2DGE generates cancer-specific isoform patterns and compositions indicative of cancer presence, tumor type, disease severity, and possibly therapeutic response. The protocol resulted in the detection of more than ten cancer-specific ENOX2 isoforms which are resolved to indicate cancer presence and tissue of origin. The ENOX2 isoforms are identified on the basis of their molecular mass and isoelectric point with detection using an ENOX2-specific recombinant antibody single chain variable region scFv fragment expressed in bacteria which recognizes all ENOX2 proteins. The latter was produced from ENOX2-specific IgG rescued from ENOX2-specific monoclonal antibody producing hybridoma cells [12].
Each of the ENOX2 isoforms signaling cervical (94 kDa), ovarian (80 kDa), prostate (75 kDa), breast (64–68 kDa), non-small cell lung (54 kDa), leukemia/lymphoma (45 kDa), and melanoma (38 kDa) is represented on 2DGE/western blots by ENOX2 antibody-reactive species of unique molecular weights. Colon, pancreatic, and small cell lung cancer share a common isoform of molecular weight 52 kDa. However, with colon cancer, the 52-kDa isoform is accompanied by a 40.5-kDa isoform with an isoelectric point of pH 3.9. For pancreatic cancer, the 52-kDa isoform exhibits an isoelectric point of pH 3.9 (compared to 4.3 for the 52-kDa isoform of colon and small cell lung cancer) and is accompanied by a 50-kDa isoform (Fig. 2g). The 40.5-kDa isoform which accompanies the 80-kDa isoform indicative of ovarian cancer appears to be the same or similar to the 40.5-kDa isoform characteristic of colon cancer. With small cell lung cancer, no obvious secondary ENOX2 isoform markers have been observed on any of the 2DGE western blots such that presence of the 52-kDa ENOX2 isoform and absence of the 40.5-kDa ENOX2 isoform serves to distinguish small cell lung from colon cancer.
Comparisons with known amounts of 34 kDa recombinant ENOX2 proteins produced in bacteria were used to demonstrate that ENOX2 proteins were absent or present at levels below the limits of detection (less than 10 pmol/ml of serum) from sera of healthy volunteers or patients with diseases other than cancer. In contrast, circulating ENOX2 has been detected in sera of more than 500 cancer patients representing all major forms of human cancer, including leukemias and lymphomas based on spectrophotometric measurements [10, 11].
The presence of the ENOX2 protein was earlier demonstrated in a number of human tumor tissues and xenografts from cytochemical studies [12]. However, serum analyses had already suggested a much broader association with cancer. ENOX2 proteins are ectoproteins reversibly bound at the outer leaflet of the plasma membrane [18]. As is characteristic of other examples of ectoproteins (sialyl and galactosyl transferases, dipeptidylamino peptidase IV, etc.), the ENOX2 proteins are shed [9, 10]. They appear in soluble form in conditioned media of cultured cells [9], in patient sera [19] and in patient urine [20]. The ENOX2 isoforms from sera of cancer patients exhibit the same degree and specificity of inhibition by anti-cancer drugs (i.e., the EC50 values for inhibition of activity are the same or similar) as do the membrane-associated forms [10, 11]. In contrast, no drug-responsive NOX activities were found with sera from healthy volunteers or sera from patients with diseases other than cancer. As such, the antitumor-responsive ENOX2 activity represents the first reported cell surface change undetected from non-cancer cells and potentially associated with most, if not all, forms of human cancer. ENOX2 isoform presence at all stages of cancer progression (I–IV) offer the potential for non-invasive, early detection of cancer as well as the monitoring of therapeutic response and cancer recurrence and other diagnostic applications.
The ENOX2 (tNOX) gene is present in the human genome as a single copy, with no obvious homologs and a single constitutive ENOX1 (CNOX) ortholog. It is not a gene mutated in cancer but is universally present. The ENOX2 splice variants all appear to be variations resulting from an exon 4 minus splicing event that allows for down-stream initiation and expression at the cell surface of the ENOX2 protein only in cancer cells [13]. Without the exon 4 deletion, mRNA derived from the gene does not appear to be translated into protein.
Analysis of ENOX2 gene transcripts and the results from the findings with the pan-ENOX2 recombinant antibody establish that most, if not all, ENOX2 isoforms share a common region of protein structure of about 25 kDa (230 amino acids) at the carboxy terminus. This region contains the region of the protein responsible for its functional enzymatic activities, its response to anticancer drugs (drug-binding site) and the determinants in exon 5 at or near the drug-binding site recognized by the recombinant antibody. These properties are shared by all of the ENOX2 isoforms recognized on western blots. However, the remainder of the protein sequence of each of the isoforms must contain sequences which are unique to that particular isoform to account for their demonstrated differences in relative molecular weight and isoelectric point. These sequences will be available to generate monoclonal or peptide antibodies that recognize only these isoform-specific regions of the ENOX2 proteins.