Truncated WT1 protein isoform expression is increased in MCF-7 cells in a long-term estrogen depletion.

Abstract

BackgroundThe WT1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer, however, its role during tumor progression is still unknown.\n\nMethodologyIn this work were analyzed the expression of WT1 isoforms (36-38 kDa and 52-54 kDa, and 17 AA (+/-) and KTS (+/-)) in breast cancer cells. On the other hand, with the purpose of mimicking the process of switch from a hormone-dependent to a hormone-independent neoplasm, an assay was performed using the MCF-7 cells cultured in long-term estrogen depletion (MCF-7 LTED cells) to determine the WT1 protein isoforms expression by western blot and RT-PCR, and Her2/neu and Estrogen receptor (ER) expression by quantitative RT-PCR assay. Growth kinetics and sensitivity to tamoxifen were performed in the MCF-7 LTED cells by trypan blue exclusion.\n\nResultsThe western blot shows the presence of the 52-54 kDa WT1 isoform in the ER (+) breast cancer cells, but not in the ER (-) cells. The 36-38 kDa WT1 isoform was detected in all the breast cancer cell lines analyzed. Using specific primers was found that 17 AA (+) / KTS (-) WT1 isoform was the most frequent in four breast cancer cell lines. During the sampling of the MCF-7 cells in estrogen depletion, an increase in the short-term of 52-54 kDa WT1 isoform was observed and this was kept until week 13, thereafter, its expression was absent; alternately, the 36-38 kDa WT1 isoform was observed from week 1 and it remained constant until week 27. MCF-7 LTED cells growth kinetic decreased 1.4 folds and were not sensitive to tamoxifen antiproliferative effect (p [≤] 0.05). Finally, were observed an increase of expression of ER and Her2/neu in the MCF-7 LTED cells.\n\nConclusionsThe 36-38 kDa WT1 isoform expression occurs during the modifications of the hormonal environment, suggesting that it may be playing an important role in its adaptation and tumor progression.