**3. Results**

### **3.1 Early detection**

Analytical 2-D gel electrophoresis and immunoblotting of ENOX proteins from a mixed population of cancer patients (cervical, breast, ovarian, lung and colon carcinomas, leukemias and lymphomas) revealed multiple species of acidic proteins of molecular weights between 34 and 100 kDa in quadrants I and IV (Fig 2B), none of which were present in sera of non-cancer patients (Fig. 2A) (Hostetler et al., 2009). Separation in the first dimension was by isoelectric focusing over the pH range of 3 to 10 and separation in the second dimension was by 10 percent SDS-PAGE. Isoelectric points of the ENOX2 transcript variants were in the range of 3.9 to 6.3. The principal reactive proteins other than the ENOX2 forms were a 53 kDa isoelectric point pH 4.l, mostly phosphorylated 1-antitrypsin inhibitor

As the 2-D-western blot protocol detects cancer early, well in advance of clinical symptoms, the opportunity to combine early detection with early intervention as a potentially curative prevention strategy for cancer by eliminating the disease in its very earliest stages is unique. The approach to early intervention is based on previous work in cell culture models showing that ENOX2 proteins are required to support the unregulated growth that typifies cancer cells. If the growth function of ENOX2 is blocked for 48 to 72 h, the cancer cells cannot enlarge following division, cannot pass the checkpoint in G1 that monitors cell size and eventually undergo programmed cell death (apoptosis) (Morré and Morré, 2003b; De Luca et al., 2010) as diagrammed in Figure 1. Among the early intervention strategies under investigation are several targeted to ENOX2, production of ENOX2-directed vaccines being one promising example. Recombinant ENOX2 peptides that exhibit cancer specificity are employed as antigens. Other forms of ENOX2-directed therapeutic interventions under study include use of dietary modulators (Morré et al., 2009b). Most advanced are studies with herbal mixtures of green tea and powders of ground chili peppers (*Capsicum* species) from efficacious pepper sources (e.g. guajillo or ancho) with levels of capsaicin, the pungent

Analytical 2-D gel electrophoresis and immunoblotting of ENOX proteins from a mixed population of cancer patients (cervical, breast, ovarian, lung and colon carcinomas, leukemias and lymphomas) revealed multiple species of acidic proteins of molecular weights between 34 and 100 kDa in quadrants I and IV (Fig 2B), none of which were present in sera of non-cancer patients (Fig. 2A) (Hostetler et al., 2009). Separation in the first dimension was by isoelectric focusing over the pH range of 3 to 10 and separation in the second dimension was by 10 percent SDS-PAGE. Isoelectric points of the ENOX2 transcript variants were in the range of 3.9 to 6.3. The principal reactive proteins other than the ENOX2 forms were a 53 kDa isoelectric point pH 4.l, mostly phosphorylated 1-antitrypsin inhibitor

principle of chili peppers, sufficiently low so as to not cause discomfort.

Cervical 18 94 kDa 5.4 Ovarian 41 80 and 40.5 kDa 4.2 and 4.1 Prostate 70 75 kDa 6.3 Breast/Uterine 55 64 to 68 kDa 4.5 Non-small cell lung 83 54 kDa 5.1 Small cell lung 22 52 kDa 4.3 Pancreatic 24 50 and 52 kDa 4.3 and 3.9 Colon 55 52 and 34 kDa 4.3 and 3.9 Lymphoma, Leukemia 16 45 kDa 3.9 Melanoma 12 38 kDa 5.1 Table 1. Sera from patients with different cancers exhibit distinct patterns of ENOX2 isoforms with characteristic molecular weights and isoelectric points (pH). Updated from

Hostetler et al. (2009).

**2.2 Early intervention** 

**3. Results** 

**3.1 Early detection** 

**Cancer Sera analyzed Molecular weight Isoelectric point, pH** 

(2-HS-glycoprotein; fetuin A) (Labeled "R" in Fig. 5) which served as a convenient loading control and isoelectric point reference and a 79-85 kDa, isoelectric point pH 6.8 serotransferrin which served as a second point of reference for loading and as an isoelectric point reference (Table 2). The two cross reactive reference proteins are present in a majority of sera and plasma of both cancer and non-cancer subjects. Albumin and other serum proteins do not react. On some blots, the recombinant scFv was weakly cross-reactive with heavy (ca. 52 kDa) and light (ca. 25 kDa) immunoglobulin chains.


Table 2. Protein sequence similarity between ENOX2 and the two reference proteins 1- anti-trypsin inhibitor and serrotransferrin reactive with the pan ENOX2 scFv recombinant antibody. Regions of similarity are restricted to a 7 amino acid sequence (underlined) adjacent in ENOX2 to the E394EMTE398 quinone inhibitor-binding site.

Sera from individual patients with various forms of cancer were analyzed by 2-D gel electrophoresis and immunoblotting to assign each of the ENOX2 isoforms of Fig. 2 to a cancer of a particular tissue of origin (Table 1). Sera of breast cancer patients contained only the 64 to 68 kDa ENOX2 (Fig. 3D; Fig. 5 arrow) and the 1-antitrypsin inhibitor reference protein (Fig. 5). Sera from cervical cancer patients contained the 94 kDa ENOX2 transcript variant (Fig. 3A). Sera from ovarian cancer patients contained ENOX1 transcript variants of 80 kDa and 40.5b kDa (Fig. 3B). Sera from patients with prostate cancer contained one or more 75 kDa ENOX2 transcript variants resulting in small variations in isoelectric points (Fig. 3C). Sera from patients with non-small cell lung carcinoma contained a 52 kDa ENOX2 transcript variant while sera from non-small cell lung carcinoma patients contained a 52 kDa ENOX2 transcript variant (Fig 3E;F; Fig 4). Simultaneous presence of ENOX2 transcript variants of both 50 and 52 kDa characterized sera of pancreatic cancer patients (Fig. 3G) whereas sera of colon cancer patients contained ENOX2 transcript variants of 52 kDa and 43 kDa (Fig. 3H). Fig. 3I from sera of a patient with non-Hodgkin's lymphoma illustrates the 45 kDa ENOX2 transcript variant of low isoelectric point characteristic of leukemias and lymphomas. Sera of patients with malignant melanoma contained an ENOX2 transcript variant of 38 kDa (Fig. 3J).

Particularly relevant are observations where the 64 to 68 kDa ENOX2 transcript variant (pH 4.5) of sera correlated with disease presence in both late (Stage IV) (Fig. 5A) and early (Stage I) (Fig. 5E) disease and in Stage IV recurrence (Fig. 5C) but was absent from sera of noncancer (normal) volunteers (Fig. 5B) or in survivors free of disease for one to five years (Fig. 5D). Additionally, the 64 to 68 kDa breast cancer-specific transcript variant does not apply to a subset of breast cancer patients but appears to be widely present. Analyses of sera of more than 55 patients with active disease including 20 Stage I and Stage II breast cancer patients all tested positive.

Unlike most published cancer markers, cancer-specific ENOX2 variants are not simply present as elevated levels of a serum constituent present in lesser amounts in the absence of cancer. The cancer-specific ENOX2 transcript variants result from cancer-specific expression of alternatively spliced mRNAs (Tang et al., 2007; 2008). Neither the splice variant mRNAs nor the ENOX2 isoform proteins are present in detectable levels in non-cancer cells or in sera of subjects without cancer (Table 1).

Early Detection: An Opportunity for Cancer Prevention Through Early Intervention 395

Fig. 5. 2-D gel electrophoretic separations and detection of ENOX2 transcript variants specific for breast cancer by western blotting of patient sera. Arrow = 66 to 68 kDa breast cancer specific transcript variant. R = 52 kDa, isoelectric point pH 4.1 1-antitrypsin

evident cancer (Petro et al., 2000) as diagrammed in Figure 6.

Findings from a separate study with small cell and non-small cell lung cancer suggest that the 2-D-western blot test detects cancer presence as early as 5 to 7 years in advance of the appearance of clinical symptoms. This supposition is based mainly on our analysis of two special cancer panels of sera obtained through the Early Detection Research Network (EDRN) of the National Cancer Institute. One panel consisted of about 20 known lung cancer patient sera and 35 control patient sera. Using the 2-D-western blot protocol to identify specific ENOX2 isoforms, we successfully identified all 20 of the known lung cancer patient sera. However, unexpectedly, a high incidence of ENOX2 presence was encountered in sera from the "control" group which were obtained from a community screening study. From additional information obtained through the EDRN, 16 of the 17 positive control subject samples where our findings specifically indicated lung cancer (the lung cancer ENOX2 markers were found) were smokers with smoking histories in the range of 15 to 88 pack-years. However, the anticipated incidence of undetected lung cancers in such a population would be in the order of 10% or less rather than nearly 50%. Since the aberrant ENOX2 transcript variants associated with lung cancer are single molecular species produced only by lung cancer, the possibility was raised that lung cancer was being detected much earlier than was currently possible by other methods. The indications might be as early as 5 to 7 years before clinical symptoms, based on the estimated 20 year development time for lung cancer expression between carcinogen exposure and a clinically

Similar results were obtained with a panel of female subjects at risk for breast and ovarian cancer. An analysis of a panel of 127 sera in a Biomarker Reference Set for Cancers in Women also provided through the Early Detection Research Network of the National Cancer Institute support our indications that the 2-D gel-western blot system is able to detect cancer presence 5 to 7 years in advance of clinical symptoms. The panel consisted of samples pooled from 441 women in 12 different gynecologic and breast disease categories plus 115 sera from age-matched control women. Of the l27 sera samples in the panel, 29 tested positive for breast cancer and another 16 tested positive for ovarian cancer. Since the aberrant transcript variants are single molecular species produced by specific cancers such as lung, breast or ovarian, the findings suggest that cancer was being detected in the control popuation much earlier than is currently possible by other methods. As estimated for lung

inhibitor reference spot.

Fig. 3. Western blots of 2-D gel electrophoresis/western blots of sera from cancer patients analyzed individually. Cancer sites are presented in the order of decreasing molecular weight of the major transcript variant present. A. Cervical cancer. B. Ovarian cancer C. Prostate cancer. D. Breast cancer. E. Non-small cell lung cancer. F. Small cell lung cancer. G. Pancreatic cancer. H. Colon cancer. I. Non-Hodgkins lymphoma. J. Melanoma. The approximate location of unreactive (at background) albumin (Ab) is labeled for comparison. Approximately 180 non-cancer patient sera were analyzed in parallel without evidence of proteins indicative of specific transcript variants. From Hostetler et al. (2009).

Fig. 4. Analytical gel electrophoresis and immunoblot of patient sera. A Sera from a patient with non-small cell lung cancer contains a 54 kDa ENOX2 transcript variant, pH 5.1 (arrow). B. Sera from a patient with small cell lung cancer contains the 52 kDa, isoelectric point pH 4.3 is transcript variant (arrow). The reference spots to the right, Mr 52 kDa and isoelectric point pH 4.l is 1-antitrypsin. Albumin and other serum proteins are unreactive.

Fig. 3. Western blots of 2-D gel electrophoresis/western blots of sera from cancer patients analyzed individually. Cancer sites are presented in the order of decreasing molecular weight of the major transcript variant present. A. Cervical cancer. B. Ovarian cancer C. Prostate cancer. D. Breast cancer. E. Non-small cell lung cancer. F. Small cell lung cancer. G.

approximate location of unreactive (at background) albumin (Ab) is labeled for comparison. Approximately 180 non-cancer patient sera were analyzed in parallel without evidence of

Fig. 4. Analytical gel electrophoresis and immunoblot of patient sera. A Sera from a patient with non-small cell lung cancer contains a 54 kDa ENOX2 transcript variant, pH 5.1 (arrow). B. Sera from a patient with small cell lung cancer contains the 52 kDa, isoelectric point pH 4.3 is transcript variant (arrow). The reference spots to the right, Mr 52 kDa and isoelectric

point pH 4.l is 1-antitrypsin. Albumin and other serum proteins are unreactive.

**A B**

Pancreatic cancer. H. Colon cancer. I. Non-Hodgkins lymphoma. J. Melanoma. The

proteins indicative of specific transcript variants. From Hostetler et al. (2009).

Fig. 5. 2-D gel electrophoretic separations and detection of ENOX2 transcript variants specific for breast cancer by western blotting of patient sera. Arrow = 66 to 68 kDa breast cancer specific transcript variant. R = 52 kDa, isoelectric point pH 4.1 1-antitrypsin inhibitor reference spot.

Findings from a separate study with small cell and non-small cell lung cancer suggest that the 2-D-western blot test detects cancer presence as early as 5 to 7 years in advance of the appearance of clinical symptoms. This supposition is based mainly on our analysis of two special cancer panels of sera obtained through the Early Detection Research Network (EDRN) of the National Cancer Institute. One panel consisted of about 20 known lung cancer patient sera and 35 control patient sera. Using the 2-D-western blot protocol to identify specific ENOX2 isoforms, we successfully identified all 20 of the known lung cancer patient sera. However, unexpectedly, a high incidence of ENOX2 presence was encountered in sera from the "control" group which were obtained from a community screening study. From additional information obtained through the EDRN, 16 of the 17 positive control subject samples where our findings specifically indicated lung cancer (the lung cancer ENOX2 markers were found) were smokers with smoking histories in the range of 15 to 88 pack-years. However, the anticipated incidence of undetected lung cancers in such a population would be in the order of 10% or less rather than nearly 50%. Since the aberrant ENOX2 transcript variants associated with lung cancer are single molecular species produced only by lung cancer, the possibility was raised that lung cancer was being detected much earlier than was currently possible by other methods. The indications might be as early as 5 to 7 years before clinical symptoms, based on the estimated 20 year development time for lung cancer expression between carcinogen exposure and a clinically evident cancer (Petro et al., 2000) as diagrammed in Figure 6.

Similar results were obtained with a panel of female subjects at risk for breast and ovarian cancer. An analysis of a panel of 127 sera in a Biomarker Reference Set for Cancers in Women also provided through the Early Detection Research Network of the National Cancer Institute support our indications that the 2-D gel-western blot system is able to detect cancer presence 5 to 7 years in advance of clinical symptoms. The panel consisted of samples pooled from 441 women in 12 different gynecologic and breast disease categories plus 115 sera from age-matched control women. Of the l27 sera samples in the panel, 29 tested positive for breast cancer and another 16 tested positive for ovarian cancer. Since the aberrant transcript variants are single molecular species produced by specific cancers such as lung, breast or ovarian, the findings suggest that cancer was being detected in the control popuation much earlier than is currently possible by other methods. As estimated for lung

Early Detection: An Opportunity for Cancer Prevention Through Early Intervention 397

Applications of the diagnostic methodology to post surgery patients is expected to help determine which patients still harbor residual disease following surgery and will require chemotherapy and which patients are free of disease where chemotherapy could be delayed or averted. The expectation is that the 2-D gel-western blot protocol will indicate that chemotherapy might be avoided or delayed in many patients (cancer survivors) where no evidence of disease is indicated. Additionally, the assay might be employed in patients with

ENOX2 was expression cloned (Chueh et al., 2002) (Genbank Accession No. AF207881) using a monoclonal antibody that recognizes only a common ENOX2 epitope near the cancer drug-binding site (Cho et al., 2002) and from which the pan ENOX2 scFv recombinant antibody was derived (Kim, 2011). This binding site contains a conserved 5 amino acid (EMTEE) motif (Table 2). Based on biochemical (drug inhibition of activity) and immunological evidence, this EEMTE drug binding motif in Exon 5 appears to be common to all ENOX2 forms and absent from the amino acid sequence of the constitutive ENOX1

The presence of ENOX2 proteins in sera of cancer patients represents an origin due to shedding from the patient's cancer (Wilkinson et al., 1996). The presence of the ENOX2 proteins has been demonstrated in a number of human tumor tissues and xenografts (mammary carcinoma, prostate cancer, neuroblastoma, colon carcinoma, and melanoma). However, serum analysis indicates a much broader association with cancer. ENOX2 proteins are ectoproteins reversibly bound at the outer leaflet of the plasma membrane (Morré, l995). As is characteristic of other examples of ectoproteins (sialyl and galactosyl transferases, dipeptidylamino peptidase IV, etc.), the ENOX2 proteins are shed, appearing in soluble form in conditioned media of cultured cells and in patient sera (Wilkinson et al., 1996; Morré et al., 1997). The ENOX2 transcript variants from sera exhibit the same degree of drug responsiveness as do the membrane-associated forms (Morré and Reust, 1997; Morré et al., 1997). With sera from more than 200 breast cancer patients, the majority (ca. 196), were found to exhibit the drug-responsive activity. In contrast, no drug-responsive activities were found with sera from healthy volunteers or sera from patients with diseases other than cancer (cardiac, arthritis and other inflammatory diseases, gastric ulceration, emphysema, various non-malignant blood disorders). As such the antitumor drug-responsive ENOX2 activities represent novel cell surface properties potentially associated with most, if not all, forms of human cancer to confirm their appropriateness as appropriate biomarkers for serum or plasma detection and diagnosis of cancer. ENOX2 proteins are robust and highly resistant to heat and protein degradation which enhances their utility as non-invasive markers for cancer detection and diagnosis (Morré and

ENOX2 proteins are absent or present at levels below the limits of detection (less than 10 picomoles/ml of serum) from sera of healthy volunteers or patients with diseases other than cancer. Circulating ENOX2 has been detected based on drug response of ENOX activity of sera of more than 500 cancer patients representing all major forms of human cancer

including leukemias and lymphomas (Morré and Reust, l997; Morré et al., l997).

no clinical evidence of disease to monitor for recurrence.

proteins characteristic of both cancer and non-cancer cells.

**4.2 ENOX2 cloning and tissue distribution** 

Morré, 2003a).

cancer, the indications might be as much as 5 to 7 years before clinical symptoms based on the development time estimated for breast as well as lung cancer expression between a cancer causing event and clinically evident disease (Weinberg, 2007).

Fig. 6. Interpretive diagram to illustrate the various stages of cancer progression (estimated to require as long as 20 y) beginning with a cancer-causing event (initiation) through development of a clinically defined malignancy.
