepatocellular-cholangiocarcinoma (cHCC-iCCA) is a rare type of primary liver cancer displaying characteristics of both hepatocytic and cholangiocytic differentiation. Summary: Because of its aggressive nature, patients with cHCC-iCCA exhibit a poorer prognosis than those with HCC. Surgical resection and liver transplantation may be considered curative treatment approaches; however, only a minority of patients are eligible at the time of diagnosis, and postoperative recurrence rates are high. For cases that are not eligible for surgery, locoregional and systemic therapy are often administered based on treatment protocols applied for HCC or iCCA. Owing to the rarity of this cancer, there are still no established standard treatment protocols; therefore, the choice of therapy is often personalized and guided by the suspected predominant component. Further, the genomic and molecular heterogeneity of cHCC-iCCA can severely compromise the efficacy of the available therapies. Key Messages: In the present review, we summarize the latest advances in cHCC-iCCA and attempt to clarify its terminology and molecular biology. We provide an overview of the etiology of cHCC-iCCA and present new insights into the molecular pathology of this disease that could contribute to further studies aiming to improve the patient outcomes through new systemic therapies.

The inhibition of YAP Signaling Prevents Chronic Biliary Fibrosis in the Abcb4-/- Model by Modulation of Hepatic Stellate Cell and Bile Duct Epithelium Cell Pathophysiology.

Primary sclerosing cholangitis (PSC) represents a chronic liver disease characterized by poor prognosis and lacking causal treatment options. Yes-associated protein (YAP) functions as a critical mediator of fibrogenesis; however, its therapeutic potential in chronic biliary diseases such as PSC remains unestablished. The objective of this study is to elucidate the possible significance of YAP inhibition in biliary fibrosis by examining the pathophysiology of hepatic stellate cells (HSC) and biliary epithelial cells (BEC). Human liver tissue samples from PSC patients were analyzed to assess the expression of YAP/connective tissue growth factor (CTGF) relative to non-fibrotic control samples. The pathophysiological relevance of YAP/CTGF in HSC and BEC was investigated in primary human HSC (phHSC), LX-2, H69, and TFK-1 cell lines through siRNA or pharmacological inhibition utilizing verteporfin (VP) and metformin (MF). The Abcb4-/- mouse model was employed to evaluate the protective effects of pharmacological YAP inhibition. Hanging droplet and 3D matrigel culture techniques were utilized to investigate YAP expression and activation status of phHSC under various physical conditions. YAP/CTGF upregulation was observed in PSC patients. Silencing YAP/CTGF led to inhibition of phHSC activation and reduced contractility of LX-2 cells, as well as suppression of epithelial-mesenchymal transition (EMT) in H69 cells and proliferation of TFK-1 cells. Pharmacological inhibition of YAP mitigated chronic liver fibrosis in vivo and diminished ductular reaction and EMT. YAP expression in phHSC was effectively modulated by altering extracellular stiffness, highlighting YAP's role as a mechanotransducer. In conclusion, YAP regulates the activation of HSC and EMT in BEC, thereby functioning as a checkpoint of fibrogenesis in chronic cholestasis. Both VP and MF demonstrate effectiveness as YAP inhibitors, capable of inhibiting biliary fibrosis. These findings suggest that VP and MF warrant further investigation as potential therapeutic options for the treatment of PSC.

Etiology-independent activation of the LTß-LTßR-RELB axis drives aggressiveness and predicts poor prognosis in HCC.

BACKGROUND AND AIMS: HCC is the most common primary liver tumor, with an increasing incidence worldwide. HCC is a heterogeneous malignancy and usually develops in a chronically injured liver. The NF-κB signaling network consists of a canonical and a noncanonical branch. Activation of canonical NF-κB in HCC is documented. However, a functional and clinically relevant role of noncanonical NF-κB and its downstream effectors is not established. APPROACH AND RESULTS: Four human HCC cohorts (total n = 1462) and 4 mouse HCC models were assessed for expression and localization of NF-κB signaling components and activating ligands. In vitro , NF-κB signaling, proliferation, and cell death were measured, proving a pro-proliferative role of v-rel avian reticuloendotheliosis viral oncogene homolog B (RELB) activated by means of NF-κB-inducing kinase. In vivo , lymphotoxin beta was identified as the predominant inducer of RELB activation. Importantly, hepatocyte-specific RELB knockout in a murine HCC model led to a lower incidence compared to controls and lower maximal tumor diameters. In silico , RELB activity and RELB-directed transcriptomics were validated on the The Cancer Genome Atlas HCC cohort using inferred protein activity and Gene Set Enrichment Analysis. In RELB-active HCC, pathways mediating proliferation were significantly activated. In contrast to v-rel avian reticuloendotheliosis viral oncogene homolog A, nuclear enrichment of noncanonical RELB expression identified patients with a poor prognosis in an etiology-independent manner. Moreover, RELB activation was associated with malignant features metastasis and recurrence. CONCLUSIONS: This study demonstrates a prognostically relevant, etiology-independent, and cross-species consistent activation of a lymphotoxin beta/LTßR/RELB axis in hepatocarcinogenesis. These observations may harbor broad implications for HCC, including possible clinical exploitation.

Concomitant Irradiation to Checkpoint Inhibitor Therapy of Hepatocellular Carcinoma Patients: A Systematic Retrospective, Single-Center Analysis.

INTRODUCTION: Immunotherapy has been established as the standard treatment option for patients with advanced hepatocellular carcinoma (aHCC). Despite the increased efficacy, disease progression occurs in a relevant proportion of patients even after an objective response. Combination concepts with locoregional therapy are currently under investigation for hepatic disease but are also in discussion for the control of distant metastasis. Radiotherapy is a highly effective treatment modality for local tumor control. It is also thought to increase the efficacy of checkpoint inhibition and sensitize distant lesions to the effects of immunotherapy, but may potentially increase adverse effects. In our center, few patients with aHCC treated with immune checkpoint inhibitors (ICIs) received concomitant radiotherapy for symptom or disease control. The aim of this study was to retrospectively analyze adverse effects and efficacy of concomitant radiotherapy in patients with aHCC treated with checkpoint inhibition. METHODS: To this aim, patients who received a combination of ICI and radiotherapy in our institution were retrospectively considered for analysis. The predefined inclusion criterion was radiotherapy after initiated checkpoint inhibition and continuation of ICI therapy for at least 8 weeks. Adverse effects and efficacy measurements were performed according to local standards. RESULTS: The database search of 2016-2021 revealed six consecutive patients fulfilling the predefined criteria for concomitant ICI and radiotherapy. Three patients received high-dose-rate brachytherapy (15 Gy) to treat progredient hepatic lesions. Two patients received stereotactic body radiotherapy (SBRT) (25-30 Gy) for symptom control, and 1 patient received brachytherapy and SBRT to treat metastases. No severe adverse events were reported in the period (<6 months) after concomitant radiotherapy. In 5 out of 6 cases, long-term tumor control could be achieved by this therapeutic combination. CONCLUSION: A good efficacy of concomitant radiotherapy and checkpoint inhibition has been achieved with no safety concerns. Further investigations should evaluate the safety, appropriate clinical context, and efficacy of this promising approach.

A hormesis-like effect of FeS on heterotrophic denitrification and its mechanisms.

To alleviate the insufficiency of carbon source in sewage, many sulfur-containing inorganic electron donors were added into traditional heterotrophic denitrification process. However, the effects of extraneous inorganic electron donors on heterotrophic denitrification were still largely unknown. In this study, a hormesis-like effect of ferrous sulfide (FeS, a representative inorganic electron donors) on Paracoccus denitrificans was observed. Total nitrogen (TN) removal efficiency of P. denitrificans rose by 15% with the increase of FeS dosage from 0 to 0.3 g L-1 (low level), whereas the TN removal significantly decreased to 53% as the dosage of FeS mounted up to 5.0 g L-1 (high level). Furthermore, the impacts of FeS on glucose utilization and bacterial growth exhibited hormesis-like effects. A subsequent mechanistic study revealed that above influences were caused by its released ions (Fe2+, Fe3+, and S2-) rather than particle size. Further study illustrated that low dosage of FeS released a small amount of Fe2+ and Fe3+, which provided sufficient electrons via promoting glucose utilization, then improved denitrification. Conversely, FeS with high dosage inhibited denitrification via its released S2-, which suppressed the activity of key denitrifying enzymes rather than influenced glucose metabolism and electron provision. Our results provide an insight into improving denitrification efficiency of the mixotrophic process coexisting with autotrophic and heterotrophic denitrifiers.

TRAIL Receptor Targeting Agents Potentiate PARP Inhibitor Efficacy in Pancreatic Cancer Independently of BRCA2 Mutation Status.

Chemotherapy, the standard treatment for pancreatic ductal adenocarcinoma (PDAC), has only a modest effect on the outcome of patients with late-stage disease. Investigations of the genetic features of PDAC have demonstrated a frequent occurrence of mutations in genes involved in homologous recombination (HR), especially in the breast cancer susceptibility gene 2 (BRCA2). Olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, is approved as a maintenance treatment for patients with advanced PDAC with germline BRCA1/2 mutations following a platinum-containing first-line regimen. Limitations to the use of PARP inhibitors are represented by the relatively small proportion of patients with mutations in BRCA1/2 genes and the modest capability of these substances of inducing objective response. We have previously shown that pancreatic cancer with BRCA2 mutations exhibits a remarkably enhanced sensitivity towards tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) receptor-stimulating agents. We thus aimed to investigate the effect of combined treatment with PARP inhibitors and TRAIL receptor-stimulating agents in pancreatic cancer and its dependency on the BRCA2 gene status. The respective effects of TRAIL-targeting agents and the PARP inhibitor olaparib or of their combination were assessed in pancreatic cancer cell lines and patient-derived organoids. In addition, BRCA2-knockout and -complementation models were investigated. The effects of these agents on apoptosis, DNA damage, cell cycle, and receptor surface expression were assessed by immunofluorescence, Western blot, and flow cytometry. PARP inhibition and TRAIL synergized to cause cell death in pancreatic cancer cell lines and PDAC organoids. This effect proved independent of BRCA2 gene status in three independent models. Olaparib and TRAIL in combination caused a detectable increase in DNA damage and a concentration-dependent cell cycle arrest in the G2/M and S cell cycle phases. Olaparib also significantly increased the proportion of membrane-bound death receptor 5. Our results provide a preclinical rationale for the combination of PARP inhibitors and TRAIL receptor agonists for the treatment of pancreatic cancer and suggest that the use of PARP inhibitors could be extended to patients without BRCA2 mutations if used in combination with TRAIL agonists.

Comparative Response of HCC Cells to TKIs: Modified in vitro Testing and Descriptive Expression Analysis.

Introduction: Although the treatment paradigm for hepatocellular carcinoma (HCC) has recently shifted in favour of checkpoint inhibitor (CPI)-based treatment options, the tyrosine kinase inhibitors (TKI) currently approved for the treatment of HCC are expected to remain the cornerstone of HCC treatment alone or in combination with CPIs. Despite considerable research efforts, no biomarker capable of predicting the response to specific TKIs has been validated. Thus, personalized approaches to HCC may aid in determining optimal treatment lines for 2nd and 3rd lines. To identify new biomarkers, we examined differential sensitivity and investigated potential transcriptomic predictors. Methods: To this aim, the sensitivity of nine HCC cell lines to sorafenib, lenvatinib, regorafenib, and cabozantinib was evaluated by a prolonged treatment scheme to determine their respective growth rate inhibition concentrations (GR50). Subgroups discriminated by GR50 values underwent differential expression and gene set enrichment analysis (GSEA). Results: The nine cell lines showed broadly different sensitivities to different TKIs. GR50 values of sorafenib and regorafenib clustered closer in all cell lines, whereas treatments with lenvatinib and cabozantinib showed diversified GR50 values. GSEA showed the activation of specific pathways in sensitive vs non-sensitive cell lines. A signature consisting of 14 biomarkers (GAGE12H, GJB6, PTCHD3, PRH1-PRR4, C6orf222, HBB, C17orf99, GOLGA6A, CRYAA, CCL23, RP11-347C12.3, RP11-514O12.4, FAM180B, and TMPRSS4) discriminates the cell lines' response into three distinct treatment profiles: 1) equally sensible to sorafenib, regorafenib and cabozantinib, 2) sensible to lenvatinib, and 3) more sensible to regorafenib than sorafenib. Conclusion: We observed diverse responses to either of the four TKIs. Subgroup analysis of TKI effectiveness showed distinct transcriptomic profiles and signaling pathways associated with responsiveness. This prompts more extensive studies to explore and validate pharmacogenomic and transcriptomic strategies for a personalized treatment approach, particularly after the failure of CPI treatment.

Advances in Pharmacotherapy of Hepatocellular Carcinoma: A State-of-the-Art Review.

BACKGROUND: Due to the number of emerging new treatment options, the systemic treatment of hepatocellular carcinoma (HCC) is rapidly changing. We provide here an overview of the current landscape of systemic treatment of HCC and discuss its potential future development. SUMMARY: HCC is a leading cause of tumor-related death worldwide. Despite the efforts aimed at reducing the prevalence of HCC through vaccination and antiviral treatment, and the implementation of screening programs for early tumor detection, most patients are diagnosed with or progress to advanced HCC. For approximately 10 years, sorafenib has been the only effective systemic treatment available for these patients. Recently, however, a number of new systemic compounds, comprising several multi-kinase inhibitors and immune-checkpoint inhibitors, have been approved for treatment of HCC. These new agents are opening a plethora of therapeutic options for the future therapy of HCC. KEY MESSAGES: The rapid progress in the treatment of HCC raises the question of the optimal combination and sequence of these agents in the treatment of patients with advanced disease. The substantial improvements in terms of objective response and survival indicate that the use of immune-checkpoint inhibitors-based treatment combinations may be extended to patients with intermediate-stage HCC.

p70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice.

BACKGROUND & AIMS: Progression of chronic liver disease (CLD) to liver cirrhosis and liver cancer is a major global cause of morbidity and mortality. Treatment options capable of inhibiting progression of liver fibrosis when etiological treatment of CLD is not available or fails have yet to be established. We investigated the role of serine/threonine kinase p70 ribosomal protein S6 kinase (p70S6K) as checkpoint of fibrogenesis in hepatic stellate cells (HSCs) and as target for the treatment of liver fibrosis. APPROACH & RESULTS: Immunohistochemistry was used to assess p70S6K expression in liver resection specimen. Primary human or murine HSCs from wild-type or p70S6K-/- mice as well as LX-2 cells were used for in vitro experiments. Specific small interfering RNA or CEP-1347 were used to silence or inhibit p70S6K and assess its functional relevance in viability, contraction and migration assays, fluorescence-activated cell sorting, and Western blot. These results were validated in vivo by a chemical model of fibrogenesis using wild-type and p70S6K-/- mice. Expression of p70S6K was significantly increased in human cirrhotic vs noncirrhotic liver-tissue and progressively increased in vitro through activation of primary human HSCs. Conversely, p70S6K induced fibrogenic activation of HSCs in different models, including the small interfering RNA-based silencing of p70S6K in HSC lines, experiments with p70S6K-/- cells, and the pharmacological inhibition of p70S6K by CEP-1347. These findings were validated in vivo as p70S6K-/- mice developed significantly less fibrosis upon exposure to CCl4. CONCLUSIONS: We establish p70S6K as a checkpoint of fibrogenesis in vitro and in vivo and CEP-1347 as potential treatment option that can safely be used for long-term treatment.

Prognostic Significance and Functional Relevance of Olfactomedin 4 in Early-Stage Hepatocellular Carcinoma.

OBJECTIVES: Hepatocellular carcinoma (HCC) is a leading cancer-related cause of death. Unfortunately, recurrence is common even after curative treatment of early-stage patients, and no adjuvant treatment has yet been established. Aberrant expression of OLFM4 in human cancers has been reported; yet, its specific function during tumor development remains poorly understood, and its role in HCC is unknown. The purpose of this study is to examine the prognostic significance of OLFM4 and its functional relevance in determining recurrence in patients with early-stage HCC. METHODS: Immunohistochemical staining to assess expression, cellular distribution, and prognostic significance of OLFM4 was performed in a tissue microarray comprising 157 HCC tissues and matched nontumor tissues. In addition, expression of OLFM4-coding mRNA was assessed in a separate patients' cohort. The findings were validated by in vitro functional studies using siRNA directed against OLFM4 to assess its effect on cell motility and proliferation. RESULTS: The fraction of HCC samples exhibiting positive OLFM4 staining was higher in comparison with that observed in hepatocytes from matched nontumor tissue (61% vs 39%). However, cytoplasmic-only staining for OLFM4 was associated with vascular invasion (P = 0.048), MMP-7 expression (P = 0.002), and poorer survival (P = 0.008). A multivariate analysis confirmed the independent significance of OLFM4 in determining patients' outcome (5-year survival [58.3% vs 17.3%; HR: 2.135 {95% confidence interval: 1.135-4.015}; P = 0.019]). Correspondingly, inhibition of OLFM4 by siRNA modulated the expression of MMP-7 and E-cadherin, causing inhibition of cell proliferation, motility, and migration. DISCUSSION: To the best of our knowledge, we provide the first report on the prognostic significance of OLFM4 in HCC and identify its mechanistic role as crucial mediator of MMP family protein and E-Cadherin in determining cell invasion and metastasis formation.

Antifibrotic effects of hypocalcemic vitamin D analogs in murine and human hepatic stellate cells and in the CCl4 mouse model.

Liver cirrhosis is a life-threatening consequence of liver fibrosis. The aim of this study was to investigate the antifibrotic potential of clinically available vitamin D analogs compared to that of calcitriol in vitro and in vivo. Murine hepatic stellate cells, Kupffer cells, and human LX-2 cells were treated with vitamin D analogs, and the profibrotic behavior of these cells was studied. In vivo liver fibrosis was induced using CCl4 until measurable fibrosis was established. Animals were then treated with calcitriol and paricalcitol. Vitamin D and its analogs showed antifibrotic effects in vitro. Treatment with active vitamin D (calcitriol, CAL) and its analogs reduced the protein expression of α-smooth muscle actin (α-SMA) in mHSC. In human LX-2 cells alfacalcidol reduced transforming growth factor-ß (TGF-ß) induced platelet-derived growth factor receptor-ß protein expression and contractility while paricalcitol (PCT), in its equipotent dose to CAL, reduced TGF-ß induced α-SMA protein expression, and ACTA2 and TGF-ß mRNA expression. No effects of a treatment with vitamin D and its analogs were observed in Kupffer cells. In vivo, PCT-treated mice had significantly lower calcium levels than CAL-treated mice. CAL and PCT reduced the hepatic infiltration of CD11b-positive cells and alanine transaminase levels, while PCT but not CAL significantly inhibited fibrosis progression, with a favorable side effect profile in the CCl4 model. We conclude that hypocalcemic vitamin D analogs should be considered in future studies investigating vitamin D for the treatment of liver fibrosis.

Predictors of ribociclib-mediated antitumour effects in native and sorafenib-resistant human hepatocellular carcinoma cells.

PURPOSE: The cyclin-dependent kinases (CDKs) CDK4 and CDK6 are important regulators of the cell cycle and represent promising targets in cancer treatment. We aimed to investigate the relevance of CDK4/6 in the development of hepatocellular carcinoma (HCC) and the potential of ribociclib, a novel orally available CDK4/6 inhibitor, as a treatment for HCC. METHODS: The effect of ribociclib was assessed in native and sorafenib-resistant HCC cell lines using viability assays, colony formation assays and FACS-based analyses. The expression of potential biomarkers of ribociclib response was assessed in cell lines and primary human hepatocytes using Western blotting. In addition, the prognostic relevance of the cyclin D-CDK4/6-retinoblastoma protein (Rb) pathway was assessed by analysing mRNA expression data from The Cancer Genome Atlas (TCGA). RESULTS: We found that ribociclib downregulated Rb and caused a profound loss of cell viability by inducing G1 cell cycle arrest in HCC cell lines exhibiting Rb-high/p16-low protein expression profiles, but not in Rb-low/p16-high cells, regardless their sensitivity to sorafenib. siRNA-based Rb silencing decreased cell proliferation, but did not diminish the sensitivity of HCC cells to ribociclib. Furthermore, we found that ribociclib synergized with sorafenib to cause cell death. mRNA analysis of primary human HCC specimens showed that CDK4 expression was correlated with patient survival and that the expression of Rb and the p16-encoding CDKN2A gene were inversely correlated. CONCLUSIONS: From our data we conclude that impairment of the cyclin D-CDK4/6-Rb pathway is a frequent feature of HCC and that it is associated with a unfavourable prognosis. We also found that ribociclib exhibits a preferential antineoplastic activity in Rb-high HCC cells. Our results warrant further investigation of Rb and p16 expression as markers of HCC sensitivity to ribociclib.

The PI3K inhibitor copanlisib synergizes with sorafenib to induce cell death in hepatocellular carcinoma.

Sorafenib, a multikinase inhibitor targeting the Ras/Raf/MAPK (mitogen-activated protein kinase) and vascular endothelial growth factor signaling pathways is an established treatment option for patients with advanced-stage hepatocellular carcinoma (HCC); however, despite its clinical benefit, chemoresistance and disease progression eventually occur almost invariably during treatment. Activation of the PI3K/AKT (phosphatidylinositol-3-kinase/serine/threonine kinase) pathway plays a role in the pathogenesis of HCC and may contribute to determine resistance to sorafenib. We thus evaluated in vitro the effects of the combination of sorafenib and copanlisib, a PI3K inhibitor recently approved for clinical use. The effects of copanlisib alone and in combination with sorafenib were assessed in several HCC cell lines by proliferation and colony formation assays, fluorescence-activated cell sorting analyses, and western blot. In addition, sorafenib-resistant cell clones were used. Copanlisib strongly reduced cell viability and colony formation in different native and sorafenib-resistant HCC cell lines by affecting cyclin D1/CDK4/6 signaling and causing cell cycle arrest. Elevation of phosphorylated (p)-AKT was observed upon incubation with sorafenib and was consistently found in six different unstimulated sorafenib-resistant cell clones. Copanlisib counteracted sorafenib-induced phosphorylation of p-AKT and synergistically potentiated its antineoplastic effect. In summary, copanlisib shows potent anticancer activity as a single agent and acts synergistically in combination with sorafenib in human HCC. Combination of sorafenib with copanlisib represents a rational potential therapeutic option for advanced HCC.

Nano-fumed silica as a novel pollutant that inhibits the algicidal effect of p-hydroxybenzoic acid on Microcystis aeruginosa.

Nanomaterials and/or contaminants are becoming more common in the developing world, but their effects on interspecific interactions are rarely reported, particularly in aquatic ecosystems. Thus, the present study determined the effects of the novel pollutant nano-fumed silica (NFS) on algal control by a macrophyte via the allelochemical p-hydroxybenzoic acid in a microcosm test. The allelochemical p-hydroxybenzoic acid caused significant decreases in chlorophyll a, but increased the amount of superoxide anion radicals ( O2∙- ) and the electric conductivity in Microcystis aeruginosa. The 50% inhibitory concentration (IC50) for p-hydroxybenzoic acid was 0.919 mmol/L in microalga during the exponential phase. Interestingly, NFS at 100-1600 mg/L had clear stimulatory effects on M. aeruginosa. When NFS at 800 mg/L was combined with different concentrations of p-hydroxybenzoic acid, the IC50 for p-hydroxybenzoic acid was 1.045 mmol/L. Thus, NFS significantly reduced the algicidal effect exhibited by p-hydroxybenzoic acid on M. aeruginosa. Furthermore, NFS might act as a silicon nutrient and enhance allelopathic resistance in M. aeruginosa to inhibit the algicidal effects of macrophytes via allelopathy. These findings suggest that before algal control is considered using macrophyte allelopathy, it is essential to remove the pollutant NFS from polluted lakes.

FEZF1-AS1/miR-107/ZNF312B axis facilitates progression and Warburg effect in pancreatic ductal adenocarcinoma.

Long non-coding RNAs (lncRNAs) play a pivotal role in pathological processes. However, little information has been published regarding the underlying functions and mechanisms of lncRNAs in pancreatic ductal adenocarcinoma (PDAC). A novel lncRNA FEZF1-AS1 and its sense-cognate gene ZNF312B were found to be highly expressed in human PDAC tissues and cell lines, which is associated with disease progression and predicts clinical outcome in PDAC patients. Of note, bioinformatics analysis, luciferase assays and RNA immunoprecipitation assays indicated that FEZF1-AS1 may act as an endogenous sponge by competing for miR-107, thereby modulating the derepression of ZNF312B. Downregulation of FEZF1-AS1 or ZNF312B significantly inhibited proliferation, colony formation, migration, and invasion of PDAC cells in vitro, whereas the miR-107 inhibitor abrogated the effect of dow-regulation of FEZF1-AS1 or ZNF312B in reducing oncogenic capacities of PDAC cells. In addition, FEZF1-AS1/miR-107/ZNF312B axis-induced promotion of PDAC cells proliferation appeared to be mediated by modulation of the apoptosis and the G1-S checkpoint. Furthermore, downregulation of FEZF1-AS1 repressed tumor growth in mouse xenograft models. In particular, our results highlight the contribution of FEZF1-AS1/miR-107/ZNF312B axis to Warburg effect maintenance of PDAC cells. Collectively, our findings demonstrate that the FEZF1-AS1/miR-107/ZNF312B axis regulatory network might provide a potential new therapeutic strategy for PDAC.

Antialgal effects of five individual allelochemicals and their mixtures in low level pollution conditions.

An effective, environmentally friendly, and eco-sustainable approach for removing harmful microalgae is exploiting the allelopathic potential of aquatic macrophytes. In this study, we simulated field pollution conditions in the laboratory to investigate algal inhibition by allelochemicals, thereby providing insights into field practices. We tested five allelochemicals, i.e., coumarin, ρ-hydroxybenzoic acid, protocatechuic acid, stearic acid, and ρ-aminobenzenesulfonic acid, and a typical green alga, Chlorella pyrenoidosa, under two conditions. In the unpolluted treatment, individual allelochemicals had strong algal inhibition effects, where coumarin and ρ-hydroxybenzoic acid had greater potential for algal inhibition than protocatechuic acid, stearic acid, and ρ-aminobenzenesulfonic acid based on the 50 % inhibitory concentration. However, when two or three allelochemicals were mixed in specific proportions, the algal inhibition rate exceeded 80 %, thereby indicating allelopathic synergistic interactions. Mixtures of four or five allelochemicals had weak effects on algal inhibition, which indicated antagonistic interactions. Furthermore, the presence of low lead pollution significantly reduced the antialgal potential of individual allelochemicals, whereas the allelopathic synergistic interactions with mixtures between two or three allelochemicals were changed into antagonistic effects by low pollution. In particular, the allelopathic antagonistic interactions between four or five allelochemicals were increased by pollution. The allelopathic performance of these five allelochemicals may depend on various factors, such as the chemical species, mixture parameters, and algal strain. Thus, we found that low level pollution reduced the allelopathic inhibition of microalgae by allelochemicals. Therefore, the control of algae by the direct addition of allelochemicals should consider various environmental factors.

A retrospective cohort study of pancreatic neuroendocrine tumors at single institution over 15 years: New proposal for low- and high-grade groups, validation of a nomogram for prognosis, and novel follow-up strategy for liver metastases.

PURPOSE: Pancreatic neuroendocrine tumors (PNETs) exhibit various tumor behaviors and symptoms that are difficult for physicians to stage and predict prognosis. We assess prognostic factors combined with staging classifications to optimal the models and try to improve follow-up strategy to monitor liver metastases after surgery. METHODS: Patients with PNETs treated at Sun Yat-sen Memorial Hospital between 2000 and 2015 were recruited. Patients were regrouped on the basis of functional status and mitotic rates. Nomograms to predict the prognostic values of classifications (AJCC, ENETS, and WHO) were constructed; the accuracy of the nomograms were quantified by the C-index and calibration plots. RESULTS: We identified 78 PNETs patients with pathological reports. Correlations with OS in univariate analysis included nonfunctional status (P = 0.002), CgA>200 ng/ml (P < 0.001), Ki-67 (3-20%, P = 0.014; >20%, P < 0.001), and mitotic rate (3-20/10HPF, P = 0.011; >20/10HPF, P < 0.001). By multivariate analysis, nonfunctional status and mitotic rate maintained significance (P = 0.039; 3-20/10HPF, P = 0.015; >20/10HPF, P = 0.007). Evaluating the new proposed system, the difference in OS between low- and high-groups was statistically significant (P = 0.001). The C-index of the regrouped nomograms were higher than that of premise ones (AJCC cohort, 0.605 v 0.576, P < 0.01; ENETS cohort, 0.73 v 0.691, P < 0.01; WHO cohort, 0.678 v 0.603, P < 0.01). CONCLUSION: An prognostic model based on mitotic rates and functional status correlates strongly with survival. PNETs should return visits every 2 months for the first half years, and every 3 months as followed until 2 years after surgery.