VHL suppresses autophagy and tumor growth through PHD1-dependent Beclin1 hydroxylation.

The Von Hippel-Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α.

The interplay of the circadian clock and metabolic tumorigenesis.

The circadian clock and cell metabolism are both dysregulated in cancer cells through intrinsic cell-autonomous mechanisms and external influences from the tumor microenvironment. The intricate interplay between the circadian clock and cancer cell metabolism exerts control over various metabolic processes, including aerobic glycolysis, de novo nucleotide synthesis, glutamine and protein metabolism, lipid metabolism, mitochondrial metabolism, and redox homeostasis in cancer cells. Importantly, oncogenic signaling can confer a moonlighting function on core clock genes, effectively reshaping cellular metabolism to fuel cancer cell proliferation and drive tumor growth. These interwoven regulatory mechanisms constitute a distinctive feature of cancer cell metabolism.

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 hepatocarcinoma (HCC) has not yet explored. APPROACH AND RESULTS: We employed adenine base editors (ABEs) to correct a 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 ETS (ETS/TCF) transcription factor family, including ETS1 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.

Glycolytic enzyme HK2 promotes PD-L1 expression and breast cancer cell immune evasion.

Immune therapies targeting the PD-1/PD-L1 pathway have been employed in the treatment of breast cancer, which requires aerobic glycolysis to sustain breast cancer cells growth. However, whether PD-L1 expression is regulated by glycolysis in breast cancer cells remains to be further elucidated. Here, we demonstrate that glycolytic enzyme hexokinase 2 (HK2) plays a crucial role in upregulating PD-L1 expression. Under high glucose conditions, HK2 acts as a protein kinase and phosphorylates IκBα at T291 in breast cancer cells, leading to the rapid degradation of IκBα and activation of NF-κB, which enters the nucleus and promotes PD-L1 expression. Immunohistochemistry staining of human breast cancer specimens and bioinformatics analyses reveals a positive correlation between HK2 and PD-L1 expression levels, which are inversely correlated with immune cell infiltration and survival time of breast cancer patients. These findings uncover the intrinsic and instrumental connection between aerobic glycolysis and PD-L1 expression-mediated tumor cell immune evasion and underscore the potential to target the protein kinase activity of HK2 for breast cancer treatment.

Creatine kinase B suppresses ferroptosis by phosphorylating GPX4 through a moonlighting function.

Activation of receptor protein kinases is prevalent in various cancers with unknown impact on ferroptosis. Here we demonstrated that AKT activated by insulin-like growth factor 1 receptor signalling phosphorylates creatine kinase B (CKB) T133, reduces metabolic activity of CKB and increases CKB binding to glutathione peroxidase 4 (GPX4). Importantly, CKB acts as a protein kinase and phosphorylates GPX4 S104. This phosphorylation prevents HSC70 binding to GPX4, thereby abrogating the GPX4 degradation regulated by chaperone-mediated autophagy, alleviating ferroptosis and promoting tumour growth in mice. In addition, the levels of GPX4 are positively correlated with the phosphorylation levels of CKB T133 and GPX4 S104 in human hepatocellular carcinoma specimens and associated with poor prognosis of patients with hepatocellular carcinoma. These findings reveal a critical mechanism by which tumour cells counteract ferroptosis by non-metabolic function of CKB-enhanced GPX4 stability and underscore the potential to target the protein kinase activity of CKB for cancer treatment.

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.

FBXW7 attenuates tumor drug resistance and enhances the efficacy of immunotherapy.

FBXW7 (F-box and WD repeat domain containing 7) is a critical subunit of the Skp1-Cullin1-F-box protein (SCF), acting as an E3 ubiquitin ligase by ubiquitinating targeted protein. Through degradation of its substrates, FBXW7 plays a pivotal role in drug resistance in tumor cells and shows the potential to rescue the sensitivity of cancer cells to drug treatment. This explains why patients with higher FBXW7 levels exhibit higher survival times and more favorable prognosis. Furthermore, FBXW7 has been demonstrated to enhance the efficacy of immunotherapy by targeting the degradation of specific proteins, as compared to the inactivated form of FBXW7. Additionally, other F-box proteins have also shown the ability to conquer drug resistance in certain cancers. Overall, this review aims to explore the function of FBXW7 and its specific effects on drug resistance in cancer cells.

Bioinformatics analysis identified RGS4 as a potential tumor promoter in glioma.

Gliomas is the most common type of intracranial primary malignant tumor and it accounts for ∼80% of primary malignant tumors of the central nervous system. At present, surgical resection with adjuvant radiotherapy and temozolomide adjuvant chemotherapy combined with radiotherapy are the only standard treatments for glioma. However, but overall survival of patients is only 15 months. Glioma is resistant to radiotherapy and chemotherapy, and this malignant behavior leads to a high recurrence rate. Therefore, the use of therapeutics is usually ineffective. As a result, patients with glioma do not significantly benefit from standard treatment. There is therefore an urgent need to develop novel diagnostic approaches and, in particular, more effective treatment strategies. The application of gene expression microarrays provides a feasible and effective way to study gliomas. The present study therefore aimed to identify the key protein-coding genes of glioma using bioinformatics methods and thereby search, for novel biomarkers and therapeutic targets for the treatment of glioma. First, mRNA microarray datasets were selected and obtained from the Gene Expression Omnibus database to identify differentially expressed genes (DEGs) between gliomas and normal tissues. The DEGs were clarified using Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), Protein-Protein Interaction (PPI) network and statistical analysis. Subsequently, reverse transcription-quantitative PCR (RT-qPCR)and western blot were used to verify the results of the bioinformatics analysis. A total of 400 DEGs were identified in glioma and they were enriched in several cancer-related GO and KEGG pathways. In the PPI network, it was observed that G-protein signal regulatory protein 4 (RGS4), thymidine phosphorylase, collagen type VI alpha-1, Src homology 2 domain-containing transforming protein1(SHC1) and ring finger protein 135 exhibited a strong protein-protein interaction. Furthermore, . Subsequently, brain damaged tissues and glioma cell lines were selected for RT-qPCR and western blotting analysis. The results demonstrated that RGS4 was highly expressed in glioma cell lines. In conclusion, RGS4 may be a key protein-coding gene in glioma. RGS4 should therefore be studied further to verify its feasibility and effectiveness as a potential glioma biomarker and therapeutic target.

CNNArginineMe: A CNN structure for training models for predicting arginine methylation sites based on the One-Hot encoding of peptide sequence.

Protein arginine methylation (PRme), as one post-translational modification, plays a critical role in numerous cellular processes and regulates critical cellular functions. Though several in silico models for predicting PRme sites have been reported, new models may be required to develop due to the significant increase of identified PRme sites. In this study, we constructed multiple machine-learning and deep-learning models. The deep-learning model CNN combined with the One-Hot coding showed the best performance, dubbed CNNArginineMe. CNNArginineMe performed best in AUC scoring metrics in comparisons with several reported predictors. Additionally, we employed CNNArginineMe to predict arginine methylation proteome and performed functional analysis. The arginine methylated proteome is significantly enriched in the amyotrophic lateral sclerosis (ALS) pathway. CNNArginineMe is freely available at https://github.com/guoyangzou/CNNArginineMe.

Recent Advances of Degradation Technologies Based on PROTAC Mechanism.

PROTAC (proteolysis-targeting chimeras), which selectively degrades target proteins, has become the most popular technology for drug development in recent years. Here, we introduce the history of PROTAC, and summarize the recent advances in novel types of degradation technologies based on the PROTAC mechanism, including TF-PROTAC, Light-controllable PROTAC, PhosphoTAC, LYTAC, AUTAC, ATTEC, CMA, RNA-PROTAC and RIBOTACs. In addition, the clinical progress, current challenges and future prospects of degradation technologies based on PROTAC mechanism are discussed.

Myocyte enhancer factor 2D promotes hepatocellular carcinoma through AMOTL2/YAP signaling that inhibited by luteolin.

Hepatocellular carcinoma (HCC) is one of the deadliest malignancies in the world. There is a lack of effective treatment. Previous studies have shown that myocyte enhancer factor 2D (MEF2D) promotes the progression of HCC. Underlying mechanisms have not been fully elucidated. In this study, we reported experimental results obtained using double luciferase. Our results showed that AMOTL2, a negative regulator of Hippo/YAP signaling, and the MEF2 cis-acting element in the upstream region of its promoter bind to MEF2D, inhibiting its transcriptional expression. Studies confirmed that MEF2D affected the protein expression level of AMOTL2 and the YAP signaling activation. It promoted the migration and proliferation of hepatoma cells. We found that luteolin, a natural flavonoid, has anti-tumor activity in HCC cells by affecting YAP signaling transduction. In conclusion, we demonstrated that AMOTL2/YAP signaling is associated with MEF2D-related HCC progression. Luteolin is a promising anti-HCC compound for regulating this signaling.

Coupling HDAC4 with transcriptional factor MEF2D abrogates SPRY4-mediated suppression of ERK activation and elicits hepatocellular carcinoma drug resistance.

Hepatocellular carcinoma (HCC) lacks effective treatment, and the patients rapidly develop the acquired resistance to sorafenib with less defined mechanisms. Here, we demonstrate that transcriptional factor myocyte enhancer factor 2D (MEF2D) overexpression is detected in sorafenib-resistant HCC specimens and HCC cell lines and predicts poor prognosis of sorafenib-treated HCC patients. Mechanistically, MEF2D in complex with histone deacetylase HDAC4 directly binds to the SPRY4 promoter regions and suppresses the transcriptional expression of SPRY4, which is a negative regulator of MAPK/ERK signaling pathway. Inhibition of HDAC4 with its clinically used inhibitor induces SPRY4 expression and inhibition of ERK activity, resulting in sensitization of HCC cells to sorafenib-induced apoptosis and greatly improved inhibition of liver tumor growth in mice with sorafenib treatment. These findings highlight the critical role of coupling HDAC4 with MEF2D in activation of ERK by suppressing SPRY4 and underscore the great potential to improve HCC treatment by combined administration of sorafenib with HDAC4 inhibitors.

Fructose and fructose kinase in cancer and other pathologies.

Fructose metabolism and fructose kinase KHK-C/A are key factors in the development of lipid oversynthesis-promoted metabolic disorders and cancer. Here, we summarize and discuss the current knowledge about the specific features of fructose metabolism and the distinct roles of KHK-C and KHK-A in metabolic liver diseases and their relevant metabolic disorders and cancer, and we highlight the specific protein kinase activity of KHK-A in tumor development. In addition, different approaches that have been used to inhibit KHK and the exploration of KHK inhibitors in clinical treatment are introduced.

Eugenol protects cells against oxidative stress via Nrf2.

Eugenol is a naturally occurring compound that is present in a variety of plants and has previous been demonstrated to exert a number of bioactivities. However, the potential effects of Eugenol on cellular protection against oxidative stress remain poorly understood. In the present study, HEK-293 cells and the mouse fibroblast cell line NIH-3T3 cells were used as models to explore the effects of eugenol on H2O2-induced damage. Among the three natural compounds tested, namely eugenol, methyleugenol and acetyleugenol, eugenol was found to increase the transcriptional activity and expression level of nuclear factor erythroid 2-related factor 2 (Nrf2), a central regulator of cellular responses to oxidative stress, in a dose-dependent manner. The mRNA levels of Nrf2 target genes glutamate-cysteine ligase modifier regulatory subunit and glutathione S-transferase A1, were also found to be upregulated following eugenol treatment. Further study revealed that eugenol enhanced the stabilization and nuclear translocation of Nrf2. Additionally, treatment with eugenol was found to reduce intracellular ROS levels while increasing cellular resistance to H2O2, in a manner that was dependent on Nrf2. In conclusion, data from the present study suggest that eugenol is a protective agent against oxidative stress that exerts its effects through a Nrf2-dependent pathway, rendering eugenol and its derivatives to be promising candidates for the future development of antioxidants.

Acetylation-dependent regulation of PD-L1 nuclear translocation dictates the efficacy of anti-PD-1 immunotherapy.

Immunotherapies that target programmed cell death protein 1 (PD-1) and its ligand PD-L1 as well as cytotoxic T-lymphocyte-associated protein 4 (CTLA4) have shown impressive clinical outcomes for multiple tumours. However, only a subset of patients achieves durable responses, suggesting that the mechanisms of the immune checkpoint pathways are not completely understood. Here, we report that PD-L1 translocates from the plasma membrane into the nucleus through interactions with components of the endocytosis and nucleocytoplasmic transport pathways, regulated by p300-mediated acetylation and HDAC2-dependent deacetylation of PD-L1. Moreover, PD-L1 deficiency leads to compromised expression of multiple immune-response-related genes. Genetically or pharmacologically modulating PD-L1 acetylation blocks its nuclear translocation, reprograms the expression of immune-response-related genes and, as a consequence, enhances the anti-tumour response to PD-1 blockade. Thus, our results reveal an acetylation-dependent regulation of PD-L1 nuclear localization that governs immune-response gene expression, and thereby advocate targeting PD-L1 translocation to enhance the efficacy of PD-1/PD-L1 blockade.

Light-induced control of protein destruction by opto-PROTAC.

By hijacking endogenous E3 ligase to degrade protein targets via the ubiquitin-proteasome system, PROTACs (PRoteolysis TArgeting Chimeras) provide a new strategy to inhibit protein targets that were regarded as undruggable before. However, the catalytic nature of PROTAC potentially leads to uncontrolled degradation that causes systemic toxicity issues, limiting the application of PROTAC in the clinic. Here, we introduce a light-inducible switch on PROTACs, thereafter termed as opto-PROTAC, to enable the degradation of protein targets in a spatiotemporal manner. By adding a photolabile caging group on pomalidomide as a parental compound and two additional PROTACs, dBET1 and dALK, we demonstrated light-inducible protein degradation. These opto-PROTACs display no activities in the dark, while the restricted degradation can be induced at a specific time and rate by ultraviolet A irradiation. Our approach provides a generalizable platform for the development of light-controlled PROTACs and enables PROTAC to be a precision medicine.

A narrative review of critical factors for better efficacy of CD19 chimeric antigen receptor T cell therapy in the treatment of B cell malignancies.

B cell malignancies are classified as different types such as B cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL) and B cell non-Hodgkin lymphoma (NHL) based on cell surface expression of various clusters of differentiation molecules. CD19 is a B cell lineage-specific antigen which is expressed on malignant B cells in patients with B-ALL, CLL and NHL. Adoptive transfer of T cells that are genetically modified to express a CD19-specific chimeric antigen receptor (CAR) represents a promising clinical strategy for patients with B cell malignancies. CD19-CAR T cell therapy has achieved high response rates and durable remissions on B cell malignancies. However, the efficacy of CAR-T therapy is still inefficient and the critical factors for better efficacy remain unclear. In this review, we summarized the critical factors for better efficacy of CD19 CAR-T cells in B-lineage malignancies including B-ALL, B-CLL and lymphoma. T cell persistence, lymphodepletion regimen, CD3/CD28 beads treatment and no IL-2 administration to T cells were positively associated with better responses. The method of enhancing the persistence of CAR-T cells need to be further optimized in order to improve the clinical efficacy in the treatment of B cell malignancies. In order to improve the therapeutic effect of CAR-T therapy, new therapeutic strategies should be developed to make factors which influence efficacy the more beneficial.