Copper chelation reduces early collagen deposition and preserves saliva secretion in irradiated salivary glands.

Radiation therapy is a first-line treatment for head and neck cancer; however, it typically leads to hyposalivation stemming from fibrosis of the salivary gland. Current strategies to restore glandular function are dependent on the presence of residual functional salivary gland tissue, a condition commonly not met in patients with extensive fibrotic coverage of the salivary gland resulting from radiation therapy. Fibrosis is defined by the pathological accumulation of connective tissue (i.e., extracellular matrix) and excessive deposition of crosslinked (fibrillar) collagen that can impact a range of tissues and given that collagen crosslinking is necessary for fibrosis formation, inhibiting this process is a reasonable focus for developing anti-fibrotic therapies. Collagen crosslinking is catalyzed by the lysyl oxidase family of secreted copper-dependent metalloenzymes, and since that copper is an essential cofactor in all lysyl oxidase family members, we tested whether localized delivery of a copper chelator into the submandibular gland of irradiated mice could suppress collagen deposition and preserve the structure and function of this organ. Our results demonstrate that transdermal injection of tetrathiomolybdate into salivary glands significantly reduced the early deposition of fibrillar collagen in irradiated mice and preserved the integrity and function of submandibular gland epithelial tissue. Together, these studies identify copper metabolism as a novel therapeutic target to control radiation induced damage to the salivary gland and the current findings further indicate the therapeutic potential of repurposing clinically approved copper chelators as neoadjuvant treatments for radiation therapy.

Regulation of Atp7a RNA contributes to differentiation-dependent Cu redistribution in skeletal muscle cells.

Cu (Cu) is essential for several biochemical pathways due to its role as a catalytic cofactor or allosteric regulator of enzymes. Its import and distribution are tightly controlled by transporters and metallochaperones and Cu homeostasis is maintained by balancing Cu uptake and export. Genetic diseases are caused by impaired Cu transporters CTR1, ATP7A, or ATP7B but little is known about the regulatory mechanisms by which these proteins meet the fluctuating demands of Cu in specific tissues. Cu is required for differentiation of skeletal myoblasts to myotubes. Here, we demonstrate that ATP7A is needed for myotube formation and that its increased abundance during differentiation is mediated by stabilization of Atp7a mRNA via the 3' untranslated region. Increased ATP7A levels during differentiation resulted in increased Cu delivery to lysyl oxidase, a secreted cuproenzyme that needed for myotube formation. These studies identify a previously unknown role for Cu in regulating muscle differentiation and have broad implications for understanding Cu-dependent differentiation in other tissues.

APOE expression and secretion are modulated by mitochondrial dysfunction.

Mitochondria influence cellular function through both cell-autonomous and non-cell autonomous mechanisms, such as production of paracrine and endocrine factors. Here, we demonstrate that mitochondrial regulation of the secretome is more extensive than previously appreciated, as both genetic and pharmacological disruption of the electron transport chain caused upregulation of the Alzheimer's disease risk factor apolipoprotein E (APOE) and other secretome components. Indirect disruption of the electron transport chain by gene editing of SLC25A mitochondrial membrane transporters as well as direct genetic and pharmacological disruption of either complexes I, III, or the copper-containing complex IV of the electron transport chain elicited upregulation of APOE transcript, protein, and secretion, up to 49-fold. These APOE phenotypes were robustly expressed in diverse cell types and iPSC-derived human astrocytes as part of an inflammatory gene expression program. Moreover, age- and genotype-dependent decline in brain levels of respiratory complex I preceded an increase in APOE in the 5xFAD mouse model. We propose that mitochondria act as novel upstream regulators of APOE-dependent cellular processes in health and disease.

FDX1-dependent and independent mechanisms of elesclomol-mediated intracellular copper delivery.

Recent studies have uncovered the therapeutic potential of elesclomol (ES), a copper-ionophore, for copper deficiency disorders. However, we currently do not understand the mechanism by which copper brought into cells as ES-Cu(II) is released and delivered to cuproenzymes present in different subcellular compartments. Here, we have utilized a combination of genetic, biochemical, and cell-biological approaches to demonstrate that intracellular release of copper from ES occurs inside and outside of mitochondria. The mitochondrial matrix reductase, FDX1, catalyzes the reduction of ES-Cu(II) to Cu(I), releasing it into mitochondria where it is bioavailable for the metalation of mitochondrial cuproenzyme- cytochrome c oxidase. Consistently, ES fails to rescue cytochrome c oxidase abundance and activity in copper-deficient cells lacking FDX1. In the absence of FDX1, the ES-dependent increase in cellular copper is attenuated but not abolished. Thus, ES-mediated copper delivery to nonmitochondrial cuproproteins continues even in the absence of FDX1, suggesting alternate mechanism(s) of copper release. Importantly, we demonstrate that this mechanism of copper transport by ES is distinct from other clinically used copper-transporting drugs. Our study uncovers a unique mode of intracellular copper delivery by ES and may further aid in repurposing this anticancer drug for copper deficiency disorders.

Ceruloplasmin as a source of Cu for a fungal pathogen.

Copper is an essential metal for virtually all organisms, yet little is known about the extracellular sources of this micronutrient. In serum, the most abundant extracellular Cu-binding molecule is the multi­copper oxidase ceruloplasmin (Cp). Cp levels increase during infection and inflammation, and pathogens can be exposed to high Cp at sites of infection. It is not known whether Cp might serve as a Cu source for microbial pathogens and we tested this using the opportunistic fungal pathogen Candida albicans. We find that C. albicans can use whole serum as a Cu source and that this Cu is sensed by the transcription factor protein Mac1. Mac1 activates expression of Mn-SOD3 superoxide dismutase and represses Cu/Zn-SOD1 during Cu starvation and both responses are regulated by serum Cu. We also show that purified human Cp can act as a sole source of Cu for the fungus and likewise modulates the Mac1 Cu stress response. To investigate whether Cp is a Cu source in serum, we compared the ability of C. albicans to use serum from wild type versus Cp-/- mutant mice. We find that serum lacking Cp is deficient in its ability to trigger the Mac1 Cu response. C. albicans did accumulate Cu from Cp-/- serum, but this Cu was not efficiently sensed by Mac1. We conclude that Cp and non-Cp Cu sources are not equivalent and are handled differently by the fungal cell. Overall, these studies are the first to show that Cp is a preferred source of Cu for a pathogen.

P2Y receptors for extracellular nucleotides: Contributions to cancer progression and therapeutic implications.

Purinergic receptors for extracellular nucleotides and nucleosides contribute to a vast array of cellular and tissue functions, including cell proliferation, intracellular and transmembrane ion flux, immunomodulation and thrombosis. In mammals, the purinergic receptor system is composed of G protein-coupled P1 receptors A1, A2A, A2B and A3 for extracellular adenosine, P2X1-7 receptors that are ATP-gated ion channels and G protein-coupled P2Y1,2,4,6,11,12,13 and 14 receptors for extracellular ATP, ADP, UTP, UDP and/or UDP-glucose. Recent studies have implicated specific P2Y receptor subtypes in numerous oncogenic processes, including cancer tumorigenesis, metastasis and chemotherapeutic drug resistance, where G protein-mediated signaling cascades modulate intracellular ion concentrations and activate downstream protein kinases, Src family kinases as well as numerous mitogen-activated protein kinases. We are honored to contribute to this special issue dedicated to the founder of the field of purinergic signaling, Dr. Geoffrey Burnstock, by reviewing the diverse roles of P2Y receptors in the initiation, progression and metastasis of specific cancers with an emphasis on pharmacological and genetic strategies employed to delineate cell-specific and P2Y receptor subtype-specific responses that have been investigated using in vitro and in vivo cancer models. We further highlight bioinformatic and empirical evidence on P2Y receptor expression in human clinical specimens and cover clinical perspectives where P2Y receptor-targeting interventions may have therapeutic relevance to cancer treatment.

Copper metabolism as a unique vulnerability in cancer.

The last 25 years have witnessed tremendous progress in identifying and characterizing proteins that regulate the uptake, intracellular trafficking and export of copper. Although dietary copper is required in trace amounts, sufficient quantities of this metal are needed to sustain growth and development in humans and other mammals. However, copper is also a rate-limiting nutrient for the growth and proliferation of cancer cells. Oral copper chelators taken with food have been shown to confer anti-neoplastic and anti-metastatic benefits in animals and humans. Recent studies have begun to identify specific roles for copper in pathways of oncogenic signaling and resistance to anti-neoplastic drugs. Here, we review the general mechanisms of cellular copper homeostasis and discuss roles of copper in cancer progression, highlighting metabolic vulnerabilities that may be targetable in the development of anticancer therapies.

P2Y2 receptors mediate nucleotide-induced EGFR phosphorylation and stimulate proliferation and tumorigenesis of head and neck squamous cell carcinoma cell lines.

OBJECTIVES: To assess functional expression of the P2Y2 nucleotide receptor (P2Y2R) in head and neck squamous cell carcinoma (HNSCC) cell lines and define its role in nucleotide-induced epidermal growth factor receptor (EGFR) transactivation. The use of anti-EGFR therapeutics to treat HNSCC is hindered by intrinsic and acquired drug resistance. Defining novel pathways that modulate EGFR signaling could identify additional targets to treat HNSCC. MATERIALS AND METHODS: In human HNSCC cell lines CAL27 and FaDu and the mouse oral cancer cell line MOC2, P2Y2R contributions to extracellular nucleotide-induced changes in intracellular free Ca2+ concentration and EGFR and extracellular signal-regulated kinase (ERK1/2) phosphorylation were determined using the ratiometric Ca2+ indicator fura-2 and immunoblot analysis, respectively. Genetic knockout of P2Y2Rs using CRISPR technology or pharmacological inhibition with P2Y2R-selective antagonist AR-C118925 defined P2Y2R contributions to in vivo tumor growth. RESULTS: P2Y2R agonists UTP and ATP increased intracellular Ca2+ levels and ERK1/2 and EGFR phosphorylation in CAL27 and FaDu cells, responses that were inhibited by AR-C118925 or P2Y2R knockout. P2Y2R-mediated EGFR phosphorylation was also attenuated by inhibition of the adamalysin family of metalloproteases or Src family kinases. P2Y2R knockout reduced UTP-induced CAL27 cell proliferation in vitro and significantly reduced CAL27 and FaDu tumor xenograft volume in vivo. In a syngeneic mouse model of oral cancer, AR-C118925 administration reduced MOC2 tumor volume. CONCLUSION: P2Y2Rs mediate HNSCC cell responses to extracellular nucleotides and genetic or pharmacological blockade of P2Y2R signaling attenuates tumor cell proliferation and tumorigenesis, suggesting that the P2Y2R represents a novel therapeutic target in HNSCC.

Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess.

Copper (Cu) is an essential, yet potentially toxic nutrient, as illustrated by inherited diseases of copper deficiency and excess. Elevated expression of the ATP7A Cu exporter is known to confer copper tolerance, however, the contribution of metal-binding metallothioneins is less clear. In this study, we investigated the relative contributions of ATP7A and the metallothioneins MT-I and MT-II to cell viability under conditions of Cu excess or deficiency. Although the loss of ATP7A increased sensitivity to low Cu concentrations, the absence of MTs did not significantly affect Cu tolerance. However, the absence of all three proteins caused a synthetic lethal phenotype due to extreme Cu sensitivity, indicating that MTs are critical for Cu tolerance only in the absence of ATP7A. A lack of MTs resulted in the trafficking of ATP7A from the trans-Golgi complex in a Cu-dependent manner, suggesting that MTs regulate the delivery of Cu to ATP7A. Under Cu deficiency conditions, the absence of MTs and / or ATP7A enhanced cell proliferation compared to wild type cells, suggesting that these proteins compete with essential Cu-dependent pathways when Cu is scarce. These studies reveal new roles for ATP7A and metallothioneins under both Cu deficiency and excess.

Elesclomol alleviates Menkes pathology and mortality by escorting Cu to cuproenzymes in mice.

Loss-of-function mutations in the copper (Cu) transporter ATP7A cause Menkes disease. Menkes is an infantile, fatal, hereditary copper-deficiency disorder that is characterized by progressive neurological injury culminating in death, typically by 3 years of age. Severe copper deficiency leads to multiple pathologies, including impaired energy generation caused by cytochrome c oxidase dysfunction in the mitochondria. Here we report that the small molecule elesclomol escorted copper to the mitochondria and increased cytochrome c oxidase levels in the brain. Through this mechanism, elesclomol prevented detrimental neurodegenerative changes and improved the survival of the mottled-brindled mouse-a murine model of severe Menkes disease. Thus, elesclomol holds promise for the treatment of Menkes and associated disorders of hereditary copper deficiency.

ATP7A delivers copper to the lysyl oxidase family of enzymes and promotes tumorigenesis and metastasis.

Lysyl oxidase (LOX) and LOX-like (LOXL) proteins are copper-dependent metalloenzymes with well-documented roles in tumor metastasis and fibrotic diseases. The mechanism by which copper is delivered to these enzymes is poorly understood. In this study, we demonstrate that the copper transporter ATP7A is necessary for the activity of LOX and LOXL enzymes. Silencing of ATP7A inhibited LOX activity in the 4T1 mammary carcinoma cell line, resulting in a loss of LOX-dependent mechanisms of metastasis, including the phosphorylation of focal adhesion kinase and myeloid cell recruitment to the lungs, in an orthotopic mouse model of breast cancer. ATP7A silencing was also found to attenuate LOX activity and metastasis of Lewis lung carcinoma cells in mice. Meta-analysis of breast cancer patients found that high ATP7A expression was significantly correlated with reduced survival. Taken together, these results identify ATP7A as a therapeutic target for blocking LOX- and LOXL-dependent malignancies.

Cu2+ selective chelators relieve copper-induced oxidative stress in vivo.

Copper ions are essential for biological function yet are severely detrimental when present in excess. At the molecular level, copper ions catalyze the production of hydroxyl radicals that can irreversibly alter essential bio-molecules. Hence, selective copper chelators that can remove excess copper ions and alleviate oxidative stress will help assuage copper-overload diseases. However, most currently available chelators are non-specific leading to multiple undesirable side-effects. The challenge is to build chelators that can bind to copper ions with high affinity but leave the levels of essential metal ions unaltered. Here we report the design and development of redox-state selective Cu ion chelators that have 108 times higher conditional stability constants toward Cu2+ compared to both Cu+ and other biologically relevant metal ions. This unique selectivity allows the specific removal of Cu2+ ions that would be available only under pathophysiological metal overload and oxidative stress conditions and provides access to effective removal of the aberrant redox-cycling Cu ion pool without affecting the essential non-redox cycling Cu+ labile pool. We have shown that the chelators provide distinct protection against copper-induced oxidative stress in vitro and in live cells via selective Cu2+ ion chelation. Notably, the chelators afford significant reduction in Cu-induced oxidative damage in Atp7a-/- Menkes disease model cells that have endogenously high levels of Cu ions. Finally, in vivo testing of our chelators in a live zebrafish larval model demonstrate their protective properties against copper-induced oxidative stress.

Family A and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions.

DNA polymerases are essential for genome replication, DNA repair and translesion DNA synthesis (TLS). Broadly, these enzymes belong to two groups: replicative and non-replicative DNA polymerases. A considerable body of data suggests that both groups of DNA polymerases are associated with cancer. Many mutations in cancer cells are either the result of error-prone DNA synthesis by non-replicative polymerases, or the inability of replicative DNA polymerases to proofread mismatched nucleotides due to mutations in 3'-5' exonuclease activity. Moreover, non-replicative, TLS-capable DNA polymerases can negatively impact cancer treatment by synthesizing DNA past lesions generated from treatments such as cisplatin, oxaliplatin, etoposide, bleomycin, and radiotherapy. Hence, the inhibition of DNA polymerases in tumor cells has the potential to enhance treatment outcomes. Here, we review the association of DNA polymerases in cancer from the A and B families, which participate in lesion bypass, and conduct gene replication. We also discuss possible therapeutic interventions that could be used to maneuver the role of these enzymes in tumorigenesis.

Isolation, Characterization and Bioactivities of an Extracellular Polysaccharide Produced from Streptomyces sp. MOE6.

A Streptomyces strain was isolated from soil and the sequence of 1471 nucleotides of its 16S rDNA showed 99% identity to Streptomyces sp. HV10. This newly isolated Streptomyces strain produced an extracellular polysaccharide (EPS) composed mainly of glucose and mannose in a ratio of 1:4.1, as was characterized by Fourier transform infrared spectroscopy (FTIR), HPLC and ¹H-NMR. The antioxidant activities of the partially purified MOE6-EPS were determined by measuring the hydroxyl free radical scavenging activity and the scavenging of 2,2-diphenyl-2-picryl-hydrazyl (DPPH) radicals. In addition, the partially purified MOE6-EPS showed high ferrous ion (Fe2+) chelation activity which is another antioxidant activity. Interestingly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays that were colorimetric assays for NAD(P)H-dependent cellular oxidoreductases and a proxy of the number of viable cells, showed that the partially purified MOE6-EPS inhibited the proliferation of the human breast cancer cells (MDA-MB-231). The scratch wound assay showed that MOE6-EPS reduced the migration of mouse breast cancer cells (4T1). This study reports the production of EPS from Streptomyces species with promising antioxidant, metal chelating and mammalian cell inhibitory activities.

A Role for The ATP7A Copper Transporter in Tumorigenesis and Cisplatin Resistance.

The ATP7A protein is a ubiquitously expressed copper-translocating P-type ATPase that controls cytoplasmic copper concentrations by mediating cellular copper egress. In vitro studies have previously demonstrated that ATP7A abundance in various tumor cell lines is correlated with increased resistance to cisplatin, a widely-used chemotherapy agent. However, to date no studies have examined a role for ATP7A in tumor growth or cisplatin sensitivity in vivo. In this study, we deleted ATP7A in H-RAS transformed tumorigenic mouse embryonic fibroblasts (MEFRAS7A-). Interestingly, loss of ATP7A was found to markedly suppress tumorigenesis in MEFRAS7A- cells relative to wild type parental cells. This was associated with hyperaccumulation of copper and sensitivity to reactive oxygen species and hypoxia. Tumor grafts lacking ATP7A were markedly more sensitive to cisplatin chemotherapy compared to ATP7A-expressing control tumors. These findings identify ATP7A at the nexus between tumorigenesis and cisplatin resistance pathways, underscoring its potential as a therapeutic target for regulating both tumor growth and the efficacy of cisplatin treatment.

Host and Pathogen Copper-Transporting P-Type ATPases Function Antagonistically during Salmonella Infection.

Copper is an essential yet potentially toxic trace element that is required by all aerobic organisms. A key regulator of copper homeostasis in mammalian cells is the copper-transporting P-type ATPase ATP7A, which mediates copper transport from the cytoplasm into the secretory pathway, as well as copper export across the plasma membrane. Previous studies have shown that ATP7A-dependent copper transport is required for killing phagocytosed Escherichia coli in a cultured macrophage cell line. In this investigation, we expanded on these studies by generating Atp7aLysMcre mice, in which the Atp7a gene was specifically deleted in cells of the myeloid lineage, including macrophages. Primary macrophages isolated from Atp7aLysMcre mice exhibit decreased copper transport into phagosomal compartments and a reduced ability to kill Salmonella enterica serovar Typhimurium compared to that of macrophages isolated from wild-type mice. The Atp7aLysMcre mice were also more susceptible to systemic infection by S Typhimurium than wild-type mice. Deletion of the S Typhimurium copper exporters, CopA and GolT, was found to decrease infection in wild-type mice but not in the Atp7aLysMcre mice. These studies suggest that ATP7A-dependent copper transport into the phagosome mediates host defense against S Typhimurium, which is counteracted by copper export from the bacteria via CopA and GolT. These findings reveal unique and opposing functions for copper transporters of the host and pathogen during infection.

Multiple di-leucines in the ATP7A copper transporter are required for retrograde trafficking to the trans-Golgi network.

The ATP7A protein is a ubiquitous copper-transporting P-type ATPase that is mutated in the lethal pediatric disorder of copper metabolism, Menkes disease. The steady-state location of ATP7A is within the trans-Golgi network (TGN), where it delivers copper to copper-dependent enzymes within the secretory pathway. However, ATP7A constantly cycles between the TGN and the plasma membrane, and in the presence of high copper concentrations, the exocytic arm of this cycling pathway is enhanced to promote a steady-state distribution of ATP7A to post-Golgi vesicles and the plasma membrane. A single di-leucine endocytic motif within the cytosolic carboxy tail of ATP7A (1487LL) was previously shown to be essential for TGN localization by functioning in retrieval from the plasma membrane, however, the requirement of other di-leucine signals in this region has not been fully investigated. While there has been some success in identifying sequence elements within ATP7A required for trafficking and catalysis, progress has been hampered by the instability of the ATP7A cDNA in high-copy plasmids during replication in Escherichia coli. In this study, we find that the use of DNA synthesis to generate silent mutations across the majority of both mouse and human ATP7A open reading frames was sufficient to stabilize these genes in high-copy plasmids, thus permitting the generation of full-length expression constructs. Using the stabilized mouse Atp7a construct, we identify a second di-leucine motif in the carboxy tail of ATP7A (1459LL) as essential for steady-state localization in the TGN by functioning in endosome-to-TGN trafficking. Taken together, these findings demonstrate that multiple di-leucine signals are required for recycling ATP7A from the plasma membrane to the TGN and illustrate the utility of large-scale codon reassignment as a simple and effective approach to circumvent cDNA instability in high-copy plasmids.