Abstract
Implementing targeted drug therapy in radio-oncologic treatment regimens has greatly improved the outcome of cancer patients. However, the efficacy of molecular targeted drugs such as inhibitory antibodies or small molecule inhibitors essentially depends on target expression and activity, which both can change during the course of treatment. Radiotherapy has previously been shown to activate prosurvival pathways, which can help tumor cells to adapt and thereby survive treatment. Therefore, we aimed to identify changes in signaling induced by radiation and evaluate the potential of targeting these changes with small molecules to increase the therapeutic efficacy on cancer cell survival. Analysis of “The Cancer Genome Atlas” database disclosed a significant overexpression of AKT1, AKT2, and MTOR genes in human prostate cancer samples compared with normal prostate gland tissue. Multifractionated radiation of three-dimensional–cultured prostate cancer cell lines with a dose of 2 Gy/day as a clinically relevant schedule resulted in an increased protein phosphorylation and enhanced protein–protein interaction between AKT and mTOR, whereas gene expression of AKT, MTOR, and related kinases was not altered by radiation. Similar results were found in a xenograft model of prostate cancer. Pharmacologic inhibition of mTOR/AKT signaling after activation by multifractionated radiation was more effective than treatment prior to radiotherapy. Taken together, our findings provide a proof-of-concept that targeting signaling molecules after activation by radiotherapy may be a novel and promising treatment strategy for cancers treated with multifractionated radiation regimens such as prostate cancer to increase the sensitivity of tumor cells to molecular targeted drugs. Mol Cancer Ther; 17(2); 355–67. ©2017 AACR.
See all articles in this MCT Focus section, “Developmental Therapeutics in Radiation Oncology.”
Introduction
Targeted drugs in combination with radiation or chemotherapy have significantly improved the survival of cancer patients (1). Although the inhibition of prosurvival pathways in malignant cells can contribute to cancer cell depletion, the eradication of solid tumors with monotherapies has shown limited success (2–4). Moreover, when compared with conventional cytotoxic drugs, the efficacy of targeted therapies is more affected by cellular signaling processes including changes in expression and activity of the target or associated proteins, which can be caused by the cancer treatment itself (2). We previously demonstrated that cells which survive radiotherapy consisting of either a single dose (SD) or multiple fractions (MF) of radiation have a significantly different expression of mRNA and miRNA, a phenomenon we call tumor adaptation (5, 6). However, whether a target can be induced by radiation making cells more susceptible to targeted therapy remains to be the key question addressed here. Because there are different types of molecular drugs such as antibodies or kinase inhibitors, radiation-induced modulations that can alter the cellular sensitivity to targeted therapeutics include both changes in protein expression levels and also posttranslational protein modifications such as phosphorylation regulating the catalytic activity of the target molecule. The use of drugs concomitantly with radiation is called radiation sensitization and is distinguished from using drugs following radiation, referred to as adaptive either after single dose (SD-adaptive) or multiple fractions (MF-adaptive).
In this study, we used prostate cancer cells as our tumor model. Prostate cancer is the most common cancer among men in developed countries. In addition to surgery, external beam radiation therapy (EBRT) or brachytherapy with implanted sources are considered to be curative treatment options (7). These may be combined with anti-androgen therapy depending on the risk group for tumor recurrence (8). EBRT is usually performed as conventional-fractionated radiotherapy with 2 Gy per fraction per day up to a total dose of 80 Gy (7). Several trials show that hypofractionated radiation or stereotactic body radiotherapy consisting of a lower number of fractions with an increased dose per fraction are equally effective and therefore serve as therapeutic options (7). In recent years, targeted drugs such as small molecule inhibitors have increasingly been used in clinical trials for the treatment of advanced or high-risk prostate cancer with encouraging results (9–11). Thus, we considered this to be a good model to investigate radiation-inducible changes given its prevalence and treatment options which include primary radiotherapy and a range of different clinical fractionation regimens.
Potential targets which are involved in the cellular radiation response of tumor cells are the prosurvival molecules EGFR, AKT, and mTOR (12–14). Targeting of EGFR before or during radiotherapy sensitizes squamous cell carcinoma or lung cancer cells to ionizing radiation and this effect is enhanced when EGFR is genetically overexpressed (15–17). The serine/threonine kinase AKT, which is downstream of EGFR (18), is involved in the regulation of essential cellular processes such as proliferation, survival, and protein translation via its interaction with mTOR (19). mTOR itself can form two functionally different complexes, mTORC1 and mTORC2. mTORC1, which contains mTOR, Raptor, and other molecules, induces mRNA translation by phosphorylation of the ribosomal S6 kinase (p70S6K). mTORC2, which comprises mTOR and Rictor, phosphorylates AKT at serine (S) 473 resulting in increased AKT activity (20). It has recently been shown that inhibition of mTOR prior to radiation enhances radiosensitivity in some tumor types (21–24). Different pathways seem to play a role in this effect including modulation of cap-dependent translation through eIF4E signaling and attenuated AKT activation after irradiation (22, 25, 26).
In this study, we focused on targeting mTOR/AKT signaling because several genes of this pathway were overexpressed in prostate cancer samples in comparison to normal prostate gland tissue. Moreover, we found a radiation-induced protein phosphorylation and activation of mTOR and AKT in prostate cancer cells in vitro and in a xenograft model. We used SD radiation as well as radiotherapy with multiple fractions reflecting the different clinical radio-oncologic regimens. For the in vitro studies, cells were cultured in a three-dimensional (3D) laminin-rich extracellular matrix that better represent the molecular signaling processes and the tumor response to targeted therapy observed in vivo (27–29).
The data show that targeting AKT and mTOR prior to multiple fractions of radiation significantly reduced survival of PC3 cells but not of DU145 cells. In contrast, treatment with AKT and mTOR inhibitors after multifractionated radiation was effective in both cell lines, indicating an increased susceptibility of drug-resistant cancer cells by radiation-induced target activation. This first proof-of-concept study presents a novel use of radiation in the precision medicine era for both improved treatment outcome and enhanced efficiency of molecular targeted agents.
Material and Methods
Antibodies
Antibodies for Western blotting included EGFR Y1068 (Invitrogen), EGFR, AKT, AKT S473, AKT T308, mTOR, mTOR S2448, p70S6K, p70S6K S371, GSK3α/β S9/21, GSK3α/β, Rictor, Raptor (Cell Signaling), β-actin (Millipore), horseradish peroxidase (HRP)–conjugated donkey anti-rabbit and sheep anti-mouse antibodies (Cell Signaling), and IRDye 800CW donkey anti-mouse and IRDye 680RD Donkey anti-rabbit antibodies (LI-COR). Antibodies for immunofluorescence staining and proximity ligation assay included AKT, mTOR (Cell Signaling), γH2AX (Millipore), Alexa488 anti-rabbit, and Alexa594 anti-mouse antibodies (Invitrogen). Antibodies for immunoprecipitation included Rictor, Raptor (Bethyl Laboratories), and rabbit IgG (Santa Cruz Biotechnology).
Cell culture and radiation exposure
DU145 and PC3 were obtained from the NCI tumor bank in 2015. A passage number of 15 was not exceeded. Asynchronously and exponentially growing cells were used in all experiments. Cells were cultured in RPMI1640 containing GlutaMAX (Invitrogen) supplemented with 10% FBS (Invitrogen). Mycoplasma testing was performed on a monthly basis.
Irradiation was delivered at room temperature using SDs or multiple fractions of 320 kV X-rays (Precision X-Ray Inc.). The dose rate was approximately 2.3 Gy/min and applied total doses ranged from 0 to 10 Gy. Multifractionated radiation was carried out either with one time 2 Gy per day or two times 1 Gy per day (with a 6 hours time interval between both radiations).
Animal experiments
A single-cell suspension of PC3 human prostate cancer cells was injected subcutaneously into the flanks of the right hind legs of athymic nude mice (NCr nu/nu; NCI Animal Production Program, Frederick, MD). Tumor growth was assessed daily with a digital caliper. When tumors had grown to an area of 5 mm × 5 mm, mice were randomized into four groups: (i) non-irradiated controls; (ii) irradiation with a SD of 10 Gy; (iii) irradiation with five fractions of 2 Gy (MF2), once a day for 5 days; (iv) irradiation with 10 fractions of 1 Gy (MF1), twice a day for 5 days (Supplementary Fig. S1). Radiation was delivered locally with animal restrained in a custom-designed lead jig. At 24 hours after the final radiation dose, tumors were excised, snap frozen in liquid nitrogen, and stored at −80°C. All animal studies were conducted in accordance with the NIH Guide for Care and Use of Animals.
Gene expression analysis in patient samples
Gene expression in prostate cancer and normal prostate was analyzed using the “The Cancer Genome Atlas” (TCGA) database. Normalized TCGA mRNA expression data for prostate cancer and normal prostate tissue were retrieved using the R package “TCGAbiolinks“ (30). The following genes were preselected for analysis: MTOR, AKT1, AKT2, AKT3, RPTOR, RICTOR, RPS6KB1, EIF4EBP1, GSK3B, FOXO1, FOXO3, FOXO4, MDM2, TSC1, TSC2, and BAD. To test for significant differences in gene expression between prostate cancer and normal prostate samples the Wilcoxon rank test including the Bonferroni adjustment for multiple testing were used. The data were plotted in boxplots for visualization.
Gene expression in in vitro and in vivo samples
For in vitro analysis, PC3 cells were cultured in a 3D laminin-rich extracellular matrix (lrECM) containing 0.5 mg/mL Matrigel (lrECM; BD) and irradiated with a single radiation dose of 10 Gy or multifractionated radiation with either 5 fractions of 2 Gy (one fraction per day) or 10 fractions of 1 Gy (2 fractions per day) with a cumulative dose of 10 Gy. At 24 h after the final radiation dose, cells were collected and total RNA was extracted from samples of three different experiments using the miRNeasy Kit (Cat # 217004, Qiagen) according to the manufacturer's protocol and as published (4). For in vivo analysis, RNA was isolated using homogenized powder from snap frozen tumor samples kept at −80°C. Extracted RNA was subjected to complementary cDNA synthesis using RT2 first-strand cDNA synthesis (Cat # 330404, Qiagen) according to the manufacturer's instructions. qRT-PCR was performed using RT2 SYBR Green qPCR Master Mix. The reaction (25 μL) along with cDNA was aliquoted into the wells of RT² Profiler PCR Array Human PI3K-AKT Signaling Pathway (PAHS-058Z, Qiagen) which contains pre-dispensed, laboratory verified, specific primer pairs. Data were analyzed using the RT2 Profiler array analysis software version 3.5 (SABiosciences). Relative gene expression was calculated by normalization to the arithmetic mean of five housekeeping genes.
Total protein extracts and Western blotting
Asynchronously and exponentially growing 3D cell cultures were lysed using cell lysis buffer (Cell Signaling) supplemented with protease inhibitors (Complete; Roche) as previously described (31). Homogenization of lysates was accomplished by four passages through a 25-gauge needle followed by centrifugation at 16,000 × g for 20 minutes. Xenograft tumors were homogenized in 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 25 mmol/L NaF, 25 mmol/L β-glycerophosphate, 0.2% Triton X-100, 0.3% Tween20, and 0.5 mmol/L sodium orthovanadate, supplemented with phosphatase inhibitor cocktails II and III (Sigma), and HALT protease inhibitor cocktail (ThermoFisher Scientific). After ultrasonic treatment, lysates were incubated for 1 hour and centrifugated at 16,000 × g for 20 minutes. Protein was quantified using the bicinchoninic acid (BCA) protein assay (Bio-Rad), separated by SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad), and probed with indicated antibodies. Bands of specific proteins were visualized with HRP-linked secondary antibodies and SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific) or with fluorescence-labeled secondary antibodies (LI-COR).
Phosphoproteomic array
For analysis of the phosphoproteome, cells were plated and irradiated with 10 Gy SD, with five fractions of 2 Gy dose per fraction, or with 10 fractions of 1 Gy dose per fraction (two fractions per day). At indicated time points after irradiation, cells were lysed with 2.5% 2-mercaptoethanol in T-PER (ThermoFisher Scientific) including protease and phosphatase inhibitors. Reverse phase protein microarrays were performed as previously published (32). In brief, samples were diluted and printed in duplicates onto nitrocellulose slides. HeLa cell lysates (with or without pervanadate) were used as positive and negative controls. Microarrays were stained with specific and validated antibodies and analyzed with a biotin-linked signal amplification system (DAKO). The total protein amount of the sample was determined with the SYPRO Ruby stain (ThermoFisher Scientific).
Immunoprecipitation
Immunoprecipitation experiments were performed as previously described (15). Protein lysates for immunoprecipitation were prepared of irradiated 3D-grown cells using cell lysis buffer (Cell Signaling) supplemented with complete protease inhibitor cocktail (Roche). Unirradiated cell cultures were used as control. Protein-G-Agarose beads (Sigma) were incubated with mTOR or Rictor antibodies (Cell Signaling) or nonspecific mouse or rabbit IgG antibody overnight. After washing of beads with PBS, protein lysates were added and incubated overnight. After washing of beads with cell lysis buffer, SDS-PAGE, and Western blotting was performed.
Immunofluorescence staining
Immunofluorescence stainings were carried out as recently published (27). At 2 hours after irradiation with 6 Gy, cells were fixed with 3% formaldehyde/PBS for 15 minutes. Permeabilization was performed with 0.25% Triton X-100/PBS for 10 minutes. After washing with PBS, samples were blocked with 3% BSA/PBS for 30 minutes. Double-staining of mTOR and AKT was carried out with specific antibodies overnight at 4°C. An 1-hour-incubation with secondary antibodies was performed after washing with PBS. Samples were covered with Vectashield/DAPI mounting medium (Alexis). Images were acquired using an AxioImager.Z1/ApoTome microscope (Zeiss).
Proximity ligation assay
The proximity ligation assay was performed according to the manufacturer's protocol and as recently described (15). In brief, cells were fixed, permeabilized, and incubated with primary antibodies overnight. After washing with PBS, samples were incubated with Duolink PLA PLUS and MINUS probes for mouse and rabbit (Olink Bioscience) for 1 hour at 37°C. After ligation with ligase for 30 min at 37°C, the amplification was carried out using polymerase and the Duolink Detection Kit (red) for 100 minutes at 37°C. Prior to covering with Vectashield/DAPI mounting medium, samples were washed multiple times with wash buffer A and B (Olink Biosciences) and dried. Images were obtained with an AxioImager.Z1/ApoTome using a 40× objective. Interactions were counted with ImageJ Cell Counter.
3D colony formation assay
3D colony formation assays were performed as previously published (33). In brief, 96-well plates were coated with 1% agarose (Sigma). Cells were trypsinized, counted, and diluted with cell culture medium. Matrigel was added to obtain a final concentration of 0.5 mg/mL. The Matrigel/cell mixture (500 cells per well) was immediately plated in the agarose-precoated wells. After an incubation time of 2 hours at 37°C, 3D cell cultures were covered with RPMI supplemented with FBS. Irradiation was started at 24 hours after plating. Cells were treated with the AKT inhibitor (AKTi) GDC-0068 (Ipatasertib; Seleckchem; ref. 34) or the mTOR inhibitor (mTORi) INK128 (Sapanisertib; Seleckchem; ref. 35) at different time points (1 hour before the start of irradiation or 2 hours after last irradiation). At 72 hours inhibitors were removed by washing the cell cultures five times with RPMI. DMSO treatment was used as control. Cells were cultured for 12 days. For counting of colonies, images were obtained using an EVOS microscope with a 2.5× objective. Cell clusters with more than 50 cells were manually counted with ImageJ Cell Counter. Results were confirmed by an automatic analysis using ImageJ and R (33). Surviving fractions were calculated as follows: (irradiated colony number/unirradiated colony number) (33).
γH2AX foci assay
The γH2AX foci assay performed as recently published (36). DU145 cells were treated with AKTi (0.5 μmol/L) or mTORi (10 nmol/L) 1 hour before or 2 hours after irradiation with three fractions of 2 Gy (2 Gy/day). DMSO-treated cells were used as control. At 24 hours after the final radiation dose, cells were fixed with 3% formaldehyde/PBS overnight at 4°C. Cells were permeabilized with 0.25% Triton X-100/PBS for 10 minutes and blocked with 3% BSA/PBS for 1 hour. γH2AX was stained with specific antibodies overnight at 4°C. After incubation with secondary antibodies, samples were covered with Vectashield/DAPI mounting medium (Alexis). Images were acquired using an AxioImager.Z1/ApoTome microscope (Zeiss). Foci of 50 cells per sample were counted for analysis.
CRISPR/Cas9 gene editing
Depletion of mTOR was accomplished by using the lentiCRISPR v2 vector (Addgene) as published (37). lentiCRISPR v2 was a gift from Feng Zhang (Addgene plasmid #52961). The design of target-specific oligonucleotides was performed with the CHOPCHOP algorithm tool (https://chopchop.rc.fas.harvard.edu/). The oligonucleotides (Supplementary Table S1) were annealed and cloned into the linearized vector (FastDigest Esp3I; ThermoFisher Scientific). After transfection of 293T cells with the lentiCRISPR plasmids, and the psPAX2 and pMD2.VSVG packaging constructs using Lipofectamine 2000 (Invitrogen), the virus-containing media was harvested and filtered through a 0.45 μmol/L PVDF syringe filter (Millipore). DU145 and PC3 cells were transduced with the virus-containing media and 8 μg/mL polybrene (Sigma). The selection was performed with puromycin (Invivogen). Knockdown of mTOR expression which was confirmed by Western blotting was only efficient in PC3 cells. Initially, five different oligonucleotide sequences were used. The constructs with the highest knockdown efficiencies were chosen for further experiments.
Data analysis
Data analysis were performed with Microsoft Excel 2010 or R. Fold change was calculated by normalization of measured values to the corresponding control. For survival and densitometric data, a logarithmic transformation was performed before statistical significance was calculated (15). To test the statistical significance, the unpaired, two-sided Student t-test or the Wilcoxon rank test was used. Results were considered statistically significant if a P value of less than 0.05 was reached. Densitometry of Western blot analyses was performed with ImageJ or with Image Studio Lite 4 (LI-COR).
Results
Genes in the mTOR/AKT signaling axis are differentially expressed in prostate cancer compared with normal tissue
Target overexpression in cancer cells compared with the corresponding normal tissue can be an important factor for the efficacy of molecular therapeutics (38). To analyze the expression of AKT1, AKT2, AKT3, MTOR, and associated genes in prostate cancer and the normal prostate gland, we used the TCGA database (NCI, NIH; ref. 39), with a total of 497 prostate cancer samples and 52 prostate normal tissue samples. We found a significant upregulation of AKT1, AKT2, MTOR, BAD, and EIF4EBP1 in cancer tissue, whereas FOXO1 and FOXO4, which are inhibited by AKT at the protein level (40), showed a decreased gene expression (Fig. 1). These results indicate an activation of the mTOR/AKT signaling axis in human prostate cancer. Therefore, targeting of these molecules might be a promising approach for cancer treatment.
AKT/mTOR signaling axis genes are differentially expressed in prostate cancer compared with normal prostate gland. Gene expression (TCGA normalized mRNA expression data, log scale) of the indicated genes in 52 normal prostate gland samples and 497 prostate cancer samples shown as box plots. For statistical analysis, the Wilcoxon rank test and the Bonferroni adjustment were used (*, P < 0.003; **, P < 0.0006).
Expression of AKT/mTOR pathway related genes is not altered after radiation in prostate cancer cells
To investigate whether the transcriptome of potential targets (Fig. 2A) can be upregulated by radiation, prostate cancer cells were either irradiated in a 3D extracellular matrix or as xenograft tumors in nude mice. RNA from in vitro and in vivo samples was collected at 24 hours after the delivery of a SD of 10 Gy or after the last dose of two different fractionated regimens (Supplementary Fig. S1). As shown in Fig. 2B, neither SD radiation nor multifractionated radiotherapy significantly affected expression of mTOR/AKT pathway-related genes (Fig. 2B and Supplementary Fig. S2). However, it has recently been shown that changes in the transcriptome after radiation do not necessarily correlate with the protein expression (26). Therefore, we next analyzed the proteome and phosphoproteome of potential targets before and after irradiation.
Radiation does not alter AKT and MTOR gene expression. A, Schematic of the AKT/mTOR signaling network, which was included in the gene array. B, mRNA expression of indicated genes in 3D-cultured PC3 cells and PC3 xenografts irradiated with a SD of 10 Gy or with a multifractionated regimen of five times 2 Gy or 10 times 1 Gy (2 Gy per day). Cells were lysed at 24 hours after the final radiation dose. After RNA isolation, cDNA was synthesized, and qRT-PCR was performed. mRNA expression was determined using the RT² Profiler PCR Array (Human PI3K-AKT Signaling Pathway) from Qiagen. Results show mean ± standard deviation (STDEV) (n = 3).
Radiation induces increased phosphorylation of the AKT/mTOR signaling axis
Analysis of the basal protein expression showed increased levels of total AKT and p70S6K in PC3 cells, whereas a higher phosphorylation of EGFR (Y1068) and p70S6K (S371) was detected in DU145 cells (Fig. 3A). Both cell lines showed a similar expression of total mTOR and EGFR and an overall low expression of phosphorylated AKT and mTOR (Fig. 3A and Supplementary Fig. S3). To determine whether the protein phosphorylation is induced by the different radiation regimens, we performed a reverse phase protein array (RPPA)-based protein pathway activation mapping analysis. The RPPA analysis disclosed a radiation-induced phosphorylation of AKT after SD radiation in both cell lines and after multifractionated radiotherapy in PC3 cells (Fig. 3B). To investigate whether these results translate to an in vivo setting, PC3 xenograft tumors were exposed to different radiation treatment regimens (Supplementary Fig. S1) and collected at 24 hours after the delivery of the last dose for protein analysis. As shown in Fig. 3C, radiation increased the phosphorylation of AKT at S437 and T308 in vivo, confirming the in vitro results (Fig. 3C). In contrast, the mTOR phosphorylation was only slightly enhanced, which could be due to different phosphorylation kinetics in xenograft tumors and in cell culture samples.
Protein phosphorylation of AKT and mTOR is increased after radiation. A, Basal protein expression and phosphorylation of 3D-grown prostate cancer cells. β-Actin served as loading control. B, RPPAs data of DU145 and PC3 cells after radiation with a SD of 10 Gy or with a multifractionated (MF) regimen of five times 2 Gy or 10 times 1 Gy (2 Gy/day). Phosphorylation of indicated proteins was determined at 2, 6, 24, and 48 h after radiation. Fold change was calculated using the corresponding unirradiated control obtained at the same time point as the irradiated sample. Red boxes indicate an increased phosphorylation (fold change > 1.5), and blue boxes indicate a decreased phosphorylation (fold change < 0.67) at at least one of the four time points (2, 6, 24, or 48 hours) after radiation. White boxes indicate no change (0.67 ≤ fold change ≤ 1.5) at any of the four time points (2, 6, 24, or 48 hours) after radiation. C, Protein phosphorylation and expression in PC3 xenografts at 24 hours after the final radiation dose. β-Actin served as loading control.
AKT/mTOR interaction is upregulated after multifractionated radiotherapy
Next, we evaluated protein phosphorylation kinetics, to determine the time point with the highest AKT/mTOR signaling activity after radiation as the start of drug exposure. We used a total dose of 6 Gy instead of 10 Gy because the cellular surviving fraction after 10 Gy was already very low making it difficult to measure additional cell killing by inhibitor treatment. At 2 hours after multifractionated radiation, we found an increase in AKT S473 phosphorylation in both cell lines, while the highest phospho-AKT expression after SD radiation was at 6 hours (Fig. 4A and B). To evaluate whether the observed increase in AKT phosphorylation is caused by changes in mTOR complex formation, co-precipitation of mTOR with either Rictor (mTORC2) or Raptor (mTORC1) was performed. As shown in Fig. 4C mTOR complex formation was not changed by irradiation indicating that the observed changes in AKT phosphorylation might be due to an enhanced interaction of mTORC2 with AKT. To test this hypothesis, we analyzed the subcellular localization and interaction of AKT and mTOR after radiation with immunofluorescence staining and proximity ligation assays. The immunofluorescence staining showed that in both cell lines, independent of radiation, AKT was located in the cytoplasm and in the nucleus, whereas mTOR was mainly expressed in the cytoplasm (Supplementary Fig. S4). The interaction of mTOR and AKT assessed with proximity ligations assays was not changed after SD radiation but significantly increased after multifractionated radiotherapy (Fig. 4D), correlating with the changes in AKT phosphorylation detected at 2 hours after radiation (Fig. 4A). These data show that multifractionated radiation provokes an interaction between mTOR and AKT resulting in an enhanced AKT phosphorylation and activation.
AKT and mTOR colocalize after multifractionated radiation. A, Western blot and B, densitometric analysis of 3D-grown DU145 and PC3 cells 2, 6, or 24 hours after irradiation with a SD of 6 Gy or after three fractions of 2 Gy. β-Actin served as loading control. Fold change was calculated by normalization to unirradiated controls (0 Gy). Results show mean ± STDEV (n = 3; *, P < 0.05; **, P < 0.01; Student t test). C, Immunoblot (IB) analysis of mTOR, Rictor, and Raptor on Rictor and Raptor immunoprecipitates (IP). Whole cell lysates of 3D-grown DU145 and PC3 cells 2 hours after multifractionated irradiation (three times 2 Gy) were used as input lysates for the immunoprecipitation. D, Proximity ligation assay using AKT and mTOR antibodies in cells irradiated with a SD of 6 Gy or three fractions of 2 Gy. Unirradiated cells were used as control. Interactions were measured using secondary antibodies labeled with single stranded DNA. After the DNA was ligated, amplified, and stained, interactions for each condition were counted using ImageJ. Results show mean ± STDEV (n = 3; **, P < 0.01; Student t test).
Pharmacological inhibition of radiation-induced AKT/mTOR activity decreases cellular survival
To assess the potential of targeting the observed radiation-induced mTOR/AKT activation, we treated the cells with INK128 (mTOR inhibitor, mTORi) or GDC-0068 (AKT inhibitor, AKTi). Both inhibitors are currently under clinical investigation and have been used in clinical trials (https://clinicaltrials.gov/). Although treatment with the AKT inhibitor resulted in decreased phosphorylation of the downstream targets GSK3β in PC3 cells and FOXO1 in both cell lines, pharmacological inhibition of mTOR diminished growth factor-induced AKT phosphorylation but failed to reduce FOXO1 phosphorylation (Fig. 5A). As shown in Fig. 5, treatment with AKTi or mTORi (Fig. 5B) significantly decreased the radiation survival of PC3 cells in a treatment time-independent manner (Fig. 5C and D). However, in DU145 cells, both inhibitors were only effective when applied after multifractionated radiation, indicating that radiation-induced activation of prosurvival pathways can be exploited to increase the susceptibility of drug-resistant cancer cells to targeted therapy. To examine whether the observed findings in DU145 cells were due to differences in radiation-induced DNA damage repair, the γH2AX foci assay was used. Although AKT and mTOR inhibition had no effect on the number of γH2AX foci in unirradiated cells, we saw a significant increase in γH2AX foci when the mTOR inhibitor was combined with multifractionated radiation (Fig. 5E).
AKT and mTOR inhibition after irradiation decreases radiation survival of prostate cancer cells. A, Western blots of serum-starved DU145 and PC3 cells after treatment with DMSO or two different doses of the AKT inhibitor (AKTi; 0.5 μmol/L, 5 μmol/L) GDC-0068 or the mTOR inhibitor (mTORi; 10 nmol/L, 50 nmol/L) INK128 for 1 hour prior to stimulation with FBS (final concentration 10%). After 15 minutes, cells were lysed. β-Actin was used as loading control. B, Treatment scheme of 3D cell cultures with inhibitors and/or irradiation. Survival data (C) and representative images (D) of 3D colony formation assays. Cells were treated as described in B with AKTi (0.5 μmol/L) or mTORi (10 nmol/L). Results show mean ± STDEV (n = 3; *, P < 0.05; **, P < 0.01; Student t test). E, γH2AX foci analysis at 24 hours after the last radiation dose of three times 2 Gy in DU145 cells. Cells were treated with AKTi (0.5 μmol/L) or mTORi (10 nmol/L) either 1 hour before radiation or 2 h after the final radiation dose. DMSO-treated cells were used as control. Results show mean ± STDEV (n = 3; *, P < 0.05; **, P < 0.01; Student t test).
mTOR depletion attenuates radiation-induced AKT signaling and modulates the cellular radiation response
To further delineate the effect of mTOR inhibition on the cellular radiation response, we used CRISPR/Cas9 gene editing. The degree of mTOR depletion was dependent on the oligonucleotide sequence (Fig. 6A and B). Cells expressing a CRISPR/Cas9 complex with a nontargeting sequence (NTS) were used as controls. As shown in Fig. 6B, crMTOR#3, which reduced the mTOR expression to 3% of the mTOR level in NTS cells, had the highest knockdown efficiency (Fig. 6A and B). Interestingly, although both Rictor and Raptor form a complex with mTOR, only Rictor expression was downregulated in mTOR-depleted cells. After fractionated radiation, AKT S473 phosphorylation was increased in NTS control cultures but not in mTOR-depleted cells (Fig. 6C and Supplementary Fig. S5). In parallel, knockdown of mTOR reduced the radiation survival of PC3 cells which was more pronounced after multifractionated radiation (Fig. 6D and Supplementary Fig. S5). These results suggest that mTOR/AKT signaling has an important role for the cellular radiation response of prostate cancer cells.
Depletion of mTOR modulates the cellular radiation response. Western blots (A) and knockdown efficiency (B) of mTOR, and expression of Rictor and Raptor after CRISPR/Cas9-mediated depletion of mTOR (crMTOR) using different targeting sequences. An NTS was used as control. β-Actin expression was evaluated to ensure equal sample loading. C, Western blots of mTOR, phospho-AKT S473, and AKT 2 hours after three fractions of 2 Gy in 3D-grown NTS and crMTOR#3 PC3 cells. D, Representative images and survival data of 3D colony formation assays. Cells were irradiated either with 6 Gy SD or with three fractions of 2 Gy (2 Gy per day). Results show mean ± STDEV (n = 3; **, P < 0.01; Student t test).
Discussion
Understanding the signaling processes and underlying molecular mechanisms in cancer cells determining tumor growth and progression has led to the development of targeted therapeutics, a novel class of oncologic drugs, which are designed to interfere with cancer-specific pathways essential for tumor survival. Ideally, the targeted molecules are overexpressed in cancer cells compared with the corresponding normal tissue to achieve a pronounced tumor response and in parallel minimize the side effects. Among the promising pathways for specifically targeting tumor cells, is the mTOR/AKT pathway. Activation of mTOR/AKT signaling is a negative prognostic marker in different cancer types (41, 42). Moreover, using human tumor sample gene expression from the TCGA database, we found an overexpression of the AKT1, AKT2, MTOR, and EIF4EBP1 genes, which is indicative of an active mTOR/AKT signaling in prostate tumor cells. Although the efficacy of molecular inhibitors is not only affected by gene expression but also by posttranslational modifications such as protein phosphorylation, the differential MTOR and AKT gene expression in cancer and normal cells support the hypothesis that inhibition of mTOR or AKT might be a promising approach to target malignant prostate tissue more specifically. Interestingly, BAD mRNA was also upregulated in prostate cancer cells compared with normal prostate epithelium which is in line with previous studies (43). Despite its well-known proapoptotic function, increased BAD expression has been found to promote tumor growth of prostate cancer xenografts and seems to have an essential role for cellular proliferation in this tumor type (44).
Our laboratory is investigating the change in phenotype of cells that survive SD and multifractionated radiation (5, 6, 45). Having demonstrated adaptive changes in gene expression, the interest is in identifying how radiation-inducible changes might be exploited to improve radiooncologic treatment. In the study presented here, we could show that radiation did not affect mRNA and total protein expression of mTOR, AKT, and their downstream targets. However, radiation significantly induced protein–protein interactions and protein phosphorylation of amino acids that are known to regulate the enzymatic activity of the proteins in a fractionation-dependent manner. Differential molecular responses after SD or multifractionated radiotherapy have been observed before and may be due to different effects on cellular survival and cell-cycle distribution (6).
In parallel with the increased AKT and mTOR phosphorylation, mTOR and AKT colocalized in the cytoplasm after multifractionated radiation. Inhibition of AKT or mTOR at this time point resulted in a significantly reduced radiation survival of DU145 cells, whereas there was no effect when the inhibitors were applied prior to radiation. These data indicate a higher susceptibility of resistant cells to mTOR or AKT inhibition when the pathway is activated by multifractionated radiation providing a novel approach for optimizing the efficiency of combined radiation/drug treatment within the precision medicine concept. In line with previous results, mTOR inhibition also led to an increase in γH2AX foci as a marker for DNA double-strand breaks after multifractionated radiation (22, 23). Because this effect occurred independently of the treatment time point and therefore did not correlate with tumor cell survival, other mechanisms seem to contribute to the observed findings.
In contrast to DU145 cells, PC3 cells were much more sensitive to AKT/mTOR inhibition in combination with radiotherapy. This could be due to the higher AKT expression in PC3 cells or distinct effects on downstream proteins such as GSK3β after inhibitor treatment. Further, different genetic backgrounds and mutations have also been shown to affect inhibitor efficacy (46). Treatment with the AKT inhibitor GDC-0068 increased the basal and growth factor-induced AKT phosphorylation but diminished AKT downstream signaling, similarly to previous data showing that this paradoxic hyperphosphorylation is caused by an mTORC2-dependent feedback mechanism (47). Surprisingly, although mTOR inhibition reduced AKT phosphorylation, phosphorylation of the AKT downstream target FOXO1 was not affected indicating that either AKT can phosphorylate FOXO1 independently of its own phosphorylation status or another kinase is phosphorylating FOXO1 in these cell lines.
The role of mTOR in the radiation response seems to be at least to some extent mediated by the mTORC2-mediated activation of AKT (23, 48). In glioma and breast carcinoma cells, inhibition of both mTORC1 and mTORC2 decreases radiation survival, while targeting mTORC1 alone has no significant effect (48). Phosphorylation of AKT after mTORC1 inhibition with rapamycin in combination with irradiation has been associated with a lack of therapeutic efficiency in non–small cell lung cancer, while combined targeting of mTORC1 and AKT results in decreased radiation survival of nonresponders (23).
Interestingly, loss of mTOR had differential effects on the protein expression of Rictor and Raptor, which indicates distinct regulatory signaling mechanisms of the two mTOR complexes. Although Raptor levels did not change after genetic mTOR depletion, we found a downregulation of Rictor implying a critical role of mTOR in Rictor stability and function. Similar effects have been described for other signaling complexes (49). In contrast, Sarbassov and colleagues observed no change of Rictor or Raptor expression after siRNA-mediated mTOR knockdown in HeLa cells (50). One possible explanation for this is that the method and the duration of mTOR knockdown plays an important role for the observed effects on Rictor expression. Although siRNA operates on the mRNA level and decreases the target expression only transiently, the CRISPR-Cas9 technique enables the definite deletion of the MTOR gene, which allows to determine also long-term effects.
Here, we show for the first time that multifractionated radiation can induce a molecular response which can be exploited to sensitize resistant cells to targeted therapy and thereby decrease tumor cell survival.
De novo or acquired drug resistance to molecular inhibitors is a highly relevant clinical problem and can be caused by multiple factors, such as decreased target expression, mutations in the target sequence resulting in impaired drug functionality but also changes in downstream molecules or related pathways allowing the cancer cell to bypass the inhibitory effects (51, 52). As the complexity of precision medicine including tumor heterogeneity becomes better understood additional strategies are necessary to enhance the clinical efficacy of molecular-targeted therapy (53, 54). Furthermore, to date, therapy decisions are primarily based on genomic analysis of the tumor prior to treatment without taking into account that the efficiency of targeted drugs is also modulated by protein modifications, which can change during the course of treatment (51). Moreover, systemic therapies including molecular inhibitors are frequently delivered before or simultaneously with multifractionated radiation in the clinical setting. This treatment concept dates back decades and is mainly based on the assumption that conventional cytotoxic drugs such as cisplatin cause radiosensitization and therefore its concurrent use results in more lethal treatment-induced DNA damage (55). Whether this concept is also a reasonable approach for biologicals is highly unclear, because these drugs have completely different mechanisms of action (56). Our results show that the timing of treatment with small molecule inhibitors can significantly affect drug efficiency. Thus, it may be possible to use radiation therapy to “set up” the tumor for enhanced drug-induced killing. In addition to the strategy of concomitant drug therapy to radiosensitize cells, this is a novel use of radiation that potentially also could be applied for immune modulation with check-point inhibitors.
The study has several limitations which need to be addressed in the future before this concept can be translated into clinical practice. Additional in vivo experiments with DU145 or other prostate cancer cells which are resistant to mTOR or AKT inhibition are warranted to elucidate whether drug treatment after multifractionated radiation results in increased tumor control or growth delay. Further, phosphoproteome data of irradiated human prostate cancer samples would help to clarify the molecular mechanisms in patients, albeit this can be very challenging because protein analysis requires greater sample amounts and especially after radiation therapy tumor cell samples are very limited due to tissue necrosis and cell death. Clearly, there is more work to be done, however, the data presented here demonstrate for the first time that it may be possible to use this new paradigm to enhance the efficacy of molecular therapeutics, also potentially enhancing return on investment. Overall, it is important for the design of clinical trials with combined radiation plus drug therapy to identify the optimal treatment schedule. Targeting of radiation-induced molecular changes may hereby be a unique approach that can increase therapy efficiency and repurpose and broaden the utility of molecular targeted precision medicine.
Disclosure of Potential Conflicts of Interest
M. Pierobon has ownership interest (including patents) in Theranostics Health and is a consultant/advisory board member of Perthera, Inc. E.F. Petricoin is a Co-Founder, Board of Directors, and Chief Scientific Officer in Perthera, Inc.; is a Co-Founder and Board of Directors in Ceres Nanosciences, Inc.; has ownership interest (including patents) in Ceres Nanosciences, Inc.; and is a consultant/advisory board member of Ceres Nanosciences, Inc. No potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: I. Eke, A.Y. Makinde, M.J. Aryankalayil, E.F. Petricoin, C.N. Coleman
Development of methodology: A.Y. Makinde, L. Liotta, E.F. Petricoin, J.M. Stommel, C.N. Coleman
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.Y. Makinde, M.J. Aryankalayil, V. Sandfort, B.H. Rath, M. Pierobon, E.F. Petricoin, M.F. Brown
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): I. Eke, A.Y. Makinde, L. Liotta, M. Pierobon, E.F. Petricoin, C.N. Coleman
Writing, review, and/or revision of the manuscript: I. Eke, A.Y. Makinde, M.J. Aryankalayil, V. Sandfort, B.H. Rath, J.M. Stommel, C.N. Coleman
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.J. Aryankalayil, S.T. Palayoor
Study supervision: I. Eke
Other (advisory role and provided critical review and inputs on this manuscript): M.M. Ahmed
Acknowledgments
This study was supported by the NIH Intramural Research Program, National Cancer Institute, Center for Cancer Research, grants ZIA BC 010670 (to C. N. Coleman) and ZIA BC 011441 (to J. M. Stommel).
The authors thank David Cerna (NCI/NIH) and Joel Levin (NCI/NIH) for excellent technical assistance, and the NIH Fellows Editorial Board for editorial assistance. The results shown here are in part based upon data generated by the TCGA Research Network (http://cancergenome.nih.gov/). We would like to thank the TCGA Research Network and the specimen donors.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Footnotes
Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).
- Received March 22, 2017.
- Revision received June 21, 2017.
- Accepted August 1, 2017.
- ©2017 American Association for Cancer Research.
References
Volume 17, Issue 2
