LANCL1, a cell surface protein, promotes liver tumor initiation through FAM49B-Rac1 axis to suppress oxidative stress.

BACKGROUND AND AIMS: HCC is an aggressive cancer with a poor clinical outcome. Understanding the mechanisms that drive tumor initiation is important for improving treatment strategy. This study aimed to identify functional cell membrane proteins that promote HCC tumor initiation. APPROACH AND RESULTS: Tailor-made siRNA library screening was performed for all membrane protein-encoding genes that are upregulated in human HCC (n = 134), with sphere formation as a surrogate readout for tumor initiation. Upon confirmation of membranous localization by immunofluorescence and tumor initiation ability by limiting dilution assay in vivo, LanC-like protein-1 (LANCL1) was selected for further characterization. LANCL1 suppressed intracellular reactive oxygen species (ROS) and promoted tumorigenicity both in vitro and in vivo. Mechanistically, with mass spectrometry, FAM49B was identified as a downstream binding partner of LANCL1. LANCL1 stabilized FAM49B by blocking the interaction of FAM49B with the specific E3 ubiquitin ligase TRIM21, thus protecting FAM49B from ubiquitin-proteasome degradation. The LANCL1-FAM49B axis suppressed the Rac1-NADPH oxidase-driven ROS production, but this suppression of ROS was independent of the glutathione transferase function of LANCL1. Clinically, HCCs with high co-expression of LANCL1 and FAM49B were associated with more advanced tumor stage, poorer overall survival, and disease-free survival. In addition, anti-LANCL1 antibodies targeting the extracellular N-terminal domain were able to suppress the self-renewal ability, as demonstrated by the sphere formation ability of HCC cells. CONCLUSIONS: Our data showed that LANCL1 is a cell surface protein and a key contributor to HCC initiation. Targeting the LANCL1-FAM49B-Rac1-NADPH oxidase-ROS signaling axis may be a promising therapeutic strategy for HCC.

Sortilin-Driven Cancer Secretome Enhances Tumorigenic Properties of Hepatocellular Carcinoma via de Novo Lipogenesis.

A growing body of evidence suggests de novo lipogenesis as a key metabolic pathway adopted by cancers to fuel tumorigenic processes. While increased de novo lipogenesis has also been reported in hepatocellular carcinoma (HCC), understanding on molecular mechanisms driving de novo lipogenesis remains limited. In the present study, the functional role of sortilin, a member of the vacuolar protein sorting 10 protein receptor family, in HCC was investigated. Sortilin was overexpressed in HCC and was associated with poorer survival outcome. In functional studies, sortilin-overexpressing cells conferred tumorigenic phenotypes, namely, self-renewal and metastatic potential, of HCC cells via the cancer secretome. Proteomic profiling highlighted fatty acid metabolism as a potential molecular pathway associated with sortilin-driven cancer secretome. This finding was validated by the increased lipid content and expression of fatty acid synthase (FASN) in HCC cells treated with conditioned medium collected from sortilin-overexpressing cells. The enhanced tumorigenic properties endowed by sortilin-driven cancer secretome were partly abrogated by co-administration of FASN inhibitor C75. Further mechanistic dissection suggested protein stabilization by post-translational modification with O-GlcNAcylation as a major mechanism leading to augmented FASN expression. In conclusion, the present study uncovered the role of sortilin in hepatocarcinogenesis via modulation of the cancer secretome and deregulated lipid metabolism.

Ephrin-A3/EphA2 axis regulates cellular metabolic plasticity to enhance cancer stemness in hypoxic hepatocellular carcinoma.

BACKGROUND & AIMS: The highly proliferative nature of hepatocellular carcinoma (HCC) frequently results in a hypoxic intratumoural microenvironment, which creates a therapeutic challenge owing to a lack of mechanistic understanding of the phenomenon. We aimed to identify critical drivers of HCC development and progression in the hypoxic microenvironment. METHODS: We performed integrative analysis of multiple transcriptomic and genomic profiles specific for HCC and hypoxia and identified the Ephrin-A3/Eph receptor A2 (EphA2) axis as a clinically relevant and hypoxia-inducible signalling axis in HCC. The functional significance and mechanistic consequences of the Ephrin-A3/EphA2 axis were examined in EFNA3- and EPHA2- knockdown/overexpressing HCC cells. The potential downstream pathways were investigated by transcriptome sequencing, quantitative reverse-transcription PCR, western blotting analysis and metabolomics. RESULTS: EFNA3 was frequently upregulated in HCC and its overexpression was associated with more aggressive tumour behaviours. HIF-1α directly and positively regulated EFNA3 expression under hypoxia. EFNA3 functionally contributed to self-renewal, proliferation and migration in HCC cells. EphA2 was identified as a key functional downstream mediator of EFNA3. Functional characterisation of the Ephrin-A3/EphA2 forward-signalling axis demonstrated a promotion of self-renewal ability and tumour initiation. Mechanistically, the Ephrin-A3/EphA2 axis promoted the maturation of SREBP1 and expression of its transcriptional target, ACLY, was significantly associated with the expression of EFNA3 and hypoxia markers in clinical cohorts. The metabolic signature of EPHA2 and ACLY stable knockdown HCC cells demonstrated significant overlap in fatty acid, cholesterol and tricarboxylic acid cycle metabolite profiles. ACLY was confirmed to mediate the self-renewal function of the Ephrin-A3/EphA2 axis. CONCLUSIONS: Our findings revealed the novel role of the Ephrin-A3/EphA2 axis as a hypoxia-sensitive modulator of HCC cell metabolism and a key contributor to HCC initiation and progression. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a fast-growing tumour; hence, areas of the tumour often have insufficient vasculature and become hypoxic. The presence of hypoxia within tumours has been shown to negatively impact on the survival of patients with tumours, including HCC. Herein, we identified the Ephrin-A3/EphA2 axis as a key functional driver of tumour initiation and progression in response to hypoxia. Additionally, we showed that SREBP1-ACLY-mediated metabolic rewiring was an important downstream effector that induced cancer stemness in response to Ephrin-A3/EphA2 forward-signalling.

Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism.

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.

Covalent targeting of the vacuolar H+-ATPase activates autophagy via mTORC1 inhibition.

Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.

Activity-Based Sensing with a Metal-Directed Acyl Imidazole Strategy Reveals Cell Type-Dependent Pools of Labile Brain Copper.

Copper is a required nutrient for life and particularly important to the brain and central nervous system. Indeed, copper redox activity is essential to maintaining normal physiological responses spanning neural signaling to metabolism, but at the same time copper misregulation is associated with inflammation and neurodegeneration. As such, chemical probes that can track dynamic changes in copper with spatial resolution, especially in loosely bound, labile forms, are valuable tools to identify and characterize its contributions to healthy and disease states. In this report, we present an activity-based sensing (ABS) strategy for copper detection in live cells that preserves spatial information by a copper-dependent bioconjugation reaction. Specifically, we designed copper-directed acyl imidazole dyes that operate through copper-mediated activation of acyl imidazole electrophiles for subsequent labeling of proximal proteins at sites of elevated labile copper to provide a permanent stain that resists washing and fixation. To showcase the utility of this new ABS platform, we sought to characterize labile copper pools in the three main cell types in the brain: neurons, astrocytes, and microglia. Exposure of each of these cell types to physiologically relevant stimuli shows distinct changes in labile copper pools. Neurons display translocation of labile copper from somatic cell bodies to peripheral processes upon activation, whereas astrocytes and microglia exhibit global decreases and increases in intracellular labile copper pools, respectively, after exposure to inflammatory stimuli. This work provides foundational information on cell type-dependent homeostasis of copper, an essential metal in the brain, as well as a starting point for the design of new activity-based probes for metals and other dynamic signaling and stress analytes in biology.

Metal-Metal and π-π Interactions Directed End-to-End Assembly of Gold Nanorods.

The end-to-end aggregation of gold nanorods (GNRs) has been demonstrated to be directed by a thioacetate-containing alkynylplatinum(II) terpyridine complex. The in situ deprotected complex is preferentially attached at the ends of the gold nanorods (GNRs) and induce the aggregation of GNRs in an "end-to-end" manner by Pt···Pt and π-π interactions, which have been characterized by electron microscopy, energy dispersed X-ray (EDX) analysis, and UV-vis absorption spectroscopy. The assembly of the nanorods into chain-like nanostructures can be controlled by the concentration of the Pt(II) complexes.

Synthesis and Electrochemical, Photophysical, and Self-Assembly Studies on Water-Soluble pH-Responsive Alkynylplatinum(II) Terpyridine Complexes.

A series of water-soluble pH-responsive alkynylplatinum(II) terpyridine complexes have been synthesized and characterized. The electronic absorption, emission, and electrochemical properties of the complexes have been studied. The self-assembly processes of representative complexes in aqueous media, presumably through Pt···Pt and/or π-π interactions, have been investigated by concentration- and temperature-dependent UV-vis absorption measurements and dynamic light scattering experiments. Interestingly, some of the complexes have been found to undergo induced self-assembly and disassembly in aqueous media through modulation of the pH value of the solutions, resulting in remarkable UV-vis absorption and emission spectral changes. The emission spectral changes have been rationalized by the change in the hydrophilicity of the complexes, electrostatic repulsion among the complex molecules, and/or the extent of photoinduced electron transfer (PET) quenching upon protonation/deprotonation of the pH-responsive groups on the complexes. By simple modifications of the chemical structures of the complexes, induced self-assembly/disassembly of the complexes can occur at different and/or multiple pH regions, thus allowing the probing of changes at the desired pH region by triplet metal-metal-to-ligand charge-transfer emission of the complexes in the near-infrared (NIR) region. Fixed-cell imaging experiments have further demonstrated the potential of this class of complexes as pH-responsive NIR luminescent probes in vitro, while the NIR emissions of the complexes from live cells have been found to show good differentiation of acidic organelles such as lysosomes from other cellular compartments.

Cyclometalated Palladium(II) N-Heterocyclic Carbene Complexes: Anticancer Agents for Potent In†Vitro Cytotoxicity and In†Vivo Tumor Growth Suppression.

Palladium(II) complexes are generally reactive toward substitution/reduction, and their biological applications are seldom explored. A new series of palladium(II) N-heterocyclic carbene (NHC) complexes that are stable in the presence of biological thiols are reported. A representative complex, [Pd(C^N^N)(N,N'-nBu2 NHC)](CF3 SO3 ) (Pd1 d, HC^N^N=6-phenyl-2,2'-bipyridine, N,N'-nBu2 NHC=N,N'-di-n-butylimidazolylidene), displays potent killing activity toward cancer cell lines (IC50 =0.09-0.5 µm) but is less cytotoxic toward a normal human fibroblast cell line (CCD-19Lu, IC50 =11.8 µm). In vivo anticancer studies revealed that Pd1 d significantly inhibited tumor growth in a nude mice model. Proteomics data and in vitro biochemical assays reveal that Pd1 d exerts anticancer effects, including inhibition of an epidermal growth factor receptor pathway, induction of mitochondrial dysfunction, and antiangiogenic activity to endothelial cells.

Conjugated Polyelectrolyte-Induced Self-Assembly of Alkynylplatinum(II) 2,6-Bis(benzimidazol-2'-yl)pyridine Complexes.

Water-soluble cationic alkynylplatinum(II) 2,6-bis(benzimidazol-2'-yl)pyridine (bzimpy) complexes have been demonstrated to undergo supramolecular assembly with anionic polyelectrolytes in aqueous buffer solution. Metal-metal-to-ligand charge transfer (MMLCT) absorptions and triplet MMLCT ((3) MMLCT) emissions have been found in UV/Vis absorption and emission spectra of the electrostatic assembly of the complexes with non-conjugated polyelectrolytes, driven by Pt⋅⋅⋅Pt and π-π interactions among the complex molecules. Interestingly, the two-component ensemble formed by [Pt(bzimpy-Et){CCC6 H4 (CH2 NMe3 -4)}]Cl2 (1) with para-linked conjugated polyelectrolyte (CPE), PPE-SO3 (-) , shows significantly different photophysical properties from that of the ensemble formed by 1 with meta-linked CPE, mPPE-Ala. The helical conformation of mPPE-Ala allows the formation of strong mPPE-Ala-1 aggregates with Pt⋅⋅⋅Pt, electrostatic, and π-π interactions, as revealed by the large Stern-Volmer constant at low concentrations of 1. Together with the reasonably large Förster radius, large HOMO-LUMO gap and high triplet state energy of mPPE-Ala to minimize both photo-induced charge transfer (PCT) and Dexter triplet energy back-transfer (TEBT) quenching of the emission of 1, efficient Förster resonance energy transfer (FRET) from mPPE-Ala to aggregated 1 molecules and strong (3) MMLCT emission have been found, while the less strong PPE-SO3 (-) -1 aggregates and probably more efficient PCT and Dexter TEBT quenching would account for the lack of (3) MMLCT emission in the PPE-SO3 (-) -1 ensemble.

Supramolecular assembly of achiral alkynylplatinum(II) complexes and carboxylic ß-1,3-glucan into different helical handedness stabilized by Pt⋠⋠⋠Pt and/or π-π interactions.

Two-component ensembles of alkynylplatinum(II) terpyridine (tpy) complexes and carboxylic ß-1,3-glucan (CurCOOH) have been investigated by using spectroscopic and microscopic techniques, as well as time-dependent UV/Vis absorption and circular dichroism (CD) experiments. Microscopic images of [Pt(tpy){C≡CC6 H4 (CH2 NMe3 -4)}](OTf)2 (1) have revealed spherical nanostructures, whereas helical fibrous structures of different lengths, depending on the concentration of complex 1 and CurCOOH, were observed. The helical assemblies have been found to show low-energy metal-metal-to-ligand charge transfer (MMLCT) absorption and triplet MMLCT ((3) MMLCT) emission, which are indicative of Pt⋅⋅⋅Pt and/or π-π interactions between the complex 1 molecules. Interestingly, the ensemble has been demonstrated to show different handedness and even a change in handedness over time under different experimental conditions. Low temperatures, low concentrations of CurCOOH, high concentrations of complex 1, or successive addition of CurCOOH into complex 1 solution favor the formation of right-handed helical assemblies, whereas high temperatures, high concentrations of CurCOOH, low concentrations of complex 1, or single-batch addition of CurCOOH into complex 1 solution result in a fast chiroptical inversion of the ensemble solution, giving rise to the left-handed helical assemblies as the dominant species. The results have been rationalized by considering the competing kinetically and thermodynamically controlled assembly-elongation of the ensemble, which leads to the formation of right-handed and left-handed helical assemblies, respectively. The change in the handedness of the ensemble has been demonstrated to stem from the formation of two-component assemblies with supramolecular interactions, in contrast to the template- induced chiral amplification commonly observed in other systems.

Induced self-assembly of platinum(II) alkynyl complexes through specific interactions between citrate and guanidinium for proof-of-principle detection of citrate and an assay of citrate lyase.

Water-soluble alkynylplatinum(II) terpyridine complexes appended with guanidinium moieties, [Pt(tpy)(C≡C-Ar)][OTf]2 (tpy=terpyridine; OTf=trifluoromethanesulfonate; Ar=C6 H4-{NHC(=NH2(+))(NH2)}-4 (1), C6 H4-{CH2 NHC(=NH2(+))(NH2)}-4 (2)), and [Pt(tBu3 tpy)(C≡CC6H4-{NHC(=NH2(+))(NH2)}-4)][OTf]2 (3; tBu3 tpy=4,4',4''-tri-tert-butyl-2,2':6',2''-terpyridine), have been synthesized and characterized. The photophysical properties of the complexes have been studied. Based on the results of UV/Vis absorption, resonance light scattering, and dynamic light scattering experiments, in aqueous buffer solutions complexes 1 and 2 undergo aggregation in the presence of citrate through strong and specific electrostatic and hydrogen-bonding interactions with citrate. The emergence of a triplet metal-metal-to-ligand charge transfer ((3 MMLCT) emission in the near-infrared (NIR) region brought on by the induced self-assembly of complex 1 has been demonstrated for proof-of-principle detection of citrate with good sensitivity and selectivity over other mono- and dicarboxylate substrates in the tricarboxylic acid (TCA) cycle as well as phosphate and lactate anions. Such a good selectivity toward citrate has been rationalized by the high charge density of citrate under physiological conditions and specific interactions between the guanidinium moiety on complex 1 and citrate. Extension of the work to citrate detection in fetal bovine serum and real-time monitoring of the activity of citrate lyase by the NIR emission of complex 1 have also been demonstrated.

Dual pH- and temperature-responsive metallosupramolecular block copolymers with tunable critical micelle temperature by modulation of the self-assembly of NIR-emissive alkynylplatinum(II) Complexes induced by changes in hydrophilicity and electrostatic effects.

Terpyridylplatinum(II)-based metallosupramolecular triblock copolymers with hydrophilic alkynyl ligands have been synthesised and characterised. As a result of the intrinsic properties of Pluronics, reversible temperature-induced micellisation occurred at high temperature leading to aggregation of the platinum(II) complex moieties through Pt⋅⋅⋅Pt and π-π interactions, resulting in significant UV/Vis absorption and near-infrared (NIR) emission spectral changes. The critical micelle temperatures of the complexes were found to vary from 21 to 30 °C due to differences in the hydrophilicity of the alkynyl ligands and the electrostatic repulsions between the positively charged platinum(II) complex moieties. One of the complexes with pH-responsive CH2NMe2 groups on the alkynyl ligand was found to show NIR emission that is sensitive to both pH and temperature. Such dual-responsive behaviour has been ascribed to the modulation of the self-assembly of the complex moieties by temperature-induced micellisation and the changes in the hydrophilicity as well as electrostatic interactions upon protonation/deprotonation of the CH2NMe2 groups on the alkynyl ligand.

N-Acryloylindole-alkyne (NAIA) enables imaging and profiling new ligandable cysteines and oxidized thiols by chemoproteomics.

Cysteine has been exploited as the binding site of covalent drugs. Its high sensitivity to oxidation is also important for regulating cellular processes. To identify new ligandable cysteines which can be hotspots for therapy and to better study cysteine oxidations, we develop cysteine-reactive probes, N-acryloylindole-alkynes (NAIAs), which have superior cysteine reactivity owing to delocalization of π electrons of the acrylamide warhead over the whole indole scaffold. This allows NAIAs to probe functional cysteines more effectively than conventional iodoacetamide-alkyne, and to image oxidized thiols by confocal fluorescence microscopy. In mass spectrometry experiments, NAIAs successfully capture new oxidized cysteines, as well as a new pool of ligandable cysteines and proteins. Competitive activity-based protein profiling experiments further demonstrate the ability of NAIA to discover lead compounds targeting these cysteines and proteins. We show the development of NAIAs with activated acrylamide for advancing proteome-wide profiling and imaging ligandable cysteines and oxidized thiols.

A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues.

Hydrogen peroxide (H2O2) is a key member of the reactive oxygen species family of transient small molecules that has broad contributions to oxidative stress and redox signaling. The development of selective and sensitive chemical probes can enable the study of H2O2 biology in cell, tissue and animal models. Peroxymycin-1 is a histochemical activity-based sensing probe that responds to H2O2 via chemoselective boronate oxidation to release puromycin, which is then covalently incorporated into nascent proteins by the ribosome and can be detected by antibody staining. Here, we describe an optimized two-step, one-pot protocol for synthesizing Peroxymycin-1 with improved yields over our originally reported procedure. We also present detailed procedures for applying Peroxymycin-1 to a broad range of biological samples spanning cells to animal tissues for profiling H2O2 levels through histochemical detection by using commercially available anti-puromycin antibodies. The preparation of Peroxymycin-1 takes 9 h, the confocal imaging experiments of endogenous H2O2 levels across different cancer cell lines take 1 d, the dot blot analysis of mouse liver tissues takes 1 d and the confocal imaging of mouse liver tissues takes 3-4 d.

Liver cancer cells with nuclear MET overexpression release translation regulatory protein-enriched extracellular vesicles exhibit metastasis promoting activity.

MET receptor tyrosine kinase is a cell surface receptor that plays important role in embryonic development and tissue regeneration. Aberrant MET activation has been widely reported in different human cancers, making MET an attractive therapeutic target. The presence of truncated MET within the nucleus (nMET) with potential novel functions poses a great challenge to the current therapeutic strategies against MET surface receptor. Previous work has demonstrated the promoting effect of nMET in aggressive properties of hepatocellular carcinoma (HCC) cells by activating TAK1/NF-κB signalling pathway. Herein, we report the role of nMET in modulating tumour microenvironment and tumour metastasis mediated by extracellular vesicles (EVs). EVs released by nMET overexpressing cells enhanced cell motility and provoked metastasis. Proteomic profiling revealed the enrichment of translational regulatory proteins in EVs derived from nMET overexpressing cells. These proteins include eukaryotic initiation factor (EIF), ribosomal protein small subunit (RPS) and ribosomal protein larger subunit (RPL) gene families. Knockdown of EIF3I, RPS3A and RPL10 diminished the promoting effect of EVs in cell migration invasiveness and metastasis. In conclusion, the findings reveal an unrecognized capacity of nMET to augment HCC through the release of EVs with oncogenic effect. Targeting these translation-related proteins may serve as an alternative treatment for patients with nMET overexpression.

Induced self-assembly and Förster resonance energy transfer studies of alkynylplatinum(II) terpyridine complex through interaction with water-soluble poly(phenylene ethynylene sulfonate) and the proof-of-principle demonstration of this two-component ensemble for selective label-free detection of human serum albumin.

The interaction of conjugated polyelectrolyte, PPE-SO(3)(-), with platinum(II) complexes, [Pt(tpy)(C≡CC(6)H(4)-CH(2)NMe(3)-4)](OTf)(2) (1) and [Pt(tpy)(C≡C-CH(2)NMe(3))](OTf)(2) (2), has been studied by UV-vis, and steady-state and time-resolved emission spectroscopy. A unique FRET from PPE-SO(3)(-) to the aggregated complex 1 on the polymer chain with Pt···Pt interaction has been demonstrated, resulting in the growth of triplet metal-metal-to-ligand charge transfer ((3)MMLCT) emission in the near-infrared (NIR) region. This two-component ensemble has been employed in a "proof-of-principle" concept for the sensitive and selective label-free detection of human serum albumin (HSA) by the emission spectral changes in the visible and in the NIR region. The spectral changes have been ascribed to the disassembly of the polymer-metal complex aggregates upon the binding of PPE-SO(3)(-) to HSA, which is rich in arginine residues and hydrophobic patches, leading to the decrease in FRET from PPE-SO(3)(-) to the aggregated platinum(II) complex. The ensemble is found to have high selectivity toward HSA over a number of polyelectrolytes, proteins and small amino acids. This has been suggested to be a result of the extra stabilization gained from the Pt···Pt and π-π interactions in addition to the electrostatic and hydrophobic interactions found in the polymer-metal complex aggregates.

Genome-wide CRISPR-Cas9 knockout library screening identified PTPMT1 in cardiolipin synthesis is crucial to survival in hypoxia in liver cancer.

Hypoxia, low oxygen (O2), is a key feature of all solid cancers, including hepatocellular carcinoma (HCC). Genome-wide CRISPR-Cas9 knockout library screening is used to identify reliable therapeutic targets responsible for hypoxic survival in HCC. We find that protein-tyrosine phosphatase mitochondrial 1 (PTPMT1), an important enzyme for cardiolipin (CL) synthesis, is the most significant gene and ranks just after hypoxia-inducible factor (HIF)-1α and HIF-1ß as crucial to hypoxic survival. CL constitutes the mitochondrial membrane and ensures the proper assembly of electron transport chain (ETC) complexes for efficient electron transfer in respiration. ETC becomes highly unstable during hypoxia. Knockout of PTPMT1 stops the maturation of CL and impairs the assembly of ETC complexes, leading to further electron leakage and ROS accumulation at ETC in hypoxia. Excitingly, HCC cells, especially under hypoxic conditions, show great sensitivity toward PTPMT1 inhibitor alexidine dihydrochloride (AD). This study unravels the protective roles of PTPMT1 in hypoxic survival and cancer development.

Fluorescein-Containing Superoxide Probes with a Modular Copper-Based Trigger.

Fluorescein-derived superoxide probes featuring a copper(II) complex that can be activated by superoxide to initiate ether bond cleavage and uncage a fluorescein reporter for imaging in live cells are described. Compared to other superoxide sensing moieties, this bond cleavage strategy can be modularly adapted to fluorescent reporters with different properties without compromising the superoxide reactivity and selectivity. A green-emitting probe and its lysosome-targeting analogue have been successfully developed. Both probes are sensitive with more than 30-fold fluorescence enhancement towards superoxide and are highly selective with no significant response towards other reactive oxygen species. A structure-activity relationship study of the copper-based superoxide trigger showed that the secondary coordination environment of the copper(II) center is important for the superoxide reactivity and selectivity. The probes have been applied in imaging changes in intracellular superoxide level in live HeLa and HEK293T cells upon menadione stimulation and also in a cellular inflammation model in RAW 264.7 cells.

Nidogen 1-Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1.

In hepatocellular carcinoma (HCC) patients with extrahepatic metastasis, the lung is the most frequent site of metastasis. However, how the lung microenvironment favors disseminated cells remains unclear. Here, it is found that nidogen 1 (NID1) in metastatic HCC cell-derived extracellular vesicles (EVs) promotes pre-metastatic niche formation in the lung by enhancing angiogenesis and pulmonary endothelial permeability to facilitate colonization of tumor cells and extrahepatic metastasis. EV-NID1 also activates fibroblasts, which secrete tumor necrosis factor receptor 1 (TNFR1), facilitate lung colonization of tumor cells, and augment HCC cell growth and motility. Administration of anti-TNFR1 antibody effectively diminishes lung metastasis induced by the metastatic HCC cell-derived EVs in mice. In the clinical perspective, analysis of serum EV-NID1 and TNFR1 in HCC patients reveals their positive correlation and association with tumor stages suggesting the potential of these molecules as noninvasive biomarkers for the early detection of HCC. In conclusion, these results demonstrate the interplay of HCC EVs and activated fibroblasts in pre-metastatic niche formation and how blockage of their functions inhibits distant metastasis to the lungs. This study offers promise for the new direction of HCC treatment by targeting oncogenic EV components and their mediated pathways.