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To determine if the mTOR inhibitor CCI-779 can sensitize head and neck squamous cell carcinoma (HNSCC) to radiotherapy (XRT) and compare the radiosensitizing effects to cisplatin with its known considerable toxicity.
Radiosensitizing effects of CCI-779 were assayed on HNSCC cell lines in vitro. CCI-779 (5mg/kg), cisplatin (1 mg/kg) and XRT (2 Gy) alone and in combination were evaluated for antitumor activity in mice bearing FaDu and SCC40 xenografts. Effects of CCI-779 on radiation-induced activation of the Akt/mTOR pathway were analyzed.
Although CCI-779 did not sensitize HNSCC cells to ionizing radiation in vitro, combination of CCI-779 and XRT significantly augmented the in vivo tumor growth inhibitory effects of XRT and CCI-779 (P<0.05). In addition CCI-779+XRT suppressed tumor growth more effectively than cisplatin+XRT (P<0.05). CCI-779+XRT significantly improved survival compared to XRT alone in both cisplatin-sensitive FaDu (P<0.01) and cisplatin-resistant SCC40 xenograft mice (P<0.05). There were no additional benefits of adding cisplatin to CCI-779+XRT. CCI-779 significantly attenuated irradiation-induced upregulation of the mTOR pathway, increased apoptosis and displayed potent antiangiogenic activity in FaDu xenografts that was further enhanced by its combination with radiotherapy (P<0.05) which may explain the mechanism of its selective radiosensitizing effects in vivo and not in vitro.
Antitumor activity of radiotherapy was enhanced when combined with CCI-779 in HNSCC xenograft model. CCI-779+XRT showed antitumor activity superior to conventional chemoradiotherapy with cisplatin. These results pave the way for clinical trials utilizing molecular targeted therapy with CCI-779 in combination with radiotherapy for HNSCC treatment.
Head and neck cancer is one of the six most frequent cancers world-wide (1). Squamous cell carcinoma of the head and neck remains a challenge because of the high locoregional recurrence rates, approximately 50% in advanced stage disease (2, 3). The combination of organ preservation cisplatin-based chemoradiotherapy is now used as standard treatment (4). However, the incidence of severe acute adverse effects was higher in the group receiving cisplatin-based chemoradiotherapy than in the radiotherapy group alone (77% vs. 34%; (4)). Cisplatin displays considerable toxicity causing significant morbidity as a result of dysphagia, prolonged use of percutaneous endoscopic gastrostomy (PEG) tube and tracheostomy tubes (5). Additionally many patients acquire resistance to cisplatin during therapy suggesting a need for other treatment strategies (6).
The use of molecularly targeted agents in combination with radiotherapy is a promising strategy. Cetuximab, an epidermal growth factor receptor (EGFR) monoclonal antibody, has been approved by the FDA for the treatment of HNSCC patients. The addition of cetuximab to radiotherapy is beneficial in a small fraction of HNSCC patients (10–15%), and some patients acquire cetuximab resistance (7–9). The presence of mutant EGFR (EGFRvIII) detected in 42% of HNSCC tumors might contribute to the limited clinical response. EGFRvIII-overexpressing cells and xenografts were more resistant to cetuximab treatment compared to cells and xenografts overexpressing wild-type EGFR (10). Moreover in models of non-small cell lung cancer and colon cancer, persistent activation of the PI3K/Akt/mTOR pathway was associated with resistance to anti-EGFR drugs, including cetuximab (11, 12). HNSCC is characterized by persistent activation of the Akt/mTOR pathway that leads to phosphorylation of p70 S6 kinase and 4E-BP1 (13). The anti-apoptotic role of Akt/mTOR accounts not only for its transforming potential, but also for the resistance of cancer cells to the action of chemotherapeutic agents and ionizing radiation (14).
The PI3K/Akt/mTOR kinase pathway is a central regulator of cell metabolism, proliferation, and survival, and is upregulated in many tumors, including HNSCC (15–17). Although radiotherapy initiates cytotoxic cellular mechanisms through the induction of DNA damage and activation of the pro-apoptotic ASK1/JNK pathway, ionizing radiation also activates the anti-apoptotic PI3K/Akt/mTOR pathway. For example, ionizing radiation activates mTOR signaling in breast cancer cells (18) and in T lymphocytes (19). Rapamycin analogues, such as CCI-779 and RAD001, effectively inhibit signal transduction downstream of mTOR, attenuating cap-dependent translation of many pro-oncogenic and pro-survival factors, suggesting that rapamycin analogues can be potentially used as radiosensitizing agents. mTOR inhibitors are well tolerated by patients with mild, manageable and reversible toxicities (20–23). The radiosensitizing effects of mTOR inhibitors have been shown in breast and prostate cancer cell models (24, 25) and on glioma xenografts in mice (26). Radiosensitizing properties of mTOR inhibitors in HNSCC have not been elucidated. Moreover, antitumoral effects of mTOR inhibitors combined with radiation therapy have not been compared to conventional cisplatin-based chemoradiotherapy in any other organ system.
The goal of this study is to determine if the mTOR inhibitor CCI-779 can sensitize HNSCC to radiotherapy in both in vitro and in vivo models and to compare the radiosensitizing effects of CCI-779 to cisplatin. In addition, the effects of radiation on pro- and anti-apoptotic pathways in HNSCC are elucidated.
The HNSCC cell line FaDu (derived from a hypopharyngeal SCC) was obtained from the American Type Culture Collection (Manassas, VA). SCC40 (tongue cancer) and SCC66 (established from locally advanced primary cancer of the floor of mouth) were kindly provided by Dr. Susanne Gollin and PCI-15a (pyriform sinus cancer) was provided by Dr. Theresa L. Whiteside (both from the University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA, USA). All cell lines were maintained in MEM (Sigma-Aldrich; St. Louis, MO, USA), supplemented with 10% bovine calf serum, non-essential amino acids (Gibco - BRL) and 100 units of penicillin with 100 µg of streptomycin. Cells were grown in monolayers and maintained in humidified 5% CO2 atmosphere at 37°C. Chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) except CCI-779 provided by Wyeth-Ayerst Research.
Radiation sensitivities of the HNSCC cell lines were determined by measuring colony formation after cells were exposed to ionizing radiation. Exponentially growing cells were pretreated with vehicle (DMSO) or CCI-779 at concentrations of 10 or 100 ng/ml or cisplatin at concentrations of 0.5 µM or 1.5 µM for 24 h. The cells were then washed, suspended at a density of 2×105 cells/ml, and irradiated with escalating doses of radiation from 0 to 8 Gy using a 137Cs γ-ray source (J.L. Shepard and Associates, San Fernando, CA). After irradiation, cells were serially diluted, and resuspended in fresh growth medium without CCI-779 or cisplatin. Cells were seeded in triplicate at various densities dependent on plating efficiency in 60-mm culture dishes to achieve about 50–100 colonies per dish. The colonies were allowed to grow for 14 days, stained with Gentian violet (1% Gentian violet, 70% ethanol, 5% formaldehyde), and colonies, defined as >50 cells, were counted. The relative surviving fraction was determined by dividing the plating efficiency of the irradiated cells by the plating efficiency of the unirradiated control in at least three independent experiments. A Student's t-test was used to compare differences in clonogenic survival between treatment groups.
The cytotoxic effect of cisplatin treatment on FaDu and SCC40 cells was evaluated using the CellTiter 96® AQueous cell proliferation assay according to the manufacturer’s instructions (Promega Corp., Madison, WI). Sensitivities of the cell lines to cisplatin were expressed as IC50 values. The growth-inhibitory effect of CCI-779 on HNSCC in vitro was tested in our laboratory previously (27).
The effects of CCI-779 treatment and ionizing irradiation on VEGF expression in FaDu and SCC40 cells was determined with the ELISA commercial kit (R&D Systems, Minneapolis, MN) as described previously (27).
In the xenograft model, Balb/c nu/nu mice (Harlan, Indianapolis, IN) were injected subcutaneously with 1×106 FaDu or SCC40 cells. These two cell lines were chosen because of their relative differences in sensitivity to cisplatin. Animals were housed in a barrier facility and maintained on a normal diet ad lib. All studies were conducted in compliance with the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee guidelines.
Tumor volume ((length × width2)/2) was determined with a digital caliper.
The flow diagram of the in vivo studies is shown in Supplementary Figure 1. In order to evaluate the early effects of CCI-779 and radiation therapy on the Akt/mTOR pathway and pro-apoptotic factors a group of mice were injected with FaDu cells. When tumors reached approximately 300–600 mm3 mice were divided randomly into several groups with 3–4 mice per group and treated either with vehicle, 5 mg/kg of CCI-779, targeted radiation at a single 2 Gy fraction or combined CCI-779 and radiation. Tumors were harvested at 0, 1, 2 and 6 hours after treatment, snap frozen in liquid nitrogen, and protein was extracted at a later time.
To determine prolonged effects of CCI-779 and XRT on the mTOR pathway six mice from each group of the FaDu xenograft experiment described below were randomly selected to be sacrificed after three weeks of treatment and prior to the survival arm of the study to assay tumors for the mechanisms of anti-tumor action of different treatment modalities.
Antitumor activities of CCI-779, cisplatin and XRT alone and in combination were evaluated in mice injected subcutaneously with FaDu or SCC40 cells. When tumors reached approximately 40 mm3 (defined as day 0), mice with established xenografts were stratified by tumor volume and randomized into experimental groups as follows: 1) control, vehicle only; 2) 5 mg/kg CCI-779 intraperitoneally (i.p.) five times a week; 3) 1 mg/kg cisplatin i.p. twice a week before irradiation; 4) CCI-779 + cisplatin; 5) XRT at 2 Gy fractions twice a week for three weeks; 6) cisplatin + XRT; 7) CCI-779 + XRT; and 8) CCI-779 + cisplatin + XRT. CCI-779 was prepared in 4% ethanol, 5.2% Tween 80, 5.2% PEG 400 and administered i.p. The dose of 5mg/kg of CCI-779 was chosen as it was the lowest dose tested in nude mice in our previous study (27). Cisplatin was prepared in sterile 0.9% saline, stored at 4°C and protected from light until use. The dose of 1 mg/kg of cisplatin i.p. was chosen as a well-tolerated dose with significant anti-tumor efficacy against human oral squamous carcinoma xenografts in nude mice (28). Maximum tolerated dose of cisplatin for nude mice is approximately 2.9 mg/kg (28). Both CCI-779 and cisplatin were injected 1 hour prior to XRT. The radiation groups received targeted irradiation delivered by an SL1 Plus linear accelerator with 6-MV photons (Elekta; Norcross, GA) at 2 Gy fractions. Each mouse was immobilized in a plastic tube during XRT. A sheet of 1 cm thick bolus (MedTec, Orange City, IA) was overlaid on animals covering the entire irradiated area, to ensure tumors receive a maximum dose of 6MV photon. All mice were treated for 3 weeks with 11–12 mice per group for the FaDu xenograft experiment and 5–6 mice per group for the SCC40 xenograft experiment. Tumors were measured twice a week. Xenograft tumor volumes were compared statistically on day 12 (FaDu) or day 17 (SCC40), the last day when all mice were still alive. In both experiments some mice in the control and cisplatin alone groups were sacrificed due to severe tumor burden before the 3-week treatment was completed. The Kruskal-Wallis one-way analysis of variance (ANOVA) test was used to determine significant differences among the treatment groups. When an overall significant difference was indicated by the test, pairwise comparisons among the treatment groups were performed using the Wilcoxon rank-sum test to determine which pairwise difference contributed to the overall significant difference.
As a surrogate marker of toxicity, body weight was measured 2–3 times a week for the duration of the experiment without any differences observed between treated and control animals (data not shown).
FaDu xenograft tumors from the mice treated for 3 weeks were fixed overnight with 10% phosphate-buffered formalin, rinsed twice with 50% ethanol and embedded in paraffin. 5µm tissue sections were deparaffinized and stained with CD31 antibody to determine microvessel counts. CD31 (PECAM-1) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at 1:300 dilution. Using low power magnification, the regions containing the most intense area of vascularization were chosen for counting in each of the tumors. Individual microvessels were counted using a 400× field (40× objective lens and 10×occular lens). Any stained endothelial cells that were clearly separate in appearance were counted as individual vessels. Six random fields within the areas of intense vascularization were viewed and counted at 400×. Results were expressed as the average number of microvessels per field. To evaluate the differences between treatment groups one-way ANOVA and Tukey's multiple comparison tests were performed.
Apoptosis was assayed by detecting terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in paraffin sections using a commercially available kit (In Situ Cell Death Detection Kit, Roche) and performed according to the manufacturer’s instructions. Slides were mounted with Vectashield mounting medium, containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). TUNEL labeling was observed using a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Inc., Gottingen, Germany). Images were acquired using AxioVision software and an AxioCam MRm digital camera (Zeiss). The number of TUNEL-positive and total number of nuclei (DAPI-positive) per field of view were counted automatically after the threshold was set manually and this procedure was repeated in at least five fields from non-necrotic regions of each tumor section. Percent of TUNEL-positive nuclei was counted separately for each image and averaged for the sample.
In the survival arm of the xenograft study, treatment was discontinued after 3 weeks and mice were followed until one of the following end points were reached: xenograft volume of 2000 mm3, mice lost over 15% of their body weight, or the maximum time point of 60 days after the treatment initiation. Kaplan-Meier curves in combination with log rank test were used for survival analysis.
Protein was extracted directly from cells and tumor tissues from the established tumor model (~5 mg) using lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4; Cell Signaling, Beverly, MA) containing 1x Protease Inhibitor Cocktail (Roche Molecular Biochemicals, IN). Western blot analysis was done according to previously published laboratory protocol (16). The following antibodies were used: anti-4E binding protein 1 (4E-BP1), anti-S6 ribosomal protein, anti-phospho S6 ribosomal protein (serine 235/236), anti-phospho-4E-BP1 (serine 65), anti-Akt, anti-phospho-Akt (serine 473), anti-phospho-Akt (threonine 308), anti-Bad, anti-phospho-Bad (serine 136), anti-β-actin and anti-poly (ADP-ribose) polymerase (PARP). All antibodies were obtained from Cell Signaling, Beverly, MA, except PARP antibody obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
To determine whether CCI-779 sensitizes HNSCC cell lines to radiation therapy, clonogenic assays were performed (Figure 1). In vehicle-treated samples the surviving fractions were 0.68±0.06, 0.64±0.12, 0.75±0.07, and 0.46±0.05 after 2 Gy of irradiation for FaDu, SCC40, PCI 15a and SCC66 respectively. There was no significant change in the surviving fractions of cells in the presence of CCI-779 at all doses tested compared to controls for all HNSCC cell lines (Figure 1; Supplementary Figure 2). The results demonstrate that neither 10 nor 100 ng/ml of CCI-779 had any significant effects on the sensitivity of the HNSCC cell lines to radiation. CCI-779 treatment also had no significant effect on plating efficiency in all three HNSCC cell lines.
We also tested the radiosensitizing effects of cisplatin on FaDu, SCC40 and PCI 15a cell lines (Figure 1). Cisplatin treatment had significant radiosensitizing effects on FaDu (P<0.05 for 0.5 µM of cisplatin at 4 and 8 Gy and for 1.5 µM of cisplatin at exposure to all levels of radiation tested). Cisplatin treatment decreased plating efficiency of FaDu by 41.6±14.4% and 96.9±1.3% at 0.5 and 1.5 µM respectively. In the PCI 15a cell line only a higher dose of cisplatin (1.5 µM) had significant radiosensitizing effects. Cisplatin treatment decreased plating efficiency of PCI 15a cells by 14.6±1.9% and 76.5±2.6% at doses of 0.5 and 1.5 µM respectively. On the contrary, cisplatin did not sensitize SCC40 cells to radiation and affected plating efficiency of SCC40 at 1.5 µM but not 0.5 µM with a decrease of plating efficiency of only 37.9±7.8% at 1.5 µM. The results of the proliferation assay confirmed that FaDu is more sensitive to cisplatin with an IC50 of 3.9±0.4 µM, while SCC40 is a relatively cisplatin-resistant cell line with an IC50 of 7.6±0.8 µM (P<0.01). Based on their differential sensitivities to cisplatin these two cell lines were selected for the in vivo studies.
The effects of 3-week CCI-779 treatment on the sensitivity of FaDu (cisplatin-sensitive) and SCC40 (cisplatin-resistant) xenografts to radiation were compared to cisplatin (Table 1). On day 0 when mice were stratified into experimental groups, tumor volumes averaged approximately 40 mm3 and were not significantly different between groups. Tumor growth curves for FaDu and SCC40 xenografts are shown in Figure 2A,B. CCI-779 treatment alone, even at the low dose of 5 mg/kg, displayed remarkable inhibition of tumor growth comparable to the effect of XRT alone in both HNSCC xenografts. The addition of CCI-779 to radiation therapy further enhanced radiation-induced tumor regression (P<0.05 vs. CCI-779 or XRT alone). CCI-779+XRT suppressed xenograft growth significantly more than cisplatin+XRT for both cisplatin-sensitive FaDu and cisplatin-resistant SCC40 cell lines (P<0.05). Notably, the addition of cisplatin to CCI-779+XRT had no additional benefit compared to CCI-779+XRT.
The Kaplan-Meier survival curves for the FaDu xenograft survival study are shown in Figure 2C. The median survival time of the control group was only 19 days (Table 2). CCI-779 significantly extended survival time compared to control (35 days; P<0.05), which was comparable to median survival time in the XRT alone group (37 days). A combination of CCI-779 with XRT significantly increased survival of FaDu xenograft mice (P<0.05 vs. CCI-779 or XRT alone). Notably CCI-779 alone prolonged the median survival time by 16 days compared to control and radiation therapy which prolonged the median survival time by 18 days compared to the control group. However the combination of CCI-779+XRT prolonged the median survival time by 37 days as compared to control, which is greater than the sum (16+18=34) of single-modality treatments, suggesting a synergistic effect of combined treatment on survival in FaDu xenograft mice. Mice bearing the relatively cisplatin-sensitive FaDu xenografts treated with the combination of CCI-779+XRT had a median survival time >56 days which represented an improved survival compared to XRT alone which, in turn, was as effective only as conventional chemoradiotherapy, i.e. cisplatin+XRT with a median survival time of 47.5 days. There was no additional benefit of combining both cisplatin and CCI-779 with XRT (median survival time 52 days).
The Kaplan-Meier survival curves for mice with the cisplatin resistant SCC40 xenografts are shown in Figure 2D. The median survival time of the control group in the SCC40 survival study was 27 days (Table 2). The combination of cisplatin with XRT (median survival time 38 days) failed to significantly improve survival of mice bearing the relatively cisplatin-resistant SCC40 xenograft tumors compared to control. However, all CCI-779-based treatment modalities significantly improved survival of SCC40 xenograft mice as compared to either control, radiotherapy alone, or chemoradiotherapy with cisplatin groups (P<0.05). The median survival time for groups treated with CCI-779 was >49 days (Table 2).
The early effects of a single treatment of ionizing radiation (2 Gy) and CCI-779 (5 mg/kg in vivo or 24 h treatment of CCI-779 at 10 ng/ml in vitro) on the Akt/mTOR pathway were analyzed. Western blot analyses of tumors from irradiated FaDu xenografts showed that ionizing radiation activates anti-apoptotic, pro-survival Akt/mTOR signaling. Although Akt phosphorylation at Ser-473 was upregulated after irradiation pAkt-Thr-308 expression did not change (Figure 3A). Western blot analysis of irradiated FaDu and SCC40 cells revealed that in contrast to the in vivo data, radiation had no effect on Akt phosphorylation in vitro (Figure 3B). We tested the effect of ionizing radiation (2 Gy) and CCI-779 treatment on downstream targets of mTOR, 4E–BP1, phospho-S6 ribosomal protein, and apoptotic markers. CCI-779 treatment effectively inhibited phosphorylation of S6 ribosomal protein at Ser235/236 (a physiological downstream target of p70 S6 kinase) (Figure 3B,C) and 4E–BP1 in HNSCC cell cultures and FaDu xenograft tumors as evidenced by a shift of the more phosphorylated ‘δ’ and ‘γ’ isoforms and to the less phosphorylated ‘β’ isoform and non-phosphorylated ‘α’ isoform after western blot analysis of total 4E–BP1 (Figure 3C). Treatment of FaDu and SCC40 cells with CCI-779 significantly inhibited VEGF secretion in the medium when compared to the control or irradiated samples (Figure 3B). CCI-779 treatment significantly inhibited expression of pBad(Ser136) in FaDu xenograft tissues (P<0.05; Figure 3C), which facilitates apoptosis. We found that PARP cleavage, a crucial early marker of apoptosis, was up-regulated in FaDu xenograft tumors at 6 hours after irradiation (increased from 6% in control to 29% at 6 hours after irradiation; Figure 3C). CCI-779 treatment alone increased the percentage of cleaved PARP to 21% and substantially augmented radiation-induced cleavage of PARP to 40%, which also occurred at an earlier time point i.e. 2 hours after irradiation (Figure 3C), suggesting that the drug can facilitate apoptotic cell death caused by radiation in tumor tissues, and therefore serve as a radiosensitizing agent.
CCI-779 treatment upregulated Akt phosphorylation at Ser-473 in both in vitro (Figure 3B) and in vivo (Figure 3C,D) settings indicating that in HNSCC experimental models mTOR inhibition is disrupting the negative feedback loop that suppresses PI3K/Akt signaling when S6K is phosphorylated. The changes in Akt-Thr-308 phosphorylation after CCI-779 were not significant (Figure 3B,C). Although ionizing radiation treatment did not upregulate Akt phosphorylation in our in vitro studies (Figure 3B) it caused an increase in pAkt-Ser-473 expression in xenograft tissue as early as two hours after irradiation (Figure 3A,C,D). Moreover, after 3 weeks of treatment all three therapeutic agents (cisplatin, CCI-779, XRT and their combinations) caused an increase of pAkt expression (both sites) in the xenograft tumor tissues when compared to control (Figure 3D). Importantly, CCI-779 treatment did not further exacerbate cisplatin- and XRT-induced upregulation of Akt phosphorylation at Ser-473 and Thr-308.
The effects of prolonged 3-week treatments with CCI-779, cisplatin, and radiotherapy on mTOR signaling were examined in the FaDu xenograft model. While we did not observe radiation-induced upregulation of 4E–BP1 and S6 ribosomal protein at early time points after irradiation (Figure 3C), 3 weeks of radiotherapy (6 fractions of 2 Gy) resulted in a 2-fold increased expression of pS6 and promoted phosphorylation of 4E–BP1 as evidenced by a shift from less phosphorylated to more phosphorylated isoforms (Figure 3D). In contrast, CCI-779 decreased pS6 and caused a shift of 4E–BP1 phosphorylated bands to the unphosphorylated and less phosphorylated isoforms and significantly attenuated radiation-induced upregulation of these mTOR downstream factors (P<0.05).
The absence of radiation sensitizing effects noted in the in vitro group but not in the in vivo groups where CCI-779 augmented the tumor growth inhibitory effects of radiotherapy led us to evaluate the effects of CCI-779 on tumor-stromal interactions. To examine these interactions we assessed the effects of CCI-779 on angiogenesis by counting the number of blood vessels in CD31-stained sections of xenograft tumors following 3 weeks treatment. At 400× magnification the average counts per field were as follows: 30.4±5.8 (mean±SD) blood vessels for control tumors, 26.3±4.7 for radiation alone and 25.2±1.4 for chemoradiotherapy with cisplatin (Figure 4). Radiotherapy and chemoradiotherapy with cisplatin did not reduce the number of vessels significantly. Treatment with CCI-779 alone significantly reduced the intratumoral microvessel density compared to control (17.6±2.3 vessels per field; P < 0.001 vs. control and P < 0.05 vs. irradiation alone). The combination of CCI-779 with radiotherapy even further enhanced the anti-angiogenic effects of CCI-779 (10.8±2.3 vessels per field; P < 0.001 vs. radiotherapy alone; P < 0.05 vs. CCI-779 alone).
Apoptosis was assessed by TUNEL staining in xenograft tumor tissues at early times after irradiation (2 h) and CCI-779 treatment (24 h) and following 3 weeks treatment. Xenograft tumor tissues from vehicle-treated mice in the early effects study had 1.52±0.27% (mean±SE) of the apoptotic TUNEL-positive nuclei per field. CCI-779 (24 h), XRT (2 h) and CCI-779+XRT treatment significantly increased the percentage of apoptotic nuclei to 3.48±0.09%, 3.41±0.21% and 3.65±0.33% correspondingly (P < 0.05 vs. control). Three weeks treatment further increased the apoptosis in the FaDu xenograft tissues (Figure 4 lower panel). The average percentage of apoptotic nuclei per field were as follows: 2.16±0.45% for control tumors, 4.41±0.57% for CCI-779 alone, 6.48±0.46% for radiotherapy, and 9.3±0.27% for chemoradiotherapy with cisplatin (Figure 4). The combination of CCI-779 and radiotherapy augmented the percentage of apoptotic nuclei to 11.34±0.92% (P < 0.001 vs. radiotherapy alone or CCI-779 alone; difference vs. cisplatin+XRT is not significant). Representative photomicrographs of TUNEL-stained tumor sections from each treatment group are shown in Figure 4.
The growth-inhibitory properties of mTOR inhibitors on various types of cancers have been well studied (29–32). We demonstrated that CCI-779 is an effective agent against HNSCC in both in vitro and in vivo studies (27). The combination of mTOR inhibitors with cytotoxic treatments, such as radiation therapy or chemotherapy, might potentiate the antitumoral effects of mTOR inhibition (33). Hence we wanted to elucidate the efficacy of using CCI-779 in combination with radiotherapy in preclinical HNSCC models and in comparison to cisplatin. The efficacy of combining radiotherapy with mTOR inhibitors have been studied in breast and prostate cancer cell models (24, 25) and in murine glioma xenografts (26). However the effects of CCI-779 and XRT have not been studied in HNSCC and have never been compared to cisplatin and XRT which is a very commonly used radiosensitizer in a number of organ systems.
In radiation therapy, activation of cytotoxic cellular mechanisms, such as induction of DNA damage and upregulation of pro-apoptotic ASK1/JNK pathway, in tumors is favorable. Additionally, irradiation further enhances the anti-apoptotic PI3K/Akt/mTOR pathway, which is already upregulated in many types of cancer, including HNSCC. In our experiments Akt (at Ser-473) and mTOR were activated in FaDu xenografts after irradiation (Figure 3A). Rapamycin analogues, such as CCI-779 and RAD001, effectively inhibit signal transduction downstream of mTOR, attenuating cap-dependent translation of many pro-oncogenic and pro-survival factors, which may explain their radiosensitizing effects.
We found that doses of CCI-779 that significantly inhibit mTOR signaling have a growth-inhibitory effect and importantly, cause significant inhibition of VEGF production in the cells, and did not sensitize HNSCC cell lines to radiation in vitro as evidenced by clonogenic assays. In contrast our in vivo studies suggest that CCI-779 enhances radiation-induced cell death by decreasing 4E–BP1, S6 and Bad(Ser136) phosphorylation while promoting radiation-induced cleavage of PARP and ultimately apoptosis as evidenced by TUNEL staining of apoptotic nuclei. Based on these promising results we hypothesize that in combination with radiation, CCI-779 would be effective for treatment of HNSCC. This is not only the first study in HNSCC preclinical models but the first study to compare cisplatin to CCI-779 as a radiosensitizer in any organ system. We found that compared to radiation therapy alone the combination of CCI-779 and radiotherapy significantly inhibited tumor burden and extended survival of mice. In both xenograft models (cisplatin sensitive and cisplatin resistant) the combination of CCI-779 and radiotherapy was significantly more effective in reducing tumor burden than conventional chemoradiotherapy with cisplatin. Although there was no significant difference in survival of cisplatin-sensitive FaDu mice when compared to CCI-779, in the cisplatin-resistant SCC40 xenografts CCI-779 and radiotherapy combined significantly exceeded survival of mice treated with cisplatin and radiotherapy. Also there was no additional benefit when all three treatments (cisplatin, CCI-779 and XRT) were combined, which is important in designing clinical trials where novel therapeutics typically are added to standard of care that already cause significant toxicity. The potent antitumoral efficacy of CCI-779 as a single agent (over 60% of tumor growth inhibition compared to control) precluded deciphering the exact mode of interaction (synergism, additive, antagonism) between CCI-779 and radiotherapy. Importantly the addition of CCI-779 to radiotherapy markedly enhanced the effects of radiation on tumor burden. Combined CCI-779+XRT treatment inhibited tumor growth by more than 90% compared to control, as well as more than doubled the survival benefit of mice compared to XRT alone in both xenograft models. Importantly the effects of combined CCI-779+XRT treatment on angiogenesis and apoptosis in xenograft tissues exceeded the sum of those by the drug or radiotherapy alone.
Head and neck cancer is not the only type of cancer in which dissonant results are observed when examining the radiosensitizing properties of mTOR inhibitors in an in vitro vs. in vivo model. For example, in a glioma model there was no increased radiosensitivity with the mTOR inhibitor RAD001 treatment in vitro, while the drug significantly enhanced radiation-induced regression of glioma xenografts (26). Conversely, in breast cancer cell lines, radiation sensitization was observed with RAD001 treatment in vitro (24). In our study Akt phosphorylation was affected differently by ionizing radiation in cell cultures compared to a xenograft model. Akt phosphorylation was not upregulated after irradiation of FaDu and SCC40 cell cultures. It is known that ionizing radiation effectively induces phosphorylation of Akt at Ser473 in the vascular endothelium within minutes of irradiation (34), which may explain the observed increase of pAkt-Ser-473 levels in the FaDu xenografts that could most likely occur in tumor vascular endothelium. Radiation-induced activation of mTOR signaling was absent in glioma GL261 cells and mTOR inhibitors did not sensitize them to radiation (26) similarly to what we observed in the HNSCC model. Radiation-induced upregulation of Akt/mTOR signaling was observed in breast cancer cells (24) and human umbilical vein endothelial cells (26). mTOR inhibition attenuated radiation-induced upregulation of Akt/mTOR signaling and sensitized breast cancer and HUVEC cells to radiation in vitro (24, 26). Also mTOR inhibition sensitized vascular endothelium to radiation injury in vivo (26). mTOR inhibition reduces microvessel density in tumor tissues by decreasing VEGF production (35–37). Our results show that CCI-779 significantly inhibited vascularization in the HNSCC xenografts. We have shown that treatment of HNSCC cells with CCI-779 decreases VEGF production that is consistent with anti-angiogenic effects of the drug. Evidence suggests that the level of VEGF might not only affect angiogenesis and radiation resistance of endothelial cells, but may also have an effect on radiation resistance of tumor cells, including HNSCC (38). Although CCI-779 treatment decreased VEGF production in HNSCC cells in vitro it did not have an effect on radiation sensitivity suggesting that a decrease in VEGF levels alone is not sufficient for radiation sensitization of HNSCC cells. We found that CCI-779 treatment upregulated Akt phosphorylation (Ser-473) in vitro and in vivo indicating that in HNSCC experimental models mTOR inhibition is disrupting the negative feedback loop that suppresses Akt phosphorylation (39, 40). However the increase in pAKT noted with CCI-779 and XRT was not significantly different from the increase in pAkt noted with all other treatments, i.e. cisplatin, XRT and the combination. Additionally, this increase did not affect the potent antitumoral effects of CCI-779 or its radioenhancing effects.
In our study CCI-779 showed potent antitumor activity against cisplatin-sensitive as well as cisplatin-resistant cell lines. Our data suggests that CCI-779 can be as effective as cisplatin in combination with radiotherapy for the treatment of head and neck cancer. Since CCI-779 is a relatively well tolerated drug with mainly manageable and reversible dermatological side effects, it is a promising therapy that can potentially replace cisplatin with its known considerable toxicity. Because the PI3K/Akt/mTOR pathway in HNSCC is a critical regulator of multiple downstream effectors including angiogenesis, mitosis, and apoptosis targeting this pathway during radiation therapy may be a key factor to blocking multiple other pathways enhancing the antitumoral action of radiotherapy.
Supported in part by National Cancer Institute grant (R01 CA 102363 to C.O. Nathan), and intramural grant from the Feist-Weiller Cancer Center, Shreveport, Louisiana.
We thank Cheryl Clark for help in the preparation of the manuscript.
Wyeth-Ayerst Research, manufacturer of CCI-779, had no role in the study design, collection, analysis, interpretation of data, writing or decision to submit the manuscript for publication.