CRC is a heterogeneous disease, especially with respect to features such as genetic and dietary interactions and the anatomic location of the tumor [2]. Taking into account the distribution of neoplasia in the colorectum, CRCs are divided into right sided and left sided bowel cancer, and the two types show differences in their clinical presentation and surgical management, as well as functional and molecular differences including the phenotypic expression of various biomarkers [2, 3]. The pathogenesis of CRC usually follows a stepwise progression from benign adenoma to malignant adenocarcinoma and distant metastasis, its sequential nature lending itself to the notion of identifying diagnostic markers.
The expression of HSPGs is markedly altered during malignant transformation and tumor progression, affecting both the PG core proteins and the GAG chains [30]. Substantial evidence in support of this has been reported in relation to various tumors, including brain, breast, lung, skin, pancreas, colon, ovarian and head and neck [30], and as such abnormal HSPG expression in cancer and stromal cells can serve as a biomarker for tumor progression and patient survival [31]. As a result of these modifications, some processes appear to be affected, such as cancer cell signaling, growth and survival, cell adhesion, and differentiation, migration and angiogenesis [31]. The HS fine structure is determined by cell-type specific expression of only certain isoforms of some of the biosynthetic enzymes, notwithstanding the existence in some specific cases of regulation at the level of translation or enzymatic catalysis [32–34]. We have therefore investigated the expression patterns of the genes involved in HSPG biosynthesis in CRCs and compared their expression patterns to that of healthy tissue from the same patients. We have centered the study on right sided tumors due to the fact that there are differences between them and left sided CRCs. In addition, we have considered the comparative analysis of two groups of tumors, both at the T3 stage where the muscularis propria is affected. Also, we included in these analyses, the absence of lymph node metastases.
In human cells, two gene families, syndecans and glypicans, account for most cell surface HSPGs, although a few more part-time proteins may appear [24]. Previous immunohistochemical studies performed with syndecans in colon cancer have demonstrated a loss of expression of syndecan-1 in CRCs, some of which have been found to correlate with tumor stage and metastasis [35–38]. However, a study analyzing mRNA transcripts of syndecan-1 did not find significant differences between normal and cancerous tissues [39], while another, using colon carcinoma cells, evidenced a decrease in syndecan-1 mRNA levels in most of samples, along with a 2–5 fold increase in syndecan-2, and a decrease in syndecan-4 mRNA which was restricted to highly metastatic cell lines [18]. In contrast, in the current work, no significant differences in levels of transcripts of any of the four isoforms were found in non-metastatic CRCs, while syndecan-1 mRNA alone appeared overexpressed in metastatic tumors. Analyzing the expression of syndecan-1 protein using immunohistochemistry, provided the noteworthy finding that non-metastatic CRCs displayed similar immunoreactivity to that detected in normal tissues from the same patients, while the metastatic tumors showed a dramatic decrease in staining. Nevertheless, examining transcription at the tissue level by means of CISH showed positive hybridization in normal as well as tumoral cells, both metastatic and non-metastatic, consistent with the qRT-PCR. These data suggests that in the expression of syndecan-1 in CRCs, additional post-transcriptional mechanisms, such as protein translation, degradation, inhibition by feedback loops or miRNA regulation may be involved. Post-transcriptional regulation of syndecan-1 expression has been indicated previously, for example in pancreatic cancer and peritoneal macrophages [34, 39, 41]. Upregulation of syndecan-1 has also been described in tumors other than those of the colon, and it has been postulated that this aberrant expression may play a key role in promoting growth factor signaling in cancer cells [30]. In contrast, other malignances showed downregulation of this molecule, indicating that this HSPG could well serve as a prognostic marker in a cancer-type-specific manner [30]. Furthermore, at the transcriptional level, syndecan-1 modulation has been related with the expression of other PGs, as well as with genes involved in biosynthesis and sulfation of HS chains [40].
The glypicans constitute a six-member family of cell surface HSPGs that are glycosylphosphatidylinositol-linked to the cell membrane. Their expression is cell-type and developmental-stage specific and they are implicated in the regulation of several signaling pathways, including Wnts, Hedgehogs, FGF, bone morphogenic protein and insulin-like growth factor, where they can either stimulate or inhibit activity depending on the biological context [41]. Because of their biological roles, they influence tumor progression and their abnormal expression has been described in various human tumors [31]. In this work, analysis of the transcription of the 6 genes in CRCs showed deregulations for isoforms 1, 3 and 6 in non-metastatic tumors, while only glypican-1 transcription was significantly altered in metastatic, and to lesser extent than in non-metastatic.
Glypican-1 is, with −3 and −5, the isoform most widely associated with the tumorigenic process. Immunohistochemical analysis of glypican-1 protein showed a very weak staining in most of the cells, although certain specific ones displaying neuroendocrine features showed intense immunostaining, On the other hand, it must be taken into account that the values of transcription quantified by PCR are not probably revealing these differences, since it must be considered that the data are affected by the decrease or absence of neuroendocrine cells with high expression levels in the tumor stroma. The disappearance of the expression of this protein has previously been described in tumor epithelial cells in prostate cancer, although with high expression being maintained in tumor stroma [42]. Conversely, glipican-1 overexpression has been described in certain tumors, although with some peculiarities depending on the neoplasia: in gliomas, where it acts to enhance FGF signaling and mitogenesis [43]; in pancreatic cancer, both in the cancer cells and the adjacent fibroblasts [44]; in high-grade breast cancer tumors [45]. Interestingly, in neuroendocrine neoplasias derived from the large bowel, GPC1 expression has been shown to be intense in well-differentiated tumors, but far less in poorly differentiated, a feature that seems to be shared with other neuroendocrine tumors, independently of their topography [46].
Glypican-3 is though to be the species for which most expression alterations in tumors have been described. In this work, this isoform appeared downregulated in all the non-metastatic CRCs analyzed, while only 50 % of metastatic tumors showed alteration in its level. Downregulation of glypican-3 has been described in many tumor types, including breast, lung, gastric, and ovarian cancers and mesothelioma [41]. However, in tumors originating from tissues where glypican-3 expression is restricted to the embryo, its expression tends to reappear with malignant transformations [41]. The effects of the loss of this protein on tumor development are compatible with its function as a tumor supressor, as this molecule can inhibit cell proliferation and also induces apoptosis [47]. However, GPC3 overexpression can act as an oncogene in some tumors, such as hepatocellular carcinomas [48].
Besides isoforms −1 and −3, 80 % of non-metastatic tumors tested here also displayed a decrease in the transcript levels of glypican-6, a percentage that was halved in CRCs with lymph node metastases. Unlike the other isoforms, relatively little is known concerning the expression or functional roles of glypican-6 in tumors. Reduced expression or loss of function of this isoform has been described in retinoblastoma and autosomal-recessive omodysplasia [49, 50]; however, breast carcinoma invasion has been reported to be promoted through induction of glypican-6 by the transcriptional factor NFAT (nuclear factor of activated T-cells) using non-canonical Wnt5a signaling [51].
In addition to glypicans and syndecans, in this study we also analyzed the expression levels of betaglycan and CD44v3. These molecules are part time membrane HSPGs, that is, they exist either with or without HS chains [24]. CD44 comprises a family of heterogeneous integral membrane PGs derived from a single gene. The HS attachment site is located on exon 8 (CD44v3) [52], and thus we designed primers to specifically detect this isoform. CD44v3 did not show statistically significant differences in any type of CRCs; this data differs from previous studies where the expression of this protein in colon tumors was found to be related to more advanced pathological stage and poorer prognosis [53]; however, further detailed observation of our analysis showed that for 60 % of patients with metastatic tumors, this protein was indeed overexpressed, the scattering of the data having lead to the negative results in the initial statistical analysis. This phenomena was not observed in tumors lacking lymph node metastases, thereby supporting the results obtained in previous studies, which suggests a role for CD44v3 in invasion and metastasis by CRC cells [53].
The other part time HSPG analyzed, betaglycan, appeared underexpressed around 8 fold in non-metastatic CRCs, and about half of the patients with metastatic tumors exhibited under-expression of the protein, a difference that was not statistically significant in this case. Betaglycan is a ubiquitously expressed membrane-bound TGF-β superfamily coreceptor, also known as type III TGF-β receptor, which acts to regulate the cellular actions of TGF-β and inhibin [54]. The expression of betaglycan in tumor cells appears to play an important role in the progression of cancer, and reduced expression of this PG has been associated with advanced stage in different types of cancers [55]. In several neoplastic cells the loss of betaglycan facilitates the epithelial–mesenchymal transition, and a suppressor role for betaglycan in epithelial carcinogenesis has been proposed [55]. However, a correlation between high levels of this PG and invasiveness has been described in breast cancer cell lines [56], and increased expression has been seen in high-grade non-Hodgkin’s lymphomas and B-cell chonic lymphocytic leukemia [57, 58], suggesting a promoting role in these cases.
Two of the three extracellular matrix HSPGs examined here showed significant downregulation in tumoral samples: perlecan and collagen XVIII. Perlecan expression was downregulated at both the mRNA and protein level in tumors, independent of the presence or absence of lymph node metatases. Perlecan is a critical regulator of growth factor-mediated signaling and angiogenesis, and is fundamental for the maintenance of basement membrane homeostasis [59], suggesting that its alteration could play an important role in CRC progression. Although expression of perlecan is enhanced in a number of tumor types, in some other cases, such as lung carcinoma and hepatocellular carcinoma cells, its levels are undetectable and in these cases, it has been suggested that the lack of perlecan may favor the diffusion of growth factors, leading to tumor growth and metastasis [60].
Collagen XVIII transcription levels were also downregulated in CRCs, both metastatic and non-metastatic. Immunohistochemical analysis of the protein revealed that its expression was mainly at arteriolar vessels in the lamina propria in healthy tissues, while in both tumor types its expression could not be detected, although they displayed positive immunostaining using anti-CD34 antibody. CD34 is a cell surface glycoprotein widely used as a marker of vascular endothelial cells [61]. Collagen XVIII expression is widespread throughout vascular basement membranes, and can negatively modulate angiogenic processes by mediating interactions between endothelial cells and underlying extracellular matrix components [62]. The expression of this HSPG has been studied in various malignances and found to vary between different cancer types; increasing in ovarian or pancreatic cancer, while diminishing in liver and oral cancer [31].
Serglycin is the only characterized PG which is located intracellularly, although it has also been documented as a secretory product that may appear incorporated into the ECM or associated with the surface of target cells [25]. In this work, transcript levels of this gene were significantly reduced, both in metastatic and non-metastatic tumors, albeit more intense in the latter. Serglycin is mainly found in hematopoietic and endothelial cells, although some reports suggest it is present in other cell types, such as pancreatic acinar or smooth muscle cells [25, 63]. Nevertheless, in all cell types, the principal GAG chains are CS, except in mast cells where the covalently attached GAG can be CS type E or heparin, depending on their origin [25, 63]. In this study, we performed immunohistochemical analyses using the antibody CD117 to detect mast cells in CRCs. The results showed a drastic reduction in the population of mast cells in tumors compared to non-tumor colon mucosa, a fact which may explain, at least in part, the observed decrease in protein expression. A small number of studies have described alterations in the expression of serglycin in non hematological tumors, such as increased expression in patients with hepatocellular and nasopharingeal carcinoma [64, 65], where it has been related to unfavorable prognosis. Also, its elevated expression in aggressive breast cancer cells has also been reported [66].
The tissue-specific expression of individual HSPGs will determine when and where HS chains are expressed [13]. However, it should be taken into account that some HSPGs can be hybrid molecules, carrying both HS and CS side chains [67]. To generate the GAG chains the regulated expression and action of multiple enzymes, mainly GTs, (which are found in the lumen of the Golgi apparatus) are required [13]. Both HS and CS chain biosynthesis begins with the formation of a tetrasaccharide linkage region that comprises xylose-galactose-galactose-GlcA. Our results revealed that transcript levels of the different enzymes involved in the transfer and modification of the xylose residue display different alterations in CRCs. First, XYLT1 and XYLT2 appeared downregulated in non-metastatic patients alone; these genes encode two different xylosyltransferases (XylT1 and XylT2) that transfer a xylose residue from UDP-xylose to the hydroxyl group of a serine on the core protein with differing efficiency and displaying different expression patterns [68]. Modulation of the expression of xylosyltransferases has been described as regulator of GAG-synthesis in rheumatoid conditions [69]. Second, the transcription of FAM20B, which encodes xylose kinase 1 that catalyzes a transient phosphorilation of the Xyl residue involved in the control of GAG biosynthesis [70], also decreased significantly, though only in non-metastatic tumors. In relation to the other enzymes involved in the synthesis of the tetrasaccharide linker, only B3GAT1, which encodes one of the three isoforms of GlcA-transferase I responsible for the transfer of the GlcA residue [28], appeared downregulated in both types of tumors, although expression was more intense in non-metastatic CRCs.
At this point, the process of GAG synthesis follows one of two divergent paths, depending on whether the addition of a GlcNAc or GalNAc residue takes place, which leads to the synthesis of HS or CS/DS respectively. EXTL2 and EXTL3 encode enzymes that possess GlcA-transferase I activity, and thus direct the pathway toward the synthesis of HS, although it has been described that EXTL2 transfer of a GlcNAc residue to a linkage region previously phosphorylated by xylose kinase 1 terminates chain elongation, suggesting that EXTL2 controls HS biosynthesis [70]. The ensuing polymerization of the chain involves the consecutive addition of alternating GlcA and GlcNAc residues, mediated by the action of two enzymes encoded by the genes EXT1 and EXT2. At the level of transcription, the only change found in this set of enzymes was a down-regulation of EXT1 in non-metastatic CRCs. EXT1 and EXT2 are tumor suppressors, associated with hereditary multiple exostoses, characterized by the development of benign skeletal tumors in patients [14]. Although in this study EXTL3 did not show any significant alterations, previous reports have described its down-regulation in CRCs associated with mucinous differentiation and caused by promoter methylation [21].
The addition of GalNA instead of GlcNAc to the linker directs the pathway towards the biosynthesis of CS. In this case, chain extension takes place through the sequential addition of alternative GlcA and GalNAc residues. Five genes, CSGALNACT1, CSGALNACT2, CHSY1, CHPF and CHSY3 encode the GTs involved in this process [71]. The expression of all except CHPF were found to decrease in the group of non-metastatic tumors analyzed, while in metastatic CRCs, CHPF alone appeared overexpressed. In previous studies of CRCs, a number of changes have been described: a gradual increase in chondroitin polymerizing factor with advancing cancer stage; an increase in CHSY1 expression in normal tissue adjacent to benign tumors compared to tumoral tissues and high expression levels during the early stages; and low expression levels of CHSY3 in both normal and tumor specimens, although it must be stressed that these studies involved colorectal cancers from different locations and not only right sided CRCs [72].
Taken together, the alterations observed in our study in the transcription of this category of genes suggests variation in the GAG chains, particularly in non-metastatic CRCs, while few changes were detected in metastatic tumors. This fact is particularly noticeable in the enzymes involved in the polymerization of CS, which appear to be more affected than those involved in HS polymerization. The decrease of the transcription of some enzymes involved in the synthesis of the tetrasaccharide linker, particularly FAM20B, could also be related. Variations in the level of GAGs for various tumor types have been described elsewhere, both increases and decreases. With respect to CRCs, a decrease in GAG production has been described, as well as reductions in levels of HS [19, 73]. Besides, some studies describe a reduction in CS levels in the neoplastic colon [73], while other reports describe an increase, albeit predominantly related to CS in the intercellular matrix produced by the tissue surrounding the tumor [74].
The fine structure of HS ultimately depends on the control of polymerization of the chains, but the expression and action of multiple sulfotransferases and one epimerase is also essential. As the polymer forms, the first reaction involved in polymer modification involves removal of acetyl groups from GlcNAc residues, followed by sulfation of the amino group which is catalyzed by four different isoforms of N-deacetylase/N-sulfotransferases [13]. NDST1 and NDST2 show broadly overlapping tissue distribution [28], while NDST3 and NDST4 are more restricted and are expressed principally during embryonic development [75]. Our analysis of colon CRCs was able to detect the existence of transcription of isoforms 1 and 2 in both tumoral and normal samples, but not of isoforms 3 and 4. NDST1 and NDST2 appeared significantly underexpressed, but only in non-metastatic tumors. These alterations are likely to involve important consequences for the structure of HS chains, since the reaction catalysed by this family of enzymes is essential for the creating of sulfated S-domains. Lower N-sulfation levels in CRCs involving modifications in both the S domains and their flanking mixed domains have been reported [20, 76], although to our knowledge there are no other studies that compare levels between tumors with or without metastasis.
Further modifications of the HS chains include the epimerization of GlcA into IdoA, catalyzed by the action of the enzyme C5-GlcA epimerase and the addition of O-sulfate groups at C2 of uronic acid and to C6 and C3 of the glucosamine residues [13, 14]. 2-O-sulfation is closely associated with epimerization, and no alteration in the transcription levels of the genes involved, GLCE and HS2ST1, could be detected in CRCs in this study. O-sulfation at C6 is carried out by enzymes encoded by the genes HS6ST1, HS6ST2 and HS6ST3, each of which differs in their substrate specificities and tissue expression [77]. HS6ST1 transcription here appeared decreased in non-metastatic tumors only; the effect of this downregulation on the sulfation at C6 should be relevant, since HS6ST1 is the principal isoform, HS3ST3 levels being found to be very low and to exhibit considerable variation between patients, while HS6ST2 transcripts were undetected. A reduction in 2-O-sulfation and an increase in 6-O-sulfation in colon carcinoma cells has been described, albeit related to HS from colon adenoma cells [77], and other studies have, in addition, detected the undersulfation of HS molecules in tumors [19].
The final family of enzymes involved in the biosynthesis of HS are the 3-O-sulfotransferases, which in fact form the largest group, comprising seven different members, and which are implicated in the formation of specific HS motifs that interact in a selective manner with specific protein ligands. Although 3-O-sulfation is a relatively rare modification, and to date very few proteins have been described that are influenced by it [78], several studies have described their alteration in different tumors. Such alterations may be upregulations, as in the case of HS3ST1 and HS3ST3A1 in, respectively, hepatocellular cancer and glioblastoma [79, 80], or subexpressions in, for example, HS3ST2 in breast, colon, lung and pancreatic cancers [22], HS3ST1 and HS3ST3A1 in chondrosarcoma cells and HS3ST4, HS3ST5 and HS3ST6 in invasive breast ductal carcinomas [81, 82]. In the present study, isoforms HS3ST3B1 and HS3ST5 appeared underexpressed in non-metastatic CRCs. Notwithstanding the implications of 3-O-sulfation decrease in tumors being as yet known, it has been suggested that certain patterns of 3-O-sulfation could impart cancerous phenotypic changes [30].
Of the different sulfate groups present on the HS chains, 6S modification is the only known sulfate moiety known to be post-synthetically edited from the chain, implying that it has special regulatory importance. Two endosulfatases that are secreted from the Golgi and localized on the cell surface, SULF1 and SULF2, selectively remove 6-O-sulfate groups on GlcN residues [27]. Alterations in the expression of these genes have been reported in various tumors, involving either up or downregulations depending on the neoplasia ivolved [17]. However, transcript levels of none of these genes were found to be altered in any type of CRCs.
As indicated earlier, some HSPGs are hybrid molecules, carrying both HS and CS side chains [67]. The alterations observed in this study in transcriptions of GTs largely point towards changes in the CS chains. In addition, changes in CSPGs associated with CRCs, for example versican or decorin, have been described [83]. CS repeating disaccharide building units can be modified by epimerization of GlcA residues and by sulfate groups at C2 of uronic acids and at C4 and/or C6 of GalNAc residues with various combinations [71]. In CRCs, the expressions of the genes encoding the enzymes that catalyze the different reactions which generate CS structures are altered, more than 60 % of them appearing underexpressed. Interestingly, and in contrast to the other groups of genes cited previously, the observed alterations were very similar in both tumor types, irrespective of presence or absence of lymph node metastasis. Furthermore, the observed alterations in expression appear to affect all modifications, except sulfation at C6 of GalNAc, including epimerization and C2 sulfation of uronic acid. In the case of sulfation at C4 of GalNAc, 3 of the 4 genes involved appeared downregulated, the only one showing no significant difference, CHST12, being the one which displayed the lowest levels of transcription. In a previous study, the transcriptions of two of these genes, CHST14 and CHST3, were analyzed and it was found that the expression of CHST3 did not differ, while CHST14 decreased as the stage of the cancer progressed [76] and, although the samples used were from tumors from different locations, these data support, at least in part, the results presented here. Disaccharide composition analysis carried out in other studies have shown an increase in 6-sulfated and non-sulfated disaccharides, while CS levels were not related with the metastatic potential of a tumor [76, 84]. Interestingly, a recent study described a decrease in the levels of CS in CRC, and this was accompanied by increases in the levels of 6S and 4S6S (CS-E) disaccharide units compared to normal tissue [73]. However, in our study we were not able to detect any transcription of CHST15, the gene which encodes the enzyme responsible for transfering sulfate to the C-6 of an already 4-O sulfated GalNAc residue.
HPSE is an endo-β-D-glucuronidase that cleaves specific β-D-glucouronosyl-N-acetyl-glucosaminyl linkages, yielding HS fragments of appreciable size which may contain biologically active HS domains [29]. HPSE expression is induced in all the principal types of human cancer and is often associated with reduced survival, increased tumor metastasis and higher density of microvessels [29]. Analysis of HPSE transcript levels in non-metastatic CRCs in this study showed an apparent decrease in 70 % of the patients analyzed, although the dispersion of values in healthy tissues was considerably higher than in tumoral tissues. In contrast, metastatic tumors showed greater variability in their transcription levels, varying from under to overexpression depending on the individual patient concerned. Conversely, other studies have found HPSE to be expressed at early stages of neoplasia, although it was not detected in the adjacent normal colon epithelium, and its expression gradually increased as the cells progressed from well differentiated to poorly differentiated colon carcinoma [85, 86]. In this study, we were able to detect the expression of HPSE in the adjacent normal-looking colon epithelium, a fact that could be explained by the existence of inflammation [87], or as due to the dynamic constant renewal of the colon mucosa, this latter being a phenomena which has been related to the unmethylated state of the HPSE promoter in the normal colon [88].
HPSE2 is a homologue of heparanase that lacks HS-degrading activity, although it is able to interact with HS with a high affinity, and is capable of modulating HPSE enzymatic activity and signaling properties, to the extent that an anti-metastatic character has been proposed for it [29, 89]. The downregulation of this protein has been described in some neoplasms [82]. In CRCs, transcripts for this gene were detected in 70 % of patients with non-metastatic tumors, which was reduced to 30 % in those cases showing lymph node metastasis, although the dispersion of the data meant that statistically significance was not reached.