**1. Introduction**

388 Cancer Prevention – From Mechanisms to Translational Benefits

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Cancer is the second leading disease cause of death in the United States. A group of more than 100 different and distinctive diseases, cancer may involve any tissue of the body. Estimates are that there were over 1.5 million cases in 2010 in the United States alone. Only a small fraction (less than 20%) of cancers are diagnosed at a localized stage where curative therapy is effective. Most cancers are diagnosed only after the primary tumor has already metastasized so that chemotherapy is required for treatment. Hence, early detection is a favored opportunity to reduce cancer mortality. By detecting cancer in its very earliest stages when perhaps only a small number of cells are present, it is possible that early intervention will be effective in preventing further development of the incipient cancer thereby resulting in what might be viewed as curative prevention.

Despite advances in early detection of major forms of human cancer (prostate, breast, lung, colon, leukemia, lymphoma), more often than not, cancers have developed to a sufficiently late stage at the time of detection to preclude most opportunities for curative therapy (Altekruse et al., 2010). The problem is exacerbated for pancreatic cancer where clinical symptoms invariably are delayed until the disease state is well advanced beyond metastatic spread. A need for early detection remains as one of the most important challenges at the forefront of cancer research, treatment and prevention.

### **2. Approach**

### **2.1 Early detection**

Ecto-Nicotinamide Adenine Dinucleotide Oxidase Disulfide-Thiol Exchanger 2 (ENOX2) (GenBank accession no. AF207881; Chueh et al., 2002) also known as Tumor-Associated Nicotinamide Adenine Dinucleotide Oxidase (tNOX) is ideally suited as a target for early diagnosis of cancer as well as for early preventive intervention (Fig. 1). The proteins are expressed on the cell surface of malignancies and detectable in the serum of patients with cancer (Cho et al., 2002). ENOX2 proteins are terminal hydroquinone oxidases of plasma membrane electron transport. From the standpoint of early intervention, they are important in the growth and enlargement of tumor cells (Morré and Morré, 2003a; Tang et al., 2007, 2008). Our approach using ENOX2, as a target for both early detection and for early interventions, is based on these properties (Cho et al., 2002; Morré and Morré, 2003a;

Early Detection: An Opportunity for Cancer Prevention Through Early Intervention 391

molecular weight and isoelectric point indicative of a particular form of cancer (Hostetler et

ENOX transcript variants of specific molecular weights and isoelectric points are produced uniquely by patients with cancer. The proteins are shed into the circulation and have the potential to serve as definitive, non-invasive and sensitive serum markers for early detection of both primary and recurrent cancer in at risk populations with a low incidence of false positives, as they are molecular signature molecules produced specifically by cancer cells

Fig. 2. 2-Dimensional gel/western blot of ENOX2 transcript variants comparing pooled non-

cancer (A) and pooled cancer representing major carcinomas plus leukemias and lymphomas (B) patient sera. The approximate location of unreactive (at background) albumin is labeled for comparison. ENOX2 reactive proteins are restricted to quadrants I and IV. Detection uses recombinant scFv-S (S-tag peptide: His-Glu-Ala-Ala-Lys-Phe--Gln-Arg-Glu-His) antibody with alkaline phosphatase linked antiS protein. The approximately 10 ENOX2 transcript variants of the pooled cancer sera are absent from non-cancer (A) and

A **B**

are cancer site-specific as indicated in Fig. 3. From Hostetler et al. (2009).

al., 2009; Table l).

and absent from non-cancer cells.

reviewed by Davies and Bozzo, 2006). While ENOX2 presence provides a non-invasive approach to cancer detection, without methodology to identify cancer site-specific ENOX2 forms, it offered no indication as to cancer type or location.

Fig. 1. Schematic representation of the utility of the ENOX2 family of cancer-specific, cell surface proteins for early diagnosis and early intervention of cancer. Cancer site-specific transcript variants of ENOX2 are shed into the serum to permit early detection and diagnosis. The ENOX2 proteins of origin at the cell surface act as terminal oxidases of plasma membrane electron transport functions essential to the unregulated growth of cancer. When the ENOX2 proteins are inhibited, as for example through EGCg/*Capsicum* synergies, the unregulated growth ceases and the cancer cells undergo programmed cell death (apoptosis).

The opportunity to simultaneously determine both cancer presence and cancer site emerged as a result of 2-dimensional gel electrophoretic separations where western blots with a pan ENOX2 recombinant single chain variable region (ScFv) antibody carrying an S tag (Fig. 2) was employed for detection (Hostetler et al., 2009; Hostetler and Kim, 2011). The antibody cross reacted with all known ENOX2 forms from hematological and solid tumors of human origin but, of itself, did not differentiate among different kinds of cancers. Analyses using this antibody, when combined with two-dimensional gel electrophoretic separation, revealed specific ENOX2 species possibly as transcript variants, each with a characteristic

reviewed by Davies and Bozzo, 2006). While ENOX2 presence provides a non-invasive approach to cancer detection, without methodology to identify cancer site-specific ENOX2

Fig. 1. Schematic representation of the utility of the ENOX2 family of cancer-specific, cell surface proteins for early diagnosis and early intervention of cancer. Cancer site-specific transcript variants of ENOX2 are shed into the serum to permit early detection and diagnosis. The ENOX2 proteins of origin at the cell surface act as terminal oxidases of plasma membrane electron transport functions essential to the unregulated growth of cancer. When the ENOX2 proteins are inhibited, as for example through EGCg/*Capsicum* synergies, the unregulated growth ceases and the cancer cells undergo programmed cell

The opportunity to simultaneously determine both cancer presence and cancer site emerged as a result of 2-dimensional gel electrophoretic separations where western blots with a pan ENOX2 recombinant single chain variable region (ScFv) antibody carrying an S tag (Fig. 2) was employed for detection (Hostetler et al., 2009; Hostetler and Kim, 2011). The antibody cross reacted with all known ENOX2 forms from hematological and solid tumors of human origin but, of itself, did not differentiate among different kinds of cancers. Analyses using this antibody, when combined with two-dimensional gel electrophoretic separation, revealed specific ENOX2 species possibly as transcript variants, each with a characteristic

death (apoptosis).

forms, it offered no indication as to cancer type or location.

molecular weight and isoelectric point indicative of a particular form of cancer (Hostetler et al., 2009; Table l).

ENOX transcript variants of specific molecular weights and isoelectric points are produced uniquely by patients with cancer. The proteins are shed into the circulation and have the potential to serve as definitive, non-invasive and sensitive serum markers for early detection of both primary and recurrent cancer in at risk populations with a low incidence of false positives, as they are molecular signature molecules produced specifically by cancer cells and absent from non-cancer cells.

Fig. 2. 2-Dimensional gel/western blot of ENOX2 transcript variants comparing pooled noncancer (A) and pooled cancer representing major carcinomas plus leukemias and lymphomas (B) patient sera. The approximate location of unreactive (at background) albumin is labeled for comparison. ENOX2 reactive proteins are restricted to quadrants I and IV. Detection uses recombinant scFv-S (S-tag peptide: His-Glu-Ala-Ala-Lys-Phe--Gln-Arg-Glu-His) antibody with alkaline phosphatase linked antiS protein. The approximately 10 ENOX2 transcript variants of the pooled cancer sera are absent from non-cancer (A) and are cancer site-specific as indicated in Fig. 3. From Hostetler et al. (2009).

Early Detection: An Opportunity for Cancer Prevention Through Early Intervention 393

(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

ENOX2 EEMTETK400ETEESA406LVS

Alpha-1-antitrypsin inhibitor GTDCVAK211EATEAA216KCN Serrotransferrin CLDGTRK589PVEEYA595NCH

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

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.

malignant melanoma contained an ENOX2 transcript variant of 38 kDa (Fig. 3J).

all tested positive.

sera of subjects without cancer (Table 1).

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

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

heavy (ca. 52 kDa) and light (ca. 25 kDa) immunoglobulin chains.


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).
