UBC9 stabilizes PFKFB3 to promote aerobic glycolysis and proliferation of glioblastoma cells.

Cancer cells prefer to utilizing aerobic glycolysis to generate energy and anabolic metabolic intermediates for cell growth. However, whether the activities of glycolytic enzymes can be regulated by specific posttranslational modifications, such as SUMOylation, in response to oncogenic signallings, thereby promoting the Warburg effect, remain largely unclear. Here, we demonstrate that phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key glycolytic enzyme, interacts with SUMO-conjugating enzyme UBC9 and is SUMOylated at K302 in glioblastoma cells. Expression of UBC9, which competitively prevents the binding of ubiquitin E3 ligase APC/C to PFKFB3 and subsequent PFKFB3 polyubiquitination, increases PFKFB3 stability and expression. Importantly, EGFR activation increases the interaction between UBC9 and PFKFB3, leading to increased SUMOylation and expression of PFKFB3. This increase is blocked by inhibition of EGFR-induced AKT activation whereas expression of activate AKT by itself was sufficient to recapitulate EGF-induced effect. Knockout of PFKFB3 expression decreases EGF-enhanced lactate production and GBM cell proliferation and this decrease was fully rescued by reconstituted expression of WT PFKFB3 whereas PFKFB3 K302R mutant expression abrogates EGF- and UBC9-regulated lactate production and GBM cell proliferation. These findings reveal a previously unknown mechanism underlying the regulation of the Warburg effect through the EGFR activation-induced and UBC9-mediated SUMOylation and stabilization of PFKFB3.

Base editing of the mutated TERT promoter inhibits liver tumor growth.

BACKGROUND AND AIMS: Base editing has shown great potential for treating human diseases with mutated genes. However, its potential for treating HCC has not yet been explored. APPROACH AND RESULTS: We employed adenine base editors (ABEs) to correct a telomerase reverse transcriptase ( TERT ) promoter mutation, which frequently occurs in various human cancers, including HCC. The mutated TERT promoter -124 C>T is corrected to -124 C by a single guide (sg) RNA-guided and deactivated Campylobacter jejuni Cas9 (CjCas9)-fused adenine base editor (CjABE). This edit impairs the binding of the E-twenty six/ternary complex factor transcription factor family, including E-twenty six-1 and GABPA, to the TERT promoter, leading to suppressed TERT promoter and telomerase activity, decreased TERT expression and cell proliferation, and increased cell senescence. Importantly, injection of adeno-associated viruses expressing sgRNA-guided CjABE or employment of lipid nanoparticle-mediated delivery of CjABE mRNA and sgRNA inhibits the growth of liver tumors harboring TERT promoter mutations. CONCLUSIONS: These findings demonstrate that a sgRNA-guided CjABE efficiently converts the mutated TERT promoter -124 C>T to -124 C in HCC cells and underscore the potential to treat HCC by the base editing-mediated correction of TERT promoter mutations.

The moonlighting function of glycolytic enzyme enolase-1 promotes choline phospholipid metabolism and tumor cell proliferation.

Aberrantly upregulated choline phospholipid metabolism is a novel emerging hallmark of cancer, and choline kinase α (CHKα), a key enzyme for phosphatidylcholine production, is overexpressed in many types of human cancer through undefined mechanisms. Here, we demonstrate that the expression levels of the glycolytic enzyme enolase-1 (ENO1) are positively correlated with CHKα expression levels in human glioblastoma specimens and that ENO1 tightly governs CHKα expression via posttranslational regulation. Mechanistically, we reveal that both ENO1 and the ubiquitin E3 ligase TRIM25 are associated with CHKα. Highly expressed ENO1 in tumor cells binds to I199/F200 of CHKα, thereby abrogating the interaction between CHKα and TRIM25. This abrogation leads to the inhibition of TRIM25-mediated polyubiquitylation of CHKα at K195, increased stability of CHKα, enhanced choline metabolism in glioblastoma cells, and accelerated brain tumor growth. In addition, the expression levels of both ENO1 and CHKα are associated with poor prognosis in glioblastoma patients. These findings highlight a critical moonlighting function of ENO1 in choline phospholipid metabolism and provide unprecedented insight into the integrated regulation of cancer metabolism by crosstalk between glycolytic and lipidic enzymes.

Tetrazine bioorthogonal chemistry makes nanotechnology a powerful toolbox for biological applications.

Bioorthogonal chemistry enables researchers to manipulate bioactive molecules in living systems. These highly selective and biocompatible reactions can be carried out in various complex environments. Over the past two decades, a considerable number of strides have been made to expand the capacities of bioorthogonal chemistry coupled with the aim to fine-tune present reactions for specific applications. The good points of bioorthogonal chemistry have pushed material chemists to integrate bioorthogonal chemistry with nanotechnologies to broaden the biological applications of nanomaterials. Notably, bioorthogonal nanotechnologies fundamentally rely on, more than half, according to our investigation, tetrazine bioorthogonal chemistry (TBC) to function as bioorthogonal handles to react with target agents owing to the extremely rapid kinetics and high selectivities of TBC. Its utilization in combination with nanotechnologies has led to developments in various areas of biomedicine, such as in situ drug activation and targeted delivery, bioimaging and biosensing, and the understanding of cell-biomolecule interactions. Given the fantastic past achievements and the rapid developments in tetrazine bioorthogonal technologies, the future is certainly very bright.

Targeting Trop2 in solid tumors: a look into structures and novel epitopes.

Trophoblast cell surface antigen 2 (Trop2) exhibits limited expression in normal tissues but is over-expressed across various solid tumors. The effectiveness of anti-Trop2 antibody-drug conjugate (ADC) in managing breast cancer validates Trop2 as a promising therapeutic target for cancer treatment. However, excessive toxicity and a low response rate of ADCs pose ongoing challenges. Safer and more effective strategies should be developed for Trop2-positive cancers. The dynamic structural attributes and the oligomeric assembly of Trop2 present formidable obstacles to the progression of innovative targeted therapeutics. In this review, we summarize recent advancements in understanding Trop2's structure and provide an overview of the epitope characteristics of Trop2-targeted agents. Furthermore, we discuss the correlation between anti-Trop2 agents' epitopes and their respective functions, particularly emphasizing their efficacy and specificity in targeted therapies.

Tetrazine bioorthogonal chemistry derived in vivo imaging.

Bioorthogonal chemistry represents plenty of highly efficient and biocompatible reactions that proceed selectively and rapidly in biological situations without unexpected side reactions towards miscellaneous endogenous functional groups. Arise from the strict demands of physiological reactions, bioorthogonal chemical reactions are natively selective transformations that are rarely found in biological environments. Bioorthogonal chemistry has long been applied to tracking and real-time imaging of biomolecules in their physiological environments. Thereinto, tetrazine bioorthogonal reactions are particularly important and have increasing applications in these fields owing to their unique properties of easily controlled fluorescence or radiation off-on mechanism, which greatly facilitate the tracking of real signals without been disturbed by background. In this mini review, tetrazine bioorthogonal chemistry for in vivo imaging applications will be attentively appraised to raise some guidelines for prior tetrazine bioorthogonal chemical studies.

METTL3 promotes tumour development by decreasing APC expression mediated by APC mRNA N6-methyladenosine-dependent YTHDF binding.

The adenomatous polyposis coli (APC) is a frequently mutated tumour suppressor gene in cancers. However, whether APC is regulated at the epitranscriptomic level remains elusive. In this study, we analysed TCGA data and separated 200 paired oesophageal squamous cell carcinoma (ESCC) specimens and their adjacent normal tissues and demonstrated that methyltransferase-like 3 (METTL3) is highly expressed in tumour tissues. m6A-RNA immunoprecipitation sequencing revealed that METTL3 upregulates the m6A modification of APC, which recruits YTHDF for APC mRNA degradation. Reduced APC expression increases the expression of ß-catenin and ß-catenin-mediated cyclin D1, c-Myc, and PKM2 expression, thereby leading to enhanced aerobic glycolysis, ESCC cell proliferation, and tumour formation in mice. In addition, downregulated APC expression correlates with upregulated METTL3 expression in human ESCC specimens and poor prognosis in ESCC patients. Our findings reveal a mechanism by which the Wnt/ß-catenin pathway is upregulated in ESCC via METTL3/YTHDF-coupled epitranscriptomal downregulation of APC.

Prognostic Impact of PCK1 Protein Kinase Activity-Dependent Nuclear SREBP1 Activation in Non-Small-Cell Lung Carcinoma.

Metabolic enzymes can perform non-metabolic functions and play critical roles in the regulation of a variety of important cellular activities. Phosphoenolpyruvate carboxykinase 1 (PCK1), a gluconeogenesis enzyme, was recently identified as an AKT-regulated protein kinase that phosphorylates INSIG1/2 to promote nuclear SREBP1-dependent lipogenesis. However, the relationship of this regulation with the progression of non-small-cell lung carcinoma (NSCLC) is unclear. Here, we demonstrate that epidermal growth factor receptor (EGFR) activation induces AKT-dependent PCK1 pS90, PCK1-mediated INSIG1 pS207/INSIG2 pS151, and nuclear SREBP1 accumulation in NSCLC cells. In addition, the expression levels of AKT pS473, PCK1 pS90, INSIG1 pS207/INSIG2 pS151, and nuclear SREBP1 are higher in 451 analyzed human NSCLC specimens than in their adjacent normal tissues and positively correlated with each other in the tumor specimens. Furthermore, the expression levels of PCK1 pS90, INSIG1 pS207/INSIG2 pS151, and nuclear SREBP1 are associated with TNM stage and progression in NSCLC. Importantly, levels of PCK1 pS90 or INSIG1 pS207/INSIG2 pS151 are positively correlated with poor prognosis in NSCLC patients, and the combined expression value of the PCK1 and INSIG1/2 phosphorylation has a better prognostic value than that of each individual protein phosphorylation value and is an independent prognostic marker for NSCLC. These findings reveal the role of PCK1-mediated nuclear SREBP1 activation in NSCLC progression and highlight the potential to target the protein kinase activity of PCK1 for the diagnosis and treatment of human NSCLC.

Association of phosphoenolpyruvate carboxykinase 1 protein kinase activity-dependent sterol regulatory element-binding protein 1 activation with prognosis of oesophageal carcinoma.

BACKGROUND: Metabolic enzymes have non-canonical functions and play vital roles in the regulation of various cellular activities. Phosphoenolpyruvate carboxykinase 1 (PCK1), a gluconeogenic enzyme, was recently identified as an AKT-dependent protein kinase and promoted sterol regulatory element-binding protein 1 (SREBP1)-dependent lipogenesis. However, association of this protein kinase activity of PCK1 with progression of oesophageal squamous cell carcinoma (ESCC) is unclear. METHODS: We examined 200 ESCC patient samples and prognosis using immunohistochemistry, multivariate Cox regression and Kaplan-Meier Plot analyses. RESULTS: We show that the expression levels of AKT pS473, AKT-regulated PCK1 pS90, PCK1-mediated INSIG1 pS207/INSIG2 pS151 and nuclear SREBP1 were higher in analysed 200 human ESCC specimens than in their adjacent non-tumour tissues; the expression levels of these proteins were significantly and positively correlated with each other in tumour specimens. In addition, the expression levels of PCK1 pS90, INSIG1 pS207/INSIG2 pS151 and SREBP1 were associated with the tumour, node and metastasis stage and progression in ESCC. Importantly, levels of PCK1 pS90 or INSIG1 pS207/INSIG2 pS151 or nuclear SREBP1 were positively correlated with poor prognosis in patients with ESCC, and the combined expression values of PCK1 pS90, INSIG1 pS207/INSIG2 pS151 and nuclear SREBP1 had a better prognostic value than that of each individual protein expression value and was an independent prognostic marker for ESCC. CONCLUSION: These findings reveal the role of PCK1 protein kinase activity-dependent SREBP1 activation in ESCC progression. The regulation of SREBP1 by AKT activation-dependent PCK1 protein kinase activity may provide the potential for the diagnosis and treatment of human ESCC.

Keratin 14-high subpopulation mediates lung cancer metastasis potentially through Gkn1 upregulation.

Metastasis is the leading cause of lung cancer-related death. Elucidating the metastasis process can provide new avenues to inhibit this malignant behavior of cancer cells. Here we found that human lung cancers with high Keratin 14 (K14) expression were associated with nodal metastasis and poor survival. Using the KrasG12D/Trp53L/L lung cancer mouse model, we confirmed that K14-high cancer cells harbored increased metastatic potential. Mechanistic investigation revealed that Gastrokine 1 (Gkn1) expression positively correlated with K14 level, cancer metastasis, and poor patient survival. Importantly, ectopic expression of Gkn1 enhanced the metastatic capability of K14-low cells in vitro and in vivo, whereas knockdown of Gkn1 did the opposite, indicating the importance of Gkn1 in mediating the metastasis of K14-high cells. Further study demonstrated that Gkn1 expression conferred K14-high cells resistance to anoikis, which is critical for cancer metastasis. Collectively, our findings demonstrate that K14-high cells contribute to lung cancer metastasis potentially through inhibition of anoikis via upregulation of Gkn1.

Cullin5 deficiency promotes small-cell lung cancer metastasis by stabilizing integrin ß1.

Metastasis is the dominant cause of patient death in small-cell lung cancer (SCLC), and a better understanding of the molecular mechanisms underlying SCLC metastasis may potentially improve clinical treatment. Through genome-scale screening for key regulators of mouse Rb1-/- Trp53-/- SCLC metastasis using the pooled CRISPR/Cas9 library, we identified Cullin5 (CUL5) and suppressor of cytokine signaling 3 (SOCS3), two components of the Cullin-RING E3 ubiquitin ligase complex, as top candidates. Mechanistically, the deficiency of CUL5 or SOCS3 disrupted the functional formation of the E3 ligase complex and prevented the degradation of integrin ß1, which stabilized integrin ß1 and activated downstream focal adhesion kinase/SRC (FAK/SRC) signaling and eventually drove SCLC metastasis. Low expression levels of CUL5 and SOCS3 were significantly associated with high integrin ß1 levels and poor prognosis in a large cohort of 128 clinical patients with SCLC. Moreover, the CUL5-deficient SCLCs were vulnerable to the treatment of the FDA-approved SRC inhibitor dasatinib. Collectively, this work identifies the essential role of CUL5- and SOCS3-mediated integrin ß1 turnover in controlling SCLC metastasis, which might have therapeutic implications.

Gossypol induces pyroptosis in mouse macrophages via a non-canonical inflammasome pathway.

Gossypol, a polyphenolic compound isolated from cottonseeds, has been reported to possess many pharmacological activities, but whether it can influence inflammasome activation remains unclear. In this study, we found that in mouse macrophages, gossypol induced cell death characterized by rapid membrane rupture and robust release of HMGB1 and pro-caspase-11 comparable to ATP treatment, suggesting an induction of pyroptotic cell death. Unlike ATP, gossypol induced much low levels of mature interleukin-1ß (IL-1ß) secretion from mouse peritoneal macrophages primed with LPS, although it caused pro-IL-1ß release similar to that of ATP. Consistent with this, activated caspase-1 responsible for pro-IL-1ß maturation was undetectable in gossypol-treated peritoneal macrophages. Besides, RAW 264.7 cells lacking ASC expression and caspase-1 activation also underwent pyroptotic cell death upon gossypol treatment. In further support of pyroptosis induction, both pan-caspase inhibitor and caspase-1 subfamily inhibitor, but not caspase-3 inhibitor, could sharply suppress gossypol-induced cell death. Other canonical pyroptotic inhibitors, including potassium chloride and N-acetyl-l-cysteine, could suppress ATP-induced pyroptosis but failed to inhibit or even enhanced gossypol-induced cell death, whereas nonspecific pore-formation inhibitor glycine could attenuate this process, suggesting involvement of a non-canonical pathway. Of note, gossypol treatment eliminated thioglycollate-induced macrophages in the peritoneal cavity with recruitment of other leukocytes. Moreover, gossypol administration markedly decreased the survival of mice in a bacterial sepsis model. Collectively, these results suggested that gossypol induced pyroptosis in mouse macrophages via a non-canonical inflammasome pathway, which raises a concern for its in vivo cytotoxicity to macrophages.

The BH3-mimetic gossypol and noncytotoxic doses of valproic acid induce apoptosis by suppressing cyclin-A2/Akt/FOXO3a signaling.

Previously we reported that valproic acid (VPA) acts in synergy with GOS to enhance cell death in human DU145 cells. However, the underlying mechanism remains elusive. In this study, we observed that such synergistic cytotoxicity of GOS and VPA could be extended to human A375, HeLa, and PC-3 cancer cells. GOS and VPA co-treatment induced robust apoptosis as evidenced by caspase-8/-9/-3 activation, PARP cleavage, and nuclear fragmentation. GOS and VPA also markedly decreased cyclin A2 protein expression. Owing to the reduction of cyclin A2, Akt signaling was suppressed, leading to dephosphorylation of FOXO3a. Consequently, FOXO3a was activated and the expression of its target genes, including pro-apoptotic FasL and Bim, was upregulated. Supporting this, FOXO3a knockdown attenuated FasL and Bim upregulation and apoptosis induction in GOS+VPA-treated cells. Furthermore, blocking proteasome activity by MG132 prevented the downregulation of cyclin A2, dephosphorylation of Akt and FOXO3a, and induction of apoptosis in cells co-treated with GOS and VPA. In mouse model, GOS and VPA combination significantly inhibited the growth of A375 melanoma xenografts. Our findings indicate that GOS and VPA co-treatment induces apoptosis in human cancer cells by suppressing the cyclin-A2/Akt/FOXO3a pathway.

Piperine metabolically regulates peritoneal resident macrophages to potentiate their functions against bacterial infection.

Pepper, a daily-used seasoning for promoting appetite, is widely used in folk medicine for treating gastrointestinal diseases. Piperine is the major alkaloid in pepper and possesses a wide range of pharmacological activities. However, the mechanism for linking metabolic and medicinal activities of piperine remains unknown. Here we report that piperine robustly boosts mTORC1 activity by recruiting more system L1 amino acid transporter (SLC7A5/SLC3A2) to the cell membrane, thus promoting amino acid metabolism. Piperine-induced increase of mTORC1 activity in resident peritoneal macrophages (pMΦs) is correlated with enhanced production of IL-6 and TNF-α upon LPS stimulation. Such an enhancement of cytokine production could be abrogated by inhibitors of the mTOR signaling pathway, indicating mTOR's action in this process. Moreover, piperine treatment protected resident pMΦs from bacterium-induced apoptosis and disappearance, and increased their bacterial phagocytic ability. Consequently, piperine administration conferred mice resistance against bacterial infection and even sepsis. Our data highlight that piperine has the capacity to metabolically reprogram peritoneal resident macrophages to fortify their innate functions against bacterial infection.

Cucurbitacin E Induces Autophagy via Downregulating mTORC1 Signaling and Upregulating AMPK Activity.

Cucurbitacins, the natural triterpenoids possessing many biological activities, have been reported to suppress the mTORC1/p70S6K pathway and to induce autophagy. However, the correlation between such activities is largely unknown. In this study, we addressed this issue in human cancer cells in response to cucurbitacin E (CuE) treatment. Our results showed that CuE induced autophagy as evidenced by the formation of LC3-II and colocalization of punctate LC3 with the lysosomal marker LAMP2 in HeLa and MCF7 cells. However, CuE induced much lower levels of autophagy in ATG5-knocked down cells and failed to induce autophagy in DU145 cells lacking functional ATG5 expression, suggesting the dependence of CuE-induced autophagy on ATG5. Consistent with autophagy induction, mTORC1 activity (as reflected by p70S6K and ULK1S758 phosphorylation) was inhibited by CuE treatment. The suppression of mTORC1 activity was further confirmed by reduced recruitment of mTOR to the lysosome, which is the activation site of mTORC1. In contrast, CuE rapidly activated AMPK leading to increased phosphorylation of its substrates. AMPK activation contributed to CuE-induced suppression of mTORC1/p70S6K signaling and autophagy induction, since AMPK knockdown diminished these effects. Collectively, our data suggested that CuE induced autophagy in human cancer cells at least partly via downregulation of mTORC1 signaling and upregulation of AMPK activity.

The critical molecular interconnections in regulating apoptosis and autophagy.

Apoptosis and autophagy are both highly regulated biological processes that have important roles in development, differentiation, homeostasis, and disease. These processes may take place independently, with autophagy being cytoprotective for preventing cells from apoptosis and apoptosis blocking autophagy. But in most circumstances, both may be induced sequentially with autophagy preceding apoptosis. The simultaneous activation of both processes has been observed not only in experimental settings but also in pathophysiological conditions. In fact, these two pathways are tightly connected with each other by substantial interplays between them, enabling the coordinated regulation of cell fates by these two pathways. They share some common upstream signaling components, and some components of one pathway may play important roles in the other, and vice versa. Such proteins represent the critical interconnections of the two pathways, which seem to determine the cell for survival or death. Here several critical molecular interconnections between apoptosis and autophagy pathways are reviewed, with their action mechanisms being highlighted.

Chloroquine differentially modulates inflammatory cytokine expression in RAW 264.7 cells in response to inactivated Staphylococcus aureus.

The anti-inflammatory property of chloroquine (CQ) has long been recognized. This study aimed to investigate the effect of CQ on proinflammatory cytokine expression in RAW 264.7 macrophages stimulated with heat-inactivated Staphylococcus aureus cells (SAC). The results showed that CQ treatment reduced the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and CC chemokine ligand 2 (CCL2) in culture medium but increased intracellular protein expression of TNF-α and CCL2 in SAC-activated cells. However, CQ showed differential effects on their messenger RNA (mRNA) levels: it reduced IL-6 mRNA expression, increased CCL2 mRNA levels, but had no effect on TNF-α mRNA. The SAC-activated inflammatory signaling pathways were slightly or minimally affected by CQ treatment. Additionally, CQ increased the accumulation of pro-TNF-α proteins within cells, suggesting an inhibition of pro-TNF-α processing and secretion. Collectively, CQ differentially modulated proinflammatory cytokine expression in Gram-positive bacterium-activated macrophages at multiple regulatory layers in the course of their biosynthesis.

Virion-associated viral proteins of a Chinese giant salamander (Andrias davidianus) iridovirus (genus Ranavirus) and functional study of the major capsid protein (MCP).

Chinese giant salamander iridovirus (CGSIV) is the emerging causative agent to farmed Chinese giant salamanders in nationwide China. CGSIV is a member of the common midwife toad ranavirus (CMTV) subset of the amphibian-like ranavirus (ALRV) in the genus Ranavirus of Iridoviridae family. However, viral protein information on ALRV is lacking. In this first proteomic analysis of ALRV, 40 CGSIV viral proteins were detected from purified virus particles by liquid chromatography-tandem mass spectrometry analysis. The transcription products of all 40 identified virion proteins were confirmed by reverse transcription polymerase chain reaction analysis. Temporal expression pattern analysis combined with drug inhibition assay indicated that 37 transcripts of the 40 virion protein genes could be classified into three temporal kinetic classes, namely, 5 immediate early, 12 delayed early, and 20 late genes. The presence of major capsid proteins (MCP, ORF019L) and a proliferating cell nuclear antigen (ORF025L) was further confirmed by Western blot analysis. The functions of MCP were also determined by small interfering RNA (siRNA)-based knockdown assay and anti-recombinant MCP serum-based neutralization testing. At low dosages of CGSIV, siRNA-based knockdown of the MCP gene effectively inhibited CGSIV replication in fathead minnow cells. The antiviral effect observed in the anti-MCP serum-based neutralization test confirms the crucial function of the MCP gene in CGSIV replication. Taken together, detailed information on the virion-associated viral proteins of ALRV is presented for the first time. Our results also provide evidence that MCP is essential for CGSIV replication in vitro.