Drug-induced increase of carcinoembryonic antigen expression in cancer cells

Abstract

Most of gastrointestinal, breast and lung cancer cells express carcinoembryonic antigen (CEA). Therefore, this protein represents a suitable target for innovative diagnostic and immunotherapeutic strategies of various tumours. Presently CEA can be involved in three main approaches concerning cancer detection and therapy, i.e. (a) detection of tumour cells in the peripheral blood, bone marrow or lymph node using reverse transcriptase-polymerase chain reaction (RT-PCR)-based measurement of CEA mRNA; (b) targeting of anticancer agents or radionuclides by tumour-selective anti-CEA monoclonal antibodies (mAbs); (c) use of antitumour vaccines capable of eliciting major histocompatibility complex (MHC)-restricted immune responses against CEA-derived peptides.

Actually, it has been shown that the expression of CEA can be up-regulated by pharmacological agents including, antineoplastic drugs (i.e. 5-fluorouracil), cytokines (i.e. interferons or interleukin-6), differentiating agents (i.e. sodium butyrate) and protein kinase inhibitors (i.e. staurosporine). Therefore, the use of drugs capable of increasing CEA expression, could amplify the sensitivity of diagnostic procedures that rely on CEA determination. Moreover, the same agents could increase the efficacy of vaccines based on immunogenic CEA-derived peptides restricted by the MHC. The purpose of this review is to describe several agents that are able to increase CEA expression and to discuss the rational bases for new strategies in cancer detection and therapy aimed at increasing the expression of tumour-associated antigens.

Introduction

The carcinoembryonic antigen (CEA) is a glycoprotein containing approximately 50% of carbohydrate with a molecular weight of 180–200 kDa [1], [2], [3]. This protein belongs to the large CEA gene family, which includes 29 genes/pseudogenes [4], [5]. All genes of the CEA family are located within the long arm of chromosome 19 [6], [7]. CEA gene family can be divided into three subgroups, i.e. the CEA subgroup containing seven expressed genes; the pregnancy-specific-glycoprotein (PSG) containing 11 expressed genes and the third untitled subgroup containing six pseudogenes. Components of the CEA subgroup are cell surface bound proteins including CEA and the CEA cross reacting molecules, i.e. non-specific cross-reacting antigen (NCA), biliary glycoprotein (BGP) and CEA gene family member (CGM-6). As a tumour-associated antigen, CEA is extensively expressed in more than 95% of colorectal, gastric and pancreatic carcinomas as well as in approximately 70% of non-small cell lung cancer, 50% of breast cancer, in mucinous ovarian carcinoma, adenocarcinoma of the penis [8] and endometrial adenocarcinoma. CEA is also present, to a lesser extent, on normal adult human colon and in some foetal tissues [9], [10], [11], [12]. In particular, it is expressed on the mature columnar epithelial cells and goblet cells of colon, on pyloric mucous cells of stomach, on squamous epithelial cells of tongue, oesophagus and cervix, in secretory epithelia and duct cells of sweat glands and epithelial cells of the prostate [13].

The molecular basis for this differential expression in different tumours and normal tissues are not completely elucidated. The molecular cloning of cDNA for the CEA and CEA-related molecules was performed by different groups [14], [15], [16], [17], [18], [19], [20], [21]. Schrewe et al. [21] reported the isolation and characterisation of a cosmid clone that contains the complete coding region of the CEA gene, including its promoter sequence. No classic TATA or CAAT boxes have been found at the expected position upstream of the transcription sites of the CEA or NCA gene [21]. Approximately 400 bp upstream from the translation start within the promoter region confers cell-type-specific expression on a reported gene. Hauck and Stanners [22] also identified several nuclear factor binding sites such as upstream stimulatory factor (USF), Sp1, and Sp1-like factor, interacting with the promoter region of the CEA gene. CEA promoter region isolated from CEA-producing human colorectal carcinoma or normal adjacent mucosa are not qualitatively different [23]. It was further demonstrated that nuclear extracts from CEA-producing colorectal carcinoma cells could equally bind to both CEA promoter region isolated from colorectal carcinoma and from normal adjacent mucosa [23]. These studies suggest that DNA-binding CEA transcription factors are not sufficient to induce high levels of CEA expression in normal tissues. Therefore, some other mechanisms appear to be involved in the notable difference of CEA found in tumours compared with normal colonic mucosa. It has been demonstrated that methylation of CEA gene [24] and/or structural changes of transcription factors that interact with the regulatory elements [22] of the CEA promoter may increase CEA gene expression. Moreover, post-transcriptional modifications could also have a modulatory role.

The analysis of the amino acid sequences of the CEA family revealed that they belong to the immunoglobulin super-family [25]. CEA has been found to play a role as adhesion molecule with an important function in signal transduction in cancer cells. Several CEA subfamily members, including CEA, possess cell adhesion properties [26], [27], [28], [29], [30], and their presence in the tumour cell membrane has been considered responsible for increasing the tumour cell dissemination [31], [32], [33], [34], [35], [36].

Some authors suggested that CEA molecules expressed on normal cells might play a role in innate immune defence, protecting the colonic mucosa from microbial invasion [37]. Recently, Soeth et al. [38] reported that over expression of CEA protects human colon cancer cells from a variety of apoptotic stimuli, including drug treatment, UV light and confluent growth.

Identification of human tumour-associated antigens (TAAs) and epitopes as target for specific immunotherapy are currently under study. The TAAs that have been characterised in recent years, derive from a broad array of intracellular molecules such as point mutated ras [39], point mutated p53 [40] c-Erb/2, prostate-specific antigen [41], MUC-1 [42], and CEA [43], [44], [45]. It is well accepted that MHC-restricted peptides originated from these molecules are capable of being recognised by T cells. For example, CEA protein is processed endogenously and subsequently short peptide segments are presented by HLA alleles on tumour cell surface. Two distinct pathways have been identified for the processing of CEA as an immunogenic protein. In the first pathway, extra cellular antigen is processed for presentation as 15–25 amino acid length peptides to CD4+ T helper (TH) lymphocytes by MHC class II molecules found on the professional antigen-presenting cells (APCs) including dendritic cells and macrophages. In the second pathway, intracellular antigen produce processed peptides of eight or nine amino acids that are presented to CD8+ cytotoxic T lymphocytes (CTL) by MHC class I molecules (i.e. A2, A3, and A24).

These findings point out that CEA could represent a potential target molecule for specific immunotherapy including recombinant vaccines. A number of preclinical studies demonstrate the possibility to generate in vivo a CEA-specific CTL response with anti-tumour activity in mice and in non-human primates. These results have been obtained by using several strategies of immunisation such as recombinant viruses (CEA gene engineered poxoviruses, adenoviruses, etc.), recombinant DNA or anti-idiotypic antibodies [46], [47], [48], [49]. A human CTL-mediated response to CEA has been demonstrated by using CEA-derived peptides with HLA-A2.1 binding anchor motifs to stimulate in vitro human peripheral blood mononuclear cells (PBMC) derived from normal donors and patients with colorectal carcinoma [45]. As a consequence of such studies, several clinical trials are currently investigating the possible use of CEA as a target antigen, according to different protocols of active specific immunotherapy of patients with colo-rectal carcinoma [50], [51], [52]. In spite of encouraging preclinical results, the use of CEA-directed vaccines has been so far unsuccessful in controlling cancer progression in humans. One of the possible explanations is that CEA is heterogeneously expressed in the tumour. Therefore, it cannot be ruled out that a fraction of the entire tumour cell population would not express adequate amount of CEA peptide to be recognised by CEA-specific CTLs.

CEA is widely used as a tumour marker for diagnostic and therapeutic purposes in various neoplasias including gastrointestinal, breast and lung cancer. Since most of CEA positive colorectal tumours shed the antigen in the plasma, measurement of CEA levels is currently used to monitor tumour progression during the post surgery follow-up of patients affected by colorectal cancer. However, it should be noted that not all CEA positive tumours shed the antigen in the serum and no correlation has been found between CEA content in the tumour and CEA levels in the serum. Therefore, in a substantial number of cases this antigen cannot be considered a suitable marker for the detection of sub-clinical residual disease.

Previously, it has been demonstrated that methods based on reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of CEA positive circulating cancer cells, are sensitive and specific [53], [54], [55], [56]. However, limitations of this technique have also been reported in terms of false positive results when RNA was extracted from whole blood [57], [58]. Therefore, it has been proposed that epithelial tumour cell enrichment by immunomagnetic beads coated with an antibody raised against a common epithelial antigen would increase specificity of tumour-associated CEA transcript detection [59], [60], [61], [62]. Recently, real-time RT-PCR approach have been introduced as an alternative to conventional RT-PCR for sensitive and quantitative detection of CEA mRNA expressing micrometastasis in the peripheral blood [63], in lymph nodes [64] and in the peritoneal cavity [65] of gastroenteric cancer patients.

CEA has been also used for cancer cell targeting of antineoplastic agents or radionuclides bound to tumour-selective anti-CEA monoclonal antibodies (mAbs) [66]. This is an attractive approach either for therapeutic or diagnostic purpose. In fact, anti-CEA mAbs conjugated with ${}^{123}\text{I}$ have been demonstrated to be safe and effective in tumour localisation in cancer patients [67]. Additional studies have shown that ${}^{131}\text{I}$ radiolabelled mAbs provide promising therapeutic effects in terms of reduction of tumour mass and metastasis [68], [69], [70], [71], [72]. Moreover, CEA expression might be involved in radioimmunoguided surgery for the recognition of tumour cells by anti-CEA radiolabelled mAbs [73], [74].

Important factors that appear to limit the use of tumour markers for antibody-based diagnosis and therapy are the low expression of target molecules, the poor penetration of whole antibodies and the formation of human anti-mouse antibodies against to the administered antibody. Therefore, agents capable of increasing CEA expression might represent suitable tools to improve sensitivity and efficacy of diagnostic and therapeutic approaches based on this tumour marker. In particular, pharmacological increment of tumour-associated CEA could be accompanied by higher chances of presenting MHC-restricted CEA-derived peptides by cancer cells. It follows that drug-mediated increase of CEA expression could facilitate the recognition of target cells by cell-mediated immune response induced by anti-CEA peptide vaccines. It was also demonstrated that agents which increase CEA expression can be used to improve either the antitumour effect of radionuclide-conjugated anti-CEA mAbs [72], [75], [76] or the immunodiagnostic procedures [77] such as radioimmunoscintigraphy or radioimmunoguided surgery based on recognition of this marker. Moreover, drug-induced enhancement of CEA might improve sensitivity of RT-PCR-based detection of circulating tumour cells from peripheral blood of cancer patients [61], [62].

A summary of the agents alone or in combination reviewed in this article and their effects on CEA expression/secretion is shown in Table 1, Table 2, respectively.