AMPK: The energy sensor at the crossroads of aging and cancer.

AMP-activated protein kinase (AMPK) is a protein kinase that plays versatile roles in response to a variety of physiological stresses, including glucose deprivation, hypoxia, and ischemia. As a kinase with pleiotropic functions, it plays a complex role in tumor progression, exhibiting both tumor-promoting and tumor-suppressing activities. On one hand, AMPK enhances cancer cell proliferation and survival, promotes cancer metastasis, and impairs anti-tumor immunity. On the other hand, AMPK inhibits cancer cell growth and survival and stimulates immune responses in a context-dependent manner. Apart from these functions, AMPK plays a key role in orchestrating aging and aging-related disorders, including cardiovascular diseases (CVD), Osteoarthritis (OA), and Diabetes. In this review article, we summarized the functions of AMPK pathway based on its oncogenic and tumor-suppressive roles and highlighted the importance of AMPK pathway in regulating cellular aging. We also spotlighted the significant role of various signaling pathways, activators, and inhibitors of AMPK in serving as therapeutic strategies for anti-cancer and anti-aging therapy.

Un-methylation of NUDT21 represses docosahexaenoic acid biosynthesis contributing to enzalutamide resistance in prostate cancer.

AIMS: The recent approval of enzalutamide for metastatic castration-sensitive prostate cancer underscores its growing clinical significance, raising concerns about emerging resistance and limited treatment options. While the reactivation of the androgen receptor (AR) and other genes plays a role in enzalutamide resistance, identifications of novel underlying mechanism with therapeutic potential in enzalutamide-resistant (EnzaR) cells remain largely elusive. METHODS: Drug-resistant prostate cancer cell lines, animal models, and organoids were utilized to examine NUDT21 function by transcriptomic and metabolomic analyses through loss-of-function and gain-of-function assays. Notably, a mono-methylation monoclonal antibody and conditional-knockin transgenic mouse model of NUDT21 were generated for evaluating its function. RESULTS: NUDT21 overexpression acts as a crucial alternative polyadenylation (APA) mediator, supported by its oncogenic role in prostate cancer. PRMT7-mediated mono-methylation of NUDT21 induces a shift in 3'UTR usage, reducing oncogenicity. In contrast, its un-methylation promotes cancer growth and cuproptosis insensitivity in EnzaR cells by exporting toxic copper and suppressing docosahexaenoic acid (DHA) biosynthesis. Crucially, NUDT21 inhibition or DHA supplementation with copper ionophore holds therapeutic promise for EnzaR cells. CONCLUSIONS: The un-methylation of NUDT21-mediated 3'UTR shortening unveils a novel mechanism for enzalutamide resistance, and our findings offer innovative strategies for advancing the treatment of prostate cancer patients experiencing enzalutamide resistance.

Protocol to analyze the role of various metabolites in impacting global RNA 5-methylcytosine levels in cultured cells by dot blot.

RNA 5-methylcytosine (m5C) modification critically impacts many biological processes. Here, we provide a protocol to analyze the role of various metabolites in impacting global RNA m5C levels in cultured cells by dot blot. We describe steps for treating cultured cells with various metabolites; extracting, quantifying, and denaturing RNA samples; and performing dot blot to detect global RNA m5C levels in cultured cells. We then detail procedures to verify the input loading by methylene blue staining and quantify using ImageJ. For complete details on the use and execution of this protocol, please refer to Chen et al.1.

Phosphate burden induces vascular calcification through a NLRP3-caspase-1-mediated pyroptotic pathway.

AIMS: The aim of this study is to clarify the role of NLRP3 inflammasome in phosphate burden-induced vascular smooth muscle cell (VSMC) calcification. MAIN METHODS: VSMC calcification was induced using a high concentration of inorganic phosphate. After pharmacological inhibition or genetic silencing of the NLRP3 inflammasome, pyroptosis, or potassium efflux, the cells were examined by RT-qPCR, immunofluorescence, and western blotting to identify the NLRP3-mediated pathway for VSMC calcification. KEY FINDINGS: Calcified VSMCs with α-smooth muscle actin (α-SMA) disarray presented features of pyroptosis, including caspase-1 maturation, cleaved gasdermin D (GSDMD), and a high supernatant level of lactate dehydrogenase A. Pharmacological inhibitions of caspase-1 and pyroptosis attenuated VSMC calcification, whereas interleukin-1ß receptor antagonism did not. Unlike canonical NLRP3 activation, osteogenic VSMCs did not upregulate NLRP3 expression. However, NLRP3 genetic silencing or inhibitions, which targets different domains of the NLRP3 protein, could ameliorate VSMC calcification by aborting caspase-1 and GSDMD activation. Furthermore, potassium efflux through the inward-rectifier potassium channel, and not through the P2X7 receptor, triggered NLRP3 inflammasome activation and VSMC calcification. SIGNIFICANCE: In the present study, we identified a potassium efflux-triggered NLRP3-caspase-1-mediated pyroptotic pathway for VSMC calcification that is unique and different from the canonical NLRP3 inflammasome activation. Therefore, targeting this pathway may serve as a novel therapeutic strategy for vascular calcification.

NSUN2 is a glucose sensor suppressing cGAS/STING to maintain tumorigenesis and immunotherapy resistance.

Glucose metabolism is known to orchestrate oncogenesis. Whether glucose serves as a signaling molecule directly regulating oncoprotein activity for tumorigenesis remains elusive. Here, we report that glucose is a cofactor binding to methyltransferase NSUN2 at amino acid 1-28 to promote NSUN2 oligomerization and activation. NSUN2 activation maintains global m5C RNA methylation, including TREX2, and stabilizes TREX2 to restrict cytosolic dsDNA accumulation and cGAS/STING activation for promoting tumorigenesis and anti-PD-L1 immunotherapy resistance. An NSUN2 mutant defective in glucose binding or disrupting glucose/NSUN2 interaction abolishes NSUN2 activity and TREX2 induction leading to cGAS/STING activation for oncogenic suppression. Strikingly, genetic deletion of the glucose/NSUN2/TREX2 axis suppresses tumorigenesis and overcomes anti-PD-L1 immunotherapy resistance in those cold tumors through cGAS/STING activation to facilitate apoptosis and CD8+ T cell infiltration. Our study identifies NSUN2 as a direct glucose sensor whose activation by glucose drives tumorigenesis and immunotherapy resistance by maintaining TREX2 expression for cGAS/STING inactivation.

Iron promotes glycolysis to drive colon tumorigenesis.

Colorectal cancer (CRC) is the third most common cancer and is also the third leading cause of cancer-related death in the USA. Understanding the mechanisms of growth and progression of CRC is essential to improve treatment. Macronutrients such as glucose are energy source for a cell. Many tumor cells exhibit increased aerobic glycolysis. Increased tissue micronutrient iron levels in both mice and humans are also associated with increased colon tumorigenesis. However, if iron drives colon carcinogenesis via affecting glucose metabolism is still not clear. Here we found the intracellular glucose levels in tumor colonoids were significantly increased after iron treatment. 13C-labeled glucose flux analysis indicated that the levels of several labeled glycolytic products were significantly increased, whereas several tricarboxylic acid cycle intermediates were significantly decreased in colonoids after iron treatment. Mechanistic studies showed that iron upregulated the expression of glucose transporter 1 (GLUT1) and mediated an inhibition of the pyruvate dehydrogenase (PDH) complex function via directly binding with tankyrase and/or pyruvate dehydrogenase kinase (PDHK) 3. Pharmacological inhibition of GLUT1 or PDHK reactivated PDH complex function and reduced high iron diet-enhanced tumor formation. In conclusion, excess iron promotes glycolysis and colon tumor growth at least partly through the inhibition of the PDH complex function.

Posttranslational regulation of liver kinase B1 in human cancer.

Liver kinase B1 (LKB1) is a serine-threonine kinase that participates in multiple cellular and biological processes, including energy metabolism, cell polarity, cell proliferation, cell migration, and many others. LKB1 is initially identified as a germline-mutated causative gene in Peutz-Jeghers syndrome and is commonly regarded as a tumor suppressor due to frequent inactivation in a variety of cancers. LKB1 directly binds and activates its downstream kinases including the AMP-activated protein kinase (AMPK) and AMPK-related kinases by phosphorylation, which has been intensively investigated for the past decades. An increasing number of studies have uncovered the posttranslational modifications (PTMs) of LKB1 and consequent changes in its localization, activity, and interaction with substrates. The alteration in LKB1 function as a consequence of genetic mutations and aberrant upstream signaling regulation leads to tumor development and progression. Here, we review current knowledge about the mechanism of LKB1 in cancer and the contributions of PTMs, such as phosphorylation, ubiquitination, SUMOylation, acetylation, prenylation, and others, to the regulation of LKB1 function, offering new insights into the therapeutic strategies in cancer.

NLRP3 inflammasome activation, metabolic danger signals, and protein binding partners.

The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is an oligomeric complex that assembles in response to exogenous signals of pathogen infection and endogenous danger signals of non-microbial origin. When NLRP3 inflammasome assembly activates caspase-1, it promotes the maturation and release of the inflammatory cytokines interleukin-1B and IL-18. Aberrant activation of the NLRP3 inflammasome has been implicated in various diseases, including chronic inflammatory, metabolic, and cardiovascular diseases. The NLRP3 inflammasome can be activated through several principal mechanisms, including K+ efflux, lysosomal damage, and the production of mitochondrial reactive oxygen species. Interestingly, metabolic danger signals activate the NLRP3 inflammasome to induce metabolic diseases. NLRP3 contains three crucial domains: an N-terminal pyrin domain, a central nucleotide-binding domain, and a C-terminal leucine-rich repeat domain. Protein-protein interactions act as a 'pedal or brake' to control the activation of the NLRP3 inflammasome. In this review, we present the mechanisms underlying NLRP3 inflammasome activation after induction by metabolic danger signals or via protein-protein interactions with NLRP3 that likely occur in metabolic diseases. Understanding these mechanisms will enable the development of specific inhibitors to treat NLRP3-related metabolic diseases.

Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation.

Activating the macrophage NLRP3 inflammasome can promote excessive inflammation with severe cell and tissue damage and organ dysfunction. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and interleukin-1ß (IL-1ß) secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves crista ultrastructure, and attenuates mitochondrial reactive oxygen species (ROS) production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. Our study suggests a non-canonical role of mitochondrial PDHK in promoting mitochondrial stress and supporting NLRP3 inflammasome activation during acute inflammation.

Protocol for detecting macrophage-mediated cancer cell phagocytosis in vitro and in vivo.

Here, we present optimized approaches to identify the efficiency of cancer cell phagocytosis by macrophages in vitro and in vivo. We describe the preparation and co-culture of macrophages and cancer cells, followed by in vitro phagocytosis assay using flow cytometry and confocal microscopy, respectively. We then detail the establishment of xenograft tumor mouse model and the in vivo detecting of phagocytosis efficiency by flow cytometry and qRT-PCR. This protocol provides a convenient way to assess macrophage-mediated phagocytosis of cancer cells. For complete details on the use and execution of this protocol, please refer to Xu et al.1.

A transformer-based deep-learning approach for classifying brain metastases into primary organ sites using clinical whole-brain MRI images.

Treatment decisions for brain metastatic disease rely on knowledge of the primary organ site and are currently made with biopsy and histology. Here, we develop a deep-learning approach for accurate non-invasive digital histology with whole-brain magnetic resonance imaging (MRI) data. Contrast-enhanced T1-weighted and fast spoiled gradient echo brain MRI exams (n = 1,582) were preprocessed and input to the proposed deep-learning workflow for tumor segmentation, modality transfer, and primary site classification into one of five classes. Tenfold cross-validation generated an overall area under the receiver operating characteristic curve (AUC) of 0.878 (95% confidence interval [CI]: 0.873,0.883). These data establish that whole-brain imaging features are discriminative enough to allow accurate diagnosis of the primary organ site of malignancy. Our end-to-end deep radiomic approach has great potential for classifying metastatic tumor types from whole-brain MRI images. Further refinement may offer an invaluable clinical tool to expedite primary cancer site identification for precision treatment and improved outcomes.

Activation of embryonic/germ cell-like axis links poor outcomes of gliomas.

BACKGROUND: It is unclear which core events drive the malignant progression of gliomas. Earlier studies have revealed that the embryonic stem (ES) cell/early PGC state is associated with tumourigenicity. This study was designed to investigate the role of ES/PGC state in poor outcomes of gliomas. METHODS: Crispr-Cas9 technology, RT-PCR and animal experiments were used to investigate whether PGC-like cell formation play crucial roles in the tumorigenicity of human glioma cells. Bioinformatic analysis was used to address the link between ES/PGC developmental axis and glioma overall outcomes. RESULTS: Here, our findings showed that germ cell-like cells were present in human gliomas and cultured glioma cells and that the formation of germ cell-like cells was essential for glioma tumours. Bioinformatic analysis showed that the mRNA levels of genes related to embryonic/germ cell development could be detected in most gliomas. Our findings showed that the activation of genes related to reprogramming or the germ cell-like state alone seemed to be insufficient to lead to a malignant prognosis, whereas increased mRNA levels of genes related to the activation of the embryonic/germ cell-like cycle (somatic PGC-EGC-like cycle and somatic parthenogenetic embryo-like cycle) were positively correlated with malignant prognoses and poor clinical outcomes of gliomas. Genes related to the embryonic/germ cell cycle alone or in combination with the WHO grade or 1p19q codeletion status could be used to subdivide gliomas with distinct clinical behaviours. CONCLUSION: Together, our findings indicated that a crucial role of germ cell-like cell formation in glioma initiation as well as activation of genes related with the parthenogenetic embryo-like cycle and PGC-EGC-like cycle link to the malignant prognosis and poor outcomes of gliomas, which might provide a novel way to better understand the nature of and develop targeted therapies for gliomas as well as important markers for predicting clinical outcomes in gliomas.

Deregulated transcription factors in cancer cell metabolisms and reprogramming.

Metabolic reprogramming is an important cancer hallmark that plays a key role in cancer malignancies and therapy resistance. Cancer cells reprogram the metabolic pathways to generate not only energy and building blocks but also produce numerous key signaling metabolites to impact signaling and epigenetic/transcriptional regulation for cancer cell proliferation and survival. A deeper understanding of the mechanisms by which metabolic reprogramming is regulated in cancer may provide potential new strategies for cancer targeting. Recent studies suggest that deregulated transcription factors have been observed in various human cancers and significantly impact metabolism and signaling in cancer. In this review, we highlight the key transcription factors that are involved in metabolic control, dissect the crosstalk between signaling and transcription factors in metabolic reprogramming, and offer therapeutic strategies targeting deregulated transcription factors for cancer treatment.

Akt: a key transducer in cancer.

Growth factor signaling plays a pivotal role in diverse biological functions, such as cell growth, apoptosis, senescence, and migration and its deregulation has been linked to various human diseases. Akt kinase is a central player transmitting extracellular clues to various cellular compartments, in turn executing these biological processes. Since the discovery of Akt three decades ago, the tremendous progress towards identifying its upstream regulators and downstream effectors and its roles in cancer has been made, offering novel paradigms and therapeutic strategies for targeting human diseases and cancers with deregulated Akt activation. Unraveling the molecular mechanisms for Akt signaling networks paves the way for developing selective inhibitors targeting Akt and its signaling regulation for the management of human diseases including cancer.

Feasible Column Chromatography-Free, Multi-Gram Scale Synthetic Process of VH032 Amine, Which Could Enable Rapid PROTAC Library Construction.

PROTACs represent a promising modality that has gained significant attention for the treatment of cancer, Alzheimer's disease, and so forth. Due to limited structural information of the POI-PROTAC-E3 ligase ternary complex, the discovery of active PROTACs relies on the screening of diversity-oriented PROTAC libraries. VH032 amine is a key building block for the synthesis of VHL E3 ligase-based PROTACs. To construct VHL PROTAC libraries rapidly, the availability of VH032 amine is crucial. In this paper, we report a column chromatography-free process which enables the production of 42.5 g of VH032 amine hydrochloride in 65% overall yield with 97% purity in a week.

Postnatal Dexamethasone Therapy Impairs Brown Adipose Tissue Thermogenesis and Autophagy Flux in Neonatal Rat Pups.

Rationale: Very preterm infants may require dexamethasone (Dex) for facilitating extubation or treating bronchopulmonary dysplasia. However, Dex may result in disturbance of metabolisms. This study was to investigate the effects of postnatal short course Dex exposure on brown adipose tissue (BAT) in neonatal rats. Method: Neonatal rats received either three consecutive doses of daily Dex (0.2 mg/kg/day) or saline from postnatal P1 to P3. We investigated the effects of Dex on BAT including thermogenesis, mitochondrial dynamics and autophagy flux. We also compared diurnal temperature variation between preterm infants who received systemic corticosteroid and their treatment-naïve controls. Results: Postnatal Dex treatment induced growth retardation, BAT whitening, UCP1 downregulation and cold intolerance in neonatal rats. BAT mitochondria were damaged, evident by loss of normal number, structure, and alignment of cristae. Mitochondrial fission-fusion balance was disrupted and skewed toward increased fusion, reflected by increased OPA1 and MFN2 and decreased DRP1, FIS1 and phosphorylated MFF protein levels. Autophagosome synthesis was increased but clearance was inhibited, indicated by accumulation of p62 protein after Dex treatment and no further increase of LC3-II after chloroquine co-treatment. While autophagy modulators, including chloroquine and rapamycin, did not improve UCP1 downregulation and BAT whitening, AMPK activators could partially rescue these damages. We also demonstrated that preterm infants had higher diurnal temperature variation during corticosteroid treatment. Conclusions: Postnatal short course Dex impaired BAT mitochondrial function and autophagy flux in rat pups. AMPK activators had the potential to rescue the damage.

Discovery of fused benzimidazole-imidazole autophagic flux inhibitors for treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) with the absence of estrogen receptor (ER), progesterone receptor (PR) and HER2 ptotein, is the highly aggressive subtype of breast cancer that exhibits poor prognosis and high tumor recurrence. It is vital to develop effective agents regulating the core molecular pathway of TNBC. Through a medium throughput screening and iterative medicinal chemistry optimization, we identified compound 7h as an autophagic flux inhibitor, which showed potent activities against human TNBC (MDA-MB-231 and MDA-MB-468) cell lines with IC50 values of 8.3 µM, and 6.0 µM, respectively, which are comparable to the potency of 5-FU and Cisplatin, the first line therapies for TNBC. Extensive investigation of mechanisms of action indicated that 7h inhibits autophagic flux and sequential accumulation of p62, leading to DNA damage and disrepair in TNBC cells. Importantly, nuclear p62 accumulation induced by compound 7h results in the inhibition of RNF168-mediated chromatin ubiquitination and the degradation of HR-related proteins in regulating the DNA damage response (DDR) process. In in vivo studies, compound 7h completely suppressed tumor growth in the MDA-MB-231 xenograft model at a dose of 15 mg/kg/q.d. Our findings indicate that compound 7h is an autophagic flux inhibitor and induced the degradation of HR-related proteins. Compound 7h could be potentially developed as an anti-cancer therapeutics for TNBC.