Impact of AKT1 on cell invasion and radiosensitivity in a triple negative breast cancer cell line developing brain metastasis

Introduction: The PI3K/AKT pathway is activated in 43-70% of breast cancer (BC)-patients and promotes the metastatic potential of BC cells by increasing cell proliferation, invasion and radioresistance. Therefore, AKT1-inhibition in combination with radiotherapy might be an effective treatment option for triple-negative breast cancer (TNBC)-patients with brain metastases. Methods: The impact of AKT1-knockout (AKT1_KO) and AKT-inhibition using Ipatasertib on MDA-MB-231 BR cells was assessed using in vitro cell proliferation and migration assays. AKT1-knockout in MDA-MB-231BR cells was performed using CRISPR/Cas9. The effect of AKT1-knockout on radiosensitivity of MDA-MB-231BR cell lines was determined via colony formation assays after cell irradiation. To detect genomic variants in AKT1_KO MDA-MB-231BR cells, whole-genome sequencing (WGS) was performed. Results: Pharmacological inhibition of AKT with the pan-AKT inhibitor Ipatasertib led to a significant reduction of cell viability but did not impact cell migration. Moreover, only MDA-MB-231BR cells were sensitized following Ipatasertib-treatment. Furthermore, specific AKT1-knockout in MDA-MB-231BR showed reduced cell viability in comparison to control cells, with significant effect in one of two analyzed clones. Unexpectedly, AKT1 knockout led to increased cell migration and clonogenic potential in both AKT1_KO clones. RNAseq-analysis revealed the deregulation of CTSO, CYBB, GPR68, CEBPA, ID1, ID4, METTL15, PBX1 and PTGFRN leading to the increased cell migration, higher clonogenic survival and decreased radiosensitivity as a consequence of the AKT1 knockout in MDA-MB-231BR. Discussion; Collectively, our results demonstrate that Ipatasertib leads to radiosensitization and reduced cell proliferation of MDA-MB-231BR. AKT1-inhibition showed altered gene expression profile leading to modified cell migration, clonogenic survival and radioresistance in MDA-MB-231BR. We conclude, that AKT1-inhibition in combination with radiotherapy contribute to novel treatment strategies for breast cancer brain metastases

H2S preconditioning of human adipose tissue-derived stem cells increases their efficacy in an in vitro model of cell therapy for simulated ischemia

Aims: A major limitation of cell-based therapies for ischemia – reperfusion injury is the excessive loss of adminis- tered cells. We investigated whether H 2 S can improve the survival and ef fi cacy of therapeutic cells in an in vitro model of cell-based therapy for simulated ischemia. Main methods: H9c2 rat cardiomyoblasts were exposed to oxygen – glucose deprivation and NaHS (3 – 30 μ M) pretreated human adipose tissue derived stem cells (hASCs) were added after reoxygenization. Viability of both cell lines was assessed with fl ow cytometry after 24 h. The effects of H 2 S on antioxidant defense, prolifera- tion, AKT and ERK1/2 phosphorylation and mitochondrial activity were analyzed in hASCs. Proliferation was evaluated using propargylglycine, an inhibitor of endogenous H 2 S synthesis. Key fi ndings: NaHS pretreatment decreased the ratio of necrotic therapeutic cells by 41.8% in case of 3 μ M NaHS and by 34.3% with 30 μ M NaHS. The ratio of necrotic postischemic cardiomyocytes decreased by 35%, but only with the use of 3 μ M NaHS. Antioxidant defense mechanisms and ERK-phosphorylation were enhanced after 3 μ M NaHS treatment while AKT-phosphorylation was suppressed. NaHS dose-dependently increased the prolif- eration of hASCs while pretreatment with propargylglycine decreased it. Signi fi cance: NaHS pretreatment can increase the survival of therapeutically used human adipose tissue-derived stemcells viaincreased antioxidant defense andimproves the postischemic cardiac derived cells' survival aswell. Proliferation ofhuman adiposetissue-derivedstemcells is enhanced by H 2 S.The underlying mechanisms involve enhanced ERK-phosphorylation and decreased AKT-phosphorylation. Pretreatment with NaHS may represent a simple pharmacological step that may enhance the ef fi cacy of cell-based therapies

Akt targeting promotes the radiosensitizing effect of rapamycin in <i>non-responder</i> cells.

<p><b>(A)</b> Cells were treated with MK2206 (A549: 5 μM; H460: 2.5 μM) for 1 h, followed by treatment with rapamycin (100 nM) for 2 h. Control cells received the appropriate concentrations of DMSO. Thereafter, P-Akt (S473, T308) and P-Akt2 (S474) levels were analyzed in protein samples by Western blotting. Blots were stripped and incubated with anti-Akt1 or anti-Akt2 antibodies. The densitometry data represent the mean ratio of phosphorylated Akt1 to Akt1 based on 2 biologically independent experiments; the control conditions were normalized to 1. The densitometry values for phospho-Akt2 represent the mean ratio of phospho-S474 to Akt2 normalized to 1 for the control condition. For colony formation assays, 24 h after pre-plating, cells were treated with MK2206 (A549: 5 μM; H460: 2.5 μM) for 1 h, followed by treatment with rapamycin (100 nM) for 2 h. Control cells received the appropriate concentrations of DMSO. The cultures were irradiated after rapamycin treatment and incubated for colony growth. Data represent the mean SF ± SD of 18 data from three biologically independent experiments in A549 cells and of 12 data from two biologically independent experiments in H460 cells. In <i>non-responder</i> A549 cells, asterisks indicate a significant difference between the radiosensitizing effect produced by the combination of MK2206 and rapamycin compared to the effects of single treatment with rapamycin alone (**, P < 0.01; ***, P < 0.001). <b>(B)</b> Cells were plated in culture dishes and were transfected with non-target-siRNA (siCON) or AKT1-siRNA (siAKT1) after 24 hours at concentrations of 50 nM (A549) and 150 nM (H460). At the indicated time points after transfection, Akt1 knockdown efficiency was tested by Western blotting. GAPDH was used as a loading control. Densitometry values represent the mean ratio of Akt1 to GAPDH from three independent experiments; the control conditions were normalized to 1. In parallel, 2 days after transfection, cells were trypsinized and seeded for clonogenic assays. Twenty-four hours later, cells were treated with rapamycin (100 nM) for 2 hours, followed by irradiation with single doses of 0 to 4 Gy. The clonogenic assay was performed as described in <i>Materials and Methods</i>. Data represent the mean SF ± SD of four biologically independent experiments (21 data) in A549 cells and 6 parallel experiments in H460 cells. In <i>non-responder</i> A549 cells, asterisks indicate a significant difference between the radiosensitizing effect produced by the combination of AKT1-siRNA with rapamycin compared to the effects of single treatment with rapamycin alone (***, P < 0.001). DMF: Dose modification factor.</p

Rapamycin-induced Akt activation accelerates the repair of radiation-induced DNA-DSBs.

<p><b>(A-B)</b> Cells grown to confluency on glass slides were treated with MK2206 (A549: 5 μM; H460: 2.5 μM) and rapamycin (100 nM) or were pretreated with MK2206 (A549: 5 μM; H460: 2.5 μM) for 1 hour and followed by treatment with rapamycin (100 nM) for 2 h. <b>(C-D)</b> Cells grown on glass slides were transfected with control-siRNA (siCON) or AKT1-siRNA (siAKT1) (A549: 50 nM; H460: 150 nM). Seventy-two hours after transfection, cells were treated with rapamycin (100 nM) for 3 hours. In the experiments described above, control cells received the appropriate concentrations of DMSO. Following the treatment procedures described above, cells were irradiated with the indicated doses of X-ray, and γ-H2AX foci assays were performed 24 h after irradiation, as described in <i>Materials and Methods</i>. The frequency of residual γ-H2AX foci was counted in indicated number of cells and experiments (exp.) per treatment condition. The mean number of foci/cell ± SEM was calculated and graphed. Asterisks indicate statistically significant enhancement of residual γ-H2AX foci under the indicated conditions (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (<b>E</b>) Data represent the mean value of GFP expression from 7 data obtained from 4 independent experiments after 24 h treatment with DMSO, MK2206 (MK /5 μM), Rapamycin (Rapa / 100 nM) or the combination of MK2206 with rapamycin in the NHEJ assay performed in A549 cells expressing NHEJ assay platform, as described in the <i>Materials and Methods</i> section (*, P < 0.05; **, P < 0.01).</p

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4.

BACKGROUND:Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today's gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo. METHODS:The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo. RESULTS:Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3. CONCLUSIONS:We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors

Rapamycin treatment induces Akt1 activation through PI3K in a cell line-dependent manner.

<p>Cells were treated with rapamycin (100 nM) for the indicated time points <b>(A)</b> or for 6 h <b>(B)</b>. (<b>C)</b> After 1 h pretreatment with LY294002 (10 μM), cells were treated with rapamycin (100 nM) for 2 hours. Control cells received the appropriate concentrations of DMSO. Thereafter, protein samples were isolated and the phosphorylation patterns of mTOR, S6, Akt, PRAS40 and GSK3α/β were analyzed by Western blotting. Blots were stripped and incubated with antibodies against total proteins. Densitometry values represent the ratio of phosphorylated protein to total protein, which was normalized to 1 for the control condition. Densitometry values represented in part B are at least from 3 independent experiments.</p

Dual Targeting of Akt and mTORC1 Impairs Repair of DNA Double-Strand Breaks and Increases Radiation Sensitivity of Human Tumor Cells

<div><p>Inhibition of mammalian target of rapamycin-complex 1 (mTORC1) induces activation of Akt. Because Akt activity mediates the repair of ionizing radiation-induced DNA double-strand breaks (DNA-DSBs) and consequently the radioresistance of solid tumors, we investigated whether dual targeting of mTORC1 and Akt impairs DNA-DSB repair and induces radiosensitization. Combining mTORC1 inhibitor rapamycin with ionizing radiation in human non-small cell lung cancer (NSCLC) cells (H661, H460, SK-MES-1, HTB-182, A549) and in the breast cancer cell line MDA-MB-231 resulted in radiosensitization of H661 and H460 cells <i>(responders)</i>, whereas only a very slight effect was observed in A549 cells, and no effect was observed in SK-MES-1, HTB-182 or MDA-MB-231 cells (<i>non-responders</i>). In <i>responder</i> cells, rapamycin treatment did not activate Akt1 phosphorylation, whereas in <i>non-responders</i>, rapamycin mediated PI3K-dependent Akt activity. Molecular targeting of Akt by Akt inhibitor MK2206 or knockdown of Akt1 led to a rapamycin-induced radiosensitization of <i>non-responder</i> cells. Compared to the single targeting of Akt, the dual targeting of mTORC1 and Akt1 markedly enhanced the frequency of residual DNA-DSBs by inhibiting the non-homologous end joining repair pathway and increased radiation sensitivity. Together, lack of radiosensitization induced by rapamycin was associated with rapamycin-mediated Akt1 activation. Thus, dual targeting of mTORC1 and Akt1 inhibits repair of DNA-DSB leading to radiosensitization of solid tumor cells.</p></div