Sabine Strommer

Everolimus (RAD001) and anti-angiogenic cyclophosphamide show long-term control of gastric cancer growth in vivo.

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-1α 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-1α 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-1α, activated caspase 3, microvascular density (MVD) and tumor necrotic area was assessed.Everolimus decreased proliferation and attenuated production of HIF-1α 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

FDG uptake is a surrogate marker for defining the optimal biological dose of the mTOR inhibitor everolimus in vivo

This study aimed to test whether [18F]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 ⩾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 ⩾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.

mTOR inhibition by everolimus counteracts VEGF induction by sunitinib and improves anti-tumor activity against gastric cancer in vivo.

VEGF receptor blockage has been reported to increase serum VEGF. We hypothesized that mTOR inhibition by everolimus counteracts VEGF induction by sunitinib resulting in an improved anti-tumor activity of sunitinib. In vitro, sunitinib in combination with everolimus did not outperform the respective monotherapies. In vivo, monotherapies reduced tumor growth by 60%, whereas the combination of sunitinib and everolimus led to an almost complete tumor growth inhibition. This superior anti-tumor activity coincided with attenuation of VEGF peaks. In conclusion mTOR inhibition by everolimus counteracts VEGF induction by sunitinib and results in significant reduction of tumor burden and long-lasting tumor growth control.

Gastric Cancer Growth Control by BEZ235 In Vivo Does Not Correlate with PI3K/mTOR Target Inhibition but with [18F]FLT Uptake

Purpose: In this study, we tested the antitumor activity of the dual phosphoinositide 3-kinase (PI3K)/mTOR inhibitor BEZ235 against gastric cancer in vitro and in vivo. Experimental Design: Gastric cancer cell lines (N87, MKN45, and MKN28) were incubated with BEZ235 and assessed for cell viability, cell cycle, and PI3K/mTOR target inhibition. In vivo, athymic nude mice were inoculated with N87, MKN28, or MKN45 cells and treated daily with BEZ235. 3′-Deoxy-3′-[18F]fluorothymidine ([18F]FLT) uptake was measured via small animal positron emission tomography (PET). Results:In vitro, BEZ235 dose dependently decreased the cell viability of gastric cancer cell lines. The antiproliferative activity of BEZ235 was linked to a G1 cell-cycle arrest. In vivo, BEZ235 treatment resulted in PI3K/mTOR target inhibition as shown by dephosphorylation of AKT and S6 protein in all xenograft models. However, BEZ235 treatment only inhibited tumor growth of N87 xenografts, whereas no antitumor effect was observed in the MKN28 and MKN45 xenograft models. Sensitivity to BEZ235 in vivo correlated with downregulation of the proliferation marker thymidine kinase 1. Accordingly, [18F]FLT uptake was only significantly reduced in the BEZ235-sensitive N87 xenograft model as measured by PET. Conclusion: In conclusion, in vivo sensitivity of gastric cancer xenografts to BEZ235 did not correlate with in vitro antiproliferative activity or in vivo PI3K/mTOR target inhibition by BEZ235. In contrast, [18F]FLT uptake was linked to BEZ235 in vivo sensitivity. Noninvasive [18F]FLT PET imaging might qualify as a novel marker for optimizing future clinical testing of dual PI3K/mTOR inhibitors. Clin Cancer Res; 17(16); 5322–32. ©2011 AACR.

Mcl-1 is a relevant molecular target for antisense oligonucleotide strategies in gastric cancer cells.

Gastric cancer is the second most common cause of death from cancer worldwide and resistant to various chemotherapeutic regimens. In gastric cancer, the anti-apoptotic Mcl-1 protein is expressed in up to 75% of all cases and associated with poor prognosis. The biological relevance of Mcl-1 expression in gastric cancer is unclear. Thus, we investigated the functional significance and potential role of Mcl-1 as a molecular target in gastric cancer 1348e treated with Mcl-1 antisense oligonucleotides. Protein expression, cell growth and apoptosis were assessed for single-agent Mcl-1 AS oligonucleotide treatment and for combinations with docetaxel or cisplatin. Treatment by Mcl-1 AS oligonucleotides resulted in approximately 50% reduction in Mcl-1 protein levels in all gastric cancer cell lines examined. Surprisingly, dose-dependent Mcl-1 downregulation produced a significant increase in apoptosis and up to 60% decrease in cell growth. Moreover, combination of Mcl-1 AS oligonucleotide with docetaxel or cisplatin displayed synergistic anti-tumor activity. In conclusion, the impressive single-agent anti-tumor activity and the synergistic effect of Mcl-1 AS oligonucleotides in combination with chemotherapy might qualify Mcl-1 as a promising molecular target for AS oligonucleotide based treatment strategies for gastric cancer in the future.

Reduction of Human Melanoma Tumor Growth in Severe Combined Immunodeficient Mice by Passive Transfer of Antibodies Induced by a High Molecular Weight Melanoma-Associated Antigen Mimotope Vaccine

Purpose: The high molecular weight melanoma-associated antigen (HMW-MAA) is an attractive target for immunotherapy of malignant melanoma. We have recently generated a vaccine based on the HMW-MAA mimotope 225D9.2+ that was able to induce anti-HMW-MAA antibodies with antitumor activity in vitro. Here, we investigated the antitumor activity of these antibodies in a human melanoma xenotransplant severe combined immunodeficient (SCID) mouse model. Experimental Design: Tumors were established by injecting the human melanoma 518A2 cells into C.B.17 SCID/SCID mice. In tumor prevention experiments, 200 μg purified total IgG antibodies were injected intravenously the same day or on day 5 in therapeutic experiments. Antibody administration was repeated every fourth day and tumor volumes were measured. Antibody specificity and tumor infiltration by macrophages were investigated by immunohistochemistry. Results: Within 35 days after cell inoculation, antibody treatment reduced tumor growth up to 40% in the therapeutic and up to 62% in the tumor prevention experiments compared with the control mice. In tumors of all groups, a similar distribution of the HMW-MAA and no differences in infiltration of macrophages were detected by immunohistochemistry. Conclusions: Here, we showed that antibodies induced by the 225D9.2+ mimotope effectively inhibited melanoma tumor growth. Additional mechanisms besides antibody-dependent cell cytotoxicity like disruption of interactions of melanoma cells mediated by extracellular matrix components seem to be involved in tumor growth inhibition. Based on our findings, we suggest that active immunization with this mimotope might be a promising strategy for treatment of melanoma.

Colistin reduces LPS-triggered inflammation in a human sepsis model in vivo: A randomized controlled trial

The previously described anti‐endotoxin effect of colistin has not been investigated in humans yet. We performed a randomized, double‐blind, placebo‐controlled crossover trial to determine the degree of colistin‐driven modulation of inflammatory response in blood of lipopolysaccharide (LPS)‐challenged healthy volunteers in a human endotoxemia model. After a single intravenous dose of 2.5 million IU colistin methanesulfonate, interleukin (IL)‐6, IL‐8, tumor necrosis factor alpha (TNF‐α), and IL‐1β concentrations as well as other biomarkers of inflammation such as C‐reactive protein, differential leukocyte counts, and body temperature were measured up to 24 h postdose. Colistin significantly decreased the inflammatory cytokine response to LPS in blood of healthy volunteers. This effect was most evident for IL‐6, IL‐8, and TNF‐α. This study is the first to confirm the anti‐endotoxin effect of colistin in humans in vivo. Further studies might increase our knowledge on the interaction between colistin and the effectors of the immune system.

Abstract A124: ERCC1 expression correlates with EGFR expression and is regulated by cisplatin, cetuximab, and dasatinib in head and neck cancer cells

Background: ERCC1 has been linked to resistance against platin‐based chemotherapy. Cetuximab sensitizes colorectal cancer cells to oxaliplatin. This sensitization coincides with ERCC1 downregulation, but the link between EGFR and ERCC1 is not fully understood. It is known that treatment with cetuximab inhibits nuclear transport of EGFR and DNA repair in response to irradiation. Moreover, cisplatin treatment results in phosphorylation at Y845 and nuclear transport of EGFR. We intended to investigate the regulation of ERCC1 by cisplatin, cetuximab and dasatinib, a src inhibitor stabilizing EGFR in the membrane, its relevance for chemosensitivity and its correlation with EGFR and its phosphorylation at Y845 in head and neck cancer cells. Material and Methods: In UM‐SCC 14c head and neck cancer cell line, ERCC1, EGFR and EGFR (pY845) expression was determined by western blot and FACS analysis. Cell viability, cell cycle and apoptosis induction was assessed by cell titer blue assay, propidium iodide and caspase 3 staining, respectively. Results: In cell viability assay, combination of cisplatin with cetuximab led to a significant (p Conclusion: 14c cells are sensitized to cisplatin treatment by adding cetuximab. ERCC1 expression correlated with phosphorylation status of EGFR (pY845) following treatment with cisplatin and cetuximab. However, treatment with the src inhibitor dasatinib before adding cisplatin and cetuximab reduced phosphorylation of EGFR (Y845), but increased ERCC1 and EGFR expression. Whether the increase of ERCC1 is a late effect due to transcriptional upregulation of EGFR after dasatinib treatment remains to be determined. However, the dose‐effect relationship and the lack of regulation of ERCC1 expression in peripheral blood leukocytes after treatment with cisplatin and cetuximab support the notion that ERCC1 expression is regulated by EGFR in 14c cells. Further studies elucidating the subcellular transport of EGFR and its transcriptional regulation after dasatinib treatment are ongoing and will be presented. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A124.