DOI 10.1515/cclm-2013-0578 Clin Chem Lab Med 2014; 52(2): 189–200

Review

Evangelos Marinakis*, Georgios Bagkos, Christina Piperi*, Paraskevi Roussou and Evanthia Diamanti-Kandarakis Critical role of RAGE in lung physiology and tumorigenesis: a potential target of therapeutic intervention?

Abstract: Lung is one of the most common malig- *Corresponding authors: Evangelos Marinakis, MD, Third nancies in the world and one of the leading causes of Department of Internal Medicine, Medical School, National and Kapodistrian University of Athens, ‘Sotiria’ Chest Diseases–General death from cancer. In the search for molecules that may Hospital, 152 Mesogion Av., 11527, Athens, Greece, Phone: +30 210 be involved in lung tumor induction and progression, 7763400, Fax: +30 210 7778838, E-mail: [email protected]; the receptor of advanced glycation end products (RAGE) and Christina Piperi, PhD, Assistant Professor, Department of comes across as a critical regulator of lung physiology. Biological Chemistry, National and Kapodistrian University of RAGE is a multiligand receptor that presents a differen- Athens, Medical School, 75 Mikras Asias Str., 11527, Goudi, Athens, tial expression pattern in lung epithelial cells compared Greece, Phone: +30 210 7462610, Fax: +30 210 8037372, E-mail: [email protected] to other cell types being gradually increased from fetal Georgios Bagkos, Paraskevi Roussou and Evanthia Diamanti- to birth and adult life. Under stress conditions, RAGE Kandarakis: Third Department of Internal Medicine, Medical School, expression and activation are rapidly elevated resulting in National and Kapodistrian University of Athens, Athens, Greece chronic inflammation, which, in turn, in many instances, promotes epithelial cell malignant transformation. RAGE overexpression in normal lung alveolar type I epithelial cells is followed by rapid downregulation upon malignant transformation, being associated with increased aggres- Introduction siveness. This is a striking paradox, since in every other The receptor for advanced glycation end products (RAGE), cell type the pattern of RAGE expression follows the oppo- also known as advanced glycosylation end product-spe- site direction, suggesting the involvement of RAGE in the cific receptor (AGER), is a transmembrane pattern recog- well-functioning of lung cells. Additionally, RAGE has nition receptor of the immunoglobulin superfamily [1, 2]. been attributed with the role of adhesion molecule, since Several RAGE isoforms have been recognized includ- it can stabilize mature alveolar epithelial cells to their ing a truncated circulating soluble form of RAGE lacking substrate (basal lamina) by interacting electrostatically the transmembrane and the cytosolic domain (sRAGE), with other molecules. However, the reduction of RAGE arising from receptor ectodomain shedding, as well as observed in lung tumorigenesis interrupts cell-to-cell and splice variant [endogenous secretory RAGE (esRAGE)] cell-to-substrate communication, which is a critical step secretion [3, 4]. These isoforms represent naturally occur- for cancer cell induction, progression and migration. This ring competitive inhibitors of RAGE signaling pathways, review addresses the differential properties of RAGE in being capable of counteracting RAGE-mediated pathogen- lung physiology and carcinogenesis, providing evidence esis and acting as potential biomarkers of endogenous of therapeutic possibilities. protective response. RAGE and its isoforms are encoded in the class III Keywords: cancer; electrostatic charge; lung; receptor for region of the major histocompatibility complex (chromo- advanced glycation end products. some 6, 6p21.3) along with other members of the innate immune system. RAGE structure consists of five domains: a short intracellular negatively charged C-terminal tail, a single transmembrane domain and three extracellular positively charged immunoglobulin-like domains (V, C1 190 Marinakis et al.: RAGE and and C2). The V domain binds most of the RAGE ligands, responses [23, 33–36]. In endothelial cells, RAGE acts just whereas the cytosolic tail is essential for downstream like an adhesion molecule interacting with leukocyte β2 signal transduction [5–7]. integrin Mac-1 [13]. When activated, immune response RAGE was firstly identified in bovine endothelial cells cells enhance RAGE expression. Increased levels of RAGE and initially characterized by its ability to bind AGEs [1, have been observed in acute and chronic inflammatory 2], a complex group of heterogeneous compounds [8–12]. diseases and cancer. Thus, the RAGE/ligand interaction However, nowadays, the list of RAGE ligands has been sig- represents the common causative mechanism that links nificantly extended, with RAGE acting either as a recep- chronic inflammation to cancer [6, 15, 16, 29, 37]. tor or as an adhesion molecule [13], being able to bind to Regarding tumorigenesis, RAGE and its ligands are amphoterin, S100 , integrin Mac-1, collagen IV, commonly overexpressed in most types of solid tumors amyloid-β and amyloid A peptides, glycosaminoglycans [18, 38–48]. For example, both amphoterin and RAGE are and others [5, 6, 13–20]. overproduced in inflammatory states, as well as in many This property of RAGE to recognize and properly malignant tumors with high metastatic capacity [42–48]. respond to myriads of diverse molecular ligands rep- However, a recent study showed that increased plasma resents a challenge to the commonly accepted view of AGE levels are associated with a protective effect over molecular recognition, where each receptor has one non-small cell lung cancer (NSCLC) tumor progression, single ligand that binds to a specific area on the receptor’s presenting a simple predictor of patients’ outcome after surface [5]. Intensive research efforts are currently focused curative surgery [49]. on the elucidation of multiligand recognition patterns by RAGE and the specificity of cellular response elicited by each of them. Furthermore, the RAGE/ligand interaction acti- The role of RAGE in lung physiology vates several diverse cellular pathways. The type of cell response is signal-tissue specific and depends on the type Mature lung cells express higher levels of RAGE than other of ligand, the particular cellular environment and the cell cell types. This suggests that RAGE has a specific role in type. Thus, RAGE downstream signaling activates several lung cells (Table 1). Existing evidence shows that, in this diverse cell activities, such as differentiation, prolifera- case, RAGE is serving lung cells as an adhesion molecule tion, adhesion and migration [7, 14, 21–24]. It is generally rather than as a real receptor. The lung alveolar epithe- believed that RAGE-induced pathways are implicated in lium consists of two cell types: alveolar type I (ATI) and the development of several common diseases [15, 25–29]. alveolar type II (ATII) epithelial cells. ATII cells are cuboi- This view is contradictory to data showing that the acti- dal in shape and, although more abundant, cover much vation and upregulation of RAGE enhance cell survival less area of the alveolar surface. They are the primary sites in cases of cell dysfunction due to increased AGE levels, of surfactant synthesis with a capacity to proliferate and oxidative stress, etc. [16]. Thus, it seems highly likely that differentiate into ATI cells. In contrast, mature ATI cells the observed RAGE upregulation in clinical disorders are thin and flat and cover more than 95% of the alveolar represents the cell defense response for dealing with the surface of the lung, being unable to differentiate. Their insulting cell damage. This is further supported by the thin and flat morphology makes them ideal for normal fact that all known RAGE ligands are able to influence cell bidirectional gas exchange in the lung [62]. Evidence functioning independent of RAGE presence; for example, shows that RAGE is mainly found at the basolateral mem- high-mobility group box 1 (HMGB1) can signal through brane of ATI cells, establishing the contact between ATI Toll-like receptors (TLR) 2 and 4, bypassing RAGE [30, 31], cells and their substrate [22, 50, 65]. whereas AGEs, among many other side effects, can inhibit In the developing rat lung, RAGE expression is gradu- the function of electron transport chain, reduce ATP syn- ally increased from fetal to birth and adult lungs [51]. This thesis and increase reactive oxygen species (ROS) levels pattern was demonstrated in both membrane and sRAGE [32]. expression, predominantly in ATI pneumocytes. In almost every cell type, RAGE expression is gradu- Neonatal rat lung, which is not fully alveolarized, exhibits ally decreased during the embryonic stages of develop- low RAGE expression, whereas the postnatal upregulation ment. Thus, it is usually downregulated in most mature of RAGE reflects ongoing alveolarization characterized by cells, including endothelial, smooth muscle cells, fibro- an expansion of type I epithelial cell population. These blasts, neurons and immune cells. RAGE has been func- observations are in accordance with the study of Demling tionally linked to almost all cell types involved in immune et al. [22], where RAGE appears to be a highly selective Marinakis et al.: RAGE and lung cancer 191

Table 1 Differential pattern of RAGE expression: impact on lung physiology and cancer.

Normal RAGE expression Suppressed RAGE expression

Normal ATI cell differentiation and phenotype [22, 50, 51] Normal: embryonic stages [50, 51] Tumorigenesis: cancer cell regression to embryonic phenotypes [37, 52–58] Normal ATI cell adhesion, spreading and stability [22] Decreased adhesiveness, increased migration, enhanced invasiveness [15, 22, 37, 59–61] Normal alveolar cell apoptosis Evasion of apoptosis [15, 16, 29, 59, 60] Normal alveolar function – gas exchange [22, 62] Malignant behavior [59, 60, 61, 63] Normal lung architecture [22, 64] Malignant tissue and tumor stroma reorganization and modification [15, 16, 29, 59–61] Normal cell-to-cell and cell-to-matrix communication Disruption of normal communication – cell isolation and autonomy

differentiation marker that promotes adherence and frequency electromagnetic signals ranging from a few to spreading of human alveolar epithelial cells. Furthermore, 300 Hz [76]. This cannot be achieved, not even approxi- the optimal regulation of RAGE expression is a prerequi- mately, by most modern electromagnetic devices. Also, site for normal lung development. Mice overexpressing a large body of evidence shows that the living organisms RAGE during lung embryogenesis show altered respiratory from cells to humans possess a huge spectrum of recep- epithelial cell differentiation, severe lung hypoplasia and tors (photoreceptors) that are able to efficiently receive exhibit 100% perinatal mortality rates [64]. These obser- electromagnetic fields (signals). The V domain of RAGE, vations suggest that tight regulation of RAGE expression which is the major contributor to ligand “binding”, is pos- is a requirement for alveolar epithelial cell differentiation. itively charged [5, 77–80]. This positive charge extends far Moreover, RAGE assists ATI cells in obtaining the flat and enough and renders RAGE an electrostatic trap for its neg- extended morphology, which is optimal for effective gas atively charged ligands [5, 77, 79–82]. Also, there is strong exchange [22]. evidence that RAGE interacts with the negatively charged At the same time, RAGE is one among many other patches of AGE-modified proteins [5, 79, 80]. In addition, receptors that assumes the role of an adhesion mole- electrostatic charge modifications alter RAGE-ligand inter- cule. RAGE inhibition results in decreased adhesiveness actions and downstream cell signaling pathways, affect- and increased cell migration and invasiveness [15, 22, 37, ing major cell functions such as adhesion, migration and 65–68]. Structural analysis revealed that the V domain proliferation [81, 82]. The existing differences in individ- of RAGE has features typical of I-set topology, which are ual electrostatic field physical properties of ligands may characteristic of cell adhesion molecules [65–68]. The represent the characteristic electrostatic identity of each specific localization of RAGE between cells and their sub- particular ligand. Given the evidence coming from differ- strate strongly suggests that RAGE is the main, or even the ent scientific fields, it is safe to assume that RAGE, just only, adhesion molecule that stabilizes alveolar cells to like many other receptors, is able to efficiently receive the their position. Lung epithelial cells, due to their role in gas extremely weak electrostatic field (signal) emitted from exchange, have to be as thin and transparent as possible. electrically charged ligands (Figure 1). RAGE is just the right choice, which ensures the optimal It has been shown that RAGE is present in cell mem- transparency and performance for these cells. The more brane in the form of RAGE oligomers [16]. Oligomerization complex molecular entities, which perform the same task, of RAGE provides a mechanism to increase the number of cannot be used because they will increase cell thickness, extracellular V-binding domains, which, in turn, enhance resulting in a reduced alveolar cell transparency and gas RAGE-ligand binding affinity [16]. Also, it is known that the exchange efficiency. clusters formed from receptor polymers or heteropolimers It is well documented that electrostatic interactions contain several different receptors and so acquire a higher play a key role in protein-protein recognition as well as sensitivity and accuracy. Thus, RAGE polymers are able in receptor/ligand recognition and binding [69–75]. More­ to efficiently recognize the extremely weak electrostatic over, it is well known that many different cells are able signals (fields) generated by their ligands and discrimi- to receive and respond to extremely weak electromagnetic nate each one of them. The selective electrostatic interac- fields emitted from different external sources [69–75]. tion between RAGE and its ligands can explain the higher In fact, due to the action of their receptors, cells are the binding affinity that is observed when the multimers of only known entities that are able to receive extremely low RAGE interact with the multimers of ligands. It can also 192 Marinakis et al.: RAGE and lung cancer

cells with basal lamina is achieved by the electrostatic interactions exerted between RAGE and basal lamina gly- cosaminoglycans and heparin sulfate chains.

RAGE expression is upregulated in most cancer tissues, except the lung

RAGE and RAGE ligands are involved in cancer develop- Figure 1 RAGE as a sensor of ligand-specific electrostatic fields. ment and migration in most types of malignant diseases. RAGE senses ligand-specific electrostatic fields. V domain (black Several detailed reviews and experimental reports clearly V shapes) is a positively charged electrostatic trap for negatively charged ligands (gray triangles). RAGE recognizes the individual show that the multiligand-RAGE axis is implicated in electrostatic field emitted by a particular ligand. These electrostatic the development of chronic inflammation, together with fields are ligand specific. regional and systemic immune response deregulation, intercellular communication derangement and tumor pro- motion [6, 15–17, 29, 37, 38, 95]. explain the difference in binding affinity between differ- RAGE activation modulates numerous intracellu- ent ligands, since each one of them possesses its own spe- lar factors, such as ROS, extracellular signal-regulated cific electrostatic signal. protein kinase (ERK1/2), p38 and stress-activated protein Touré et al. showed that neutrophils exhibit enhanced kinase/Jun N-terminal kinase, mitogen activated protein adhesiveness and decreased migration on AGE-modified kinases (MAPKs), p21ras and Rho GTPases (Rac1, Cdc42), collagen in comparison to non-modified collagen [68]. phosphoinositol-J kinase, janus kinase/signal transducer Consequently, collagen glycoxidation may inhibit normal and activator of transcription pathway, nuclear factor neutrophil migration in patients with diabetes and kidney kappa B (NF-κB), activator protein-1 (AP-1) and activa- disease, i.e., in conditions that are characterized by tor of transcription 3 (STAT-3) [6, 15–17, 37, 38]. That is, increased levels of AGEs and RAGE. Furthermore, RAGE RAGE activation has been considered to promote hypoxia antibody inhibits neutrophil adhesion and enhances neu- resistance, angiogenesis of tumor vasculature, tumor trophil migration on glycoxidated collagen [68]. Based growth and invasion, and modulation of the host immune on the electrostatic viewpoint, the increased adhesion of response, contributing to the immunosuppressive state neutrophils to AGE-modified collagen can be explained seen in cancer patients [15, 16, 29]. by the AGE-modification process itself, during which In most cancer types, a parallel increase in the levels negative charge is added to collagen and, thus, collagen of RAGE and RAGE ligands has been observed. RAGE acquires a higher electrostatic reactivity with RAGE. ligands act in an autocrine fashion and induce direct acti- Regarding the normal lung, RAGE mediates the adher- vation of cancer cells by stimulating proliferation, inva- ence of alveolar cells on basal lamina (basal membrane). sion and . Moreover, they act in a paracrine Alveolar basal lamina consists of numerous anionic (nega- manner over RAGE-positive cells within the tumor micro- tively charged) sites that contain heparan sulfate proteogly- environment. Thus, it is not clear whether the observed cans [83]. The heparin sulfate proteoglycan family includes, cellular responses are due to RAGE activation or to the among others, collagen XVIII, which is abundant in the direct influence of its ligands. lung, although less than collagen IV [84]. The electronega- tivity enables heparin sulfate chains to interact with vari- able specificity with several growth factors and receptors and, thus, to affect many diverse cellular functions [85–91]. Role of RAGE in lung cancer Heparan sulfate plays a crucial role in normal lung devel- opment. A decrease in heparan sulfate level is related to Lung cancer, one of the most invasive malignancies, lung hypoplasia and other developmental malformations expresses low levels of RAGE [37], which correlate posi- with increased perinatal mortality [92, 93]. In the lung, tively to increased tumor growth and invasiveness [59, 60]. glycosaminoglycans and heparan sulfate bind strongly to Furthermore, RAGE may have a role in the diagnosis RAGE and RAGE ligands such as HMGB1 [19, 20, 94]. Thus it of lung neoplasms, since they can be employed for the dis- is highly likely that the observed adhesion of lung epithelial crimination between normal and malignant lung tissues Marinakis et al.: RAGE and lung cancer 193

[96, 97]. In addition, sRAGE and esRAGE could also serve augments the proliferation of cancer cells, both in mono­ as potential diagnostic and prognostic biomarkers for layer and in spheroid culture models. By contrast, the lung cancer [61, 98]. ­proliferative stimulus of fibroblasts is reduced in lung Investigation of the RAGE expression between normal cancer cells overexpressing full-length RAGE [60]. These lung and NSCLC tissues demonstrated a strongly reduced results show that the influence of fibroblasts in cancer or absent expression in NSCLC tissues, both at the tran- cells is not mediated through RAGE but through other scriptional and at the protein level, compared to normal receptors like the Wnt, Notch and Hedgehog. Fibroblast- [63]. Furthermore, a study of lung specimens from tumor induced cancer cell proliferation was associated with and paired non-tumor tissues of 34 NSCLC patients, who increased activation of particular MAP kinases (p42/44) in underwent pulmonary resection surgery, showed that the lung cancer cells. The behavior of ΔcytoRAGE-expressing reduced mRNA level of RAGE in NSCLC correlated with cells was investigated as well. Curiously, these cells often higher tumor stages [59]. Since the differences were even tended to a higher stimulation of p44/42 MAPKs (ERK1/2). more prominent at the protein level, further post-tran- Moreover, in three-dimensional cultures, they form the scriptional regulation must take place. The same reduc- largest spheroids in response to fibroblasts impact, thus tion was also observed in squamous cell lung carcinomas behaving like mock-transfected cells [60]. and adenocarcinomas [59] as well as in benign lung neo- Whereas RAGE expression itself is downregulated in plasms (hamartomas). Additionally, in metastatic lesions lung cancer, RAGE ligands are widely overexpressed [37]. originating from the kidney and colon, the magnitude of Overexpression of S100 family members is related to worse RAGE reduction is comparable to the reduction observed prognosis and poor survival in patients with lung cancer in primary lung cancer specimens [59]. [99–101]. Significant alterations in tissue and serum levels There is evidence that downregulation of RAGE in of HMGB1 (amphoterin) in lung cancer have also been human NSCLC may contribute to the loss of normal cell reported [102–105]. differentiation and epithelial structure organization with The study of the RAGE variants and lung cancer rela- concomitant oncogenic transformation (Table 1). Cultures tions provide interesting results. sRAGE is a RAGE isoform of NCI-H358 cells on collagen layers exhibited a spread- that lacks the transmembrane and cytoplasmic domains. ing epithelial-like growth of both RAGE- and ΔcytoRAGE- Since it carries the V domain, sRAGE has the ability to bind expressing cells (i.e., cells expressing RAGE without ligands, acting as a decoy receptor that blocks RAGE signal- cytoplasmic domain), which was not seen in NCI-H358 ing. Serum sRAGE levels are significantly decreased in lung control cells. In particular, RAGE-deficient lung cancer cancer patients compared with healthy controls, and they cells exhibited a multicellular-complexed proliferation are negatively correlated with lymph node involvement [98]. pattern. Immunocytochemistry revealed a diffused mem- Similarly, the expression of cytoplasmic esRAGE, which is a brane localization of RAGE in NCI-H358 single cells and the splice variant of RAGE ubiquitously present in normal lung redistribution of the receptor towards intercellular contact tissue, decreases or disappears in most NSCLC tissues. Lack sites at higher cell densities. Interestingly, ΔcytoRAGE- of expression is related with poor histological differentia- expressing cells, although lacking the cytosolic domain, tion, higher Ki67 index, increased tumor invasiveness and exhibited preferential localization of the truncated recep- decreased survival after surgical resection in patients with tor at cell-cell contact sites as well. Consequently, forma- stage I lung cancer [61]. However, esRAGE and RAGE expres- tion of intercellular contacts requires either the full-length sion may have different effects on cell proliferation and or the truncated receptor, and RAGE localization is inde- tumor growth, and may be independently regulated [61]. pendent of the intracellular domain [59]. Malignancies share a number of major characteristics Regarding cell migration, it has been observed in that include (a) cell dedifferentiation and acquisition of ΔcytoRAGE transfection that overexpression of RAGE embryonic phenotype, (b) disruption of normal cell-to-cell did not induce cell migration in vitro, suggesting that and cell-to-matrix communication with concomitant cell activation of RAGE per se does not contribute to tumor isolation – autonomy and derangement of normal tissue cell migration and metastases. In contrast, primary lung architecture, (c) avoidance of apoptosis and (d) enhanced cancer and metastatic lesions demonstrate reduced RAGE proliferation, invasiveness and metastatic potential. In expression, indicating that neither RAGE downregulation order to acquire the above major characteristics, cancer nor RAGE overexpression contributes to malignant pheno- cells accomplish a large number of structural, functional type behavior per se [59]. and metabolic changes. The outcome of these changes is the Co-cultivation of RAGE-deprived human lung cancer return of cells to embryonic stages. To this sense, lung cancer cells (H358, a NSCLC cell line) with lung fibroblasts (WI38) cells re-program the expression of numerous receptors, 194 Marinakis et al.: RAGE and lung cancer such as RAGE and RAGE-ligands, epidermal growth factor receptor, vascular endothelial growth factor, β1 integrins, TLRs, Wnt, dishevelled, Notch receptors, connexins and others. In NSCLC, upregulation of expression has been reported for integrin α5β1, S100A8/A9, HMGB1, TLR4/9, dishevelled, Wnt-1, Wnt-2 and Notch 3 [52–55, 106, 107]. Interestingly, the same group of receptors is also overexpressed in embryonic stages of lung development. Their expression is suppressed in normal adult lung, only to be once again upregulated as part of the pathogenesis of malignant lung diseases. For example, Wnt, Notch and hedgehog receptors are normally expressed in embryonic lung cells and are downregulated in mature lung cells. Then, they are once more fully expressed in lung cancer cells [52, 53, 55, 106]. That is, the pattern of Wnt, Notch and hedgehog expression is linearly negatively correlated Figure 2 RAGE expression and alveolar cell phenotype: (A) normal with the corresponding pattern of RAGE isoform expres- alveolar cells; (B) malignant cells ( : RAGE). sion. When one group of receptors is upregulated the (A) Normal RAGE expression is related to normal alveolar type I epi- thelial cell phenotype (ATI), which is characterized by an extremely other is downregulated. Thus, it is possible that, in lung thin and flat morphology and normal adhesion to substrate. Normal cells, each one of these groups can efficiently substitute RAGE expression is essential for the preservation of normal cell the other as signal receptors – although these receptors do phenotype and tissue architecture, ensuring efficient gas exchange not substitute RAGE in its role as an adhesion molecule. functioning (diffusion of oxygen and carbon dioxide) in the alveolar In lung ATI cells, RAGE acts both as a receptor and cells. (B) RAGE downregulation is related to a dedifferentiated abnormal cell phenotype with enhanced capacities of migration and as an adhesion molecule serving differentiation and invasion, disruption of normal contacts with substrate and adjacent adhesion. When these cells are malignantly transformed, cells, and disruption of normal lung tissue organization and architec- RAGE presence is no longer required. Thus, lung cancer ture. Thus, downregulation of RAGE is a critical step in the process of cells reduce RAGE production in order to break the inter- cell regression to a dedifferentiated embryonic malignant pheno- cellular and cell-matrix contacts and get ready to prolifer- type, with a concomitant disruption of lung tissue architecture. ate, migrate and invade. That is, downregulation of RAGE along with the concurrent upregulation of Wnt, Notch and However, in terms of RAGE-axis inhibition as a poten- hedgehog reflects the regression of lung cancer cells to tial therapeutic multi-tool, one cannot draw a general embryonic phenotype (Figure 2). conclusion that would be widely applicable. The view of RAGE as a receptor that mediates purely damaging effects and, consequently, the therapeutic approaches that aim RAGE as a potential target of thera- in its blockade or inhibition present a rather oversimpli- fied and misleading assumption [111]. Although there peutic intervention: evidence and is evidence that RAGE-axis inhibition may be beneficial concerns in certain occasions, experimental data exist that show RAGE blockade inducing deleterious side effects, whereas Animal models have demonstrated that blockade of RAGE increased expression of RAGE or RAGE variants being ben- signaling with various experimental methods, such as eficial [59, 60, 61, 66, 98]. sRAGE antagonists, anti-RAGE antibodies and RAGE According to evidence coming from in vitro and in silencing, may be beneficial under certain circumstances. vivo experimental and clinical studies on lung cancer Potential therapeutic applications of RAGE-axis blockade (especially NSCLC), RAGE and RAGE variants are down- include prevention of diabetic complications, treatment of regulated in lung cancer tissues. Nevertheless, this obser- acute or chronic inflammatory diseases and neurodegen- vation is not specific for lung malignancies, since RAGE erative diseases, management of myocardial and pulmo- expression is suppressed in both non-malignant lung nary ischemia-reperfusion injury, organ transplantations, tumors and lung metastatic disease from other primary acute lung injury and lung fibrosis therapy, as well as tar- sites [59]. geted cancer therapy and prevention of metastases forma- Cell line and in vivo studies have demonstrated that tion to the lung [19, 26, 56–58, 61, 68, 107, 108–110]. RAGE overexpression has an inhibitory effect on the Marinakis et al.: RAGE and lung cancer 195 proliferation and migration of lung cancer cells and tumor by a remarkably lower 82SS and 82GS genotype than growth [59–61]. However, there are significant differences non-responders. between observations seen in monolayer and spheroid Another finding of the study was that 82GS and 82SS cultures, as well as among particular studies, leading to carriers had a significantly increased risk for NSCLC. the conclusion that RAGE re-expression per se does not Therefore, polymorphisms of the RAGE could be inhibit tumor growth. A reasonable therapeutic approach used as a screening diagnostic test to identify subjects at would be the controlled upregulation of RAGE expression high risk for lung cancer, as well as prognostic markers in lung cancer tissues; however, the precise and specific that could modify treatment decisions in patients with in vivo regulation of the expression of this ubiquitous NSCLC [116]. receptor represents a great challenge. Moreover, although sRAGE and tissue esRAGE may prove to be useful bio- maintenance of RAGE expression appears to be a reason- markers for the diagnosis of lung cancer, as well as for the able way of inhibiting lung tumorigenesis, it still remains assessment of prognosis [61, 98]. Serum concentration of theoretical. Consequently, considering the regulation of sRAGE is markedly reduced in patients with lung cancer, RAGE expression (and re-expression) as a potential thera- independent of histologic type, and lower serum levels peutic tool for lung cancer therapy is a very complicated being correlated to lymph node infiltration [98]. Fur- issue. What may be more feasible as a therapeutic inter- thermore, immunohistochemical studies in 182 NSCLC vention is the prevention and treatment of lung metastatic surgical specimens demonstrated that downregulation disease arising from other primary tumors, such as mela- of cytoplasmic esRAGE is an independent prognostic noma. Both in vitro and in vivo studies demonstrate that factor of survival, especially in patients with TNM stage ligand-RAGE axis inactivation restricts metastatic forma- I. Additionally, absence of esRAGE immunoreactivity was tion to the lung [19, 20, 112–114]. associated with significantly increased mortality, with a This observation applies to the inhibition of RAGE 5-year survival in patients with NSCLC stage I below 20% activation by classic ligands and several other molecules [61, 116]. like glycosaminoglycans. In vivo studies have shown that glycosaminoglycan-RAGE interactions are promising targets in the prevention and management of pulmonary metastases [19, 20]. Administration of the derivative 2-O, Conclusions and perspective 3-O-desulfated heparin significantly reduces the forma- tion of lung metastasis after intravenous injection of mel- RAGE occupies a central position in developmental and anoma cells [113]. The mechanism of protein inhibition adult lung physiology and disease. Both as a receptor and by heparin and non-anticoagulant heparin derivatives as an adhesion molecule, RAGE is related to alveolar cell dif- is exerted by electrostatic interactions between heparin ferentiation and preservation of normal lung architecture and positively charged proteins [115]. Thus the beneficial and function. The distinct property of RAGE to recognize effects of heparin may also apply to RAGE interaction with and interact with numerous and diverse ligands can only these compounds, since the V-domain binding region of be explained through an electrostatic viewpoint, accord- RAGE is characterized by its highly positive charge. RAGE/ ing to which RAGE senses the electrostatic fields (signals) ligand binding inhibition by heparin and heparin deriva- emitted by its ligands. This view may also apply to RAGE- tives can be explained by the competition between these mediated alveolar cell adhesion to alveolar basal lamina. compounds and the classic RAGE ligands for electrostatic Deregulated RAGE expression contributes to lung binding within the cationic V-domain of RAGE. morphogenic disorders, as well as to the development of Tissue and serum measurements of RAGE and RAGE inflammatory and neoplastic diseases in adult lung. RAGE variants in lung cancer patients could have diagnostic is markedly downregulated in lung tumors, both at the and prognostic significance, thus influencing indirectly mRNA and at the protein levels. treatment decisions [59, 61, 98, 116]. RAGE genetic poly- Lung cancer cells downregulate RAGE by detaching morphisms were investigated in a prospective study with from the surroundings and then retaining a low differen- 562 patients with NSCLC and 764 healthy subjects. The tiation state, whereas they interrupt normal intercellular single-nucleotide polymorphism G82S (rs 2070600) is and cell-matrix crosstalk in order to efficiently migrate located in RAGE gene exon 3, in a putative ligand binding and invade. Therefore, suppression of RAGE is considered site [117]. Patients with advanced NSCLC and the 82G/S as a critical step in lung tumorigenesis, occurring within polymorphisms showed significant differences in chemo- the context of malignant tissue reorganization and cell therapy response, with responders being characterized regression to embryonic phenotype. 196 Marinakis et al.: RAGE and lung cancer

The regulation of RAGE expression as a potential ther- binding with the V domain, thus inhibiting RAGE-axis apeutic tool for lung cancer sounds reasonable. However, activation and preventing migration and metastasis. although RAGE downregulation is related to lung tumori­ genesis, re-expression of RAGE is not per se inhibitory Conflict of interest statement for lung cancer development. Moreover, the precise in vivo regulation of RAGE in lung tissue represents a great Authors’ conflict of interest disclosure: The authors ­challenge. A more promising therapeutic approach can be stated that there are no conflicts of interest regarding the the prevention and treatment of lung metastases derived publication of this article. from other primary sites. In this context, RAGE/ligand Research funding: None declared. interactions could be potential therapeutic targets. More- Employment or leadership: None declared. over, a new arrow in the quiver of treatment options for Honorarium: None declared. lung cancer could be the design of electrostatic inhibitors of RAGE/ligand interactions. These charged molecules Received July 22, 2013; accepted August 20, 2013; previously pub- could compete with classic RAGE ligands for electrostatic lished online October 9, 2013

References

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basis of Polycystic Ovarian Syndrome. Her research focuses on the role of advanced glycation end-products in metabolic diseases such as Polycystic Ovarian Syndrome and Diabetes Mellitus type 2, aiming in elucidating the intracellular molecular mechanisms of their action. She is the author or co-author of more than 90 peer-reviewed publications.

Evangelos Marinakis, MD, studied Medicine in Aristotle University of Thessaloniki, Greece and he is a consultant of Internal Medicine in the Third Department of Medicine, Medical School, National and Kapodistrian University of Athens, Greece. His PhD is focused on the role of advanced glycation end-products (AGEs) and their receptor (RAGE) in space-occupying lesions of the lung. His research interests focus on the crosstalk of advanced glycation and Paraskevi Roussou, MD, PhD, obtained her degree in Medicine in bioenergetics, oxidative stress and cancer biology. He currently 1975 and her specialty in Internal Medicine (1981) and Hematology participates in several collaborative projects in the Department of (1985). She received her PhD in 1984 from the National and Biological Chemistry, of the University of Athens Medical School. Kapodistrian University of Athens, Medical School. In 1983 she was trained in column chromatography in the Laboratory of Hematology of the Royal Free Hospital in London. She was a visiting fellow in the Laboratory of Cellular Biology of F.D.A. - N.I.H. (Federal Drug Administration - National Institute of Health, Bethesda), with primary interest in the investigation of B-cell differentiation and the role of IL-6 (1987–1988). She currently holds the position of the Assistant Professor of Medicine and Hematology at the 3rd University Department of Internal Medicine of “Sotiria” Hospital of Athens being also the Director of the Haematology Unit. Her current research efforts focus on RAGE biological functions with emphasis George Bagkos MD, PhD, obtained his degree in Medicine (1973), in immune system regulation. She has co-authored 44 peer- specialty of Internal Medicine (1980) and his PhD (1981) from the reviewed publications. Medical School, National and Kapodistrian University of Athens, Greece. He is an experienced consultant in Internal Medicine with research interests in mechanisms of ATP production and molecules involved in cell communication and cell migration. He is currently working on RAGE biological functions with emphasis on electrostatic interactions. Further interests include scientific writing of books related to the methodology of Science and Nature of Human Mind.

Professor Evanthia Diamanti-Kandarakis MD, PhD received her MD from Medical School of Athens and her PhD in Experimental Endocrinology on the effects of androgens in hypophysectomised rats, from the same University. She was trained in Internal Medicine in the UK (1974–1980) and in Endocrinology and Metabolism in the USA (1980–1986). Currently she holds a chairman position in the Third Department of Medicine, Medical School-University of Athens. Her research interests focus for the last 15 years on clinical, molecular and environmental aspects of metabolic and hormonal Christina Piperi, PhD, completed her studies in Medical abnormalities in Polycystic Ovarian syndrome. Her research on Biochemistry in the University of Surrey, UK (1995) and received endocrine disruptors was the first to detect elevated endogenous her PhD in Molecular Immunology at the Sir William Dunn School advanced glycation end-products in women with Polycystic Ovary of Pathology of the University of Oxford, UK (1999). Dr Piperi Syndrome (2005) and implicate them in the pathophysiology is a recipient of the Gordon Piller studentship from Leukaemia of the syndrome. Her work aims in elucidation of the molecular Research Fund (1996) and the L’Oreal-UNESCO award (Hellas) for mechanisms underlying advanced glycation end-products and their Women in Science (2010). She now holds a position of Assistant receptors in metabolic diseases. She is the author or co-author of Professor at the Department of Biological Chemistry of the more than 132 peer-reviewed publications. University of Athens Medical School. She has received several awards and distinctions for her studies in the field of molecular