Estudo da via PI3k/AKT e da perda de Heterozigosidade do gene supressor tumoral Pten em lesões potencialmente malignas e Carcinoma epidermoide oral

Abstract

Oral Potentially Malignant Lesions (PML) is a group of lesions that present a risk for the development of Oral Squamous Cell Carcinoma (OSSC), including Oral Epithelial Dysplasia (OED). PTEN is a tumor suppressor gene of the PI3K / AKT pathway, which is one of the more deregulated pathways of cancer. However, it is still unknown how this pathway is involved in the malignant transformation process of PML. The heterozygosity loss (LOH) profiles were validated as predictors of malignant transformation of OED and in carcinomas from several locations. In addition, it is not known whether the allelic loss of PTEN also occurs in malignant lesions in the early stage and, as well, is involved as one of the mechanisms of oral carcinogenesis. In addition, it is suggested that allelic loss of PTEN also occurs in the early stages of malignant lesions and is involved in the mechanisms of oral carcinogenesis. Thus, a better understanding of the role of pathway in the process of oral carcinogenesis is necessary, searching for possible molecular markers for oral cancer. Objective: To evaluate the PI3K/AKT pathway in the malignant transformation process of PML and CEOs, through the immunoexpression of pAKT, pJNK, FoxO3a and Ki-67, besides performing immunomolecular evaluation of the PTEN tumor suppressor gene in DEO and CEO samples, with different histological types of malignancy, through the analysis of LOH in two chromosomal regions and immunohistochemical evaluation of the PTEN protein. Materials and methods: Tissue samples of 20 cases of OSCCs, 20 OEDs and 5 cases of normal oral mucosa were subjected to immunohistochemistry reactions for anti-p-AKT, anti-p-JNK, anti-FoxO3a and anti-Ki-67 antibodies. It was analyzed quantitative (number of immunostained cells) and qualitative (immunostaining intensity) parameters in different cell immunostaining sublocations. For molecular analysis, formalin-fixed paraffin-embedded samples of 19 OED and 16 OSCC were included to immunohistochemistry and LOH analysis. For the immunohistochemical study, 5 random fields with greater immunoreactivity were photomicrographed and it was done the count of keratinocytes which showed cytoplasmic and nuclear staining. For LOH analysis, 2 polymorphic microsatellite markers (AFMA086WG9 and D10S1765) localizing to chromosomes 10 were used to detect LOH. Results: Nuclear p-AKT was observed significantly greater immunostaining in CCEOs (21.2 ± 19.0) than in dysplasias (7.9 ± 8.1) and control (1.8 ± 4.7) (p = 0.002). Immunostaining of strong nuclear p-JNK was greater in controls (48.3 ± 13.7) than in OEDs (11.0 ± 10.3) and OSCCs (1.1 ± 1.3) (p<0.001). Strong nuclear immunostaining of FoxO3a proved to be absent in OSCCs (0.0 ± 0.1) with little staining on dysplasia (3.2 ± 5.4) and increased expression in controls (13.5 ± 4.8) (p<0.001). The mean Ki- 67 positive cells were directly associated with the degree of cell differentiation (p <0.001). Molecular analysis of LOH, it was demonstrated that for marker AFMA086WG9 allelic retention in 89.5% of OEDs cells and in 100% of OSCC cells, in the analysis of marker D10S1765 LOH was demonstrated in 57.2% of OEDs and 50 % of OSCCs. Higher nuclear immunostaining was detected in cases of OSCCs when compared to OEDs and control group (p <0.001) and strong cytoplasmic immunostaining was more present in OSCCs (p <0.045). Conclusion: The PI3K / AKT pathway appears to be involved in the process of malignancy of DEOs in this research, especially p-JNK and FoxO3a with antitumor activity in both OED and OSCCs analyzed. We provide evidence that the allelic loss of PTEN is present in premalignant oral lesions e OSCCs, however the LOH in this gene did not influence its protein expression in the evaluated cases. In addition, the PTEN immunoexpression in DEO and CEO was also evidenced, being independent of its histopathological staging, proving that PTEN does not seem to influence the tumorigenesis of CEOs.