Disruption of sugar nucleotide clearance is a therapeutic vulnerability of cancer cells.

Identifying metabolic steps that are specifically required for the survival of cancer cells but are dispensable in normal cells remains a challenge1. Here we report a therapeutic vulnerability in a sugar nucleotide biosynthetic pathway that can be exploited in cancer cells with only a limited impact on normal cells. A systematic examination of conditionally essential metabolic enzymes revealed that UXS1, a Golgi enzyme that converts one sugar nucleotide (UDP-glucuronic acid, UDPGA) to another (UDP-xylose), is essential only in cells that express high levels of the enzyme immediately upstream of it, UGDH. This conditional relationship exists because UXS1 is required to prevent excess accumulation of UDPGA, which is produced by UGDH. UXS1 not only clears away UDPGA but also limits its production through negative feedback on UGDH. Excess UDPGA disrupts Golgi morphology and function, which impedes the trafficking of surface receptors such as EGFR to the plasma membrane and diminishes the signalling capacity of cells. UGDH expression is elevated in several cancers, including lung adenocarcinoma, and is further enhanced during chemoresistant selection. As a result, these cancer cells are selectively dependent on UXS1 for UDPGA detoxification, revealing a potential weakness in tumours with high levels of UGDH.

HNRNP A1 Promotes Lung Cancer Cell Proliferation by Modulating VRK1 Translation.

THeterogeneous nuclear ribonucleoprotein (HNRNP) A1 is the most abundant and ubiquitously expressed member of the HNRNP protein family. In recent years, it has become more evident that HNRNP A1 contributes to the development of neurodegenerative diseases. However, little is known about the underlying role of HNRNP A1 in cancer development. Here, we report that HNRNP A1 expression is significantly increased in lung cancer tissues and is negatively correlated with the overall survival of patients with lung cancer. Additionally, HNRNP A1 positively regulates vaccinia-related kinase 1 (VRK1) translation via binding directly to the 3' untranslated region (UTR) of VRK1 mRNA, thus increasing cyclin D1 (CCND1) expression by VRK1-mediated phosphorylation of the cAMP response element-binding protein (CREB). Furthermore, HNRNP A1 binding to the cis-acting region of the 3'UTR of VRK1 mRNA contributes to increased lung cancer cell proliferation. Thus, our study unveils a novel role of HNRNP A1 in lung carcinogenesis via post-transcriptional regulation of VRK1 expression and suggests its potential as a therapeutic target for patients with lung cancer.

Comparative interactomes of SIRT6 and SIRT7: Implication of functional links to aging.

Sirtuins are NAD(+) -dependent deacetylases that regulate a range of cellular processes. Although diverse functions of sirtuins have been proposed, those functions of SIRT6 and SIRT7 that are mediated by their interacting proteins remain elusive. In the present study, we identified SIRT6- and SIRT7-interacting proteins, and compared their interactomes to investigate functional links. Our interactomes revealed 136 interacting proteins for SIRT6 and 233 for SIRT7 while confirming seven and 111 proteins identified previously for SIRT6 and SIRT7, respectively. Comparison of SIRT6 and SIRT7 interactomes under the same experimental conditions disclosed 111 shared proteins, implying related functional links. The interaction networks of interactomes indicated biological processes associated with DNA repair, chromatin assembly, and aging. Interactions of two highly acetylated proteins, nucleophosmin (NPM1) and nucleolin, with SIRT6 and SIRT7 were confirmed by co-immunoprecipitation. NPM1 was found to be deacetylated by both SIRT6 and SIRT7. In senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased levels of SIRT6 and SIRT7, suggesting that the acetylation of NPM1 could be regulated by SIRT6 and SIRT7 in the aging process. Our comparative interactomic study of SIRT6 and SIRT7 implies important functional links to aging by their associations with interacting proteins. All MS data have been deposited in the ProteomeXchange with identifiers PXD000159 and PXD000850 (http://proteomecentral.proteomexchange.org/dataset/PXD000159, http://proteomecentral.proteomexchange.org/dataset/PXD000850).

From odor to oncology: non-canonical odorant receptors in cancer.

Odorant receptors, traditionally associated with olfaction as chemoreceptors, have been increasingly recognized for their presence and diverse functions in various non-nasal tissues throughout the body. Beyond their roles in sensory perception, emerging evidence suggests a compelling interplay between odorant receptors and cancer progression as well. Alongside the canonical GPCR odorant receptors, dysregulation of non-canonical odorant receptors such as trace amine-associated receptors (TAARs), formyl peptide receptors (FPRs), and membrane-spanning 4A family (MS4As) has been observed in various cancer types, suggesting their contributions to cancer progression. The roles of these non-canonical chemoreceptors in cancer are complex, with some receptors promoting tumorigenesis and others acting as tumor-suppressing factors upon activation, depending on the cancer type. These findings shed light on the potential of non-canonical odorant receptors as therapeutic targets and prognostic markers in cancer, inviting further exploration to unravel their precise mechanisms of action and implications in cancer biology. In this review, we provide a comprehensive overview of the intricate relationships between these chemoreceptors and various types of cancer, potentially paving the way for innovative odor-based therapeutics. Ultimately, this review discusses the potential development of novel therapeutic strategies targeting these non-canonical chemoreceptors.

Extracellular flux assay (Seahorse assay): Diverse applications in metabolic research across biological disciplines.

Metabolic networks are fundamental to cellular processes, driving energy production, biosynthesis, redox regulation, and cellular signaling. Recent advancements in metabolic research tools have provided unprecedented insights into cellular metabolism. Among these tools, the extracellular flux analyzer stands out for its real-time measurement of key metabolic parameters: glycolysis, mitochondrial respiration, and fatty acid oxidation, leading to its widespread use. This review provides a comprehensive summary of the basic principles and workflow of the extracellular flux assay (the Seahorse assay) and its diverse applications. We highlight the assay's versatility across various biological models, including cancer cells, immunocytes, Caenorhabditis elegans, tissues, isolated mitochondria, and three-dimensional structures such as organoids, and summarize key considerations for using extracellular flux assay in these models. Additionally, we discuss the limitations of the Seahorse assay and propose future directions for its development. This review aims to enhance the understanding of extracellular flux assay and its significance in biological studies.

Combinatorial expression of neurexin genes regulates glomerular targeting by olfactory sensory neurons.

Precise connectivity between specific neurons is essential for the formation of the complex neural circuitry necessary for executing intricate motor behaviors and higher cognitive functions. While trans -interactions between synaptic membrane proteins have emerged as crucial elements in orchestrating the assembly of these neural circuits, the synaptic surface proteins involved in neuronal wiring remain largely unknown. Here, using unbiased single-cell transcriptomic and mouse genetic approaches, we uncover that the neurexin family of genes enables olfactory sensory neuron (OSNs) axons to form appropriate synaptic connections with their mitral and tufted (M/T) cell synaptic partners, within the mammalian olfactory system. Neurexin isoforms are differentially expressed within distinct populations of OSNs, resulting in unique pattern of neurexin expression that is specific to each OSN type, and synergistically cooperate to regulate axonal innervation, guiding OSN axons to their designated glomeruli. This process is facilitated through the interactions of neurexins with their postsynaptic partners, including neuroligins, which have distinct expression patterns in M/T cells. Our findings suggest a novel mechanism underpinning the precise assembly of olfactory neural circuits, driven by the trans -interaction between neurexins and their ligands.

Selenium reduction of ubiquinone via SQOR suppresses ferroptosis.

The canonical biological function of selenium is in the production of selenocysteine residues of selenoproteins, and this forms the basis for its role as an essential antioxidant and cytoprotective micronutrient. Here we demonstrate that, via its metabolic intermediate hydrogen selenide, selenium reduces ubiquinone in the mitochondria through catalysis by sulfide quinone oxidoreductase. Through this mechanism, selenium rapidly protects against lipid peroxidation and ferroptosis in a timescale that precedes selenoprotein production, doing so even when selenoprotein production has been eliminated. Our findings identify a regulatory mechanism against ferroptosis that implicates sulfide quinone oxidoreductase and expands our understanding of selenium in biology.

REST-dependent expression of TRF2 renders non-neuronal cancer cells resistant to DNA damage during oxidative stress.

REST is a neuronal gene silencing factor ubiquitously expressed in non-neuronal tissues. REST is additionally believed to serve as a tumor suppressor in non-neuronal cancers. Conversely, recent findings on REST-dependent tumorigenesis in non-neuronal cells consistently suggest a potential role of REST as a tumor promoter. Here, we have uncovered for the first time the mechanism by which REST contributes to cancer cell survival in non-neuronal cancers. We observed abundant expression of REST in various types of non-neuronal cancer cells compared to normal tissues. The delicate roles of REST were further evaluated in HCT116 and HeLa, non-neuronal cancer cell lines expressing REST. REST silencing resulted in decreased cell survival and activation of the DNA damage response (DDR) through a decrease in the level of TRF2, a telomere-binding protein. These responses were correlated with reduced colony formation ability and accelerated telomere shortening in cancer cells upon the stable knockdown of REST. Interestingly, REST was down-regulated under oxidative stress conditions via ubiquitin proteasome system, suggesting that sustainability of REST expression is critical to determine cell survival during oxidative stress in a tumor microenvironment. Our results collectively indicate that REST-dependent TRF2 expression renders cancer cells resistant to DNA damage during oxidative stress, and mechanisms to overcome oxidative stress, such as high levels of REST or the stress-resistant REST mutants found in specific human cancers, may account for REST-dependent tumorigenesis.

Kinetic characterization of on-chip DNA ligation on dendron-coated surfaces with nanoscaled lateral spacings.

We analyzed the enzymatic profiles of on-chip DNA ligation as we controlled the lateral spacing of surface-immobilized DNA substrates using dendron molecules with different sizes at the nanoscale. Enzymatic on-chip DNA ligation was performed on the dendron-coated surface within 20 min with no need for post-ligation gel electrophoresis. The enzymatic DNA repair was assessed by the fluorescence intensity at the repaired DNA duplex after thermally dissociating the unligated Cy3-labeled DNA from the DNA duplex, in which the Cy3-labeled DNA was hybridized prior to the on-chip DNA ligation. The rate of the nick-sealing reaction on the 27-acid dendron surface was 3-fold higher than that on the 9-acid dendron surface, suggesting that the wider lateral spacing determined by the larger dendron molecule could facilitate the access of DNA ligase to the nick site. The performance of on-chip DNA ligation was dropped to 10% and 3% when the nick was replaced by one- and two-nucleotide-long gaps, respectively. The 5' terminal phosphorylation of DNA strands by polynucleotide kinase and the on-chip DNA cleavage by endonucleases were also quantitatively monitored throughout the on-chip DNA ligation on the dendron-coated surface. A better understanding of the enzymatic kinetics of on-chip DNA ligation will contribute to a more reliable performance of various on-chip DNA ligation-based assays.

Toxic Metabolites and Inborn Errors of Amino Acid Metabolism: What One Informs about the Other.

In inborn errors of metabolism, such as amino acid breakdown disorders, loss of function mutations in metabolic enzymes within the catabolism pathway lead to an accumulation of the catabolic intermediate that is the substrate of the mutated enzyme. In patients of such disorders, dietarily restricting the amino acid(s) to prevent the formation of these catabolic intermediates has a therapeutic or even entirely preventative effect. This demonstrates that the pathology is due to a toxic accumulation of enzyme substrates rather than the loss of downstream products. Here, we provide an overview of amino acid metabolic disorders from the perspective of the 'toxic metabolites' themselves, including their mechanism of toxicity and whether they are involved in the pathology of other disease contexts as well. In the research literature, there is often evidence that such metabolites play a contributing role in multiple other nonhereditary (and more common) disease conditions, and these studies can provide important mechanistic insights into understanding the metabolite-induced pathology of the inborn disorder. Furthermore, therapeutic strategies developed for the inborn disorder may be applicable to these nonhereditary disease conditions, as they involve the same toxic metabolite. We provide an in-depth illustration of this cross-informing concept in two metabolic disorders, methylmalonic acidemia and hyperammonemia, where the pathological metabolites methylmalonic acid and ammonia are implicated in other disease contexts, such as aging, neurodegeneration, and cancer, and thus there are opportunities to apply mechanistic or therapeutic insights from one disease context towards the other. Additionally, we expand our scope to other metabolic disorders, such as homocystinuria and nonketotic hyperglycinemia, to propose how these concepts can be applied broadly across different inborn errors of metabolism and various nonhereditary disease conditions.

Examining xCT-mediated selenium uptake and selenoprotein production capacity in cells.

The production of selenoproteins in cancer cells is dependent on uptake of selenium and processing via the selenocysteine biosynthesis pathway. Both the uptake and processing of selenium has recently shown to be upregulated in subsets of cancer cells due to their increased expression of xCT transporter, and the resulting increased expression of selenoproteins such as GPX4 can play multiple roles in cancer cells such as providing protection against ferroptotic insults. Here, we describe a set of protocols designed to measure this process in cancer cell culture-the measurement of xCT transporter expression and activity, the intracellular uptake of selenium in cancer cells, and the expression of selenoproteins as the final functional readout of this process. The successful measurement of xCT requires non-denaturing western blotting of xCT subunits, while its activity is determined by the measurement of reduced thiol groups that accumulate over time, as determined by Ellman's reagent. Selenium uptake is determined by supplementing a selenium source and then measuring total intracellular selenium levels, which is determined from digested cellular material using a reactive fluorescent probe or via inductively coupled plasma mass spectrometry. Finally, specific tips for efficiently determining the expression level of a set of "indicator" selenoproteins is provided. These parameters allow one to determine the "selenophilicity" of cells, i.e., the ability of cells to utilize selenite to upregulate their selenoprotein production and thus antioxidant defenses.

De novo sphingolipid biosynthesis necessitates detoxification in cancer cells.

Sphingolipids play important signaling and structural roles in cells. Here, we find that during de novo sphingolipid biosynthesis, a toxic metabolite is formed with critical implications for cancer cell survival. The enzyme catalyzing the first step in this pathway, serine palmitoyltransferase complex (SPT), is upregulated in breast and other cancers. SPT is dispensable for cancer cell proliferation, as sphingolipids can be salvaged from the environment. However, SPT activity introduces a liability as its product, 3-ketodihydrosphingosine (3KDS), is toxic and requires clearance via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). In cancer cells, but not normal cells, targeting KDSR induces toxic 3KDS accumulation leading to endoplasmic reticulum (ER) dysfunction and loss of proteostasis. Furthermore, the antitumor effect of KDSR disruption can be enhanced by increasing metabolic input (via high-fat diet) to allow greater 3KDS production. Thus, de novo sphingolipid biosynthesis entails a detoxification requirement in cancer cells that can be therapeutically exploited.

xCT-Driven Expression of GPX4 Determines Sensitivity of Breast Cancer Cells to Ferroptosis Inducers.

Inducers of ferroptosis such as the glutathione depleting agent Erastin and the GPX4 inhibitor Rsl-3 are being actively explored as potential therapeutics in various cancers, but the factors that determine their sensitivity are poorly understood. Here, we show that expression levels of both subunits of the cystine/glutamate antiporter xCT determine the expression of GPX4 in breast cancer, and that upregulation of the xCT/selenocysteine biosynthesis/GPX4 production axis paradoxically renders the cancer cells more sensitive to certain types of ferroptotic stimuli. We find that GPX4 is strongly upregulated in a subset of breast cancer tissues compared to matched normal samples, and that this is tightly correlated with the increased expression of the xCT subunits SLC7A11 and SLC3A2. Erastin depletes levels of the antioxidant selenoproteins GPX4 and GPX1 in breast cancer cells by inhibiting xCT-dependent extracellular reduction which is required for selenium uptake and selenocysteine biosynthesis. Unexpectedly, while breast cancer cells are resistant compared to nontransformed cells against oxidative stress inducing drugs, at the same time they are hypersensitive to lipid peroxidation and ferroptosis induced by Erastin or Rsl-3, indicating that they are 'addicted' to the xCT/GPX4 axis. Our findings provide a strategic basis for targeting the anti-ferroptotic machinery of breast cancer cells depending on their xCT status, which can be further explored.

Endogenous toxic metabolites and implications in cancer therapy.

It is well recognized that many metabolic enzymes play essential roles in cancer cells in producing building blocks such as nucleotides, which are required in greater amounts due to their increased proliferation. On the other hand, the significance of enzymes in preventing the accumulation of their substrates is less recognized. Here, we outline the evidence and underlying mechanisms for how many metabolites normally produced in cells are highly toxic, such as metabolites containing reactive groups (e.g., methylglyoxal, 4-hydroxynonenal, and glutaconyl-CoA), or metabolites that act as competitive analogs against other metabolites (e.g., deoxyuridine triphosphate and l-2-hydroxyglutarate). Thus, if a metabolic pathway contains a toxic intermediate, then we may be able to induce accumulation and poison a cancer cell by targeting the downstream enzyme. Furthermore, this poisoning may be cancer cell selective if this pathway is overactive in a cancer cell relative to a nontransformed cell. We describe this concept as illustrated in selenocysteine metabolism and other pathways and discuss future directions in exploiting toxic metabolites to kill cancer cells.

Selenium detoxification is required for cancer-cell survival.

The micronutrient selenium is incorporated via the selenocysteine biosynthesis pathway into the rare amino acid selenocysteine, which is required in selenoproteins such as glutathione peroxidases and thioredoxin reductases1,2. Here, we show that selenophosphate synthetase 2 (SEPHS2), an enzyme in the selenocysteine biosynthesis pathway, is essential for survival of cancer, but not normal, cells. SEPHS2 is required in cancer cells to detoxify selenide, an intermediate that is formed during selenocysteine biosynthesis. Breast and other cancer cells are selenophilic, owing to a secondary function of the cystine/glutamate antiporter SLC7A11 that promotes selenium uptake and selenocysteine biosynthesis, which, by allowing production of selenoproteins such as GPX4, protects cells against ferroptosis. However, this activity also becomes a liability for cancer cells because selenide is poisonous and must be processed by SEPHS2. Accordingly, we find that SEPHS2 protein levels are elevated in samples from people with breast cancer, and that loss of SEPHS2 impairs growth of orthotopic mammary-tumour xenografts in mice. Collectively, our results identify a vulnerability of cancer cells and define the role of selenium metabolism in cancer.

Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging.

Cell protrusion is morphodynamically heterogeneous at the subcellular level. However, the mechanism of cell protrusion has been understood based on the ensemble average of actin regulator dynamics. Here, we establish a computational framework called HACKS (deconvolution of heterogeneous activity in coordination of cytoskeleton at the subcellular level) to deconvolve the subcellular heterogeneity of lamellipodial protrusion from live cell imaging. HACKS identifies distinct subcellular protrusion phenotypes based on machine-learning algorithms and reveals their underlying actin regulator dynamics at the leading edge. Using our method, we discover "accelerating protrusion", which is driven by the temporally ordered coordination of Arp2/3 and VASP activities. We validate our finding by pharmacological perturbations and further identify the fine regulation of Arp2/3 and VASP recruitment associated with accelerating protrusion. Our study suggests HACKS can identify specific subcellular protrusion phenotypes susceptible to pharmacological perturbation and reveal how actin regulator dynamics are changed by the perturbation.

Comparative Interactomes of VRK1 and VRK3 with Their Distinct Roles in the Cell Cycle of Liver Cancer.

Vaccinia-related kinase 1 (VRK1) and VRK3 are members of the VRK family of serine/threonine kinases and are principally localized in the nucleus. Despite the crucial roles of VRK1/VRK3 in physiology and disease, the molecular and functional interactions of VRK1/VRK3 are poorly understood. Here, we identified over 200 unreported VRK1/VRK3-interacting candidate proteins by affinity purification and LC-MS/MS. The networks of VRK1 and VRK3 interactomes were found to be associated with important biological processes such as the cell cycle, DNA repair, chromatin assembly, and RNA processing. Interactions of interacting proteins with VRK1/VRK3 were confirmed by biochemical assays. We also found that phosphorylations of XRCC5 were regulated by both VRK1/VRK3, and that of CCNB1 was regulated by VRK3. In liver cancer cells and tissues, VRK1/VRK3 were highly upregulated and its depletion affected cell cycle progression in the different phases. VRK3 seemed to affect S phase progression and G2 or M phase entry and exit, whereas VRK1 affects G1/S transition in the liver cancer, which could be explained by different interacting candidate proteins. Thus, this study not only provides a resource for investigating the unidentified functions of VRK1/VRK3, but also an insight into the regulatory roles of VRK1/VRK3 in biological processes.

DISC1 Modulates Neuronal Stress Responses by Gate-Keeping ER-Mitochondria Ca2+ Transfer through the MAM.

A wide range of Ca2+-mediated functions are enabled by the dynamic properties of Ca2+, all of which are dependent on the endoplasmic reticulum (ER) and mitochondria. Disrupted-in-schizophrenia 1 (DISC1) is a scaffold protein that is involved in the function of intracellular organelles and is linked to cognitive and emotional deficits. Here, we demonstrate that DISC1 localizes to the mitochondria-associated ER membrane (MAM). At the MAM, DISC1 interacts with IP3R1 and downregulates its ligand binding, modulating ER-mitochondria Ca2+ transfer through the MAM. The disrupted regulation of Ca2+ transfer caused by DISC1 dysfunction leads to abnormal Ca2+ accumulation in mitochondria following oxidative stress, which impairs mitochondrial functions. DISC1 dysfunction alters corticosterone-induced mitochondrial Ca2+ accumulation in an oxidative stress-dependent manner. Together, these findings link stress-associated neural stimuli with intracellular ER-mitochondria Ca2+ crosstalk via DISC1, providing mechanistic insight into how environmental risk factors can be interpreted by intracellular pathways under the control of genetic components in neurons.

Myricetin improves endurance capacity and mitochondrial density by activating SIRT1 and PGC-1α.

Robust mitochondrial respiration provides energy to support physical performance and physiological well-being, whereas mitochondrial malfunction is associated with various pathologies and reduced longevity. In the current study, we tested whether myricetin, a natural flavonol with diverse biological activities, may impact mitochondrial function and longevity. The mice were orally administered myricetin (50 mg/kg/day) for 3 weeks. Myricetin significantly potentiated aerobic capacity in mice, as evidenced by their increased running time and distance. The elevated mitochondrial function was associated with induction of genes for oxidative phosphorylation and mitochondrial biogenesis in metabolically active tissues. Importantly, myricetin treatment led to decreased PGC-1α acetylation through SIRT1 activation. Furthermore, myricetin significantly improved the healthspan and lifespan of wild-type, but not Sir-2.1-deficient, C. elegans. These results demonstrate that myricetin enhances mitochondrial activity, possibly by activating PGC-1α and SIRT1, to improve physical endurance, strongly suggesting myricetin as a mitochondria-activating agent.

Cancer Metabolism: Fueling More than Just Growth.

The early landmark discoveries in cancer metabolism research have uncovered metabolic processes that support rapid proliferation, such as aerobic glycolysis (Warburg effect), glutaminolysis, and increased nucleotide biosynthesis. However, there are limitations to the effectiveness of specifically targeting the metabolic processes which support rapid proliferation. First, as other normal proliferative tissues also share similar metabolic features, they may also be affected by such treatments. Secondly, targeting proliferative metabolism may only target the highly proliferating "bulk tumor" cells and not the slower-growing, clinically relevant cancer stem cell subpopulations which may be required for an effective cure. An emerging body of research indicates that altered metabolism plays key roles in supporting proliferation-independent functions of cancer such as cell survival within the ischemic and acidic tumor microenvironment, immune system evasion, and maintenance of the cancer stem cell state. As these aspects of cancer cell metabolism are critical for tumor maintenance yet are less likely to be relevant in normal cells, they represent attractive targets for cancer therapy.