Μεταβολές των γλυκοζαμινογλυκανών και πρωτεογλυκανών σε ασθενείς με επιθηλιακό καρκίνωμα του λάρυγγα

Abstract

Larynx is a part of the respiratory system. It is strategically located so that it plays a significant role in the respiratory and vocal physiology. It comprises mostly from two kinds of tissue: epithelium and connective tissue. The main connective tissue present in the larynx is cartilage. Cartilage is a highly specialized connective tissue which is particularly rich in extracellular matrix (ECM) up to 90% of the dry weight of the tissue. Studies have revealed the degradation of cartilage during cancer and the loss of its ability to act as a barrier for the migration and dispersion of cancer cells. Studies of our laboratory have revealed the structural and chemical modifications occurring in ECM molecules during laryngeal cancer. More specifically, a significant loss of the predominant PG of the cartilage, aggrecan, was observed and an increase in the PGs versican and decorin in the tumor associated stroma. Structural and chemical changes were also observed in the GAG chains which are covalently attached to the PGs.The propose of this study was to extend our previous work and examine in more details the changes of the above listed molecules occurred during malignancy and understand their role in laryngeal cancer. Firstly we investigated in details the GAGs on both fine chemical and structural levels in the separated cartilaginous and epithelial laryngeal tissues at different stages of laryngeal cancer. Overall chemical analyses of the basic GAG components, i.e. uronic acid and hexosamine, in the laryngeal tissues indicated stage-related countertrend alterations. These alterations implied significant and different macromolecular remodeling between cartilaginous and epithelial laryngeal tissues during the progression of laryngeal cancer. Enzymatic treatment with chondroitinase ABC and heparinases, which degrade all GAG types apart from KS, indicated that the CS/DS PGs and HA underwent the major quantitative changes. In particular, the amounts of CS and KS, as intrinsic constituents of the large cartilage-derived PG aggrecan, in the cartilaginous tissues were dramatically decreased with the progression of laryngeal cancer revealing the dramatic loss of aggrecan that in turn leads to the degeneration and destruction of laryngeal cartilages. On the other hand, a remarkable increase in the amounts of CS and DS in the non-cartilaginous tissues was observed as expected, since these GAG moieties are intrinsic components of versican and decorin, two CS/DSPGs with contradictory roles in the pathophysiology of cancer. The increased presence of CS/DS has been reported in many studies on tumor progression. In laryngeal cancer, on the other hand, an inverse distribution of CS/DS was observed since the larger increase was detected for the amounts of DS rather than those of CS. This finding may be of particular interest for patients with laryngeal cancer since it has been reported that decorin bearing DS side chains was 20-fold more effective that decorin bearing CS side chains in inhibiting the migration of bone tumor cells, highlighting the importance of the post-translational modifications of PGs to their effects on cancer progression. On structural level, the composition of sulfated and non-sulfated disaccharides of the CS/DS chains was also significantly altered. The molar ratios of 4-sulfated/6-sulfated disaccharides in both cartilaginous and epithelial tissues showed a signifigant increase with the progression of laryngeal cancer. This finding revealed that in tumor-associated CS/DS chains of both tissue-types 4-sulfated disaccharides predominated. This result is very interesting since in various gastric adenocarcinomas the 6-sulfated disaccharides predominated. It is possible that the different sulfation pattern is related with the malignant phenotype of the various cancer types. Moreover, the molar ratios of non-sulfated/sulfated disaccharides in both cartilaginous and epithelial tissues showed also a significant increase, implying the presence of a significant proportion of non-sulfated disaccharide units in the tumor-associated CS/DS chains.The CS/DS and KS are modified by variable sulfation and in the case of DS, by epimerization of some of the glucuronic to iduronic resulting in more flexible polysaccharide structures. These modifications provide for multiple structural variations that in turn provide a high degree of specificity in function. In general, the functions of matrix HA and the matrix PGs relate to interactions with other matrix substances such as collagen to provide scaffolding and shape. Their variable structures provide specificity of function for particular tissues, and specific aspects of interaction with other matrix molecules.Hyaluronan in its native state is a high molecular mass polymer, but during tissue damage, inflammantory diseases, and aggressive forms of tumors, it occurs in a variety of sizes, that have a vast array of properties. Several studies have suggested that high and low molecular weight species of HA exhibit different biological effects on cells and in tissues, some of which appear to be contradictory. The very large HA polymers are extracellular, space-occupying and anti-angiogenic and have an array of regulatory and structural functions. The small polymer fragments are angiogenic, inflammatory, and immuno-stimulatory. These properties of HA molecules provide cancer cells with a microenvironment that facilitates their invasion and migration and promotes their aggressiveness.In the present study, considerable changes in the amounts and the molecular mass of HA molecules were detected between the cartilaginous and the epithelial tissues of human larynx as well as between the normal and the cancerous laryngeal specimens. In HNL, the LCT was much richer in HA content, about 10-fold, compared to LET. In addition, the vast majority of the HA molecules in LCT was found to be of high molecular mass, which excluded by the column, compared to HA molecules in LET. These findings are in agreement with the common phenomenon in which the HA content and its molecular mass depend on both tissue source and on physiological conditions. In contrast, the HA content of the LCT in laryngeal cancer dramatically decreased, about 20-fold, while in LET the HA amount significantly increased, about 10-fold, being the predominant GAG type in this tissue. On molecular-size level, in both tissue-types a significant decrease of high molecular size HA molecules was observed. Of particular interest was the great increase of the amount of low molecular mass HA in LET which ranged from 330 to 890 kDa. The way of how these low molecular mass HA were generated is not known, nor it is established whether the enzymes of HA synthesis and degradation are involved in maintaining proper polymer sizes and concentration. However, it is possible that the accumulation of low molecular-size HA observed in the cancerous non-cartilaginous laryngeal tissues, especially in stage IV of laryngeal cancer, is most likely due to overexpression of Hyal-1, as reported in previous study. Moreover the case of an increased activity of Hyaluronan Synthase 3 (HAS3) should not be excluded, since it is known that this enzyme is the most active isoform and drives the synthesis of short HA chains (100-1000 kDa), where its expression seems to be activated in order to produce large amounts of low molecular weight HA. In addition, it is known that HAS3 appears to favour the malignant phenotype in many types of malignancies.As previously referred cartilage PGs go through quantitative and qualitative alterations during laryngeal cancer. Therefore, another purpose of the present study was to confirm aggrecan, versican and decorin expression both in normal human larynx and LSCC at the mRNA level and to examine whether the alterations in these cartilage components occurred at the expression level. In an initial set of experiments, we examined the expression of aggrecan, versican and decorin in normal and cancerous (stage IV) specimens. The results indicated a significant but disproportionate increase of expression of both versican and decorin in cancerous specimens compared to normal ones, whereas aggrecan expression totally ceased in cancerous specimens, indicating either the total absence of chondrocytes from the tissue or their inability to express aggrecan under such conditions. The work then focused on the expression of versican and decorin in laryngeal cancer of different stages. The results indicated that in normal tissues the expression levels of decorin and its accumulation were very much higher than those of versican, approximately 27-fold. In comparison with the normal tissue, the expression levels of decorin in the cancerous samples of stages II and III presented a stable increase in both stages of approximately two-fold with an additional increase in the stage IV of approximately three-fold. Correspondingly, the expression levels of versican in the cancerous samples presented a characteristic stage-related increase, i.e., 50-, 90- and 140-fold for stage II, III and IV, respectively. The overexpression of versican in comparison to decorin led the former to predominate in cancerous specimens of stages III and IV. Moreover, a simultaneous stage-related increase of accumulation of versican was also observed On a quantitative level, the findings of this study indicated that the expression and accumulation of both versican and decorin in advanced laryngeal cancer (stage IV) were to about the same extent with light predominance of versican. LSCC amounts for approximately one-fourth of all head and neck SCCs. More than one-half of LSCCs are present as local disease without metastasis, one fourth are present as local disease with regional metastasis, and approximately 15% are first seen at an advanced stage with or without distant metastasis. The mild aggressive potential of LSCC, apart from the extensive presence of laryngeal cartilages, which act as physiological barriers to the spread of carcinoma cells, could be explained, at least in part, by both positive and negative roles that have been proposed for versican and decorin. The dramatic decrease of the main cartilage PG aggrecan, in laryngeal cancer, is probably due to the action of catabolic enzymes. Enzymes that are consider responsible for aggrecan catabolism, and for that are called aggrecanases, are molecules from the ADAMTS family of metalloproteinases. Seven members of this family are identified as aggrecanases. In the present work our main interest was the investigation of the expression of the three aggrecanases, ADAMTS-1, -4 and -5 in mRNA and protein level. Expression was examined in healthy larynx and LSCC by using RT-PCR and western blotting. All three aggrecanases were found to be expressed in healthy larynx, with ADAMTS-1 and ADAMTS-4 being expressed 3 and 6.5 times more than ADAMTS-5. In LSCC, characteristic alterations occurred. The expression of ADAMTS-1 in the primary cancer stages (II and III) was in lower levels than the healthy tissue while in stage IV it increased and reached the levels of HNL. ADAMTS-4 in LSCC had the same expression levels as HNL with exception of cancer stage III in which it presented expression levels twice as much of the HNL. ADAMTS-5 presented stage related increase in the cancer stages II and III while it dramatically decreased in stage IV. In AANC tissues its expression was the same in all cancer stages but it was four times as much as that of the HNL.In order to examine the expression of aggrecanases in protein level, the tissues have been subjected to sequential extraction. ADAMTS-1 was identified in cartilage and epithelium as a 34 kDa band. Knowing that the antibody used is against the C-terminal region of the enzyme we can conclude that the 34kDa band is a result of C-terminal processing. In LSCC and AANC the same results have been obtained and in addition one 87 kDa band (full length active molecule) was identified which was more intense at primary cancer stages. ADAMTS-4 in HNL both in cartilage and epithelium was identified as a 68kDa band that corresponded to the full-length active molecule. In LSCC and AANC epithelium 100 kDa and 75 kDa bands were identified which corresponded to zymogene and full length molecule respectively. Finally 50 kDa bands were track but they were not identified and they should be more investigated. ADAMTS-5 was identified in HNL as a 73kDa band (full length active molecule) and 50kDa and 40 kDa band (results of C-terminal processing). In LSCC the same bands were track but in AANC the band of 73 kDa is absent. The results suggested that the removal of aggrecan from laryngeal cartilage occurred due to both the decrease of aggrecan expression and the increased production of aggrecanases. In addition, aggrecanases might be responsible for the observed degradation of versican, which became major proteoglycan in LSCC. However, the increased biosynthesis of versican overcame this degradation. ADAMTS-5 seemed to be the enzyme responsible for aggrecan removal in LSCC and this might be a target for medical treatment in early stages of cancer.

Ο λάρυγγας αποτελεί μέρος του αναπνευστικού συστήματος και παίζει σημαντικό ρόλο στις λειτουργίες της αναπνοής και της φώνησης. Αποτελείται από δύο είδη ιστών τον επιθηλιακό και τον συνδετικό. Ο τελευταίος παρουσιάζεται κυρίως υπό τη μορφή χόνδρων, οι οποίοι αποτελούν το στηρικτικό σκελετό του λάρυγγα και είναι πλούσιοι σε εξωκυττάρια ουσία. Έχει αναφερθεί ότι σε καρκινικούς ιστούς οι αλλαγές στη δομική σύσταση της εξωκυττάριας ουσίας βοηθούν στην αποσύνδεση και μετανάστευση των καρκινικών κυττάρων καθώς και στην είσοδό τους στα αιμοφόρα και λεμφικά αγγεία. Σε προηγούμενες μελέτες που πραγματοποιήθηκαν στο εργαστήριό μας αναφέρθηκαν τόσο ποσοτικές όσο και δομικές αλλαγές σε μόρια του ECM σε καρκινικούς ιστούς λάρυγγα, όπως οι PGs aggrecan, versican, decorin αλλά και των ομοιοπολικά συνδεδεμένων σε αυτές GAG αλυσίδων. Λαμβάνοντας υπόψη τα παραπάνω κρίθηκε σκόπιμο όπως η παρούσα διατριβή προχωρήσει σε περαιτέρω και σε βάθος έρευνα των προαναφερθέντων μορίων καθώς και άλλων συσχετιζόμενων με αυτά για να κατανοηθεί περισσότερο ο ρόλος τους στο καρκίνο του λάρυγγα. Έτσι ερευνήθηκαν σε λεπτομέρεια οι GAGs τόσο σε χημικό όσο και δομικό επίπεδο στο χόνδρο και επιθήλιο του λάρυγγα σε διάφορα καρκινικά στάδια. Τα αποτελέσματα έδειξαν σημαντική μείωση όλων των τύπων GAGs στο καρκινικό χόνδρο σε σχέση με τους υγιείς, σε αντίθεση με τους επιθηλιακούς ιστούς που παρουσίασαν σημαντική αύξηση στο καρκίνο. Όσον αφορά στη χημική τους δομή, οι μοριακοί λόγοι των 4S/6S και 0S/totalS δισακχαριτών στον καρκινικό χόνδρο και στο επιθήλιο παρουσίασαν αύξηση. Σε επίπεδο μοριακού μεγέθους, στο καρκινικό λάρυγγα, η χρωματογραφική συμπεριφορά των θειωμένων GAG αλυσίδων και στα δύο είδη ιστών έδειξε μόρια μικρότερου Mr με μεγαλύτερη πολυδιασπορά σε σχέση με τα υγιή. Επιπρόσθετα και στους δύο ιστούς παρατηρήθηκε σημαντική μείωση του HA υψηλού μοριακού μεγέθους. Ακολούθως ερευνήθηκε η έκφραση των PGs aggrecan, versican και decorin σε επίπεδο mRNA σε υγιείς και καρκινικούς ιστούς λάρυγγα ανθρώπου. Παρατηρήθηκε σημαντική και ταυτόχρονα δυσανάλογη αύξηση των versican και decorin στα καρκινικά δείγματα σε σχέση με τα υγιή, ενώ η έκφραση της aggrecan δεν ανιχνεύθηκε στα καρκινικά δείγματα. Τα επίπεδα έκφρασης της decorin στους υγιείς ιστούς ήταν υψηλότερα από αυτά της versican σχεδόν 27 φορές. Σε σχέση με τα υγιή δείγματα τα επίπεδα έκφρασης της decorin στα καρκινικά στάδια II και III παρουσίασαν σταθερή αύξηση (διπλασιασμός) με επιπρόσθετη αύξηση στο στάδιο IV (τριπλασιασμός). Αντίστοιχα τα επίπεδα έκφρασης της versican στα καρκινικά δείγματα παρουσίασαν χαρακτηριστική σταδιοεξαρτόμενη αύξηση, 50-, 90- και 140-φορές στα στάδια II, III και IV, αντίστοιχα. Η υπερέκφραση της versican σε σχέση με την decorin οδήγησε την πρώτη να υπερισχύει στα καρκινικά στάδια III και IV. Οι μεταβολές αυτές παρατηρήθηκαν τόσο στα καρκινικά δείγματα, όσο και στα γειτονικά μακροσκοπικά φισιολογικά δείγματα που λήφθηκαν από τους ίδιους ασθενείς. Στη παρούσα διατριβή μελετήθηκε επίσης η έκφραση των τριών πιο δραστικών αγγρικανασών, ADAMTS-1, ADAMTS-4 και ADAMTS-5 τόσο σε επίπεδο πρωτεΐνης όσο και mRNA. Μετά από RT-PCR ανάλυση παρατηρήθηκε ότι στους υγιείς ιστούς εκφράζονταν και οι τρεις, με τις ADAMTS-1 και ADAMTS-4 να υπερέχουν της ADAMTS-5 στο επίπεδο των 3 και 6.5 φορών αντίστοιχα. Στους καρκινικούς ιστούς η έκφραση των ADAMTS-1 και ADAMTS-4 δεν έδειξε κάποια σημαντική αλλαγή σε σχέση με τα υγιή. Η ADAMTS-5 είχε την πιο σημαντική μεταβολή με σταδιακή αύξηση στα καρκινικά στάδια II και III σε σχέση με τους υγιείς ενώ στο στάδιο IV μειωνόταν σημαντικά. Στα μακροσκοπικά φυσιολογικά δείγματα σε όλα τα καρκινικά στάδια έδειξε ίδια έκφραση η οποία ήταν τετραπλάσια εκείνης των υγιών. Σε επίπεδο πρωτεΐνης, η ADAMTS-1 στους υγιείς ιστούς ανιχνεύθηκε τόσο στο χόνδρο όσο και στο επιθήλιο στη μορφή μίας ζώνης των 34kDa. Δεδομένου ότι το αντίσωμα που χρησιμοποιήθηκε ανιχνεύει το καρβοξυτελικό άκρο του ενζύμου, το μόριο των 34 kDa προφανώς αντιπροσωπεύει το καρβοξυτελικά απομακρυσμένο μέρος της περιοχής του βραχίονα. Τα δείγματα των ασθενών έδειξαν παρόμοια εικόνα μεταξύ τους, αλλά σχετικά διαφορετική από τους υγιείς. Ανιχνευόταν στο PBS εκχύλισμα η ζώνη των 34kDa και στο 4M GdnHCl η ίδια ζώνη, αλλά και δεύτερη στην περιοχή των 87kDa η οποία αντιστοιχεί σε ολόκληρο το ενεργό μόριο και η οποία ήταν πιο έντονη στα πρώτα καρκινικά στάδια..Η ΑDAMTS-4 στους υγιείς ιστούς ανιχνευόταν τόσο στο χόνδρο όσο και στο επιθήλιο με τη μορφή των 68 kDa που αντιστοιχεί στο πλήρως ώριμο ένζυμο. Στα καρκινικά και στα μακροσκοπικά φυσιολογικά δείγματα έδειξε παρόμοια συμπεριφορά με αυτή των υγιών με τη διαφορά ότι στο εκχύλισμα του 1Μ GndHCl από επιθηλιακό ιστό εμφανίζονταν οι μορφές των 100 kDa και 75 kDa που αντιστοιχούν στο ζυμογόνο και στο ολόκληρο ενεργό μόριο αντίστοιχα. Τέλος ανιχνεύθηκαν ζώνες των 50 kDa οι οποίες χρήζουν περαιτέρω διερεύνησης αφού δεν έχουν αναφερθεί ξανά και πιθανό να είναι αποτέλεσμα δράσης άλλων ενζύμων. Η ADAMTS-5 στους υγιείς ιστούς ανιχνεύτηκε και στα δύο είδη ιστών στη μορφή των 73 kDa (ολόκληρο ενεργό ένζυμο), 50 kDa και 40 kDa. Στα καρκινικά δείγματα τόσο στο χόνδρο όσο και στο επιθήλιο ανιχνεύθηκαν οι ίδιες ζώνες όπως αυτές των υγιών, στο μεν χόνδρο στο εκχύλισμα με 4Μ GdnHCl, ενώ στο επιθήλιο σε εκείνο του PBS. Αξιοσημείωτο είναι ότι στους μακροσκοπικά φυσιολογικούς ιστούς ενώ ανιχνεύονται και πάλι οι μορφές των 50 και 40 απουσιάζει η ζώνη των 73 kDa. Συμπερασματικά βλέπουμε πως η αποδιάταξη του ECM φαίνεται να είναι βασικός παράγοντας στην ανάπτυξη του καρκίνου. Παρατηρήθηκαν σημαντικές αλλαγές στην δομή, συγκέντρωση αλλά και έκφραση πολλών κύριων συστατικών του όπως γλυκοζαμινογλυκανών, πρωτεογλυκανών αλλά και των υπεύθυνων για τον καταβολισμό τους ενζύμων.