Em diversos casos, o câncer bucal é precedido por lesões pré-malignas, como por exemplo, a leucoplasia, sendo que o tabaco é um fator de risco. No entanto, apesar deste potencial negativo, há estudos limitados em fumantes e não fumantes sobre o desenvolvimento de displasia para carcinoma. Sabe-se que o principal componente do factor de transcrição AP-1, a proteína c-Jun e a sua forma fosforilada (p-c-Jun), participam do ciclo celular e que sua inibição compromete a proliferação celular. A proteína p27 tem sido demonstrada como inibidora de quinase, e sabe-se que tem sua expressão diminuída durante a carcinogênese. A expressão diminuída de p27 tem sido relacionada a um pior prognóstico em carcinoma epidermóide. O objetivo deste estudo foi verificar a expressão das proteínas c-Jun, p-c-Jun e p27, em lesões potencialmente malignas diagnosticadas clinicamente como leucoplasia de pacientes fumantes e não fumantes. Foram selecionados 73 casos diagnosticados clinicamente como leucoplasias, os quais foram divididos nos seguintes grupos: leucoplasia com e sem displasia epitelial, e entre fumantes e não fumantes (perfazendo 4 grupos). Cortes histológicos das lesões foram classificados segundo o sistema de graduação binário, e subsequentemente submetidos à técnica imunohistoquímica da estreptoavidina-biotina. Avaliou-se também a associação da proliferação celular pela c-Jun, p-c-Jun e p27 com o tabagismo, presença e ausência de displasia, localização e gênero. O presente estudo verificou que ocorrência de displasia epitelial em baixo e alto risco não teve associação com o hábito de fumar do paciente (p= 0,5430). Avaliando apenas a imunorreatividade das proteínas c-Jun, p-c-Jun e p27 individualmente observouse que a expressão destas não teve associação com a condição fumante do paciente (p> 0,05). Porém, ao compararmos a imunomarcação de c-Jun e p-c-Jun entre si, no total da amostra (p=0,0448) e somente para displasia em fumantes (p=0,0165), nota-se significativamente menor expressão de p-c-Jun. Comparando c-Jun e p27 nas displasias em não fumantes, houve significativamente maior expressão de c-Jun e menor expressão de p27 (p=0,0079). A análise do Teste binomial de duas proporções revelou que as regiões de língua e assoalho bucal estavam associadas à ocorrência de lesões displásicas quando comparadas a lesões não displásicas (p<0,0001). Concluiu-se que não houve correlação entre fumo e grau de displasia. A expressão das proteínas c-Jun, p-c-Jun e p27 foi independente da condição fumante do paciente. / In Several cases, the oral cancer is preceded by-malignant lesions potentially, such as leukoplakia, and that tobacco is a risk factor. However, despite this negative potential, there are limited studies in smokers and nonsmokers on the development of dysplasia to carcinoma. It is known that the main component of the transcription factor AP-1, c-Jun protein and its phosphorylated form (pc-Jun), participate in cell cycle inhibition and their committed cell proliferation. The p27 protein has been shown to inhibit kinase, and it is known that their expression is decreased during carcinogenesis. The decreased expression of p27 has been linked to poor prognosis in squamous cell carcinoma. The aim of this study was to evaluate the expression of proteins c-Jun, pc-Jun and p27 in potentially malignant lesions from smokers and non-smokers, diagnosed clinically as leukoplakiaa in smokers and nonsmokers. We selected 73 cases diagnosed clinically as leukoplakia, which were divided into the following groups: leukoplakia with and without dysplasia, and between smokers and nonsmokers (totaling 4 groups). Histological lesions were classified according to the binary grading system, and subsequently subjected to immunohistochemistry technique streptavidin-biotin. We also evaluated the association of cell proliferation of c-Jun, pc-Jun and p27 with smoking, presence and absence of dysplasia, location and gender. This study found that the occurrence of epithelial dysplasia in low-and high-risk had no association with the patient\'s smoking habit (p = 0,5430). When evaluating the immunoreactivity of the proteins c-Jun, p27 and pc-Jun alone it was observed that the expression of them was also independent of the condition of the patient smoking (p> 0,05). However, when comparing the immunostaining of c-Jun and pc-Jun together, for the total sample (p = 0,0448) and only for dysplasia in smokers (p = 0,0165), there was significantly lower expression of pc-Jun. Comparing p27 and c-Jun in dysplasias in nonsmokers, there was significantly higher expression of c-Jun and reduced expression of p27 (p = 0,0079). The analysis of two binomial proportions test revealed that the lesions in the tongue and floor of the mouth were more prone to be dysplastic (p <0.0001). It was concluded that there was no correlation between smoking and degree of dysplasia. The expression of c-Jun, p27 and pc-Jun proteins was independent of smoking habit of the patient. The anatomoclinical aspects combined with the expressions of c-Jun and p27 may assist the pathologist in directing the histopathological diagnosis.

Em diversos casos, o câncer bucal é precedido por lesões pré-malignas, como por exemplo, a leucoplasia, sendo que o tabaco é um fator de risco. No entanto, apesar deste potencial negativo, há estudos limitados em fumantes e não fumantes sobre o desenvolvimento de displasia para carcinoma. Sabe-se que o principal componente do factor de transcrição AP-1, a proteína c-Jun e a sua forma fosforilada (p-c-Jun), participam do ciclo celular e que sua inibição compromete a proliferação celular. A proteína p27 tem sido demonstrada como inibidora de quinase, e sabe-se que tem sua expressão diminuída durante a carcinogênese. A expressão diminuída de p27 tem sido relacionada a um pior prognóstico em carcinoma epidermóide. O objetivo deste estudo foi verificar a expressão das proteínas c-Jun, p-c-Jun e p27, em lesões potencialmente malignas diagnosticadas clinicamente como leucoplasia de pacientes fumantes e não fumantes. Foram selecionados 73 casos diagnosticados clinicamente como leucoplasias, os quais foram divididos nos seguintes grupos: leucoplasia com e sem displasia epitelial, e entre fumantes e não fumantes (perfazendo 4 grupos). Cortes histológicos das lesões foram classificados segundo o sistema de graduação binário, e subsequentemente submetidos à técnica imunohistoquímica da estreptoavidina-biotina. Avaliou-se também a associação da proliferação celular pela c-Jun, p-c-Jun e p27 com o tabagismo, presença e ausência de displasia, localização e gênero. O presente estudo verificou que ocorrência de displasia epitelial em baixo e alto risco não teve associação com o hábito de fumar do paciente (p= 0,5430). Avaliando apenas a imunorreatividade das proteínas c-Jun, p-c-Jun e p27 individualmente observouse que a expressão destas não teve associação com a condição fumante do paciente (p> 0,05). Porém, ao compararmos a imunomarcação de c-Jun e p-c-Jun entre si, no total da amostra (p=0,0448) e somente para displasia em fumantes (p=0,0165), nota-se significativamente menor expressão de p-c-Jun. Comparando c-Jun e p27 nas displasias em não fumantes, houve significativamente maior expressão de c-Jun e menor expressão de p27 (p=0,0079). A análise do Teste binomial de duas proporções revelou que as regiões de língua e assoalho bucal estavam associadas à ocorrência de lesões displásicas quando comparadas a lesões não displásicas (p<0,0001). Concluiu-se que não houve correlação entre fumo e grau de displasia. A expressão das proteínas c-Jun, p-c-Jun e p27 foi independente da condição fumante do paciente. / In Several cases, the oral cancer is preceded by-malignant lesions potentially, such as leukoplakia, and that tobacco is a risk factor. However, despite this negative potential, there are limited studies in smokers and nonsmokers on the development of dysplasia to carcinoma. It is known that the main component of the transcription factor AP-1, c-Jun protein and its phosphorylated form (pc-Jun), participate in cell cycle inhibition and their committed cell proliferation. The p27 protein has been shown to inhibit kinase, and it is known that their expression is decreased during carcinogenesis. The decreased expression of p27 has been linked to poor prognosis in squamous cell carcinoma. The aim of this study was to evaluate the expression of proteins c-Jun, pc-Jun and p27 in potentially malignant lesions from smokers and non-smokers, diagnosed clinically as leukoplakiaa in smokers and nonsmokers. We selected 73 cases diagnosed clinically as leukoplakia, which were divided into the following groups: leukoplakia with and without dysplasia, and between smokers and nonsmokers (totaling 4 groups). Histological lesions were classified according to the binary grading system, and subsequently subjected to immunohistochemistry technique streptavidin-biotin. We also evaluated the association of cell proliferation of c-Jun, pc-Jun and p27 with smoking, presence and absence of dysplasia, location and gender. This study found that the occurrence of epithelial dysplasia in low-and high-risk had no association with the patient\'s smoking habit (p = 0,5430). When evaluating the immunoreactivity of the proteins c-Jun, p27 and pc-Jun alone it was observed that the expression of them was also independent of the condition of the patient smoking (p> 0,05). However, when comparing the immunostaining of c-Jun and pc-Jun together, for the total sample (p = 0,0448) and only for dysplasia in smokers (p = 0,0165), there was significantly lower expression of pc-Jun. Comparing p27 and c-Jun in dysplasias in nonsmokers, there was significantly higher expression of c-Jun and reduced expression of p27 (p = 0,0079). The analysis of two binomial proportions test revealed that the lesions in the tongue and floor of the mouth were more prone to be dysplastic (p <0.0001). It was concluded that there was no correlation between smoking and degree of dysplasia. The expression of c-Jun, p27 and pc-Jun proteins was independent of smoking habit of the patient. The anatomoclinical aspects combined with the expressions of c-Jun and p27 may assist the pathologist in directing the histopathological diagnosis.

Ο καρκίνος της ουροδόχου κύστης είναι η τέταρτη συχνότερη κακοήθεια στους άνδρες και η δέκατη στις γυναίκες και η ετήσια επίπτωσή του αυξάνει συνεχώς στις ανεπτυγμένες χώρες. Oι προγνωστικοί παράγοντες που χρησιμοποιούνται σήμερα δεν μπορούν να προβλέψουν με βεβαιότητα την μακροπρόθεσμη έκβαση του ουροθηλιακού καρκίνου και έτσι προκύπτει η ανάγκη αναγνώρισης δεικτών με δυνατότητα πρόγνωσης της συμπεριφοράς των καρκινωμάτων. Επιπλέον, δεδομένων των περιορισμένων δυνατοτήτων των σημερινών θεραπευτικών επιλογών (χειρουργική αντιμετώπιση, χημειοθεραπεία ή ανοσοθεραπεία και ακτινοθεραπεία), απαιτούνται νέες θεραπευτικές στρατηγικές. Μία τέτοια στρατηγική είναι η στόχευση σε μεταγραφικούς παράγοντες όπως οι πυρηνικοί υποδοχείς και οι upstream ενεργοποιητές τους. Η διαταραχή αυτών των μεταγραφικών παραγόντων είναι κομβικό σημείο της έναρξης και διατήρησης του κακοήθους φαινοτύπου. O πυρηνικός υποδοχέας PPARγ εμπλέκεται στον έλεγχο του μεταβολισμού, την κυτταρική ανάπτυξη, την αγγειογένεση και την ανοσολογική και φλεγμονώδη απάντηση. Επιπρόσθετα, υπάρχουν ενδείξεις ότι ρυθμίζει τους μηχανισμούς καταστολής αλλά και προαγωγής της καρκινογένεσης. Ο RXRα είναι επίσης μέλος της υπεροικογένειας των πυρηνικών υποδοχέων και ετεροδιμερίζεται με τον PPARγ προς σχηματισμό του συμπλόκου που αλληλεπιδρά με το DNA. Οι προσδέτες των RXR υποδοχέων έχουν ήδη χρησιμοποιηθεί στη χημειοπρόληψη διαφόρων μορφών καρκίνου. Ο μεταγραφικός παράγoντας AP-1, απαρτίζεται από διμερή των Fos και Jun πρωτεϊνών και η δράση του σχετίζεται με την πρόοδο της καρκινογένεσης. Υπάρχουν πάντως και ενδείξεις για προ-αποπτωτική δράση του. Η CBP είναι ένας απ’ τους σημαντικότερους ολοκληρωτές σημάτων της μεταγραφής. Ο ανταγωνισμός μεταξύ των PPARγ και AP-1 για τη CBP είναι ένας απ’ τους μηχανισμούς που εξηγούν την αρνητική «συνομιλία» (cross-talk) μεταξύ των PPARγ και AP-1. Στην παρούσα μελέτη εξετάσαμε τόσο ξεχωριστά όσο και σε συνδυασμό μεταξύ τους, την έκφραση των πέντε μοριακών παραγόντων (PPARγ, RXRα, p-c-Jun, c-Fos, CBP) στο φυσιολογικό ουροθήλιο, τις προκαρκινικές αλλοιώσεις και τα ουροθηλιακά καρκινώματα (ΟΚ). Τα ιστικά δείγματα προήλθαν από 88 ασθενείς οι οποίοι υπέστησαν διαγνωστική βιοψία ή θεραπευτική κυστεκτομή, νεφρεκτομή ή ουρητηρεκτομή. Εφαρμόστηκε η ανοσοϊστοχημική μέθοδος σε τομές παραφίνης και εκτιμήθηκε η σχετική έκφραση των μελετώμενων παραγόντων στα ενδοκυττάρια διαμερίσματα, τις ενδοεπιθηλιακές στιβάδες και τις φυσιολογικές ή παθολογικές ιστολογικές βαθμίδες. Όλοι οι παράγοντες παρουσίασαν κυρίως πυρηνική εντόπιση. Η έκφραση του p-c-Jun ελαττώνεται στους ασθενείς άνω των 70 ετών σε σχέση με τους νεώτερους, ενώ κανένα άλλο απ’ τα μελετώμενα μόρια δε φαίνεται να επηρεάζεται από την ηλικία. Η έκφραση των PPARγ, CBP, p-c-Jun και c-Fos σημειώνει αύξηση κατά την πορεία προς τον καρκίνο. Όσο αφορά στα ΟΚ, οι PPARγ και CBP παρουσιάζουν αρνητική συσχέτιση με την αποδιαφοροποίηση. Επιπλέον ο PPARγ συσχετίζεται αρνητικά με την απόκτηση χαρακτήρων διήθησης στα ΟΚ. Αντιθέτως, η έκφραση του RXRα δεν διακυμαίνεται στατιστικώς σημαντικά σε όλη την πορεία της καρκινογένεσης. Η ανάλυση της συνδυασμένης έκφρασης των πέντε παραγόντων έγινε με σκοπό την αποκάλυψη ενδεχόμενων αλληλεπιδράσεων μεταξύ τους. Η προστατευτική δράση του PPARγ στο ουροθήλιο συνοδεύεται από ταυτόχρονη μέτρια ή ισχυρή έκφραση των RXRα, p-c-Jun και c-Fos. Αναλυτικά, η αυξανόμενη έκφραση του p-c-Jun συμπίπτει με ενίσχυση της θετικής συσχέτισης του PPARγ με καλύτερα διαφοροποιημένους, λιγότερο διηθητικούς όγκους, ενώ ο c-Fos φαίνεται να εξασθενίζει ήπια την ευνοϊκή δράση του PPARγ στη διαφοροποίηση του ουροθηλίου. Η αυξανόμενη έκφραση της CBP έδειξε να εξασθενίζει και τελικά να εκμηδενίζει τη στατιστικά σημαντική αύξηση του PPARγ στην πορεία προς τον καρκίνο και την επαγωγή του στους μη διηθητικούς όγκους σε σύγκριση με τους διηθητικούς. Ταυτόχρονα, η αρνητική σχέση της CBP με την αποδιαφοροποίηση και την αύξηση της κακοήθειας των ΟΚ επηρεάζεται από την παρουσία των PPARγ και AP-1, επιβεβαιώνοντας την υπόθεση της συνομιλίας αυτών των μοριακών παραγόντων. Ενδιαφέρουσα είναι η παρατήρηση ότι οι περισσότερες από τις αναφερθείσες πιο πάνω συσχτίσεις μεταξύ των μοριακών παραγόντων ίσχυαν για μεγαλύτερους των 70 ετών αλλά όχι πάντα για τους νεώτερους ασθενείς. Τα αποτελέσματα της παρούσας μελέτης μπορούν πιθανόν να οδηγήσουν σε συμπεράσματα με εφαρμογή σε χημειοπροληπτικές και θεραπευτικές στρατηγικές για τον ουροθηλιακό καρκίνο. / Bladder cancer is the fourth and tenth most common malignancy in men and women, respectively, and its incidence is increasing annually in the developed countries. Current prognostic parameters cannot predict with certainty the long-term outcome of bladder cancer and as a result there is a need to identify markers that may predict tumor behavior. Furthermore, given the limitations of current therapeutic options (surgery, chemotherapy or immunotherapy and radiotherapy), novel treatment strategies are very much needed. One such strategy targets transcription factors such as nuclear receptors and their upstream activators. Disruption of these transcription factors is a key element in the initiation and maintenance of a malignant phenotype. The nuclear receptor PPARγ is involved in controlling metabolism, cell growth, angiogenesis, and immune and inflammatory responses. In addition, it has also been suggested that it regulates tumor suppression as well as tumor promotion. RXRα is another member of the nuclear receptor superfamily, that partners PPARγ to form the DNA-binding complex. RXR ligands are already being used as chemopreventive agents in various types of cancer. The transcription factor AP-1 is formed by dimerization of Jun and Fos proteins and its activity is often associated with tumor progression. On the other hand, there is also evidence that AP-1 may enhance apoptosis. CBP is one of the most important transcriptional integrators. The competition of PPARγ and AP-1 for CBP is one of the multiple mechanisms that explain the negative PPARγ/AP-1 cross-talk. In the present study, we assessed separate and concurrent expression of the five factors (PPARγ, RXRα, p-c-Jun, c-Fos, CBP) in normal urothelium, precancerous lesions and urothelial carcinomas (UC). Clinical samples were derived from 88 patients who had undergone diagnostic biopsy or therapeutic excision of the bladder, the kidney or the ureter. Parafin section immunohistochemistry was utilized and relative expression was estimated in intracellular compartments, intraepithelial layers and histologic categories of urothelium. All five factors had mainly nuclear pattern of expression. P-c-jun was downregulated in patients older than 70 years old compared to younger ones, whereas age did not affect the expression of the rest four factors. PPARγ, CBP, p-c-Jun and c-Fos were upregulated towards tumorigenesis. PPARγ and CBP showed an inverse relationship with carcinoma level of differentiation. Moreover, PPARγ expression downregulated significantly in invasive tumors compared to non-invasive ones. On the contrary, RXRα expression did not vary significantly along the carcinogenesis course. The following correlations were based on coexpression analysis to reveal molecular interactions between the five factors. The established protective effect of PPARγ on urothelium was accompanied by concomitant RXRα, p-c-Jun and c-Fos moderate or strong expression. In detail, p-c-Jun’s increasing expression strengthened the positive relation of PPARγ with better differentiated, less invasive tumors, whereas c-Fos seemed to mildly lessen PPARγ’s favourable effect in urothelium differentiation. Statistically significant PPARγ upregulation in malignant tissues compared to normal urothelium and in non-invasive tumors compared to invasive ones is suppressed and finally cancelled by CBP’s increasing expression. PPARγ and AP-1 seemed to influence the negative relation of CBP with loss of differentiation and increase of malignant potential in UC, an observation that denotes a cross-talk between these molecular factors. Interestingly, most of the aforementioned correlations were noticed in patients older than 70 years old, but not all of them were plausible in younger patients. The results from the present study could lead to conclusions possibly applicable in chemoprevention and therapy strategies for urothelial carcinomas.