Dynamic Tuning of Galectins and Their Binding Sites During

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Chapter 11

Dynamic Tuning of Galectins and Their Binding Sites During Mammary Tumor Progression and Metastasis Joana T. de Oliveira and Fátima Gärtner* Institute of Molecular Pathology and Immunology (IPATIMUP), and Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), University of Porto, Porto, Portugal *E-mail: [email protected]

Galectins are essentially bridging molecules and as such their biological effect will be dependent on their levels of expression, binding sites availability on their ligands, presence of other competitive galectins or even members of other families of endogenous lectins. Classically, glycosylation patterns were believed to be fairly constant in cells of the same tissue origin and the process was not thought to be a quickly modulated one able to transduce external signals. Nevertheless, a growing amount of data now suggests glycosylation to be a much more dynamic microenvironment-related mechanism. The consequent formation of different glycans alters the array of lectins which are able to recognize them. Therefore, rather discrete and transient changes in interacting glycans, glycan carriers and glycan receptors may profoundly influence the fate of an invasive tumor cell and ultimately its ability to metastasize to distant sites. This mini-review will focus on the dynamic interplay between galectins and their binding sites in a spontaneous model of mammary tumor progression and invasion, canine mammary tumors (CMT).

© 2012 American Chemical Society In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Introduction


Integrins (1), cadherins (2–7), caveolins (8), metalloproteinases (9), hormone receptors (10), growth factors (11), growth factor receptors (12) and mucins (13, 14) putatively modulate the interactions between mammary tumour cells and their surrounding microenvironment, at least in part, by their well-known interplay with galectin-3 via its carbohydrate recognition domain [reviewed in (15)]. However, the interplay between these ligands and galectin-3 has been shown to be modulated not only by the levels of expression of the lectin but also by differential glycosylation of the ligands in several settings (16–20).

Galectins’ Levels of Expression: Adapting to Presenting Microenvironments in CMT Progression Galectin-3 is down-regulated during the process of malignant transformation of canine mammary glands. Its expression being significantly decreased in malignant when compared with benign canine mammary tumors (CMT) (21). Galectin-3 is a marker of progression in other types of tumors presenting increased expression in non-aggressive tumors and being reduced in aggressive ones, the opposite having also been observed (22–25). Galectin-1 is on the other hand overexpressed in malignant CMT, and found in the nucleus and cytoplasm of tumor cells (21). Several examples of dynamic and differential expression of galectins-1 and -3 in physiological and pathological contexts suggesting they play opposing roles can be found in the literature (26–28). Among other examples, in lymphoma cells, galectin-1 induces apoptosis while galectin-3 induces cell-cell aggregation (29) and blocks the execution of the cell surface apoptotic signal (30). In accordance to its pro-adhesive effects, it comes as no surprise that galectin-3 is highly expressed by vessel-invading tumor cell subpopulations in both primary and metastatic CMT while in well-established metastatic lesions, there is galectin-3 staining almost only in tumor cells surrounding necrotic areas, a pattern resembling that observed in the primary malignant CMT (21). On the opposite galectin-1 is down-regulated in tumour emboli and up-regulated in well-established primary and metastatic lesions (unpublished results by the authors of this paper). Accordingly, galectin-1 serum levels are not elevated in cancer patients (31) while those of galectin-3 have been found to be increased (32). Cytoplasmic staining of galectin-3 is associated with increased aggressiveness in CMT whereas significant down-regulation of nuclear galectin-3 expression is observed in malignant when compared with benign tumors (21). The cytoplasmic subcellular distribution of galectin-3 is an important feature related to malignancy and is suggested to be responsible for increasing apoptosis resistance of tumor cells that migrate and/or are shed into the circulation (33–35). Indeed, xenografts from a CMT cell line with an in vitro homogeneous cytoplasmic-only expression of galectin-3, quickly metastasize. However, a heterogeneous pattern with specific galectin-3 positive areas is observed in well-established xenografts suggesting a role for microenvironment in the regulation of galectin-3 expression. In primary tumor xenografts, cells staining for galectin-3 were specifically located in necrosis182 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


surrounding regions and tumor emboli, as observed in spontaneously occurring malignant CMT. Likewise, metastatic lesions displayed low galectin-3 expression which was mainly present in necrosis-surrounding areas and inside tumor vessels (21). The reasons why intravascular tumor cells consistently overexpress galectin-3 when compared to sedentary tumor cells still need clarification. However a few hypothesis are to be considered. Galectin-3 is a hypoxia regulated protein (36). Hypoxia also leads to activation of epidermal growth factor receptor (EGFR) kinase function and hence may enhance tumor cell migration (37, 38). Interestingly, EGFR expression is up-regulated in CMT intravascular tumor cells and viable cells adjacent to necrotic areas paralleling that of galectin-3. This indicates a possible joint role of EGFR and galectin-3 in the survival and invasion process of tumor cells under stress conditions in malignant CMT (39). Adding to that, high level of activity in necrotic areas was found when analyzing EGFR by autoradiography (Berns and Rutteman, unpublished data, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, the Netherlands) and its levels were significantly associated to the presence of necrosis in CMT (39). Since vessel-invading tumor cells express both galectin-3 and EGFR, the hypothesis that tumor cells which intravasate, are mainly those exposed to stress conditions seems very plausible. Furthermore, as galectin-3, EGFR has also been associated to angiogenesis which could in addition aid in providing an escape pathway for these stress-exposed cells (15, 40). Another line of thinking arises from the fact that galectin-3 expression has been associated to cell differentiation in several contexts (41). Epithelial to mesenchymal transition (EMT) has been suggested to be crucial for cells to acquire invasive characteristics, migrate throughout the ECM and ultimately intravasate (42, 43). In the absence of galectin-3 there is decreased TGFβ-induced EMT, myofibroblast activation and collagen production with reduced β-catenin phosphorylation and translocation to the nucleus (44). Accordingly, in low galectin-3-expressing malignant CMT, there is little nuclear expression of β-catenin (45, 46). This further points to galectin-3 re-expression being implicated in acquisition of aggressive characteristics in stress-exposed cells, such as those under hypoxic conditions. Mixed type malignant CMT (carcinosarcomas) present a common histogenesis between epithelial and mesenchymal cells (47). These mixed malignant mammary tumours are considered a naturally occurring model of EMT [reviewed in (48)]. Galectin-1 was consistently co-expressed with mesenchymal while galectin-3, when present, was co-expressed with epithelial markers in this natural model of EMT (unpublished data). However, the differentiation status of galectin-3-positive intravascular tumor cells could not always be associated with loss or gain of membrane expression of E-cadherin since both E-cadherin-negative and E-cadherin-positive tumor emboli were found in malignant CMT. Interestingly however, β-catenin expression was often found at the cell membrane, in contact sites between some intravascular tumor cells indicating a correct assembly of the adherens junctions complex at these sites (21). Since the hallmark of EMT is loss of E-cadherin expression (49), current findings 183 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


do not support the assumption that all intravascular tumor cells found in CMT have undergone complete EMT and/or the inverse process mesenchymal to epithelial transition (MET) but rather point to the presence of several cells in hybrid states inside vessels (21). The isolated expression either of anti-adhesion, lymph node metastasis-associated, glycan Sialyl Lewis X (50) or the adherens junction E-cadherin at the cell surface of CMT emboli cells, seems to corroborate this assumption (51). Despite this it is the authors’ opinion that MET is crucial for cell-cell aggregation inside vessels insuring anoikis survival for the 2% of the circulating tumor cells that in fact successfully arrive at distant sites and metastasize (52). Thus overexpression of galectin-3 seems to be a characteristic of potentially metastatic tumor cells in malignant CMT but does not appear to be exclusively associated to a differentiation status.

Glycosylation of Galectin-Ligands: Modulating Galectin-Mediated CMT Cell-Cell Adhesion Mucins have been associated to malignancy (13, 14, 53) and are important carriers of tumour-associated glycan antigens (54, 55). Variable glycoforms of MUC1 have been found to be involved in carcinogenesis (56, 57). MUC1 is involved in cell-cell aggregation, (its sialylation being particularly important for the mucin’s anti-aggregation effect) (58, 59); and cell-ECM adhesion (58, 60, 61) in which MUC1 specific O-glycosylation plays a relevant role (62). MUC1 is overexpressed and significantly associated to vascular invasion and distant metastases in malignant CMT (21, 39). In primary tumour cells, the mucin is present in a cytoplasmic vesicular pattern and all around the cell membrane. This high level of MUC1 expression is significantly associated with the above mentioned galectin-3 generalized down-regulation. Furthermore, the two molecules are not co-expressed in primary tumor sedentary cells (39). Despite the described overexpression of its well-known ligand, galectin-3-binding sites are expressed at the tumor cell surface only in moderately differentiated tumor areas, their expression being low in the majority of sedentary primary tumor cells. However, in malignant CMT, intravascular tumor cells and tumor vasculature strongly express galectin-3-binding sites pointing to the existence of galectin-3-mediated cell–cell interactions, which could thus facilitate anoikis survival and metastatic spread (21, 63). Interestingly, in these vessel-invading cell tumor subpopulations MUC1 expression is focally localized at the cell membrane where it co-expresses with galectin-3 (39). Different glycosylation patterns of MUC1 mucin in normal mucosa and colon cancer tissues correlate well with galectin-3-binding sites expression (64). Sialylation thus acts as an important on/off switch mechanism modifying galectin-3 binding to its ligands during tumor progression in malignant CMT. The differential presence of α2, 6- linked sialic acid in sedentary tumor cells and certain tumor subpopulations, such as invasive fronts, may account for differences observed in galectin-3-binding site expression and modulate galectin-3-mediated adhesion between tumor cells at these locations (39). Up- or down-regulating 184 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


of sialyltransferases is a well-known mechanism of motility tuning, turning galectin-3-binding positive cells into negative ones and vice-versa, allowing cells to migrate in a physiological context. Indeed there is an increase of both galectin-1 and -3 binding following alpha 2,6 neuraminidase treatment of glycans at the cell surface in vitro (65). The Thomsen-Friedenreich antigen (T antigen) is one of the most common glycoforms of MUC1 found in breast cancer patients. MUC1 interaction with galectin-3 in vitro via T antigen, causes clustering of the mucin at the cell surface with consequent exposure of smaller adhesion molecules. This has been proven to increase E-cadherin mediated cell-cell adhesion and ICAM-1 mediated cellendothelial adhesion (17, 66). A similar effect has been observed for galectins-2, 4 and -8 by interaction with MUC1 via T antigen (31). Most cells of primary malignant CMT show high levels of sialylated T antigen when compared to the un-substituted form of the antigen which is expressed in low levels and is often negative. However, surprisingly, intravascular tumor cells express mainly the unsubstituted form of the T antigen. This allows a proven physical interaction in vivo between galectin-3 and MUC1-carried T antigen, therefore supporting an important role in metastasis (39). Alterations in the glycosylation patterns of its ligands, namely increased disialylated core 1 O-glycan structures, have been implied to alter galectin-3 affinity at the cell surface with consequent differences in cell biological behaviour (67). Core 1 biosynthesis is dependent on the activity of core 1 beta1,3 Gal-transferase (68) which has been found to be impaired by mutations in the molecular chaperone Cosmc leading to increased expression of Tn and sialyl Tn antigens (69). In order to have the unsubstituted core 1 (T antigen), there must not be action of ST6GalNacI and II (70). The action of these enzymes leads to the biosynthesis of Sialyl Tn and/or Sialyl-6T antigen. ST6GalNAc-I is the key-enzyme leading to sialyl-Tn biosynthesis in MUC1-Tn glycoform (71) while ST6GalNAc-II sialylates better the T antigen (70). MUC1 is also a natural carrier of Sialyl Tn (72). It is of note that ST6GalNacI and II enzymes perform very fast (70). Their subcellular localization has been suggested to influence their activity in breast cancer cells (73) as has the tumour microenvironment which was found to importantly modulate Sialyl-Tn expression (74). Galectin-3 has been implicated in the apical sorting of several proteins (75) but in the absence of galectin-3 there is still apical targeting of MUC1. However, this can be blocked by overexpression of an alpha 2,6 sialyltransferase (76) further corroborating the importance of sialylation in the interplay between MUC1 and the galectins family. In a normal context MUC1 is localized at the apical cell membrane while EGFR is localized basolaterally. MUC1-EGFR interaction in a non-tumour context is a sign of temporary polarization disruption and mediates cell survival programs. These are thought to be persistently present in several types of carcinoma where there is loss of polarization with permanent interaction of transmembrane mucins and growth factor receptors (77). In breast cancer cells this interaction is at least in part mediated by galectin-3. In vitro and in vivo results show the lectin’s co-expression with both of its ligands in intravascular tumour cells, further supporting its importance for cell survival in this context (39, 78). 185 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Microenvironment seems therefore to play a vital role in the availability of ligands for galectin-3-binding in CMT. This is corroborated in the CMT metastatic cell line which shows a homogeneous expression of galectin-3-binding sites in vitro (21) which turns to a heterogeneous one in tumor xenografts (39). Also, galectin-3-binding site expression is low in the vast majority of sedentary primary tumor cells to the opposite of invasive fronts where it is high akin to spontaneously occurring CMT. In addition, binding of galectin-3 is again increased, following neuraminidase treatment in galectin-3-binding sites negative areas, resembling the observations in the spontaneous model (39). Other than downregulation of sialyltransferases which can at least in part mask galectin-3-ligands in these cells, there may be higher activity in invading tumor cells of specific glycosyltranferases (79) some of which responsible for assembling ligands for galectin-3, such as the metastasis-related GnTV and concomitant lower activity of competitive GnTIII, which leads to the production of N-glycans not recognized by the lectin (80, 81) Indeed, recently GnT-III-mediated glycosylation, was found to be altered upon EMT/MET-inducing microenvironment changes (82).

Glycosylation of Galectin-Ligands: Modulating Galectin-Mediated CMT Cell-ECM Adhesion Downregulation of galectin-3-binding sites in the ECM parallels the malignant transformation of canine mammary glands. In fact, galectin-3-binding sites are significantly decreased in the ECM of malignant tissue when compared with that of normal-adjacent glands. In most benign lesions the ECM also presents strong expression of galectin-3-binding sites. A coordinated decrease of galectin-3-binding sites in the ECM may further account for the loss of galectin-3-mediated cell–ECM adhesion in the tumor microenvironment (21). The decreased galectin-3 binding to the ECM is attributable, at least in part, to binding sites occupancy by other galectins and altered stromal glycosylation. Although there are substantial differences in the type of glycans recognized by galectins-1 and 3 they often compete for the same ligands (83). Galectin-3 affinity for the T antigen, for instance, is two times higher than that of galectin-1 (84). These differences might account at least in part for their divergent biological functions. Galectin-1 is scattered throughout the tumor stroma and could thus be leading to galectin-3-binding site occupancy (21). The decrease in galectin-3-binding sites is also most likely due to differential ECM glycosylation in malignant CMT. Sialylation is a prevalent type of glycosylation in tumour cells but not in the ECM of malignant CMT (85). However, in normal adjacent mammary tissue, both ECA and PNA bind to matrix glycoproteins in tissue stroma and gland mucus secretion in addition to the apical border of luminal cells while in the ECM of malignant CMT, there is a striking decrease in ECA- and PNA-binding sites pointing to an overall decrease in galactosylation (21). Collagen glycosylation is known to affect tumor cell adhesion to and spreading on collagen IV (86). In normal mammary glands, the collagen-galactosylating enzyme, GLT25D1 (87), is expressed with slightly 186 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

higher levels than in benign CMT. However, the enzyme expression levels decrease considerably in malignant CMT (21). Galectin-3 knockdown causes a decrease in GLT25D1 mRNA levels in vitro. This suggests that downregulation of galectin- 3 may be at least partially responsible for the decreased GLT25D1 mRNA expression levels found in spontaneously occurring malignant CMT (21).


Concluding Remarks Circulating tumour cells (88), micrometastases (89) and well-established metastases (90) may coexist in the patient. In these, changes in the expression of galectins, glycans and the glycoproteins which carry them are at least in part microenvironment related. Galectin-3 has been shown to play a crucial role in cancer drug resistance [reviewed in (91)]. Inhibition of galectin-3/T antigen interaction was shown to reduce experimental metastatic disease (92). However, other galectins seem to be able to step up and perform its functions. Novel potential specific galectin inhibitors and galectin-targeted therapy are increasingly being studied (93–95) but there is an increasing need to broaden their galectin-specter of action and take into account the relevance of the glycosylation status of their ligands in other to achieve therapeutic efficacy.

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