Abstract A51: Evaluation of Akt molecular targets in colorectal tumors after radiotherapy and MK2206 treatment
Authors: Fernandes Jennifer Marx , Camargo Anamaria A. , Koyama Fernanda C. 
Published: May 2019

Introduction: Colorectal cancer is the third most frequent cause of cancer-related mortality in the world. The available treatment comprises surgical resection and pre- or postoperative chemoradiotherapy. Neoadjuvant radiochemotherapy (RQTn) has been widely used in the treatment of some types of cancers, including rectum cancer, which leads to complete remission of the tumor in some cases. However, there is a lack of response to this treatment in a significant number of patients (approximately 60%). Previous data from our group suggest that activation of the Akt pathway plays an important role in the response to chemoradiotherapy treatment as observed by differential gene expression followed by pathway enrichment analysis and immunohistochemistry between rectal tumors that are responsive or not to RQTn. As Akt is involved in many biologic functions, we evaluated Akt signaling in order to understand molecular effectors that could promote radioresistance in rectal tumors.

Materials and Methods: Colorectal cancer cell lines, sensitive (SW48) and resistant (SW480) to RQTn, respectively, were compared regarding basal Akt signaling status. We also assessed Akt signaling after treatment of the resistant cell line SW480 with radiation and 5-FU combined with the Akt inhibitor MK2206. To evaluate the phosphorylation of some proteins in the Akt pathway, we performed the immunoarray assay using the PathScan® Akt Signaling Antibody Array Kit Chemiluminescent Readout (Cell Signaling), following the protocol provided by the manufacturer. The images were acquired by revelation in photographic film, and the subsequent analysis of densitometry was performed by the software Image J. To validate the phosphoarray data we carried out immunoblots using both antibodies to phosphorylated AMPKα (Thr172) and total AMPK (Cell Signaling Technology, Beverly, Massachusetts, USA) with both lineages.

Results and Conclusions: Using the phosphoarray immunoassay for Akt pathway, it was observed that some proteins such as AMPKα, GSK3α, PTEN, PDK1, and PRAS40 showed different levels of basal phosphorylation in SW48 and SW480 cells. Interestingly, according to our data there is a higher basal phosphorylation of AMPKα in SW48 cells than in SW480.The treatment with MK2206 in SW480 cells demonstrated efficiency in Akt inhibition, culminating in different levels of phosphorylation of some substrates of this protein, especially AMPKα. It is possible to suggest a prosurvival role of Akt since when inhibited, phosphorylation of AMPKα, which acts as a tumor suppressor, increases, and PRAS40, an inhibitor of apoptosis through mTOR regulation, decreases. It is emphasized that the study of the components of the PI3K/Akt pathway are promising targets for therapeutic intervention, since this pathway has the ability to inhibit several types of tumor suppressors.

Citation Format: Jennifer Marx Fernandes, Anamaria A. Camargo, Fernanda C. Koyama. Evaluation of Akt molecular targets in colorectal tumors after radiotherapy and MK2206 treatment [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 A51.