CDK1-mediated BCL9 phosphorylation inhibits clathrin to promote mitotic Wnt signalling.

Uncontrolled cell division is a hallmark of cancer. Deregulation of Wnt components has been linked to aberrant cell division by multiple mechanisms, including Wnt-mediated stabilisation of proteins signalling, which was notably observed in mitosis. Analysis of Wnt components revealed an unexpected role of B-cell CLL/lymphoma 9 (BCL9) in maintaining mitotic Wnt signalling to promote precise cell division and growth of cancer cell. Mitotic interactome analysis revealed a mechanistic role of BCL9 in inhibiting clathrin-mediated degradation of LRP6 signalosome components by interacting with clathrin and the components in Wnt destruction complex; this function was further controlled by CDK1-driven phosphorylation of BCL9 N-terminal, especially T172. Interestingly, T172 phosphorylation was correlated with cancer patient prognosis and enriched in tumours. Thus, our results revealed a novel role of BCL9 in controlling mitotic Wnt signalling to promote cell division and growth.

Hypoxia-induced modulation of glucose transporter expression impacts 18F-fluorodeoxyglucose PET-CT imaging in hepatocellular carcinoma.

PURPOSE: To investigate the molecular mechanisms underlying the variable standard uptake value (SUV) of 18F-fluorodeoxyglucose positron emission tomography-computed tomography (18F-FDG PET-CT) imaging in hepatocellular carcinoma (HCC) and whether hypoxia-induced glucose transporter expression contributes to the progression of HCC and the rate of glycolysis in HCC cells. MATERIALS AND METHODS: Sixteen HCC specimens obtained from patients who underwent pre-treatment staging with 18F-FDG PET-CT imaging were divided into high maximum SUV (SUVmax > 8) and low SUVmax (SUVmax < 5) groups and employed for whole-genome gene expression profiling using GeneChip Human Genome U133 Plus 2.0 Arrays. The relationship between SUVmax and the expression of glucose transporters 1 and 3 (GLUT1 and GLUT3) was further validated using immunohistochemical analysis. The expression of GLUT1 and GLUT3 in different HCC cells under hypoxia and normoxia conditions were monitored by quantitative reverse transcription PCR (RT-qPCR). Glycolysis and FDG uptake by HCC cells were measured using the Seahorse XF glycolysis stress test and 18F-FDG PET-CT imaging. The effect of GLUT1 and GLUT3 on glucose uptake in HCC cells was examined using the fluorescent D-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) followed by detection of fluorescence produced by the cells using flow cytometry. RESULTS: Glucose transporters are differentially expressed between samples from HCC patients with high and low SUVmax. In particular, over-expression of GLUT1 and GLUT3 in high SUVmax patients was correlated with high glucose uptake and overall survival. The expression of GLUT1 and GLUT3 was significantly induced by hypoxia in different HCC cells. High expression of GLUT1 and GLUT3 in HCC cells were correlated with high rates of glycolysis and 18F-FDG uptake. Therefore, our data suggested that hypoxia-induced glucose transporters expression could result in the variations of 18F-FDG PET-CT imaging and progression of HCC, contributing to more aggressive disease phenotypes like large tumor size, recurrence, and poor survival. CONCLUSION: Over-expression of GLUT1 and GLUT3 significantly increase glucose uptake in HCC cells. Hypoxia-induced glucose transporters expression may therefore be a contributing variable in 18F-FDG PET-CT imaging and progression in HCC.

Oral administration of protein nanoparticles: An emerging route to disease treatment.

Over the last two decades, developments in nanomedicine have resulted in technical advances with application to clinical science. Both organic and inorganic nanoparticles (NPs) have shown tolerability, pharmacologic specificity and biodegradability. A subclass of NPs, protein NPs, have garnered recent attention due to the inherent biocompatibility of protein substrates. Protein NPs are currently being employed widely in pharmaceuticals development with applications in nasal, pulmonary, intravenous, ocular and oral delivery. Despite the distinct advantages of orally administered pharmaceuticals, the development of oral delivery systems has been comparatively limited. Therefore, this review attempts to discuss the most recent experimental and pre-clinical findings in the development of protein NPs for oral delivery, while envisioning upcoming challenges.

The microtubule-associated protein PRC1 promotes early recurrence of hepatocellular carcinoma in association with the Wnt/ß-catenin signalling pathway.

OBJECTIVES: Hepatocellular carcinoma (HCC) is the second leading cause of cancer mortality worldwide. Alterations in microtubule-associated proteins (MAPs) have been observed in HCC. However, the mechanisms underlying these alterations remain poorly understood. Our aim was to study the roles of the MAP protein regulator of cytokinesis 1 (PRC1) in hepatocarcinogenesis and early HCC recurrence. DESIGN: PRC1 expression in HCC samples was evaluated by microarray, immunoblotting and immunohistochemistry analysis. Molecular and cellular techniques including siRNA-mediated and lentiviral vector-mediated knockdown were used to elucidate the functions and mechanisms of PRC1. RESULTS: PRC1 expression was associated with early HCC recurrence and poor patient outcome. In HCC, PRC1 exerted an oncogenic effect by promoting cancer proliferation, stemness, metastasis and tumourigenesis. We further demonstrated that the expression and distribution of PRC1 is dynamically regulated by Wnt3a signalling. PRC1 knockdown impaired transcription factor (TCF) transcriptional activity, decreased Wnt target expression and reduced nuclear ß-catenin levels. Mechanistically, PRC1 interacts with the ß-catenin destruction complex, regulates Wnt3a-induced membrane sequestration of this destruction complex, inhibits adenomatous polyposis coli (APC) stability and promotes ß-catenin release from the APC complex. In vivo, high PRC1 expression correlated with nuclear ß-catenin and Wnt target expression. PRC1 acted as a master regulator of a set of 48 previously identified Wnt-regulated recurrence-associated genes (WRRAGs) in HCC. Thus, PRC1 controlled the expression and function of WRRAGs such as FANCI, SPC25, KIF11 and KIF23 via Wnt signalling. CONCLUSIONS: We identified PRC1 as a novel Wnt target that functions in a positive feedback loop that reinforces Wnt signalling to promote early HCC recurrence.

Nanoencapsulation as a General Solution for Lyophilization of Labile Substrates.

Protein macromolecules occur naturally at the nanoscale. The use of a dedicated nanoparticle as a lyophilization excipient, however, has not been reported. Because biopolymeric and lipid nanoparticles often denature protein macromolecules and commonly lack the structural rigidity to survive the freeze-drying process, we hypothesized that surrounding an individual protein substrate with a nanoscale, thermostable exoshell (tES) would prevent aggregation and protect the substrate from denaturation during freezing, sublimation, and storage. We systematically investigated the properties of tES, including secondary structure and its homogeneity, throughout the process of lyophilization and found that tES have a near 100% recovery following aqueous reconstitution. We then tested the hypothesis that tES could encapsulate a model substrate, horseradish peroxidase (HRP), using charge complementation and pH-mediated controlled assembly. HRP were encapsulated within the 8 nm internal tES aqueous cavity using a simplified loading procedure. Time-course experiments demonstrated that unprotected HRP loses 95% of activity after 1 month of lyophilized storage. After encapsulation within tES nanoparticles, 70% of HRP activity was recovered, representing a 14-fold improvement and this effect was reproducible across a range of storage temperatures. To our knowledge, these results represent the first reported use of nanoparticle encapsulation to stabilize a functional macromolecule during lyophilization. Thermostable nanoencapsulation may be a useful method for the long-term storage of labile proteins.

Simultaneous silencing of ACSL4 and induction of GADD45B in hepatocellular carcinoma cells amplifies the synergistic therapeutic effect of aspirin and sorafenib.

Sorafenib is currently the only US Food and Drug Administration (FDA)-approved molecular inhibitor for the systemic therapy of advanced hepatocellular carcinoma (HCC). Aspirin has been studied extensively as an anti-inflammation, cancer preventive and therapeutic agent. However, the potential synergistic therapeutic effects of sorafenib and aspirin on advanced HCC treatment have not been well studied. Drug combination studies and their synergy quantification were performed using the combination index method of Chou-Talalay. The synergistic therapeutic effects of sorafenib and aspirin were evaluated using an orthotopic mouse model of HCC and comprehensive gene profiling analyses were conducted to identify key factors mediating the synergistic therapeutic effects of sorafenib and aspirin. Sorafenib was determined to act synergistically on HCC cells with aspirin in vitro. Using Hep3B and HuH7 HCC cells, it was demonstrated that sorafenib and aspirin acted synergistically to induce apoptosis. Mechanistic studies demonstrated that combining sorafenib and aspirin yielded significant synergistically anti-tumor effects by simultaneously silencing ACSL4 and the induction of GADD45B expression in HCC cells both in vitro and in the orthotopic HCC xenograft mouse model. Importantly, clinical evidence has independently corroborated that survival of HCC patients expressing ACSL4highGADD45Blow was significantly poorer compared to patients with ACSL4lowGADD45Bhigh, thus demonstrating the potential clinical value of combining aspirin and sorafenib for HCC patients expressing ACSL4highGADD45Blow. In conclusion, sorafenib and aspirin provide synergistic therapeutic effects on HCC cells that are achieved through simultaneous silencing of ACSL4 and induction of GADD45B expression. Targeting HCC with ACSL4highGADD45Blow expression with aspirin and sorafenib could provide potential synergistic therapeutic benefits.

Single-layer MoS2 nanosheet grafted upconversion nanoparticles for near-infrared fluorescence imaging-guided deep tissue cancer phototherapy.

A multifunctional nanostructure is prepared by covalently grafting upconversion nanoparticles (UCNPs) with chitosan functionalized MoS2 (MoS2-CS) and folic acid (FA) and then loading phthalocyanine (ZnPc) on the surface of MoS2, which integrates photodynamic therapy (PDT) with photothermal therapy (PTT) and upconversion luminescence imaging into one system for enhanced antitumor efficiency.

MELK is an oncogenic kinase essential for early hepatocellular carcinoma recurrence.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Many kinases have been found to be intimately involved in oncogenesis and the deregulation of kinase function has emerged as a major mechanism by which cancer cells evade normal physiological constraints on growth and survival. Previously, we have performed gene expression profile analysis on HCC samples and have identified a host of kinases that are remarkably overexpressed in HCC. Among these, the Maternal Embryonic Leucine Zipper Kinase (MELK) is highly overexpressed in HCC and its overexpression strongly correlates with early recurrence and poor patients' survival. Silencing MELK inhibited cell growth, invasion, stemness and tumorigenicity of HCC cells by inducing apoptosis and mitosis. We further showed that the overexpression of MELK in HCC samples strongly correlated with the cell cycle- and mitosis-related genes which are directly regulated as part of the forkhead transcription factor FoxM1-related cell division program. Together, our data establish MELK as an oncogenic kinase involved in the pathogenesis and recurrence of HCC and could provide a promising molecular target to develop therapeutic strategies for patients with advanced HCC.