Abstract 3347: Post-translational modifications of Akt isoform in chemoresistance of endometrial cancer
Authors: Grenier-Naud Jérôme , Parent Sophie , Asselin Eric 
Published: May 2015

Endometrial cancer is ranked fourth in importance among all types of cancer in women. This cancer is the most prevalent gynecological cancer. Akt is an inactive cytosolic serine/threonine kinase that plays a crucial role in cell fate by promoting cellular survival and proliferation. There are three Akt isoforms (1,2 and 3). This protein acts as a key regulator of numerous cellular phenotypes associated with cancer, such as cell proliferation, growth, metabolism, angiogenesis, malignant transformation and chemoresistance. It has been shown that Akt polyubiquitination is increased in cells treated with IGF-1. This ubiquitination is associated with an augmentation in the phosphorylation status of Akt. More and more evidence show that ubiquitination plays a pivotal role in kinase activation. In this study we observed ubiquitinated Akt (Ub-Akt) in the nucleus following IGF-1 treatment in the nucleus of endometrial cell lines (Ishikawa and KLE). We have established the ubiquitination pattern for the three Akt isoforms and their location in the cells. We also showed a cleaved fragment of Akt following cisplatin treatment in the same cell lines.

Using western blot analyses, we observed a band at 60kDa corresponding to Akt and also other upper bands corresponding to Ub-Akt, in the nucleus of different endometrial cell lines. We have analyzed the pattern of two endometrial carcinoma (KLE and Ishikawa) and a cervical cancer cell line (HeLa). We found that both Akt1 and Akt3 were ubiquitinated in most of the cell lines. On the other hand, Akt2 was not ubiquitinated in any cell lines. We have used siRNA technology to knockdown each Akt isoform and characterized the loss of each isoform on the ubiquitination pattern for each cell line. We also treated cell lines with cisplatin to see which one of the three isoforms could be involved in chemoresistance and if Akt ubiquitination status could be involved in this process. We have separated fractions of the cytosol from the nucleus for each sample to investigate distribution of Ub-Akt isoforms in each cell line and confirmed the presence of Ub-Akt1 and -3 in the nucleus. We have also co-immunoprecipitated each Akt isoform and Ubiquitin to determine the exact ubiquitination pattern in cancer cells. KLE and Ishikawa had similarities in their pattern. Using immunoprecipitation, we observed that Akt1 was cleaved following cisplatin treatment. We observed that chemosentisitive cells had more cleaved Akt then chemoresistant lines. In conclusion, we are the first to establish a difference in the ubiquitination pattern for the different Akt isoform in the nucleus of endometrial cancer. We are also the first to show a cleavage of Akt isoforms linked to chemosensitive status of endometrial cancer. This study will form the basis for future investigations to determine the role of specific post-translationnal modifications of Akt isoforms in the endometrial cancer.

Citation Format: Jérôme Grenier-Naud, Sophie Parent, Eric Asselin. Post-translational modifications of Akt isoform in chemoresistance of endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3347. doi:10.1158/1538-7445.AM2014-3347