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ENOX2 (ecto-NOX disulfide-thiol exchanger 2)

Written2014-01Xiaoyu Tang, Dorothy M Morré, D James Morré
MorNuCo, Inc., 1201 Cumberland Avenue, Ste. B, Purdue Research Park,.West Lafayette, IN 47906 USA

(Note : for Links provided by Atlas : click)


Alias (NCBI)APK1
HGNC Alias symbAPK1
HGNC Alias nametumor-associated NADH oxidase
 ecto-NADPH oxidase disulfide-thiol exchanger 2
HGNC Previous nameCOVA1
HGNC Previous namecytosolic ovarian carcinoma antigen 1
LocusID (NCBI) 10495
Atlas_Id 40134
Location Xq26.1  [Link to chromosome band Xq26]
Location_base_pair Starts at 130623381 and ends at 130903317 bp from pter ( according to GRCh38/hg38-Dec_2013)  [Mapping ENOX2.png]
Fusion genes
(updated 2017)
Data from Atlas, Mitelman, Cosmic Fusion, Fusion Cancer, TCGA fusion databases with official HUGO symbols (see references in chromosomal bands)
ENOX2 (Xq26.1)::OSBPL5 (11p15.4)ENOX2 (Xq26.1)::TXN2 (22q12.3)
Note Also termed APK1 antigen, or cytosolic ovarian carcinoma antigen 1, or tumor-associated hydroquinone oxidase (tNOX).
ECTO-NOX2 = Ecto-Nicotinamide Dinucleotide Oxidase Disulfide Thiol Exchange 2.


  Figure 1. ENOX2 mRNA.
Description The human ENOX2 gene is located on the reverse strand of chromosome X (bases 4918 to 284856); according to NCBI Refseq Gene Database (gene ID: 10495, RefSeq ID: NG_012562.1), and is comprised of 279939 bp.
ENOX2 is composed of 13 protein-coding exons between 71 bp and 2066 bp in length and 14 introns which vary greatly in length (1781 bp to 117994 bp). It has a 501 bp 5' untranslated region and a long 3' UTR (approximately 1935 bp).
Transcription According to NCBI the human ENOX2 gene encodes a 4036 bp mRNA transcript, the coding sequence (CDS) located from base pairs 356 to 2101 (NM_001281736.1). The CDS from the Ensembl genome browser database (ENST00000370927, transcript length 3788 bp) and NCBI (NM_001281736.1) are identical. Transcripts NM_001281736.1 and ENST 00000370927 are also included in the human CCDS set (CCDS14626) and encode a 610 aa long protein.
Pseudogene None known.


  Figure 2. Deduced amino acid sequence and functional motifs of the bacterially expressed 46 kDa enzymatically active C-terminus of ENOX2.
Description ENOX2 transcription variants all appear to be variations that include an exon 4 minus splicing event that allows for down-stream initiation and expression of the ENOX2 protein at the cell surface of malignant cells (Tang et al., 2007a; Tang et al., 2007b). Without the exon 4 deletion, mRNA derived from the gene does not appear to be translated into protein. Thus, the exon 4 deletion is the basis for the cancer specificity of the ENOX2 transcription variants. An hnRNP splicing factor directs formation of the Exon 4 minus variants of ENOX2 (Tang et al., 2011). The fully processed 34 kDa generic ENOX2 protein found on the cell surface of HeLa cells and in sera of about 23% of early cancer patients retains full-functional activity. The deduced amino acid sequence of a bacterially expressed 46 kDa functional C-terminus of ENOX2 exhibits the same characteristics of alternation of the two activities and drug response as the cell surface and generic serums forms. Identified functional motifs include a quinone binding site, an adenine nucleotide binding site, a CXXXXC cysteine motif as a potential disulfide-thiol interchange site and two copper binding sites, one of which is conserved with superoxide dismutase. ENOX2 proteins lack flavin and only one of the two C-X-X-X-X-C motifs characteristic of flavoproteins are present in ENOX2. Yet the protein effectively carries out protein disulfide interchange. The motif C569-X-X-X-X-X-C575, alone or together with a downstream histidine (H582) provides an additional potential active site for protein disulfide-thiol interchange (Morré and Morré, 2013).
The signature ENOX2 motif is that of the potential drug/antibody binding site E394EMTE. Antisera directed to this portion of the protein act as competitive inhibitors to drug binding. The sequence provides a putative quinone or sulfonylurea-binding site with four of the five amino acids in at least one other putative quinone site in the same relative positions.
The correctness of the various assignments has, for the most part, been confirmed by site-directed mutagenesis (Chueh et al., 2002). While amino acid replacements that block oxidation of reduced pyridine nucleotide by ENOX2 also eliminated protein disulfide-thiol interchange and vice versa (Chueh et al., 2002), the two activities appear to occur independently. One can be measured in the absence of the other.
The ENOX2 proteins have properties of prions and are protease resistant (Kelker et al., 2001) and N-terminal sequencing. Concentrated solutions aggregate and form amyloid-like filaments.
Expression Widely expressed in malignant cells but only as exon 4 minus splicing variants (Tang et al., 2007b).
Localisation External cell surface (Morré, 1995).
Function ENOX2 is a member of a family of cell surface metalocatalysts with binuclear copper centers that oscillate. They catalyze both NAD(P)H and hydroquinone oxidation in one configuration and carry out protein disulfide-thiol interchange in a second configuration (Figure 3). The two activities alternate creating a regular 22 min period to impart a time-keeping function (Morré and Morré, 2003). The oscillations are highly synchronous and phased by low frequency electromagnetic fields.
Functionally ENOX2 proteins of cancer cells act as terminal oxidases of plasma membrane electron transport (PMET) whereby electrons coming from cytosolic NAD(P)H are transferred to membrane-located coenzyme Q with eventual transfer of electrons and proteins to oxygen to form water (Figure 4). The released energy is presumably utilized to drive cell enlargement. The protein disulfide-thiol interchange part of the cycle carries out a function essential to the cell enlargement mechanism (Morré et al., 2006). The phenotype of unregulated accelerated growth is recapitulated in a transgenic mouse strain over expressing human ENOX2 (Yagiz et al., 2006).
  Figure 3. Diagrammatic representation of the functional unit of the ENOX2 proteins which is a dimer, each monomer of which contains two copper centers. During the oxidative portion of the ENOX cycle on the right, the net result is the transfer of 4 electrons plus 4 protons to molecular oxygen to from 2H2O. The left portion of the diagram illustrates the protein disulfide-thiol interchange activity portion of the cycle where the result is an interchange of protons and electrons that results in the breakage and formation of disulfide bonds important to cell enlargement.
Homology RNA recognition motif (RRM) in the cell surface Ecto-NOX disulfide-thiol exchanger (ECTO-NOX or ENOX) proteins. This subgroup corresponds to the conserved RNA recognition motif (RRM) in ECTO-NOX proteins (also termed ENOX), comprising a family of plant and animal NAD(P)H oxidases exhibiting both oxidative and protein disulfide-like activities.
The ENOX2 gene is present in the human genome as a single copy, with no obvious homologs and a single constitutive ENOX1 (CNOX) ortholog with 64% identity and 80% similarity (Jiang et al., 2008).


Somatic No reports of mutations leading to inactivation of or inability to express ENOX2.

Implicated in

Entity Various cancers
Note The ENOX2 protein is universally associated with malignancies. It is not the result of an oncogenic mutation but appears to be similar if not identical to a form of ENOX protein with characteristics of an oncofetal protein important to maintenance of unregulated growth in very early development that may be re-expressed in malignancy (Cho and Morré, 2009). Re-expression as an oncofetal protein helps explain the role of ENOX2 of cancer cells in acquiring the well-known characteristic of uncontrolled growth. Consistent with this interpretation are observations that the malignant phenotypes of invasiveness and growth on soft agar of cancer cells in culture are lost when cells are transfected with ENOX2 antisense (Chueh et al., 2004; Tang et al., 2007a). ENOX2 is the first reported cell surface change absent from non-cancer cells and associated with most, if not all, forms of human cancer (Morré and Morré, 2013). As such, ENOX2 emerges as a potential universal molecular cancer marker and, being an ecto-protein at the cell surface and shed into the circulation, a reliable cancer diagnostic marker both for cancer presence and tissue of cancer origin (Figure 4).
ENOX2 proteins are expressed differently by different tissues of cancer origin within the body with each type of cancer being characterized by one, two, three or more tissue specific transcript variants of characteristic molecular weights and isoelectric points (Morré and Morré, 2012). ENOX2 proteins are absent or reduced to below the limits of detection from sera of healthy individuals or patients with diseases other than cancer. Circulating ENOX2 has been detected in sera of patients representing all major forms of human cancer including leukemias and lymphomas. All ENOX2 transcript variants appear to share the common antigenic determinant recognized both by an ENOX2-specific monoclonal antibody (Cho et al., 2002) and a corresponding scFv single chain variable region recombinant antibody expressed in bacteria and derived from the monoclonal antibody-producing hybridoma cells with analysis by 2-D-gel electrophoresis and western blot (Hostetler et al., 2009).
Figure 4. Schematic representation of the utility of the ENOX2 proteins as cancer-specific cell surface proteins for diagnosis and therapeutic intervention in cancer. Modified from Morré and Morré (2013).
Entity Breast cancer
Note Sera of breast cancer patients contains an ENOX2 transcript variant of 64 to 69 kDa, isoelectric point 4.2 to 4.9.
Entity Lung cancer
Note Sera of patients with non-small cell lung cancer contain a 53 to 56 kDa ENOX2 transcript variant, isoelectric point pH 4.7 to 5.3 while sera of small cell lung cancer contain a transcript variant of 52 kDa, isoelectric point pH 4.1 to 4.6.
Entity Prostate cancer
Note Sera of patients with prostate cancer contain a 71 to 88 kDa ENOX2 transcript variant, isoelectric point pH 5.1 to 6.5.
Entity Cervical cancer
Note Sera of cervical cancer patients contain a 90 to 100 kDa transcript variant, isoelectric point pH 4.2 to 5.4.
Entity Malignant melanoma
Note Sera from malignant melanoma patients contain an ENOX2 transcript variant of 37 to 41 kDa, isoelectric point pH 4.6 to 5.3.
Entity Leukemias, lymphomas and myelomas
Note Sera of patients with leukemia, lymphoma or myeloma, cancers having blood as the common tissue of origin, all contain ENOX2 transcript variants of 38 to 48 kDa and low isoelectric point pH 3.6 to 4.5.
Entity Ovarian cancer
Note Sera from ovarian carcinoma patients contain two ENOX2 transcript variants of 72 to 90 kDa and 37 to 47 kDa, both having similar isoelectric points in the range of pH 3.7 to 5.0.
Entity Bladder cancer
Note Sera of patients with carcinoma of the bladder contain two ENOX2 transcript variants of 63 to 66 kDa and 42 to 48 kDa with isoelectric points of 4.2 to 5.8 and 4.1 to 4.8, respectively.
Entity Uterine cancer
Note Sera of patients with uterine carcinoma contain two ENOX2 transcript variants of 64 to 69 kDa and 36 to 48 kDa with isoelectric points of pH 4.2 to 4.9 and pH 4.5 to 5.6.
Entity Colorectal cancer
Note Sera of patients with colorectal cancer contain at least two of three possible ENOX2 transcript variants of 80 to 96 kDa, isoelectric point pH 4.5 to 5.3, 50 to 60 kDa, isoelectric point pH 4.2 to 5.1 and 33 to 46 kDa, isoelectric point pH 3.8 to 5.2.
Entity Other cancers
Note Unique patterns of ENOX2 transcript variant expression (number, molecular with and isoelectric point) have been found as well associated with brain, endometrial, esophageal, gastric, hepatocellular renal cell, squamous cell, testicular germ cell and thyroid cancer as well as mesothelioma and sarcomas.
Entity Endometriosis
Note Invasive endometriosis is the only non-malignant disorder thus far characterized by the presence of unique ENOX2 transcript variants.
Entity As a cancer therapeutic drug target
Note ENOX2 is responsive to differentiating agents such as calcitriol and anticancer retinoids and inhibited by anticancer drugs such as doxorubicin, the anticancer sulfonylureas, the vanilloid capsaicin, the catechin EGCg and the cancer isoflavene phenoxodiol, all of which appear to function as quinone site inhibitors directed toward the EEMTE drug binding motif of ENOX2 (Morré and Morré, 2013; Hanau et al., 2014). The possibility of ENOX2 as a drug target is enhanced by the external location of the ENOX2 protein in a position to be readily available to drugs or antibodies conjugated to impermeant supports. As the growth involvement of ENOX2 proteins is in cell enlargement, ENOX2 inhibitors also block cell proliferation. The blocked cells, unable to enlarge, also fail to divide and eventually undergo apoptosis (Figure 4).


Monoclonal antibody to a cancer-specific and drug-responsive hydroquinone (NADH) oxidase from the sera of cancer patients.
Cho N, Chueh PJ, Kim C, Caldwell S, Morre DM, Morre DJ.
Cancer Immunol Immunother. 2002 May;51(3):121-9. Epub 2002 Feb 27.
PMID 11941450
Early developmental expression of a normally tumor-associated and drug-inhibited cell surface-located NADH oxidase (ENOX2) in non-cancer cells.
Cho N, Morre DJ.
Cancer Immunol Immunother. 2009 Apr;58(4):547-52. doi: 10.1007/s00262-008-0577-3. Epub 2008 Aug 15.
PMID 18704408
Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface.
Chueh PJ, Kim C, Cho N, Morre DM, Morre DJ.
Biochemistry. 2002 Mar 19;41(11):3732-41.
PMID 11888291
tNOX is both necessary and sufficient as a cellular target for the anticancer actions of capsaicin and the green tea catechin (-)-epigallocatechin-3-gallate.
Chueh PJ, Wu LY, Morre DM, Morre DJ.
Biofactors. 2004;20(4):235-49.
PMID 15706060
Cancer prevention trial of a synergistic mixture of green tea concentrate plus Capsicum (CAPSOL-T) in a random population of subjects ages 40-84.
Hanau C, Morre DJ, Morre DM. Clin Proteomics 2014: 11(2).
Cancer site-specific isoforms of ENOX2 (tNOX), a cancer-specific cell surface oxidase.
Hostetler B, Weston N, Kim C, Morre DM, Morre DJ. Clin Proteomics 2009:5:46-51.
Molecular cloning and characterization of a candidate human growth-related and time-keeping constitutive cell surface hydroquinone (NADH) oxidase.
Jiang Z, Gorenstein NM, Morre DM, Morre DJ.
Biochemistry. 2008 Dec 30;47(52):14028-38. doi: 10.1021/bi801073p.
PMID 19055324
Cancer isoform of a tumor-associated cell surface NADH oxidase (tNOX) has properties of a prion.
Kelker M, Kim C, Chueh PJ, Guimont R, Morre DM, Morre DJ.
Biochemistry. 2001 Jun 26;40(25):7351-4.
PMID 11412089
ATP-dependent and drug-inhibited vesicle enlargement reconstituted using synthetic lipids and recombinant proteins.
Morre DJ, Kim C, Hicks-Berger C.
Biofactors. 2006;28(2):105-17.
PMID 17379941
ECTO-NOX Proteins.
Morre DJ, Morre DM.
ISBM 978-1-4614-3957-8. Springer, New York, 2013, 507 pp.
NADH oxidase activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea (LY181984) at an external site.
Morre DJ.
Biochim Biophys Acta. 1995 Dec 13;1240(2):201-8.
PMID 8541291
hnRNP F directs formation of an exon 4 minus variant of tumor-associated NADH oxidase (ENOX2).
Tang X, Kane VD, Morre DM, Morre DJ.
Mol Cell Biochem. 2011 Nov;357(1-2):55-63. doi: 10.1007/s11010-011-0875-5. Epub 2011 May 28.
PMID 21625959
Antisense experiments demonstrate an exon 4 minus splice variant mRNA as the basis for expression of tNOX, a cancer-specific cell surface protein.
Tang X, Morre DJ, Morre DM.
Oncol Res. 2007a;16(12):557-67.
PMID 18351130
Alternative splicing as the basis for specific localization of tNOX, a unique hydroquinone (NADH) oxidase, to the cancer cell surface.
Tang X, Tian Z, Chueh PJ, Chen S, Morre DM, Morre DJ.
Biochemistry. 2007b Oct 30;46(43):12337-46. Epub 2007 Oct 9.
PMID 17924659
Transgenic mouse line overexpressing the cancer-specific tNOX protein has an enhanced growth and acquired drug-response phenotype.
Yagiz K, Morre DJ, Morre DM.
J Nutr Biochem. 2006 Nov;17(11):750-9. Epub 2006 Jan 25.
PMID 16517149


This paper should be referenced as such :
X Tang, DM Morré, DJ Morré
ENOX2 (ecto-NOX disulfide-thiol exchanger 2)
Atlas Genet Cytogenet Oncol Haematol. 2014;18(9):632-637.
Free journal version : [ pdf ]   [ DOI ]

External links


HGNC (Hugo)ENOX2   2259
Entrez_Gene (NCBI)ENOX2    ecto-NOX disulfide-thiol exchanger 2
AliasesAPK1; COVA1; tNOX
GeneCards (Weizmann)ENOX2
Ensembl hg19 (Hinxton)ENSG00000165675 [Gene_View]
Ensembl hg38 (Hinxton)ENSG00000165675 [Gene_View]  ENSG00000165675 [Sequence]  chrX:130623381-130903317 [Contig_View]  ENOX2 [Vega]
ICGC DataPortalENSG00000165675
TCGA cBioPortalENOX2
Genatlas (Paris)ENOX2
SOURCE (Princeton)ENOX2
Genetics Home Reference (NIH)ENOX2
Genomic and cartography
GoldenPath hg38 (UCSC)ENOX2  -     chrX:130623381-130903317 -  Xq26.1   [Description]    (hg38-Dec_2013)
GoldenPath hg19 (UCSC)ENOX2  -     Xq26.1   [Description]    (hg19-Feb_2009)
GoldenPathENOX2 - Xq26.1 [CytoView hg19]  ENOX2 - Xq26.1 [CytoView hg38]
Genome Data Viewer NCBIENOX2 [Mapview hg19]  
Gene and transcription
Genbank (Entrez)AF207881 AI540639 AK000353 AK289812 AK289837
RefSeq transcript (Entrez)NM_001281736 NM_001382516 NM_001382517 NM_001382518 NM_001382519 NM_001382520 NM_001382521 NM_001382522 NM_006375 NM_182314
Consensus coding sequences : CCDS (NCBI)ENOX2
Gene ExpressionENOX2 [ NCBI-GEO ]   ENOX2 [ EBI - ARRAY_EXPRESS ]   ENOX2 [ SEEK ]   ENOX2 [ MEM ]
Gene Expression Viewer (FireBrowse)ENOX2 [ Firebrowse - Broad ]
GenevisibleExpression of ENOX2 in : [tissues]  [cell-lines]  [cancer]  [perturbations]  
BioGPS (Tissue expression)10495
GTEX Portal (Tissue expression)ENOX2
Human Protein AtlasENSG00000165675-ENOX2 [pathology]   [cell]   [tissue]
Protein : pattern, domain, 3D structure
UniProt/SwissProtQ16206   [function]  [subcellular_location]  [family_and_domains]  [pathology_and_biotech]  [ptm_processing]  [expression]  [interaction]
NextProtQ16206  [Sequence]  [Exons]  [Medical]  [Publications]
With graphics : InterProQ16206
Catalytic activity : Enzyme1.-.-.- [ Enzyme-Expasy ]   1.-.-.-1.-.-.- [ IntEnz-EBI ]   1.-.-.- [ BRENDA ]   1.-.-.- [ KEGG ]   [ MEROPS ]
Domaine pattern : Prosite (Expaxy)RRM (PS50102)   
Domains : Interpro (EBI)ENOX    ENOX_RRM    Nucleotide-bd_a/b_plait_sf    RBD_domain_sf    RRM_dom   
Domain families : Pfam (Sanger)RRM_1 (PF00076)   
Domain families : Pfam (NCBI)pfam00076   
Domain families : Smart (EMBL)RRM (SM00360)  
Conserved Domain (NCBI)ENOX2
AlphaFold pdb e-kbQ16206   
Human Protein Atlas [tissue]ENSG00000165675-ENOX2 [tissue]
Protein Interaction databases
IntAct (EBI)Q16206
Ontologies - Pathways
Ontology : AmiGOnucleic acid binding  extracellular space  cytosol  ultradian rhythm  external side of plasma membrane  protein-disulfide reductase activity  electron transport chain  regulation of growth  
Ontology : EGO-EBInucleic acid binding  extracellular space  cytosol  ultradian rhythm  external side of plasma membrane  protein-disulfide reductase activity  electron transport chain  regulation of growth  
NDEx NetworkENOX2
Atlas of Cancer Signalling NetworkENOX2
Wikipedia pathwaysENOX2
Orthology - Evolution
GeneTree (enSembl)ENSG00000165675
Phylogenetic Trees/Animal Genes : TreeFamENOX2
Homologs : HomoloGeneENOX2
Homology/Alignments : Family Browser (UCSC)ENOX2
Gene fusions - Rearrangements
Fusion : MitelmanENOX2::OSBPL5 [Xq26.1/11p15.4]  
Fusion : QuiverENOX2
Polymorphisms : SNP and Copy number variants
NCBI Variation ViewerENOX2 [hg38]
dbSNP Single Nucleotide Polymorphism (NCBI)ENOX2
Exome Variant ServerENOX2
GNOMAD BrowserENSG00000165675
Varsome BrowserENOX2
ACMGENOX2 variants
Genomic Variants (DGV)ENOX2 [DGVbeta]
DECIPHERENOX2 [patients]   [syndromes]   [variants]   [genes]  
CONAN: Copy Number AnalysisENOX2 
ICGC Data PortalENOX2 
TCGA Data PortalENOX2 
Broad Tumor PortalENOX2
OASIS PortalENOX2 [ Somatic mutations - Copy number]
Somatic Mutations in Cancer : COSMICENOX2  [overview]  [genome browser]  [tissue]  [distribution]  
Somatic Mutations in Cancer : COSMIC3DENOX2
Mutations and Diseases : HGMDENOX2
LOVD (Leiden Open Variation Database)[gene] [transcripts] [variants]
DgiDB (Drug Gene Interaction Database)ENOX2
DoCM (Curated mutations)ENOX2
CIViC (Clinical Interpretations of Variants in Cancer)ENOX2
NCG (London)ENOX2
Impact of mutations[PolyPhen2] [Provean] [Buck Institute : MutDB] [Mutation Assessor] [Mutanalyser]
Genetic Testing Registry ENOX2
NextProtQ16206 [Medical]
Target ValidationENOX2
Huge Navigator ENOX2 [HugePedia]
Clinical trials, drugs, therapy
Protein Interactions : CTDENOX2
Pharm GKB GenePA162385106
Clinical trialENOX2
DataMed IndexENOX2
PubMed42 Pubmed reference(s) in Entrez
GeneRIFsGene References Into Functions (Entrez)
REVIEW articlesautomatic search in PubMed
Last year publicationsautomatic search in PubMed

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