Abstract A80: Differential expression of claudin-3 during colorectal tumorigenesis and its role in modulation and interaction with other tight junction proteins

Abstract

Abstract Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths in the world. According to the International Agency for Research on Cancer data, Brazil belongs to the group of countries that presented an increase in both incidence and mortality average of the CRC in the last decade. The loss of the apical-basal polarity can lead to cancer progression, since neoplastic cells shows deficiency in the apical junctional complexes (AJC), exhibiting a malign potential. Tight junction (TJ) is one of the junctions found in the AJC. It is a structure that helps to maintain the polarity and also presents a barrier function, which regulates the transit of important molecules through the paracellular flow. Claudin protein family are the main components of the TJ and are composed of transmembrane proteins. These proteins can interact with other TJ proteins such as occludins or scaffold proteins, essentially ZO-1/2. Our group has already showed that patients with CRC overexpress claudin-3 in the tumor tissue. Our goal was to evaluate the importance of the expression and interaction among claudin-3, ZO-1, and occludin proteins during the progression of CRC. For the molecular analysis, samples were obtained from biopsy after patients were submitted to surgery at the Brazilian National Cancer Institute. Adenocarcinoma tissue and normal adjacent region were obtained through colectomy and classified by the TNM staging of tumor. The Immunoblotting essay was performed to quantify the expression of claudin-3 and occludin proteins in those patients in different stages of the disease. Moreover, the interaction between these proteins was identified through immunoprecipitation essay. In vitro assay was performed using transfected HT-29 human adenocarcinoma cells overexpressing claudin-3. In these cells we evaluated the expression levels of claudin-3 and ZO-1, and we also analyzed the subcellular distribution of claudin-3, ZO-1, and occludin by immunofluorescence. This study is being carried out with approval of the INCA Research Ethics Committee (Protocol 84/04). The analysis by immunoblotting of claudin-3 expression showed that the patients can be segregated in two groups: one where the level of this protein in the tumor is low and other group where its levels are high. No alteration in the levels of occludin was observed when comparing the tumor and the normal adjacent tissue. However, the patients in whom the tumor presented high levels of claudin-3 showed less interaction with occludin. In vitro assay using transfected HT-29 cells that overexpress claudin-3 allowed us to observe that there was a reduction in the protein levels of ZO-1 as well as a reduced colocalization between these in the regions of cell-cell contact. Associated with these results, we also observed claudin-3 and occludin colocalization in an intermittent fashion in the regions of cell-cell contact, being more evident in the region of claudin-3 accumulation in HT-29 cells. Altogether, our data suggest that in CRC there are groups of patients that show elevated level of claudin-3 in the tumor and other group that shows low level. Further, it was observed that the increase of claudin-3 expression can be related to the decrease of the ZO-1 expression and a decrease in the interaction between claudin-3 and both, occludin and ZO-1. Our results can help to improve the understanding of the molecular mechanisms involved in the colorectal tumorigenesis, and point out potential prognostic markers aiming a therapeutic design to specific targets. Financial support: FAPERJ, CAPES and MS. Citation Format: Waldemir Fernandes de Souza, Perôny da Silva Nogueira, Maria Teresa dos Santos Guedes, Bruno Kaufmann Robbs, Joao Paulo de Biaso Viola, José Andrés Morgado-Díaz. Differential expression of claudin-3 during colorectal tumorigenesis and its role in modulation and interaction with other tight junction proteins [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A80.