PO-270 Signalling pathways WNT, hedgehog and NOTCH in breast cancer with presence and absence of cancer stem cells

Abstract

IntroductionEndometrial carcinoma is the most common malignancy of the female genital tract in developed countries. These tumours, usually are early diagnosed and associated to a good prognosis, although 20% of the cases, present an aggressive and invasive behaviour. Cancer stem cells (CSC) are defined as an undifferentiated tumour cell subpopulation involved in tumour initiation, resistance to therapy and metastatic phenotype. Several mechanisms are associated to resistance to therapy, such as pathways of aldehyde dehydrogenase (ALDH), an enzyme related with tumour size, lymphatic nodal invasion, recurrent disease, and poor prognosis. In endometrial cancer, the ALDH1 expression was related to tumorigenesis and chemoresistance. In our previous work, ALDH expression was associated with endometrial CSC phenotype.Material and methodsRL95-2, a human endometrial cancer cell line, was submitted to ALDH inhibitors, diethylaminobenzaldehyde (DEAB) 50–100 µM, all-trans retinoic acid (ATRA) 5–10 µM and JQ1 100–250 nM for 48 hours. The cytotoxicity was evaluated through MTT assay. ALDH expression was evaluated through western blot. Sphere formation assay was performed, cells were cultured in coated plates with poly(2-hydroxyethyl-methacrylate), with serum-free DMEM F12 medium with or without 100 µM DEAB, for 5 days. Every two days, spheres were supplemented with EGF and bFGF at a concentration of 10 ng/mL. To evaluate the sphere projection area, spheres obtained were photographed at a magnification of 400x and analysed with Image J software.Results and discussionsThe metabolic activity in of RL95-2 cells was not influenced by the presence of ALDH inhibitors. Cells submitted to 50 and 100 µM DEAB presented a decrease in ALDH relative expression compared with the control cells, of about 36% and 27%, respectively. Regarding the evaluation of sphere projection area, preliminary results showed that spheres submitted to 100 µM DEAB appears to harbour decreased dimensions than control spheres.ConclusionDEAB was the most promising ALDH inhibitor in endometrial cancer cells. This inhibitor also seems to influence the sphere profile considering spheres dimensions. In further studies, we aim to evaluate its influence in the response to cytostatics.FundingFCT (Portugal) PEst-UID/NEU/04539/2013 and FEDER-COMPETE (FCOMP-01–0124-FEDER-028417 and POCI-01–0145-FEDER-007440), Bolsa Liga Portuguesa Contra o Cancro/CIMAGO, CIMAGO n° 02/2017, Sociedade Portuguesa de Ginecologia/Bayer.