Background
The human carcinoembryonic antigen (CEA) family has 7 genes belonging to the CEACAM subgroup. These subgroup members are mainly associated with the cell membrane and show a complex expression pattern in normal and cancerous tissues. The CEACAM5 gene, also known as CD66e, codes for the protein, CEA [
1,
2]. CEACAM5 was first described in 1965 as a gastrointestinal oncofetal antigen [
3], but is now known to be overexpressed in a majority of carcinomas, including those of the gastrointestinal tract, the respiratory and genitourinary systems, and breast cancer [
4‐
8]. CEACAM6 (also called CD66c or NCA-90) is a non-specific cross-reacting glycoprotein antigen that shares some antigenic determinants with CEACAM5 [
9]. CEACAM6 also is expressed on granulocytes and epithelia from various organs, and has a broader expression zone in proliferating cells of hyperplastic colonic polyps and adenomas, compared with normal mucosa [
10], as well as by many human cancers [
10‐
12]. Relatively high serum levels of CEACAM6 are found in patients with lung, pancreatic, breast, colorectal, and hepatocellular carcinomas. The amount of CEACAM6 does not correlate with the amount of CEACAM5 expressed [
11].
Expression of CEACAM6 in colorectal cancer correlates inversely with cellular differentiation [
13] and is an independent prognostic factor associated with a higher risk of relapse [
14]. Both CEACAM5 and CEACAM6 have a role in cell adhesion, invasion and metastasis. CEACAM5 has been shown to be involved in both homophilic (CEA to CEA) and heterophilic (CEA binding to non-CEA molecules) interactions [
15‐
17], suggesting to some that it is an intercellular adhesion molecule involved in cancer invasion and metastasis [
18‐
20]. These reactions were completely inhibited by the Fab' fragment of an anti-CEACAM5 antibody [
16]. CEACAM6 also exhibits homotypic binding with other members of the CEA family and heterotypic interactions with integrin receptors [
17]. Antibodies that target the N-domain of CEACAM6 interfere with cell-cell interactions [
21]. We have reported previously that many breast, pancreatic, colonic and non-small-cell lung cancer (NSCLC) cell lines express CEACAM6, and that anti-CEACAM6 antibody inhibits in vitro migration, invasion, and adhesion of antigen-positive cells [
22]. Therefore, the ability to interfere with CEACAM6-mediated homotypic and heterotypic binding might have beneficial anti-metastatic effects.
The goals of the current study were to: (1) use tissue microarray analysis to compare the relative expression of CEACAM5 and CEACAM6 in different histotypes of solid tumors, and (2) develop additional supportive evidence for a role for CEACAM6 in metastasis by comparing expression between primary sites and matched metastases in the same patients. This is the first such comparison of these two CEACAM antigens in such matched patient specimens.
Discussion
CEACAM5 and CEACAM6 are two tumor-associated antigens that play important regulatory roles in cell adhesion and in tumor cell chemosensitivity [
36‐
38]. CEACAM6 overexpression independently predicts poor overall survival and poor disease-free survival, whereas CEACAM5 has not been related significantly to these outcomes [
39].
Studies have shown that CEACAM5 affects expression of various groups of cancer-related genes, especially cell cycle and apoptotic genes, protecting colonic tumor cells from various apoptotic stimuli, such as treatment with 5-fluorouracil [
40]. Therefore, CEACAM5 expression may be a means for cancer cells to overcome apoptosis-inducing therapies. Ordonez et al. have reported that expression of both CEACAM5 and CEACAM6 plays a role in inhibiting apoptosis of cells when deprived of their anchorage to the extracellular matrix, a process known as anoikis [
41]. Increased expression of CEACAM6 correlates with a decrease in sensitivity to drugs, like gemcitabine [
30]. Targeting CEACAM5 and/or CEACAM6 may therefore be a novel method of modulating cancer cell chemosensitivity and apoptosis. It has been reported that siRNA to CEACAM6 impairs resistance to anoikis and increases caspase-mediated apoptosis of xenografted tumors [
31]. Antibody-directed targeting of CEACAM6 may provide a clinically feasible alternative to RNA interference silencing to enhance responsive to chemotherapeutic agents in those tumors that express CEACAM6.
To determine which solid tumors and histological types would be most amenable to antibody blocking of CEACAM5 and CEACAM6, we studied expression of these antigens using tissue microarray analysis. To date, pancreatic and colonic cancer have been the focus of CEACAM6 expression in the literature [
35,
42]. Here, we have further explored the expression of CEACAM6 in a panel of solid tumors: breast, lung, ovary and prostate cancer, in addition to expanding on pancreatic and colonic tumors, and used tissue microarrays to further define tumors that are CEACAM6+ as a function of histological type in all six solid tumor categories. Our results show that expression is strongly dependent on the histotype of the tumor. Antigen expression in some subtypes is 2–4-fold higher than in normal tissues, while in others, expression is similar to non-neoplastic tissues. Other investigators have reported differences in the expression of select tumor antigens as a function of histotype, e.g., TAG-72 in lung cancer [
43], VEGF in skin cancer [
44], and BER EP4 and CA-125 in ovarian cancer [
45]. However, the results in this study are the first to explore differences in both CEACAM5 and CEACAM6 as a function of tumor histotype across six tumor tissues.
The demonstration of higher CEACAM6 expression compared with CEACAM5 across most solid tumors, and the differential expression as a function of histotype, are important observations for translating anti-CEACAM6 therapy to patients. However, we appreciate that additional supportive evidence from Western blots, RT-PCR/Northern blots is needed. This semi-quantitative analysis is intended only as an initial step towards elucidating the importance of CEACAM6 as a tumor target in a variety of solid tumors that extend the many important studies reported for pancreatic cancer [
28‐
35]. It also reveals that expression level varies as a function of tumor histotype. Since some histotypes only had 3–6 core samples and considerable variability in antigen expression within the histotype was noted, it is appropriate to include additional core tissues and to provide more quantitative support with other techniques on biopsy tissues in future studies.
We have also addressed the expression pattern of CEACAM6 in primary tumors and in matched metastases in the same patients. Our results show that in half of the clinical specimens, liver metastases had a much higher expression of CEACAM6 than the primary colorectal tumors, suggesting that in such patients, blocking adhesion and invasion that results from CEACAM6 expression might have influenced the ability of tumor cells to metastasize, as we have in fact shown experimentally [
4]. However, CEACAM6 expression in lymph node metastases was similar to the amount of antigen in primary breast, colon or lung tumor samples. The mechanism by which malignant tumors invade lymphatics and metastasize to regional lymph nodes appears to be regulated by VEGF-C and VEGF-D induced lymphogenesis [
46] and a chemokine gradient. Directional movement is related to chemokine receptor expression on tumor cells [
47], but does not involve members of the CEACAM family. In contrast, CEACAM6 plays an important role in migration, invasion and adhesion [
31,
34], steps that are important in the metastatic spread to secondary tissue sites other than lymph nodes [
48]. In fact, anti-adhesive molecules that disrupt cell-matrix and cell-cell attachments have been proposed as potential cancer therapeutics based on their ability to interfere with motility, adhesion, and metastatic progression [
22,
36,
49].
We have recently reported that the humanized anti-CEA (CEACAM5) antibody, MN-14, can enhance the therapeutic effects of two cytotoxic drugs used frequently in colorectal cancer therapy, fluorouracil and CPT-11, in both subcutaneous and metastatic human colonic tumor cells propagated in nude mice [
50]. In another high CEA-expressing human medullary thyroid cancer xenograft, we have also shown that MN-14 anti-CEA IgG can inhibit tumor cell growth and also augment the effects of dacarbazine, a drug that is active in this cancer type [
51]. One explanation may involve a role in antibody blocking adhesion [
38] and thereby chemosensitizing the tumor cells.
In a series of provocative studies, Duxbury and associates have shown that silencing CEACAM6 by siRNA: (a) enhances cell anoikis, (b) increases caspase activation in response to anchorage independent conditions, (c) downregulates the Akt cell survival pathway, (d) inhibits metastasis in vivo, and (e) enhances gemcitabine induced chemosensitivity [
30,
31,
33‐
35]. Thus, in addition to CEACAM5, CEACAM6 may also represent a useful therapeutic target. Blocking CEACAM6-mediated homotypic and/or heterotypic adhesion may have anti-metastatic and chemosensitizing effects. In ongoing preclinical therapy studies, we are examining the therapeutic effects of unconjugated anti-CEACAM6 antibody alone or combined with standard chemotherapeutic agents in colon, breast, and lung metastasis models. An alternative approach is to develop an anti-CEACAM6 immunoconjugate as a therapeutic agent for CEACAM6+ tumors, as described by Duxbury et al. [
32]. In vitro targeting with an anti-CEACAM6 antibody, followed by secondary saporin-conjugated immunoglobulin (IgG), induced marked cytotoxicity via caspase-mediated apoptosis. In an in vivo nude mouse xenograft model, this indirect immunotoxin approach markedly suppressed pancreatic adenocarcinoma tumor growth and enhanced tumor apoptosis.
Competing interests
Drs Goldenberg and Hansen have management roles and financial interest (stock) in Immunomedics, which owns the antibodies tested in this study. All other authors declare that they have no potential competing interests.
Authors' contributions
RDB conceived and designed the studies, analyzed the data and drafted the manuscript. EL performed all immunohistochemistry. HH was involved in revising the manuscript and influencing critical intellectual content. DMG was involved in data interpretation and presentation, revising the manuscript, and influencing critical intellectual content. All authors read and approved the final manuscript.