Role Of ATP7A Research Articles

Copper transporter gene ATP7A: A predictive biomarker for immunotherapy and targeted therapy in hepatocellular carcinoma.

ATP7A is an important copper transporter that regulates numerous cellular biological processes. However, the role of ATP7A in immunotherapy and targeted therapy, especially for hepatocellular carcinoma (HCC), remains unknown. We analyzed ATP7A expression and its effect on digestive system tumor prognoses, assessed its expression in tissue microarrays from 319 HCC patients, and investigated the relationship between ATP7A expression and tumor immunity. Specifically, we evaluated the possible association between ATP7A and programmed death ligand 1 (PD-L1) expression in human HCC tissues. Finally, we analyzed the effect of ATP7A on sorafenib efficacy in HCC. ATP7A is generally highly expressed in digestive system tumors but related to poor prognosis only in HCC. ATP7A levels are positively associated with immune cell infiltration and immune checkpoint expression (especially PD-L1). HCC patients coexpressing APT7A and PD-L1 demonstrate poor prognoses. Moreover, HCC patients with high ATP7A levels were more sensitive to sorafenib and demonstrated higher survival rates after sorafenib treatment. This study provides insights into the correlation between ATP7A levels and tumor immune infiltration and immune checkpoint function in HCC, sheds light on the significance of ATP7A in cancer progression, and provides guidance for more effective and general therapeutic strategies.

Abstract 12879: Endothelial Cu Exporter Atp7a Deficiency Promotes Cu-Dependent Endothelial-to-Mesenchymal Transition and Atherosclerosis via Orchestrating Glycolysis and Ros Signaling

Introduction: Oxidative stress, inflammation and metabolic reprogramming contribute to endothelial cell (EC) dysfunction and atherosclerosis. Copper (Cu), an essential micro-nutrient, promotes atherosclerosis via unknown mechanism. Since excess Cu is toxic, bioavailability of intracellular Cu is tightly controlled by Cu exporter ATP7A. However, the role of ATP7A and its molecular linkage with inflammation, oxidative stress and metabolism in EC dysfunction and atherosclerosis are entirely unknown. Results: Here we found a dramatic reduction of ATP7A expression in human and mouse atherosclerotic aorta. Surprisingly, ATP7A knockdown (KD) in human aortic ECs using shRNA changed the cell morphology from EC to mesenchymal types. This was confirmed by RNAseq/qPCR showing reduced EC markers and increased mesenchymal markers, indicating endothelial to mesenchymal transition (EndMT). Mechanistically, loss of ATP7A in ECs increased [Cu]i and PFKFB3-dependent glycolysis (1.8- fold by ECAR in Seahorse assay). Overexpression of PFKFB3 or constitutively active PFK2, which enhances glycolysis, or direct Cu treatment induced EndMT. In parallel, ATP7A KD in ECs increased mitochondrial (mito)ROS (9.8 fold)-histone demethylase JMJD2B, which induced inflammatory EndMT inducer TGFβ2-EndMT transcription factor Snail (2 fold) axis. Of note, ATP7A KD-induced glycolysis as well as mitoROS- TGFβ2-Snail axis involved in EndMT were inhibited by Cu chelator TTM. In vivo, Cu transport dysfunctional ATP7A mutant/ApoE -/- mice showed enhanced atherosclerosis and CD31 + /αSMA + cells representing EndMT in aorta compared to control ApoE -/- mice, which was further confirmed by EC-specific ATP7A -/- /mTmG reporter mice. Conclusion: Our study demonstrates that ATP7A dysfunction in ECs drives EndMT by orchestrating mitoROS-JMJD2B-TGFβ as well as PFKFB3-glycolysis axis in a Cu dependent manner, leading to Snail induction to accelerates atherosclerosis. Thus, ATP7A is a potential therapeutic target for treatment of inflammatory vascular disease.

Abstract 9930: Exercise-Induced ATP7A, A Cu Transporter for SOD3, Restores Endothelial Dysfunction in Type2 Diabetes via Cysteine Oxidation of Protein Kinase G

Background: Exercise training protects against type2 diabetes (T2D)-induced endothelial dysfunction which is mediated through eNOS uncoupling-induced O 2 -, but its underlying mechanisms are not fully understood. Cu transporter ATP7A is required for full activity of antioxidant Cu enzyme extracellular SOD (SOD3) that scavenges O 2 - to generate H 2 O 2 that has been proposed to function as a signaling molecule to mediate endothelium-dependent vasorelaxation (EDR) in T2D instead of nitric oxide (NO). However, role of ATP7A and its link with H 2 O 2 signaling in exercise-induced restoration of endothelial function in T2D is entirely unknown. Results: Here we show that ATP7A protein (49%), SOD3 activity (51%) and extracellular H 2 O 2 levels as well as EDR in blood vessels of high fat diet-induced T2D mice were significantly decreased compared to those of control C57Bl6 mice, which were restored by volunteer wheel exercise (2 weeks) or in T2D/ATP7A overexpressing mice. Of note, exercise-induced restoration of EDR in T2D mice was inhibited by PEG-catalase infusion or direct application of catalase to vessels, or in ATP7A dysfunctional ATP7A mut mice or T2D/SOD3 knockout (KO) mice, but not by eNOS inhibitor, L-NAME. This suggests that exercise-induced ATP7A-SOD3 axis-derived H 2 O 2 , but not eNOS/NO, plays a role in this effect. We then examined downstream targets of exercise-induced H 2 O 2 in blood vessels and found that protein kinase G (PKG)1α that is shown to be activated by exogenous H 2 O 2 via oxidation at Cys42 was sulfenylated (CysOH formed)(2.3-fold increase) in response to exercise in aorta of T2D, but not control, mice. This T2D-specific exercise-induced CysOH formaton of PKG1a in vascular tissue was inhibited by T2D/ ATP7A mut mice or T2D/SOD3 KO mice or PEG-catalase treatment. Functionally, “redox-dead” PKG1a Cys42Ser knock-in mutant mice blocked exercise-induced restoration of EDR in T2D, not control mice. Conclusion: Exercise-induced ATP7A promotes SOD3-mediated out-side in H 2 O 2 signaling, which contributes to restore EC dysfunction in T2D via PKG1α oxidation at Cys42.

Abstract 246: Role for ATP7A copper transporter in cisplatin resistance, tumorigenesis and metastasis

Abstract Copper is a trace metal with a ready capacity to gain or donate electrons. This property is harnessed by numerous enzymes to perform vital functions in the body. In humans, copper is required for various biochemical processes including cellular respiration, connective tissue development, iron transport, and pigmentation. The same redox property which makes copper useful can also have deleterious effects if the proper balance is not maintained. Hence a delicate balance of copper should be maintained in the body. Cellular copper homeostasis is maintained by several different proteins. Ctr1 (copper transporter 1) has high affinity for copper and aids in copper uptake into the cell. ATP7A and ATP7B are copper exporting ATPases, which share 60% identity, are functionally homologous. Under low intracellular copper concentrations, ATP7A is localized to the TGN (trans-Golgi network), where it transports copper to newly synthesized cuproenzymes. Under elevated copper concentrations, ATP7A traffics to post TGN vesicles and to the plasma membrane, subsequently releasing the copper load by fusion with the plasma membrane. There are emerging studies implicating a role of ATP7A in resistance against the platinum-based chemotherapy agent, cisplatin, which is widely used to treat cancer. Previous studies have correlated ATP7A expression to the sensitivity of cultured cells to cisplatin. Here, we report that deletion of the Atp7a gene in isogenic RAS-transformed mouse embryonic fibroblast (MEF) cells not only confers sensitivity to cisplatin, but markedly prevents tumor formation in the absence of chemotherapy agent. Deletion of Atp7a in the breast cancer cell line, 4T1, was also found to suppress tumorigenesis and markedly reduce metastasis to the lungs. The ATP7A knockout 4T1 cells showed a significant decrease in Lysyl Oxidase (LOX) activity as compared to the 4T1WT cells. Given the numerous studies showing that LOX family involved in breast tumor metastasis, ATP7A might play a role in tumor progression by virtue of its requirement for metallating LOX. The ATP7A knockout 4T1 cells exhibited reduced survival as compared to the 4T1WT cells in the presence of physiological concentrations of hydrogen peroxide or hypoxia, suggesting that hyper-accumulation of copper sensitizes these cells to ROS production. Taken together, these findings identify the ATP7A copper transporter at the nexus of platinum-drug resistance, tumorigenesis and metastatic pathways, underscoring its potential as a therapeutic drug target at multiple stages of carcinogenesis. Citation Format: Vinit Shanbhag, Nikita Gudekar. Role for ATP7A copper transporter in cisplatin resistance, tumorigenesis and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 246.

Abstract 13521: Novel Protective Role of Cu Transporter ATP7A Against Cardiac Fibrosis by Inhibiting Endothelial-Mesenchymal Transition Mediated Through Snail and Antioxidant-1

Cardiac fibrosis is attributed to excess pathological deposition of extracellular-matrix (ECM) components; however, it is also mediated through endothelial-mesenchymal transition (EndMT) and fibroblast-myofibroblast transition (FMT) with unknown mechanisms. Copper (Cu), essential micronutrient, has been implicated in fibrosis. Bioavailability of Cu is tightly controlled by Cu-transporter ATP7A which obtains Cu from Cu chaperone Antioxidant-1 (Atox1), and delivers Cu to the ECM secretory Cu enzyme lysysl oxidase (LOX) or exclude Cu. Atox1 also functions as a Cu-dependent transcription factor. However, role of ATP7A in cardiac fibrosis remains unknown. Here we show that high dose angiotensin II infusion (740ng/kg/min for 10 days) to ATP7A mutant (ATP7Amut) mice with reduced Cu transport function significantly increased cardiac fibrosis (200%) and Cu (170%) compared to wild type mice. Blood pressure and heart/body weight were similar between each group. ATP7Amut mice showed increased Isolectin B4+/αSMA+ cells, indicating EndMT in fibrosis region, which was associated with increase in fibrosis-related genes (αSMA, TGFβ1/2/3, CTGF). In ATP7Amut fibroblasts, although LOX activity was reduced, phenotypic conversion to αSMA+ myofibroblasts (FMT) was accelerated. In ECs isolated from ATP7Amut mice or ATP7A-depleted HUVECs, EC phenotype was converted to mesenchymal phenotype with reduced expression of ECs markers (VE-cadherin, VEGFR2) and increased mesenchymal markers (αSMA, TGFβ2) and Cu levels. Functionally, conditioned media from ATP7A-depleted HUVECs promoted cardiac fibroblast differentiation to myofibroblasts (FMT). Mechanistically, ATP7A knockdown in ECs increased expression of EndMT master regulatory transcription factor Snail as well as Atox1 (2 fold). Atox1 overexpression by adenovirus increased EndMT-inducing genes (αSMA, TGFβ2, Hey2) via binding to their promoters at Atox1 binding sites (GAAAGA) without affecting EC markers or Snail which is shown to reduce EC markers. In summary, ATP7A limits cardiac fibrosis by inhibiting EndMT and FMT via suppressing transcription factors Snail and Atox1. Thus, ATP7A is a potential therapeutic target for fibrosis-dependent cardiovascular diseases.

Effects of ATP7A overexpression in mice on copper transport and metabolism in lactation and gestation

Placentae and mammary epithelial cells are unusual in robustly expressing two copper “pumps”, ATP7A and B, raising the question of their individual roles in these tissues in pregnancy and lactation. Confocal microscopic evidence locates ATP7A to the fetal side of syncytiotrophoblasts, suggesting a role in pumping Cu towards the fetus; and to the basolateral (blood) side of lactating mammary epithelial cells, suggesting a role in recycling Cu to the blood. We tested these concepts in wild‐type C57BL6 mice and their transgenic counterparts that expressed hATP7A at levels 10–20× those of endogenous mAtp7a. In lactation, overexpression of ATP7A reduced the Cu concentrations of the mammary gland and milk ~50%. Rates of transfer of tracer 64Cu to the suckling pups were similarly reduced over 30–48 h, as was the total Cu in 10‐day ‐old pups. During the early and middle periods of gestation, the transgenic litters had higher Cu concentrations than the wild‐type, placental Cu showing the reverse trend; but this difference was lost by the first postnatal day. The transgenic mice expressed ATP7A in some hepatocytes, so we investigated the possibility that metalation of ceruloplasmin (Cp) might be enhanced. Rates of 64Cu incorporation into Cp, oxidase activity, and ratios of holo to apoceruloplasmin were unchanged. We conclude that in the lactating mammary gland, the role of ATP7A is to return Cu to the blood, while in the placenta it mediates Cu delivery to the fetus and is the rate‐limiting step for fetal Cu nutrition during most of gestation in mice.

Multiple Menkes copper ATPase (Atp7a) transcript and protein variants are induced by iron deficiency in rat duodenal enterocytes

The Menkes copper ATPase (Atp7a) pumps copper into the trans-Golgi for cuproenzyme synthesis, and translocates to the basolateral membrane of enterocytes for copper export. Recent studies demonstrated that three 5′ end splice variants of the Atp7a transcript exist in rat duodenum, all of which are strongly induced during iron deprivation. To explore a possible role for Atp7a (and copper) in intestinal iron absorption, the current studies were undertaken to test the hypothesis that multiple Atp7a transcript and protein variants exist in intestinal epithelial cells. Northern blot analyses using probes generated from the full-length Atp7a cDNA revealed several specific hybridization bands, all of which were more intense in RNA samples extracted from duodenal enterocytes isolated from iron-deficient rats. A PCR-based approach, using forward primers specific for the alternative 5′ end splice variants and a reverse primer in exon 23, demonstrated that 3 full-length transcripts exist in rat IEC-6 cells. To identify possible Atp7a protein variants, three distinct polyclonal antisera were utilized. The specificity of the antisera was first established by western blotting and immunoprecipitation studies using samples derived from isolated rat enterocytes and Atp7a knockdown IEC-6 cells. Several specific immunoreactive bands were documented, and a unique Atp7a protein distribution in cytosolic vesicle-like structures was noted. In conclusion, multiple Atp7a transcript and protein variants exist in rodent intestinal epithelial cells and are induced by dietary iron deprivation. Further studies will be designed to determine the subcellular distribution of Atp7a protein variants and possible unique functions of each.

Human Macrophage ATP7A is Localized in the trans-Golgi Apparatus, Controls Intracellular Copper Levels, and Mediates Macrophage Responses to Dermal Wounds

The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound.