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Background: In this report we investigated the combination of epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) pathway inhibition as a possible new therapeutic strategy for small cell lung cancer (SCLC)., Methods: EGFR, p-AKT, p-ERK, p-mTOR and p-p70s6K protein expressions were studied by immunohistochemistry in 107 small cell lung carcinomas and correlated with clinicopathological parameters. Cells of SCLC were treated with erlotinib+/-RAD001 and analysed for cell viability, proliferation, autophagy, and pathway regulation., Results: Epidermal growth factor receptor, p-AKT, p-ERK, p-mTOR, and p-p70s6K were expressed in 37, 24, 13, 55 and 91% of the tumour specimens of all SCLC patients, respectively, and were not associated with disease-free or overall survival. The expression of EGFR was lower in neoadjuvant-treated patients (P=0.038); mTOR pathway activation was higher in the early stages of disease (P=0.048). Coexpression of EGFR/p-mTOR/p-p70s6K was observed in 28% of all patients . EGFR immunoreactivity was associated with p-ERK and p-mTOR expression (P=0.02 and P=0.0001); p-mTOR immunoreactivity was associated with p-p70s6K expression (P=0.001). Tumour cells comprised a functional EGFR, no activating mutations in exons 18-21, and resistance to RAD001 monotherapy. We found synergistic effects of erlotinib and RAD001 combination therapy on the molecular level, cell viability, proliferation and autophagy., Conclusions: The combined inhibition of EGFR/mTOR pathways could be a promising approach to treat SCLC.

This study aimed to test whether [(18)F]fluoro-D-glucose (FDG) uptake of tumours measured by positron emission tomography (PET) can be used as surrogate marker to define the optimal biological dose (OBD) of mTOR inhibitors in vivo. Everolimus at 0.05, 0.5, 5 and 15 mg kg(-1) per day was administered to gastric cancer xenograft-bearing mice for 23 days and FDG uptake of tumours was measured using PET from day 1 to day 8. To provide standard comparators for FDG uptake, tumour volume, S6 protein phosphorylation, Ki-67 staining and everolimus blood levels were evaluated. Everolimus blood levels increased in a dose-dependent manner but antitumour activity of everolimus reached a plateau at doses >or=5 mg kg(-1) per day (tumour volume treated vs control (T/C): 51% for 5 mg kg(-1) per day and 57% for 15 mg kg(-1) per day). Correspondingly, doses >or=5 mg kg(-1) per day led to a significant reduction in FDG uptake of tumours. Dose escalation above 5 mg kg(-1) per day did not reduce FDG uptake any further (FDG uptake T/C: 49% for 5 mg kg(-1) per day and 52% for 15 mg kg(-1) per day). Differences in S6 protein phosphorylation and Ki-67 index reflected tumour volume and changes in FDG uptake but did not reach statistical significance. In conclusion, FDG uptake might serve as a surrogate marker for dose finding studies for mTOR inhibitors in (pre)clinical trials.

Background: Inhibition of mTOR complex 1 (mTORC1) with rapamycin leads to phosphorylation of AKT in some cancer cells, with unknown biological consequences. The role of this phosphorylation in melanoma is unknown, although preliminary clinical data indicate poor activity of rapalogues in melanoma., Objectives: We aimed at elucidating the role of AKT phosphorylation after mTORC1 inhibition in melanoma cells., Methods: Western blotting, apoptosis assays, cell cycle analyses and viability assays were performed to analyse the effects of rapamycin and LY294002 treatment on melanoma cells. For suppression of mTOR complex 2 (mTORC2) an siRNA directed against rictor was used., Results: Rapamycin showed limited effects on cell viability but resulted in strong and lasting AKT phosphorylation in melanoma cells. Combined PI3K/mTOR inhibition with LY294002 had pronounced effects on viability but also led to increased AKT phosphorylation after prolonged treatment. In contrast, combination of rapamycin plus LY294002 suppressed AKT phosphorylation. Suppression of AKT phosphorylation did not correlate with decreases in cell viability. Inhibition of mTORC2 led to reduced levels of phosphorylated AKT., Conclusions: mTORC1 inhibition with rapamycin and with LY294002 can lead to AKT phosphorylation in melanoma cells via mTORC2. Combination of rapamycin and LY294002 suppresses AKT phosphorylation but without significant effect on treatment efficacy.

Background: Gastric cancer is a highly chemoresistant tumor. Previous studies suggest that cancer cells can be sensitized to standard chemotherapy, and especially alkylating agents, by inhibition of mammalian target of rapamycin (mTOR) signaling. The work presented here shows that the mTOR inhibitor everolimus, in combination with cyclophosphamide, exhibits synergistic antitumor activity in gastric cancer xenografts., Materials and Methods: Treatment with everolimus at the minimal effective dose was studied in combination with cyclophosphamide at maximum tolerated dose in a human gastric cancer severe combined immunodeficient (SCID) mouse xenograft model. Besides tumor size, biomarker expression for proliferation (Ki-67), hypoxia (HIF-12alpha), apoptosis (activated caspase 3), angiogenesis (microvascular density, MVD) and levels of circulating endothelial progenitors (CEPs) were assessed., Results: Everolimus single agent treatment significantly inhibited tumor growth relative to control and cyclophosphamide treatment (T/C 19%, p<0.01). This antitumor activity was linked to a significant decrease in tumor cell proliferation (p<0.01) and a trend towards lower tumor MVD, HIF-1alpha expression and levels of CEPs. Notably, the combination of everolimus with cyclophosphamide resulted in synergistic anti-tumor activity (T/C 9%, p<0.01). This antitumor activity coincided with a statistically significant decrease in MVD (p<0.01). Whereas treatment with everolimus was well tolerated, cyclophosphamide at maximum tolerated dose (MTD) showed significant toxicity both as monotherapy and in combination with everolimus., Conclusion: The antiangiogenic activity of everolimus combined with a high dose of cyclophosphamide shows synergistic antitumor activity against gastric cancer in vivo. In potential future clinical trials, the toxicity of cyclophosphamide in combination regimens with everolimus deserves careful evaluation.

Metronomic dosing of cytotoxic drugs such as cyclophosphamide has shown anti-angiogenic activity, most likely by inducing hypoxia in tumors. Hypoxia leads to activation of escape mechanisms allowing tumor cell survival. This potentially limits the activity of anti-angiogenic strategies. We hypothesized that mTORC1 inhibition by everolimus (RAD001) leads to suppression of HIF-1alpha and VEGF resulting in synergistic anti-tumor activity in combination with anti-angiogenically dosed cyclophosphamide. In vitro, effects of everolimus on mTORC1 signaling, proliferation, cell cycle, HIF-1alpha expression and VEGF secretion were evaluated in two gastric cancer cell lines. In vivo, anti-tumor activity of everolimus in combination with metronomic cyclophosphamide was studied in a NCI-N87 human gastric cancer SCID mouse xenograft model. Expression of Ki-67 and HIF-1alpha, activated caspase 3, microvascular density (MVD) and tumor necrotic area assessed. Everolimus decreased proliferation and attenuated production of HIF-1alpha as well as VEGF in gastric cancer cells in vitro. In vivo, everolimus significantly inhibited tumor growth. This anti-tumor activity was linked to a significant increase in tumor necrotic area (p < 0.02) and trends for decreased proliferation, increased apoptosis, decreased HIF-1alpha and lower tumor MVD (p = n.s.). The combination of everolimus and cyclophosphamide resulted in a striking and highly significant long-term tumor growth control compared to monotherapy (p < 0.001), which was associated with a sharp increase in central tumor necrosis (p < 0.001). In conclusion, the combination of everolimus and metronomic cyclophosphamide showed synergistic anti-tumor activity. Depriving cancer cells by everolimus of factors necessary for their survival under hypoxia induced by anti-angiogenic chemotherapy appears to be a promising approach for treatment of gastric cancer.

This study compares the antineoplastic potential of a novel treatment strategy combining cell cycle inhibitor-779 (CCI-779) plus dacarbazine (DTIC) versus DTIC monotreatment, the current chemotherapeutic mainstay in combating metastatic melanoma. A controlled four-group parallel study design comprising 24-40 mice per tumor cell line was used in a severe combined immunodeficiency (SCID)-mouse xenotransplantation model. SCID mice were injected with 518A2, Mel-JUSO, or 607B human melanoma cells. After they developed tumors, mice received daily CCI-779 or solvent over 14 days. From treatment day 4-8 mice were additionally injected with DTIC or saline. Treatment with CCI-779 plus DTIC was superior to single agent DTIC in two out of three cell lines (P<0.05). The tumor weight reduction was 44+/-17 and 61+/-6% compared with DTIC monotreatment in Mel-JUSO and 607B melanomas, respectively (P<0.05). In contrast, in 518A2 xenotransplants, CCI-779 plus DTIC treatment was as effective as DTIC monotreatment. CCI-779 monotherapy exerted no statistically significant antitumor effect. Collectively, these data indicate that CCI-779 has the potential to increase the chemotherapeutic efficacy, as the combination of CCI-779 plus DTIC proved to be more efficacious compared to DTIC monotherapy in two out of three melanoma cell lines in vivo.