Arsenic trioxide overcomes rapamycin-induced feedback activation of AKT and ERK signaling to enhance the anti-tumor effects in breast cancer

Abstract

Inhibitors of the mammalian target of rapamycin (mTORi) have clinical activity; however, the benefits of mTOR inhibition by rapamycin and rapamycin-derivatives (rapalogs) may be limited by a feedback mechanism that results in AKT activation. Increased AKT activity resulting from mTOR inhibition can be a result of increased signaling via the mTOR complex, TORC2. Previously, we published that arsenic trioxide (ATO) inhibits AKT activity and in some cases, decreases AKT protein expression. Therefore, we propose that combining ATO and rapamycin may circumvent the AKT feedback loop and increase the anti-tumor effects. Using a panel of breast cancer cell lines, we find that ATO, at clinically-achievable doses, can enhance the inhibitory activity of the mTORi temsirolimus. In all cell lines, temsirolimus treatment resulted in AKT activation, which was decreased by concomitant ATO treatment only in those cell lines where ATO enhanced growth inhibition. Treatment with rapalog also results in activated ERK signaling, which is decreased with ATO co-treatment in all cell lines tested. We next tested the toxicity and efficacy of rapamycin plus ATO combination therapy in a MDA-MB-468 breast cancer xenograft model. The drug combination was well-tolerated, and rapamycin did not increase ATO-induced liver enzyme levels. In addition, combination of these drugs was significantly more effective at inhibiting tumor growth compared to individual drug treatments, which corresponded with diminished phospho-Akt and phospho-ERK levels when compared with rapamycin-treated tumors. Therefore, we propose that combining ATO and mTORi may overcome the feedback loop by decreasing activation of the MAPK and AKT signaling pathways.

Figure 1. Arsenic enhances temsirolimus-induced growth inhibition of selective breast cancer cell lines.

(A) MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10^-6 N NaOH and 2.5 x 10^-5 % EtOH in PBS), 2 µM arsenic trioxide (ATO), 5 ng/ml temsirolimus, or the combination for 48 hours and relative cell number assessed by SRB assay. (B&C) Cell lines were exposed to vehicle control (Vh), 1 µM ATO, 5 ng/ml temsirolimus, or the combination for 48 hours and stained with propidium iodide and assessed by flow cytometry for cell cycle analyses (B) or percentage of cells with fragmented DNA (C). Annexin V staining was performed in SkBr3 cells and the apoptotic cells were determined as those staining positive for annexin V, but negative fot 7AAD. Statistical significance is denoted as follows: * = p<0.05, ** = p<0.01, and *** = p<0.001. Experiments were performed at least three times with technical triplicates.

Figure 2. Arsenic growth inhibition correlates with decreased temsirolimus-induced phospho-AKT.

MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10^-6 N NaOH and 2.5 x 10^-5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for phospho-S473-AKT, phospho-T308-AKT, AKT, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.

Figure 3. Arsenic and temsirolimus combination treatment results in decreased phospho-S6 and ERK activation.

MDA-MB-468, MCF-7, SkBr3, and T47D cell lines were exposed to vehicle control (Vh; 5.3 x 10^-6 N NaOH and 2.5 x 10^-5 % EtOH in PBS), 1-2 µM ATO, 0.5-5 ng/ml temsirolimus and the combinations for 24 hours. Whole cell extracts were used in immunoblotting experiments for phospho-S6, S6, phospho-ERK, ERK, and β-actin. Immunoblots shown are representative of experiments performed at least 3 times.

Figure 4. Combination treatment of ATO and rapamycin significantly inhibits tumor growth in an MDA-MB-468 xenograft model.

Mice were implanted with MDA-MB-468 cells in the mammary fat pad and once tumors reached 100-200 mm³, mice were treated with vehicle (Vh; 20% PEG 400 and 20% TWEEN 80), 7.5mg/kg rapamycin, 7.5 mg/kg ATO, or rapamycin plus ATO every other day. Tumor size was monitored (A) and specific growth rate of the tumor (B) was calculated from days 37-62 and is expressed as change in tumor volume in mm³/day. Data are expressed as mean with standard error bars (n = 8-9 mice per group). Tumors were stained with antibodies detecting the proliferative marker Ki67. Representative pictures at low and high magnification of each treatment group are shown (C). Percent cells with high intensity staining and positive Ki67 staining per tumor area were calculated using algorithms provided with the ImageScope software. * = p<0.05 Tumors from individual mice are represented with mean and standard error bars (n = 4-5 mice).

Figure 5. Addition of ATO decreases rapamycin-induced AKT activation in vivo.

Tumor samples were stained with antibody against phospho-S473-AKT. Representative pictures of each treatment are shown (A). Quantification of staining intensity was performed using algorithms provided with the ImageScope software (B). Individual animals are represented with mean and standard error bars (n=4-5 mice). (C-D) Tumors at the end of the experiment were analyzed in immunoblotting experiments with antibodies against phospho-S473-AKT (C), phospho-T308-AKT (D), AKT, and β-actin. Band densitometry was performed and the relative intensity of phospho-AKT/AKT/β-actin was calculated. Individual animals are represented with mean and standard error bars (n= 8-9 mice).

Figure 6. Addition of ATO decreases rapamycin-induced phospho-ERK and phospho-S6.

(A-B) Tumors were analyzed in immunoblotting experiments with antibodies against phospho-ERK, ERK, and β-actin. Band densitometry was performed and the relative intensity of phospho-ERK/ ERK/β-actin was calculated. Individual animals (n = 9) are represented with mean and standard error bars. (B) Representative immunoblots of tumors from 4 individual animals per treatment. (C) Tumors were analyzed in immunoblotting experiments with antibodies against phospho-S6, S6, and β-actin. Immunoblots from tumors from 4 individual animals per treatment are shown.