Abstract
AKT, a serine threonine kinase, exists in three different isoforms and is known for regulating several biological processes including tumorigenesis. In this study, we investigated the expression and net effect of the individual isoforms in triple negative breast cancers and response to cisplatin treatment using cellular, mice models and clinical samples. Interestingly, analysis of the expressions of AKT isoforms in clinical samples showed relatively higher expression of AKT1 in primary tissues; whereas lung and liver metastatic samples showed elevated expression of AKT2. Similarly, triple-negative breast cancer cell lines, BT-549 and MDA-MB-231, with high proliferative and invasive properties, displayed higher expression levels of AKT1/2. By modulating AKT isoform expression in MCF-10A and BT-549 cell lines, we found that presence of AKT2 was associated with invasiveness, stemness and sensitivity to drug treatment. It was observed that the silencing of AKT2 suppressed the cancer stem cell populations (CD44high CD24low, ALDH1), mammosphere formation, invasive and migratory potential in MCF-10A and BT-549 cells. It was further demonstrated that loss of function of AKT1 isoform is associated with reduced sensitivity towards cisplatin treatment in triple-negative breast cancers cellular and syngeneic mice models. The decrease in cisplatin treatment response in shAKT1 cells was allied with the upregulation in the expression of transporter protein ABCG2, whereas silencing of ABCG2 restored cisplatin sensitivity in these cells through AKT/SNAIL/ABCG2 axis. In conclusion, our study demonstrated the varied expression of AKT isoforms in triple-negative breast cancers and also confirmed differential role of isoforms in stemness, invasiveness and response towards the cisplatin treatment.
Highlights
Breast cancer is the second-most lethal cancer in women around the world [1]
Receptor tyrosine kinases are phosphorylated in response to ligand stimulation, which activates phosphatidylinositol 3-kinase (PI3K) to drive subsequent phosphorylation by PDK1 on a threonine residue in the catalytic domain (T308 on AKT1, T309 on AKT2 and T305 on AKT3), and requires phosphorylation on a serine residue which is located at the hydrophobic C-terminal region (S473 on AKT1, S474 on AKT2 and S472 on AKT3) by the mammalian target of rapamycin complex 2; other protein kinases have been found capable of phosphorylating the same Ser residues thereby activating AKT and other downstream signalling loop [5, 6]
We further evaluated the expression of AKT isoforms in different breast cancer cell lines and found that AKT1 and AKT2 were strongly expressed in the cells of Triple-negative cancer subtype, BT-549 and MDA-MB-231, compared to their corresponding expression in luminal MCF-7 and non-tumorigenic MCF10A cells (Supplementary Figure 1A–1C)
Summary
Breast cancer is the second-most lethal cancer in women around the world [1]. Despite recent advances in tumor therapy, the main concern remains metastasis and relapse. Targeting dysregulated tumor driving pathways in human cancer has been a promising tool in cancer therapy and one such is the PI3K/AKT/mTOR signalling pathway. Receptor tyrosine kinases are phosphorylated in response to ligand stimulation, which activates phosphatidylinositol 3-kinase (PI3K) to drive subsequent phosphorylation by PDK1 on a threonine residue in the catalytic domain (T308 on AKT1, T309 on AKT2 and T305 on AKT3), and requires phosphorylation on a serine residue which is located at the hydrophobic C-terminal region (S473 on AKT1, S474 on AKT2 and S472 on AKT3) by the mammalian target of rapamycin complex 2 (mTORC2); other protein kinases have been found capable of phosphorylating the same Ser residues thereby activating AKT and other downstream signalling loop [5, 6]. It is well known that all the three AKT isoforms are highly homologous in their amino acid sequences (~80%) and display similar substrate specificity namely an N-terminal pleckstrin homology (PH) regulatory domain, a catalytic domain in the middle, and a C-terminal region necessary for the induction and maintenance of kinase activity but are encoded by separate genes [7, 8]
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