Impact of AKT1 on cell invasion and radiosensitivity on breast cancer brain metastasis
Background: Triple negative breast cancer (TNBC) is an aggressive tumor, which is associated with local recurrences and distant metastases. The pathway of phosphoinositide3-kinase/serine/threonine kinase(Pi3K/AKT) is activated in 43-70% of the patients and promotes the metastatic potential of cancer cells by increasing cell proliferation, invasion and radioresistance. Therefore, AKT1-inhibition in combination with radiotherapy might be an effective treatment option for TNBC-patients with brain metastases.   Methods: Cell viability, migration and clonogenic potential of the TNBC brain-seeking cell line MDA-MB-231 BR were evaluated by pharmacological AKT-inhibition with Ipatasertib as well as by AKT1 knock out (AKT1_k.o.) using CRISPR/Cas-9. The influence of AKT1-inhibition on radiosensitivity was further evaluated in the MDA-MB-231 BR and the parental cell line MDA-MB-231. Additionally, whole exome sequencing and RNA sequencing were performed in two different AKT1_k.o. clones.  Results: High concentration (20µM) of the pan-AKT inhibitor Ipatasertib led to a significant reduction of cell viability, but did not impact cell migration. Moreover, Ipatasertib sensitized only MDA-MB-231 BR cells to radiation. Furthermore, both AKT1_k.o. clones showed reduced cell viability in comparison to control cells (AKT_e.v.), but the effect was only statistically significant in one clone. Surprisingly, in this clone, k.o. of AKT1 led to increased cell migration. This unexpected effect was also observed in the clonogenic potential of both MDA-MB-231 BR AKT1_k.o. clones. In this context, RNAseq analysis revealed upregulation of CTSO, CYBB and GPR68 and downregulation of CEBPA, ID1, ID4, METTL15, PBX1 and PTGFRN and these genes were related to the increased cell migration, higher clonogenic survival and decreased radiosensitivity as a consequence of the AKT1 k.o in MDA-MB-231 BR.   Conclusions: Our results demonstrate that pharmacological AKT-inhibition leads to radiosensitization and reduced cell proliferation of the in MDA-MB-231 BR cell line. In contrast, Ipatasertib did not influence the radiosensitivity of the brain-seeking TNBC cell line MDA-MB-231 BR.