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Sep 7, 2011 - REVIEW. Carcinoembryonic antigen (CEA) and its receptor hnRNP M are mediators of metastasis and the inflammatory response in the liver.
Clin Exp Metastasis (2011) 28:923–932 DOI 10.1007/s10585-011-9419-3

REVIEW

Carcinoembryonic antigen (CEA) and its receptor hnRNP M are mediators of metastasis and the inflammatory response in the liver Peter Thomas • R. Armour Forse • Olga Bajenova

Received: 29 November 2010 / Accepted: 15 August 2011 / Published online: 7 September 2011 Ó Springer Science+Business Media B.V. 2011

Abstract This article discusses the role of carcinoembryonic antigen (CEA) as a facilitator of the inflammatory response and its effect on colorectal cancer hepatic metastasis. Colorectal cancer accounts for 11% of all cancers in the United States and the majority of deaths are associated with liver metastasis. If left untreated, median survival is only six to 12 months. Resection of liver metastases offers the only chance for cure. Of the small number of patients who have operable cancer most will have further tumor recurrence. The molecular mechanisms associated with colorectal cancer metastasis to the liver are largely unknown. However CEA production has been shown both clinically and experimentally to be a factor in an increased metastatic potential of colorectal cancers to the liver. CEA also has a role in protecting tumor cells from the effects of anoikis and this affords a selective advantage for tumor cell survival in the circulation. CEA acts in the liver through its interaction with its receptor (CEAR), a protein that is related to the hnRNP M family of RNA binding proteins. In the liver CEA binds with hnRNP M on Kupffer cells and causes activation and production of pro- and anti-inflammatory cytokines including IL-1, IL-10, IL-6 and TNF-a. These cytokines affect the upregulation of adhesion molecules on the hepatic sinusoidal endothelium and protect the tumor cells against cytotoxicity by nitric oxide (NO) and other reactive oxygen radicals. HnRNP M signaling in Kupffer cells appears to be controlled by beta-adrenergic receptor activation. The cells will respond to the b-adrenergic receptor agonist terbutaline resulting in reduced TNF-a and increased IL-10 and P. Thomas (&)  R. A. Forse  O. Bajenova Department of Surgery, Creighton University, 2500 California Plaza, Criss III Rm 376, Omaha, NE 68178, USA e-mail: [email protected]

IL-6 production following CEA activation. This has implications for the control of tumor cell implantation and survival in the liver. Keywords Carcinoembryonic antigen  hnRNP M  Metastasis  Inflammation  Epithelial mesenchymal transition  Cytokines  Adhesion molecules

Introduction Cancer of the large intestine (colon, rectum and anus) is a major public health problem world wide, with approximately 147,000 new cases and 50, 000 deaths annually in the US alone. This accounts for 11% of all US cancers with a similar incidence for men and women [1]. The incidence and mortality from colorectal cancer has remained constant over the past decade [2]. A majority of deaths from colorectal cancer are associated with liver metastasis and approximately 25% of patients have liver metastasis at the time of presentation [3, 4]. If metastatic disease is left untreated, median survival is six to 12 months with a 5 year survival of only 9% [2]. Resection of liver metastases offers the only chance for cure, although *70% of patients who undergo a hepatic lobectomy will have further tumor recurrence (5 year survival ranges between 25 and 39%) [5]. This raises the possibility of early tumor-cell spread at the time of, or before, surgical resection of the primary tumor [6]. Understanding the mechanisms involved in liver metastasis would provide the opportunity to intervene before resection of the primary tumor. The role of the inflammatory response in the metastatic events of cancer has recently been the subject of intense study [7, 8]. It is apparent that metastatic spread cannot be solely explained by mechanical trapping of cancer cells in

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capillary beds or by the seed and soil hypothesis which states that tumor cells will only thrive where the microenvironment is compatible. While both these mechanisms factor into metastatic progression there is also an interaction between adhesion molecules, chemokines and their receptors and normal cells at the secondary site [9]. In addition there is recruitment of tumor cells to the target organ by chemotactic factors such as epidermal growth factor (EGF) [10, 11]. EGF gradients can be generated by stromal cells in the tumor microenvironment and guide the tumor cells towards blood vessels. At the primary site interactions between tumor cells and extracellular matrix including cells present in the stroma can result in a response that activates the epithelial mesenchymal transition (EMT), a process that results in loss of epithelial differentiation and a progression towards a mesenchymal phenotype [12–14]. This with the associated release of degradative enzymes [15] allows the tumor cells to detach from each other and invade the surrounding tissue. This increase in tumor cell motility allows migration toward blood vessels and lymphatics. In addition release of proteases permits breakdown of basement membranes such that tumor cells can enter the vessels and be released into the general circulation. While the EMT is an attractive hypothesis to explain tumor cell behavior it is not without its detractors [16, 17]. Most work has focused on the invasion and extravasation events occurring at the primary site but much less is known about the mechanisms that occur at the secondary site that allow implantation and growth of tumor cells in what is often an hostile environment. It is suggested that on reaching a secondary site cancer cells can reverse the EMT producing a mesenchymal epithelial transition (MET) [18]. The mechanism and the factors initiating this reversal are not understood but an inflammatory response within the organ of invasion plays an important role [19] (Fig. 1). The mechanisms responsible for colorectal cancer metastasis to the liver are the primary focus of this article. While mechanical entrapment of tumor cells in the first capillary bed they encounter is an obvious hypothesis for site-specific spread, it does not explain the differences in metastatic potential between various colorectal cancer cells [20]. These differences may be explainable, in part, by the varying abilities of tumors to elicit inflammatory responses within the liver and thus enrich the environment for attachment, invasion and growth. Thus, by changing the environment in a way that is favorable to growth and survival the tumor cell prepares the soil. Paget’s ‘‘seed and soil’’ hypothesis states that the environment must be favorable for tumor growth. The concept that tumors can alter the soil to ensure their survival extends this hypothesis [21]. Activation of stromal cells including macrophages by tumor products may cause a localized inflammatory

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response and chronic activation leads to continuous tissue damage [22]. This can be tissue specific and may account at least in part for organ specific metastasis. Cell stress and death can also activate immune cells via the release of damage-associated molecular pattern molecules (DAMPs) such as the high mobility group box protein HMGB1 [23] and calprotectin and its subunits S100A8 and S100A9 [24] causing a cycle of inflammation. Release of the glycoprotein, carcinoembryonic antigen (CEA) from colorectal cancer cells entering the liver both by normal secretory mechanisms and by cell death also results in a cytokine response (see below). In such situations CEA appears to act as a DAMP.

Inflammation and metastasis The host microenvironment is a major factor in determining the ability of tumor cells to grow in a foreign tissue. For example, colon cancers grow best in liver. Breast and prostate cancers prefer bone, while melanoma is attracted to brain etc. [25, 26]. The micro-environment can therefore aid tumor dissemination providing essential growth factors and protection against factors such as hypoxia induced cytotoxicity. [27]. A more recent study has indicated that tumors with a low proportion of tumor cells to stromal cells have a worse prognosis, thus again emphasizing the importance of the microenvironment in cancer invasion and metastasis [28]. The microenvironment can also be detrimental to the growth of cancer cells. Colon cancers rarely metastasize to bone for example, though isolated cells are often found there in cases of advanced disease [29] one study showed that 28 of 88 colorectal cancer patients had tumor cells in their bone marrow [30]. Thus, while osseous metastasis are rare (1–4%) in colorectal cancer [31, 32], these data suggest that bone marrow is not conducive to their growth, though both breast and prostate cancers grow well at that site [27]. So what makes a good environment for cancer cell growth? One major factor seems to be the presence of an inflammatory response produced by the adjacent stromal cells or recruited macrophages. For example, mice that are unable to recruit macrophages to mammary tumors due to their inability to make colony stimulating factor-1 (CSF-1) are not capable of forming metastasis, while mice with the ability to produce CSF-1 retained the capacity to produce metastasis [33]. Tumor associated macrophages (TAMs) can also produce pro-angiogenic cytokines including TNF-a and IL8. They may also secrete angiogenic growth factors like vascular endothelial growth factor (VEGF) as well as modulating enzymes such as the matrix metalloproteinases (MMP’s) [34, 35]. These changes are probably tissue specific and occur in response to tumor associated stimuli.

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Fig. 1 A Interactions of CEA in the hepatic sinusoid. CEA released by the tumor cell binds with hnRNP M (CEAR) on the Kupffer cell surface resulting in release of the cytokines IL-1, IL-6, IL-10 and TNF-a. B Effect of CEA induced cytokines on tumor cell interactions in the hepatic sinusoid. Cytokines IL-1, IL-6, IL-10 and TNF-a produced by Kupffer cells have a number of effects on the tumor cell microenvironment. These include up-regulation of adhesion molecules on hepatic sinusoidal endothelial cells. The most important of these seem to be E-selectin and ICAM-1

For example, Kupffer cells respond to CEA while resting peritoneal macrophages do not [36]. This type of phenomenon may also occur in bone and may explain why CEA production is not a factor in colorectal cancer growth in bone but plays a role in metastasis to the liver. This may at least in part account for differences in patterns of metastasis in the solid tumors. It has also been suggested recently that an assessment of the type, density and location of immune cells associated with colorectal primary cancers can offer a more precise prognosis than even the anatomic extent of the disease [37]. Macrophages in particular play a prominent part in invasion, promoting both cancer cell motility and angiogenesis [7, 8]. However, relatively little is known regarding the signals that regulate

the activity of TAMs at the primary site [8]. Even less is known regarding the activity of macrophages influencing the microenvironment at the site of a distant metastasis. In this regard we suggest that CEA is an important factor in altering macrophage activity both at the primary (TAMs) and at the secondary sites in the liver and lung (Kupffer cells and alveolar macrophages).

Carcinoembryonic antigen Carcinoembryonic antigen was first discovered in 1965 by Gold and Freeman [38]. It is a glycoprotein that is a member of a large gene family that consists of 29 genes

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divided into three subgroups that include the CEA-like glycoproteins and the pregnancy-specific glycoproteins (PSGs). All these proteins are members of the much larger immunoglobulin supergene family [39]. CEA is also known as CD66 or CEACAM5 and the nomenclature for the entire CEA and PSG families can be found in Beauchemin et al. [40]. CEA has a molecular mass of 180–200 kD depending on the extent of its glycosylation [41]. The protein consists of a series of immunoglobulinlike domains, at the N-terminus is 108 amino acid v-domain followed by three pairs of c2-like domains each of 178 amino acids. Each c2 loop domain is held in conformation by a single disulphide bond. There is a small hydrophobic C-terminal domain (26 amino acids) that is modified to give a GPI-linked membrane anchor. This anchor can be cleaved by phospho-inositol specific phospholipases C and D to release CEA in a soluble form [41, 42]. The structural features of the protein also allow up to 28 tetra-anntenary complex carbohydrate chains of the N-linked type. The complete gene for CEA has been cloned and includes a promoter region that appears to confer organ and cell type-specific expression [43]. Since its discovery in 1965 [38] a very large number of studies have been carried out to determine the effectiveness of CEA as a clinically useful tumor marker. Serum elevations of CEA are seen in about 60% of presenting colorectal cancer patients [44]. While not considered useful as a cancer screen for the general population due to high false positive and false negative rates, CEA is used to monitor tumor recurrence following surgery and as a marker for prognosis. A small rise in CEA can be predictive of recurrence following curative surgery for colorectal cancer [45] and can result in detection of recurrence up to a year before the onset of clinical symptoms. A recent study has even suggested that elevated serum CEA levels in people over 50 are predictive of increased mortality [46]. CEA mRNA expression has recently been shown to be useful as an early marker for recurrence in pancreatic cancer [47]. Other members of the CEA gene family (e.g. CEACAM6 or NCA-90) can also be used for prognosis and as predictors of tumor recurrence [48–50]. CEA is widely used as a target antigen for radio-immunodetection of occult metastasis and for radio-immunotherapy in patients with CEA producing cancers [51]. More recently it has been used as a preferred antigen for the development of anticolorectal cancer vaccines. Elevated serum CEA levels are associated with liver metastasis and are used as a prognostic indicator [44]. In the normal colon CEA is produced by mature colonocytes and is localized to the apical membrane. In colon cancers and in fetal gut polarity is lost and CEA is found on all surfaces of the cell [52, 53]. The normal function of CEA has been a mater of some debate. There is a large body of evidence that shows that it can act

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as a receptor for bacteria including Neisseria gonorrhoea and Escherichia coli [54–56] CEA family members expressed in rodents also act as receptors for viruses, including mouse hepatitis virus [57, 58]. It seems unlikely that such a large complex glycoprotein (180 kD with 28 complex tri-antennary carbohydrate chains) would be secreted into the gut without a purpose. Thus a role for CEA in protecting the gut mucosa by binding potentially harmful bacteria seems to be a possible function in the normal individual.

CEA and the liver Input of CEA into the circulation is controlled by production rate of the tumor: its location and stage, its size, differentiation and vascularity: and the presence or absence of distant metastases. However, the serum level is also controlled by the rate of CEA elimination [41, 59]. Because CEA is eliminated by the liver elevated levels are found in patients with benign liver disease such as cirrhosis, hepatitis and obstructive biliary diseases [60]. The cell involved in CEA clearance from the circulation is the liver-fixed macrophage or Kupffer cell [41]. The function of the Kupffer cell in promoting hepatic metastasis is controversial [61]. These cells can have tumoricidal activity but can also aid liver colonization and tumor cell survival depending on the experimental system studied [61]. The removal of CEA from the circulation occurs through binding to an 80 kD Kupffer cell surface protein that was cloned from both human and rat liver [62]. The binding protein is related to the hnRNP family of proteins [62] and is identical to the heterogeneous nuclear RNAbinding protein M (hnRNP M). Following binding of CEA to hnRNP M, Kupffer cells respond by internalizing the complex and also transducing a signal that results in the production of cytokines that have the potential to change the microenvironment within the liver sinusoids. Following endocytosis CEA is partially degraded in the Kupffer cell by removal of sialic acid from the termini of its carbohydrate chains, and its breakdown is completed after transfer of the partly degraded molecule to the hepatocyte using the asialoglycoprotein receptor [41].

The CEA receptor (CEAR) The CEAR (hnRNP M) is expressed as a nuclear protein in most if not all cells but only as a cell surface protein in Kupffer cells [63], and other terminally differentiated macrophages and in some cancer cells including the human colorectal cancer cell line HT-29. The protein exists as four splice variants in macrophage cell lines (unpublished data),

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two of which are expressed in Kupffer cells [62, 63]. Both these forms of CEAR are capable of binding CEA [62]. The cellular distribution, level of expression, and relative amount of hnRNP M isoforms determines cellular specificity for CEA binding [62, 63]. The expression ratios of alternative spliced forms of hnRNP M are also tissue and cell-specific [63]. HnRNP M belongs to a large family of RNA binding proteins that have been described as ‘‘the histones of RNA’’. It was initially cloned by Datar et al. and was unusual in that it contained a methionine arginine rich repeat motif [64]. It was later identified as a monomer of the N-acetylglucosamine-specific receptor for the thyroid hormone NAGR1 [65]. Later studies showed that this putative receptor was identical to hnRNP M4 [66] and doubt was cast on its ability to function as an N-acetyl glucosamine-specific thyroglobulin receptor [66]. The CEAR exhibits a unique function in Kupffer cells and other terminally differentiated macrophages such as the alveolar macrophage in the lung. HnRNP M acts as a cell surface receptor for proteins that contain the pentapeptide sequence PELPK [41, 67]. Proteins with this motif include members of the CEA gene family though not the PSG proteins [40–42]. The PELPK sequence is also present in complement subcomponent C1s precursor (amino acids 416–420) [68] and a number of non-mammalian proteins. In CEA and other members of its family this sequence occurs at the hinge region between the N and the first immunoglobulin loop domain (amino acids 108–112 for CEA). Patients who have a mutation in the region coding for this peptide have extremely high circulating CEA levels and their CEA has an impaired clearance rate from the circulation in experimental animals [69]. It has been proposed that these high levels in patients with the mutations are due to the inability of Kupffer cells to clear the protein from the blood and are a direct consequence of the mutation in the PELPK motif [69]. In colorectal cancers in partnership with CEA, hnRNP M may also play a role in the regulation of anoikis a form of apoptosis caused by detachment from the extra-cellular matrix [70–73]. This may also be a survival mechanism during metastasis. The mechanism of CEA inhibition of anoikis occurs by CEA binding to the death receptor DR-5 and reducing caspase 8 activity. This binding requires the PELPK sequence and therefore suggests that hnRNP M4 may also be involved though the details of this interaction are not known [73]. HnRNP M4 is a ubiquitous multifunctional protein in mammals and in general is localized to the nucleus: there is also evidence that it can shuttle between the nucleus and cytoplasm carrying RNA [74, 75]. In HEK 293 cells it is associated with Drosha, a protein from the microprocessor complex that mediates production of micro RNAs [74, 75]. It also acts as a splicing regulatory protein [76].

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There are four known variants of the hnRNP M protein and there may be more. Two of these (the full-length protein and a variant with a 39 amino acid deletion near the second RNA binding motif) are the variants that so far have been identified as CEARs on Kupffer cells [62]. These cells are important in the inflammatory response to invading colon cancer cells in the liver and can produce a microenvironment conducive to invasion and the growth of metastasis [41]. HnRNP M has many functions and these seem to be dependent on the cell type in which the protein is expressed. Function may also be altered by the ratio of splice variants present. The major human hnRNP proteins have been shown to be important participants in mRNA processing and the control of alternative splicing particularly of apoptotic genes [76]. A number of other hnRNP proteins have also been implicated in tumor development and progression [77]. Other interactions of hnRNPs including hnRNP M involve actin binding proteins including alpha actinin 4 [78]. Other studies have also demonstrated interactions between hnRNP, CEA and actin [79]. Interaction between actin and the CEA family member CEACAM1 have also been documented [80] implying a role for CEA family members in the structural integrity of the cell. HnRNP proteins in general are also associated with processing micro RNAs (miRNAs) [81] and hnRNP M in particular is a regulator of splicing [82]. Recently Derry et al. reported that hnRNP M is involved in the regulation of the splice variants of the CEA family member CEACAM-1 [83]. A detailed review of the function of hnRNP proteins including hnRNP M has been recently published [84]. Unlike Kupffer cells or lung alveolar macrophages hnRNP M does not seem to bind CEA on circulating monocytic cells, due to lack of expression on the cell surface. However, Ganguly et al. [85] have described a CEA binding protein of a different size on circulating monocytes and has suggested that this protein may function in TAMs recruited to the primary tumor [85].

CEA and liver metastasis Current post-genomic strategies are being applied to identify genes/proteins that function in tumor invasion and metastasis that can be used as tumor markers for prognosis or targets for therapy. These markers include MMP’s, angiogenic factors such as VEGF, integrins, selectins and other adherence molecules. Many genes have been associated with metastasis based on expression levels in metastatic versus non-metastatic cell lines and by comparisons between primary and metastatic tumor tissue [86–90]. Few of these genes have been confirmed as significant to the development of metastasis by use of functional assays,

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such as the examination of changes in cell behavior when these genes are over-expressed. Fewer still have been implicated in metastasis by clinical studies. The CEA gene with its protein product has been shown by all these criteria to have a role in metastasis development. There is an increasing body of evidence that human Kupffer cells can interact with CEA-producing cancer cells to increase retention and survival of human colon cancer cells in experimental models [44]. A number of clinical studies have shown correlations between serum CEA levels and advanced colorectal cancer, in particular the presence of liver metastasis [41, 44, 59]. Experimentally, CEA has been implicated in the development of hepatic metastases from colorectal cancers based on a direct relationship between CEA production and metastatic potential of human colorectal cancer cell lines [20, 91]. This is supported by the observation that injection of CEA into mice prior to injection of weakly metastatic cancer cells increases liver metastasis [92]. Similarly poorly metastatic colon cancer cell lines become highly metastatic when transfected with the cDNA coding for CEA [93, 94]. CEA-producing colon cancers are also retained within the liver for a longer time than non-CEA producing cells resulting in an increased metastatic potential to the liver [95]. This is consistent with the idea that increased adhesion to the endothelium may be the key rate-limiting step in hepatic metastasis [96, 97]. Gangopadhyay et al. have shown that CEA binding will cause activation of Kupffer cells, both in vitro [98] and in vivo [99, 100] accompanied by increased secretion of cytokines that include IL-1-a, IL-1-b, IL-6, IL-10 and tumor necrosis factor (TNF-a) [98, 100]. This activation is the key to CEA’s role in liver metastasis. Further evidence for the involvement of CEA in liver metastasis comes from studies with the cotton top tamarin. These new world monkeys develop spontaneous colon cancer but very few progress to metastasis to the liver. The cotton top tamarin produces CEA like molecules that have sequence changes in the PELPK region of the molecule and also express low levels of hnRNP M in the liver [101]. This further supports the idea that CEA/hnRNP M interactions are important for the development of liver metastasis by CEA expressing colorectal cancers. The activation of Kupffer cells by CEA is also affected by the b-adrenergic receptor agonist terbutaline [102]. Indeed the response of macrophages to many outside stimuli is regulated by b-adrenergic signals [103–105]. Adrenergic responses are involved through cyclic AMP (cAMP) signaling in the repression of TNF-a and enhancement of IL-10 production through promoters that bind cAMP responsive element binding protein (CREB) and the CCAAT enhancer binding protein C/EBP) [106– 108]. This results in increased levels of IL-10 and a

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decrease in TNF-a production. Exposure of Kupffer cells to CEA also causes an up-regulation of the b-adrenergic receptor mRNA, as well as changes in the cytokine responses mentioned above [102]. Interestingly Blaxall et al. also showed interactions between the b-adrenergic receptor and members of the hnRNP family of proteins including hnRNP A [109]. It is not known if similar interactions occur with hnRNP M. These results suggest that manipulation of the b-adrenergic system could change the hepatic microenvironment and alter tumor cell behavior. Production of pro-inflammatory cytokines IL-6, IL-1a and b and TNF-a in the localized environment of the hepatic sinusoid can result in a multitude of biological effects. An immediate effect occurs on the sinusoidal endothelium. Using a multi-cell system with human Kupffer cells, endothelial cells, and CEA-producing colorectal cancer cells, it was shown that there was up-regulation of endothelial-cell adhesion molecules: in particular, ICAM-1, VCAM-1 and E-selectin [110]. Minami et al. [99] also showed that Kupffer cells respond to CEA and produce IL-1b and TNF-a. These cytokines increased colon cancer cell adhesion to an endothelial cell monolayer and this could be blocked by the cytokine inhibitor FR 167653 [111]. Recently, Khatib et al. have shown similar results with increases in E-selectin produced by the pro-inflammatory cytokine TNF-a [112, 113]. Tumor cells arresting in the liver also produce toxic levels of NO and reactive oxygen species (ROS) due to the creation of local ischemic/reperfusion injury [114]. The release of the antiinflammatory cytokine IL-10 by Kupffer cells activated by CEA also plays a role in tumor cell survival by inhibiting inducible nitric oxide synthetase (iNOS) and the production of NO and ROS, thus aiding tumor cell survival [115, 116]. Hypoxia also up-regulates CEA expression in colorectal and breast cancers [117] this could be a defensive mechanism as release of CEA would activate Kupffer cells to produce more IL-10 and protect the cancer cells from the cytotoxic effects of NO and ROS. Elevated serum IL-6 is also correlated with both lymph node and hepatic metastasis [118] and IL-6 has been identified as a negative prognostic factor in colorectal cancers [119–121]. IL-6 is thought to work through hepatocyte growth factor (HGF) to promote metastasis, though other mechanisms including up-regulation of adhesion molecules may all play a role [122]. Recent studies have shown a direct relationship between CEA and TRAIL-R2 signaling causing a decrease in anoikis. Although the involvement of hnRNP M in this mechanism has not been proven [73]. Using site-directed mutagenesis Samara et al. showed that the interaction requires the PELPK signaling motif [73]. Thus the requirement of the PELPK motif suggests that hnRNP M together with CEA interacts with the TRAIL-R2 signaling

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pathway. Interactions between CEA and TGF-b Receptor have also been reported [123]. CEA inhibits TGF signaling through an unknown mechanism and increases tumor cell proliferation. This suggests an additional mechanism by which CEA may promote tumor survival at the metastatic site.

Conclusions Survival from the events that occur following the arrival of cancer cells at the distant site represents the most challenging aspect of the metastasis cascade. The environment of a foreign tissue is not normally compatible with implantation and growth (colonization). How tumor cells overcome these obstacles is one of the major questions to be answered in the attempt to understand the metastatic process [122]. In this article we have shown that in addition to its importance in the early stages of the metastatic cascade, stimulation of macrophages at the secondary site can also profoundly influence the development of metastasis. While the ability to elicit an inflammatory response at the site of metastasis is important many other mechanisms are also operating including the ability to invade, elicit angiogenesis and inhibit apoptosis. These many mechanisms work together to ensure the survival of the cancer cell. Loss of any of them would reduce or perhaps eliminate the cells metastatic potential. We have suggested a mechanism specific to the liver and possibly the lung in which binding of CEA to Kupffer cells (or lung alveolar macrophages) alters the microenvironment of the hepatic sinusoid by activation of Kupffer cell function and the induction of IL-1a and b, IL-6, IL-10 and to a lesser degree TNF-a [98, 100]. Production of these cytokines in the sinusoid may affect the tumor cells themselves or other cells in the liver (e.g. the sinusoidal endothelial cells) and change, for example, their ability to interact with invading cancer cells. Cytokines (IL-6, IL-10) levels are often elevated in a patient’s blood at surgery [118, 119], and may promote the survival of cancer cells circulating in the blood [119, 120]. Direct correlations between CEA levels and circulating IL-6 have also been reported in colorectal-cancer patients [120] while preoperative CEA levels alone are an independent prognostic indicator [124]. The ability to inhibit the local secretion of cytokines in the liver sinusoids prior to curative surgery for a colorectal primary tumor has the potential to reduce recurrences in the liver and possibly also in the lungs. Changes in the tumor micro-environment such as up-regulation of endothelial cell adhesion molecules (E-selectin, VCAM-1, ICAM-1) could also occur under the influence of local increases in cytokines and can account for the increased retention of tumor cells in the liver seen

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after CEA treatment [95, 110]. Cytokines (IL-1, TNF-a) can also induce stromal cells to produce and secrete metalloproteinases [125, 126] and this effect could also increase the ability of the tumor cells to extravasate through the sinusoids and invade the hepatic parenchyma. Cytotoxicity by hepatic non-parenchymal cells also plays a part and the observation that NO induction in endothelial cells is an important cytotoxic event suggests that binding interactions between tumor cells and endothelial cells are crucial for either survival in the case of moderate to welldifferentiated tumor cells or destruction in the case of the poorly differentiated colorectal cancer cells [110]. The study of the molecular mechanisms involved in the progression of colorectal cancer hepatic metastasis and the role of CEA and its receptor may therefore, provide new therapeutic targets for non-resectable colorectal cancer metastasis. The development of methods to block CEAR function in vitro and in vivo and relate this to livermetastasis growth and development will increase our understanding of the molecular and biological mechanisms involved in the progression of colorectal cancers to overt metastasis in the liver. This information should allow the development of rational therapies that will be a useful addition to those currently available.

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