- Email: [email protected]
809 Treatment With MEK Inhibitors Promotes Emergence of a Stem-like Population in Resistant Melanoma Cells
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
807 Experimental Therapeutic Approach of Orthotopic and Subcutaneous Human Retinoblastoma Xenografts by the Human Anti-VEGF Antibody Bevacizumab, Alone or in Combination With Carboplatin 1 D. Decaudin1 , F. Assayag1 , N. Cassoux2 , O. Chouchane-Mlik3 , F. Nemati ´ , A. Thuleau1 , I. Aerts4 , F. Doz4 , S. Piperno-Neumann5 , L. Desjardins2 . 1 Institut Curie, Translational Research, Paris Cedex 05, France, 2 Institut Curie, Ophthalmology, Paris Cedex 05, France, 3 Institut Curie, Tumor Biology, Paris Cedex 05, France, 4 Institut Curie, Pediatry, Paris Cedex 05, France, 5 Institut Curie, Medical Oncology, Paris Cedex 05, France
Introduction: Treatment of retinoblastoma (Rb) could require enucleation in unfavorable disease. In order to assess innovative therapeutic approaches of Rb, we have developed preclinical orthotopic and subcutaneous human Rb xenografts into immunodeficient mice. Material and Methods: Three subcutaneous Rb models (Rb102, Rb111, and Rb200) have previously been developed and characterized (Aerts et al, Photodiagnosis Phtotodyn Ther 2010;7:275−83). The different in vivo treatments were (10 mice/group): Isotype control antibody (15 mg/kg twice a week, 4 weeks, IP); bevacizumab (15 mg/kg Twice a week, 4 weeks, IP); carboplatin (66 mg/kg once a week, 4 weeks, IP); and carboplatin + bevacizumab (doses as mentioned above). Tumor growth was then assessed by measuring the tumor volumes and mice were sacrificed when their tumor reached a volume of 2500 mm3 . Orthotopic models were developed from subcutaneous Rb xenografts. Mice bearing xenografts were sacrificed and tumors were dissected to obtain a suspension of fresh tumor cells at a concentration of 8000 cells/ml in DMEM serum-free medium. Under intraperitoneal anesthesia, 2 ml of cell suspension was injected into the subretinal space of the right eye for 3 groups of mice using a 32G needle via a Hamilton syringe. Using the Rb200 model, we have evaluated the efficacy of 2 ml intraocular administration of bevacizumab (25 mg/kg/week for 4 weeks) or carboplatin (100 mg/kg/week for 4 weeks). After sub-retinal injection, ophthalmic examination of the mice was performed weekly. When tumor cells invaded vitreal cavity and anterior chamber, the mice were sacrificed. Results: When administered alone, bevacizumab induced a tumor growth inhibition (TGI) of 63%, 40%, and 28% in the Rb102, Rb111, and Rb200, respectively. Similarly, carboplatin alone induced a TGI of 72%, 0%, and 33% in the Rb102, Rb111, and Rb200, respectively. In contrast, when bevacizumab and carboplatin were combined, we observed a TGI of 98%, 53%, and 78% in the Rb102, Rb111, and Rb200, respectively. In the orthotopic Rb200 model, we have observed a high efficacy of intraocular administration of carboplatin (17% of intraocular tumors), but not bevacizumab (100%). Finaly, assessment of efficacy between subcutaneously and orthotopic transplanted Rb200 model was concordant for both bevacizumab and carboplatin. Conclusion: As bevacizumab and carboplatin were not highly efficient when administered alone, their combination induced a significantly better TGI in the 3 tested Rb models. Moreover, we have developed relevant and available preclinical models of human Rb that allow pharmacological assessment of intraocular standard therapies. 808 Non-invasive Prognostic Tools for Phototherapeutic Response in Murine Tumors M. Krzykawska1 , J.M. Dabrowski2 , M. Szczygiel1 , G. Stochel2 , L.G. Arnaut3 , M.M. Pereira3 , K. Urbanska1 , M. Elas1 . 1 Jagiellonian University, Biochemistry Biophysics and Biotechnology, Krakow, Poland, 2 Jagiellonian University, Chemistry, Krakow, Poland, 3 Univerity of Coimbra, Chemistry, Coimbra, Portugal Introduction: Photodynamic therapy (PDT) is minimally invasive modality approved for clinical treatment for several types of cancer. In PDT, a photosensitizer (PS) is accumulated in targeted tissue, then is activated by light and generating reactive oxygen species (ROS) responsible for tumor regression. Bacteriochlorin derivative (F2 BMet) is an effective photosensitizer in in vitro and in vivo because absorbs light in the NIR where human tissues are the most transparent and generates ROS with high yield. Although PDT is characterized by high anti-tumor efficiency, tumor response to treatment depends on: oxygen level, vasculature physiology, concentration of PS and light doses. To provide therapeutic success, partial pressure of oxygen (pO2 ) and blood perfusion measurements were performed using Electron Paramagnetic Resonance (EPR) and Laser Doppler Perfusion Imaging (LDPI). To investigate level of PS before illumination, the PS fluorescence (F) measurements in vivo were performed. All tested methods are non-invasive, direct and quantitative. Material and Method: Model: S-91 melanoma and Lewis Lung Carcinoma tumors were grown in of DBA/2 and C57 mice. Experiments were approved by the Committee for Ethics of Experiments on Animals (permission no. 114/2009). PDT : The bacteriochlorin (2 mg/kg) were administered intravenously into DBA/2 and C57. 15 min − 72 h later tumors were irradiated with 100 J/cm2 of light at l = 750 nm.
S193
EPR: Oxymetry probes were injected into the tumor tissue when tumors reached 3 mm of mean diameter. The pO2 was determined from the line width of the LiPc spectrum measured with EPR. LDPI: The tumor area were measured and compare to the area in non-treated contralateral legs. F: Fiber optic and spectrophotometer were used to collect PS fluorescence from tumor surface. Results and Discussion: Changes in pO2 and blood perfusion in the PDTtreated tumors were consistent with destruction of vasculature, resulting in a decrease in pO2 and perfusion. The value of pO2 in the tumors immediately after vascular targeting PDT correlated with effectiveness of the therapy, i.e. both tumor volume on day 5−7 and time to tumor volume 400 mm3 . Surprisingly, a decrease in blood flow was seen also after cellular-targeted PDT, and seemed to correlate with tumor regrowth. Conclusion: Lasting inhibition of tumor growth was achieved only if PDT caused very strong hypoxia, whereas partial dysfunction of blood flow does not guarantee the good therapy effect and might even have a stimulatory effect on tumor growth. 809 Treatment With MEK Inhibitors Promotes Emergence of a Stem-like Population in Resistant Melanoma Cells E.R. Dorris1 , P. Smyth1 , R. Dunne1 , J.J. O’Leary1 , O. Sheils1 . 1 Trinity College Dublin, Histopathology, Dublin 8, Ireland Background: The MEK/ERK pathway is mutated in approximately 30% of human cancers and thus it represents a promising target for drug development. Extracellular ligands signal via RAS to recruit RAF kinase and activate MEK and its downstream effectors. Mutations within this pathway, such as activating mutations in the BRAF gene, can lead to its constitutive activation and result in a signalling addiction to this pathway. Cancer Stem Cells are reputed to exist in many human cancers and are believed to confer resistance to chemotherapy and be associated with more aggressive phenotypes. The objective of this study was to investigate the effect of MEK inhibition on a panel of cell lines, to monitor the efficacy of treatment, to assess any cell survival and characterise the differences displayed by cells surviving MEK inhibition compared with untreated counterparts. Materials and Methods: The optimal IC50 and length of treatment for the MEK inhibitor PD0325901 for each cell line (N-Thy-Ori (BRAFWTWT ), 8505c (anaplastic BRAFMUTMUT ), Sk-Mel-28 (BRAF MUTMUT ) and Sk-Mel31(BRAF MUTWT )) was determined using MTT assays. Cell lines were subjected to PD0325901 (IC50 ) or media-only treatment and surviving cells were harvested for mRNA extraction. A relative expression study of MEK pathway genes MAP2K1, MAP2K2, MAPK3, MAPK1, ELK1, stem cell markers NANOG and POU5F1 and adhesion genes CTNNB1 and CDH1 was performed using TaqMan® assays. Results: In the anaplastic thyroid cell line 8505c all 5 MEK pathway genes were significantly down-regulated in surviving PD0325901 treated cells. This pattern (of MEK gene expression inhibition) was not seen with the N-Thy-Ori thyroid cell line or the melanoma cell lines. In the melanoma cell lines the stem cell markers (NANOG and POU5F1) displayed significant upregulation in surviving PD0325901 treated cells. A statistically significant difference in expression was not seen in the 8505c cells. Conclusions: The data infer that sub-populations within cell lines display chemotherapeutic resistance. Moreover, we show that upregulation of stemness factors NANOG and OCT4 is associated with resistance to PD0325901 MEK inhibition. The lower comparative difference in expression of stem cell markers observed in the anaplastic cell line most likely reflects a higher ab initio stem cell component in this cell line. Whether the upregulation observed in melanoma cell lines is an adaptive response to MEK inhibition or due to an inherent stem cell sub-population remains to be determined. 810 Simvastatin Inhibits Leptin-activated Signaling Pathways That Promote Migration and Invasiveness of Ovarian Cancer Cells M. Cuello-Fredes1 , S. Kato1 , D. Diaz1 , A. Vecchiola1 , J. Branes ˜ 1, M.I. Barriga1 , R. Gejman2 , G.I. Owen3 . 1 Pontificia Universidad Catolica de Chile, Division of Obstetrics and Gynecology Faculty of Medicine, Santiago, Chile, 2 Pontificia Universidad Catolica de Chile, Pathology Faculty of Medicine, Santiago, Chile, 3 Pontificia Universidad Catolica de Chile, Physiological Sciences Faculty of Biological Sciences, Santiago, Chile Background: Recently, higher levels of leptin and its receptor have been correlated with less survival in epithelial ovarian cancer patients. Leptin, a peptidic hormone produced by adipocytes, usually present at higher serum levels in obese women, induced migration and invasiveness in certain epithelial cancers through JAK/STAT pathway activation. Key regulators of these pathways are involved in cell motility, cell adhesion, cell cycle progression, gene expression, and apoptosis. Previously, we have shown that statins, HMGCoA reductase inhibitors, used to reduce cholesterol synthesis, are also
european journal of cancer 48, suppl. 5 (2012) S25–S288
807 Experimental Therapeutic Approach of Orthotopic and Subcutaneous Human Retinoblastoma Xenografts by the Human Anti-VEGF Antibody Bevacizumab, Alone or in Combination With Carboplatin 1 D. Decaudin1 , F. Assayag1 , N. Cassoux2 , O. Chouchane-Mlik3 , F. Nemati ´ , A. Thuleau1 , I. Aerts4 , F. Doz4 , S. Piperno-Neumann5 , L. Desjardins2 . 1 Institut Curie, Translational Research, Paris Cedex 05, France, 2 Institut Curie, Ophthalmology, Paris Cedex 05, France, 3 Institut Curie, Tumor Biology, Paris Cedex 05, France, 4 Institut Curie, Pediatry, Paris Cedex 05, France, 5 Institut Curie, Medical Oncology, Paris Cedex 05, France
Introduction: Treatment of retinoblastoma (Rb) could require enucleation in unfavorable disease. In order to assess innovative therapeutic approaches of Rb, we have developed preclinical orthotopic and subcutaneous human Rb xenografts into immunodeficient mice. Material and Methods: Three subcutaneous Rb models (Rb102, Rb111, and Rb200) have previously been developed and characterized (Aerts et al, Photodiagnosis Phtotodyn Ther 2010;7:275−83). The different in vivo treatments were (10 mice/group): Isotype control antibody (15 mg/kg twice a week, 4 weeks, IP); bevacizumab (15 mg/kg Twice a week, 4 weeks, IP); carboplatin (66 mg/kg once a week, 4 weeks, IP); and carboplatin + bevacizumab (doses as mentioned above). Tumor growth was then assessed by measuring the tumor volumes and mice were sacrificed when their tumor reached a volume of 2500 mm3 . Orthotopic models were developed from subcutaneous Rb xenografts. Mice bearing xenografts were sacrificed and tumors were dissected to obtain a suspension of fresh tumor cells at a concentration of 8000 cells/ml in DMEM serum-free medium. Under intraperitoneal anesthesia, 2 ml of cell suspension was injected into the subretinal space of the right eye for 3 groups of mice using a 32G needle via a Hamilton syringe. Using the Rb200 model, we have evaluated the efficacy of 2 ml intraocular administration of bevacizumab (25 mg/kg/week for 4 weeks) or carboplatin (100 mg/kg/week for 4 weeks). After sub-retinal injection, ophthalmic examination of the mice was performed weekly. When tumor cells invaded vitreal cavity and anterior chamber, the mice were sacrificed. Results: When administered alone, bevacizumab induced a tumor growth inhibition (TGI) of 63%, 40%, and 28% in the Rb102, Rb111, and Rb200, respectively. Similarly, carboplatin alone induced a TGI of 72%, 0%, and 33% in the Rb102, Rb111, and Rb200, respectively. In contrast, when bevacizumab and carboplatin were combined, we observed a TGI of 98%, 53%, and 78% in the Rb102, Rb111, and Rb200, respectively. In the orthotopic Rb200 model, we have observed a high efficacy of intraocular administration of carboplatin (17% of intraocular tumors), but not bevacizumab (100%). Finaly, assessment of efficacy between subcutaneously and orthotopic transplanted Rb200 model was concordant for both bevacizumab and carboplatin. Conclusion: As bevacizumab and carboplatin were not highly efficient when administered alone, their combination induced a significantly better TGI in the 3 tested Rb models. Moreover, we have developed relevant and available preclinical models of human Rb that allow pharmacological assessment of intraocular standard therapies. 808 Non-invasive Prognostic Tools for Phototherapeutic Response in Murine Tumors M. Krzykawska1 , J.M. Dabrowski2 , M. Szczygiel1 , G. Stochel2 , L.G. Arnaut3 , M.M. Pereira3 , K. Urbanska1 , M. Elas1 . 1 Jagiellonian University, Biochemistry Biophysics and Biotechnology, Krakow, Poland, 2 Jagiellonian University, Chemistry, Krakow, Poland, 3 Univerity of Coimbra, Chemistry, Coimbra, Portugal Introduction: Photodynamic therapy (PDT) is minimally invasive modality approved for clinical treatment for several types of cancer. In PDT, a photosensitizer (PS) is accumulated in targeted tissue, then is activated by light and generating reactive oxygen species (ROS) responsible for tumor regression. Bacteriochlorin derivative (F2 BMet) is an effective photosensitizer in in vitro and in vivo because absorbs light in the NIR where human tissues are the most transparent and generates ROS with high yield. Although PDT is characterized by high anti-tumor efficiency, tumor response to treatment depends on: oxygen level, vasculature physiology, concentration of PS and light doses. To provide therapeutic success, partial pressure of oxygen (pO2 ) and blood perfusion measurements were performed using Electron Paramagnetic Resonance (EPR) and Laser Doppler Perfusion Imaging (LDPI). To investigate level of PS before illumination, the PS fluorescence (F) measurements in vivo were performed. All tested methods are non-invasive, direct and quantitative. Material and Method: Model: S-91 melanoma and Lewis Lung Carcinoma tumors were grown in of DBA/2 and C57 mice. Experiments were approved by the Committee for Ethics of Experiments on Animals (permission no. 114/2009). PDT : The bacteriochlorin (2 mg/kg) were administered intravenously into DBA/2 and C57. 15 min − 72 h later tumors were irradiated with 100 J/cm2 of light at l = 750 nm.
S193
EPR: Oxymetry probes were injected into the tumor tissue when tumors reached 3 mm of mean diameter. The pO2 was determined from the line width of the LiPc spectrum measured with EPR. LDPI: The tumor area were measured and compare to the area in non-treated contralateral legs. F: Fiber optic and spectrophotometer were used to collect PS fluorescence from tumor surface. Results and Discussion: Changes in pO2 and blood perfusion in the PDTtreated tumors were consistent with destruction of vasculature, resulting in a decrease in pO2 and perfusion. The value of pO2 in the tumors immediately after vascular targeting PDT correlated with effectiveness of the therapy, i.e. both tumor volume on day 5−7 and time to tumor volume 400 mm3 . Surprisingly, a decrease in blood flow was seen also after cellular-targeted PDT, and seemed to correlate with tumor regrowth. Conclusion: Lasting inhibition of tumor growth was achieved only if PDT caused very strong hypoxia, whereas partial dysfunction of blood flow does not guarantee the good therapy effect and might even have a stimulatory effect on tumor growth. 809 Treatment With MEK Inhibitors Promotes Emergence of a Stem-like Population in Resistant Melanoma Cells E.R. Dorris1 , P. Smyth1 , R. Dunne1 , J.J. O’Leary1 , O. Sheils1 . 1 Trinity College Dublin, Histopathology, Dublin 8, Ireland Background: The MEK/ERK pathway is mutated in approximately 30% of human cancers and thus it represents a promising target for drug development. Extracellular ligands signal via RAS to recruit RAF kinase and activate MEK and its downstream effectors. Mutations within this pathway, such as activating mutations in the BRAF gene, can lead to its constitutive activation and result in a signalling addiction to this pathway. Cancer Stem Cells are reputed to exist in many human cancers and are believed to confer resistance to chemotherapy and be associated with more aggressive phenotypes. The objective of this study was to investigate the effect of MEK inhibition on a panel of cell lines, to monitor the efficacy of treatment, to assess any cell survival and characterise the differences displayed by cells surviving MEK inhibition compared with untreated counterparts. Materials and Methods: The optimal IC50 and length of treatment for the MEK inhibitor PD0325901 for each cell line (N-Thy-Ori (BRAFWTWT ), 8505c (anaplastic BRAFMUTMUT ), Sk-Mel-28 (BRAF MUTMUT ) and Sk-Mel31(BRAF MUTWT )) was determined using MTT assays. Cell lines were subjected to PD0325901 (IC50 ) or media-only treatment and surviving cells were harvested for mRNA extraction. A relative expression study of MEK pathway genes MAP2K1, MAP2K2, MAPK3, MAPK1, ELK1, stem cell markers NANOG and POU5F1 and adhesion genes CTNNB1 and CDH1 was performed using TaqMan® assays. Results: In the anaplastic thyroid cell line 8505c all 5 MEK pathway genes were significantly down-regulated in surviving PD0325901 treated cells. This pattern (of MEK gene expression inhibition) was not seen with the N-Thy-Ori thyroid cell line or the melanoma cell lines. In the melanoma cell lines the stem cell markers (NANOG and POU5F1) displayed significant upregulation in surviving PD0325901 treated cells. A statistically significant difference in expression was not seen in the 8505c cells. Conclusions: The data infer that sub-populations within cell lines display chemotherapeutic resistance. Moreover, we show that upregulation of stemness factors NANOG and OCT4 is associated with resistance to PD0325901 MEK inhibition. The lower comparative difference in expression of stem cell markers observed in the anaplastic cell line most likely reflects a higher ab initio stem cell component in this cell line. Whether the upregulation observed in melanoma cell lines is an adaptive response to MEK inhibition or due to an inherent stem cell sub-population remains to be determined. 810 Simvastatin Inhibits Leptin-activated Signaling Pathways That Promote Migration and Invasiveness of Ovarian Cancer Cells M. Cuello-Fredes1 , S. Kato1 , D. Diaz1 , A. Vecchiola1 , J. Branes ˜ 1, M.I. Barriga1 , R. Gejman2 , G.I. Owen3 . 1 Pontificia Universidad Catolica de Chile, Division of Obstetrics and Gynecology Faculty of Medicine, Santiago, Chile, 2 Pontificia Universidad Catolica de Chile, Pathology Faculty of Medicine, Santiago, Chile, 3 Pontificia Universidad Catolica de Chile, Physiological Sciences Faculty of Biological Sciences, Santiago, Chile Background: Recently, higher levels of leptin and its receptor have been correlated with less survival in epithelial ovarian cancer patients. Leptin, a peptidic hormone produced by adipocytes, usually present at higher serum levels in obese women, induced migration and invasiveness in certain epithelial cancers through JAK/STAT pathway activation. Key regulators of these pathways are involved in cell motility, cell adhesion, cell cycle progression, gene expression, and apoptosis. Previously, we have shown that statins, HMGCoA reductase inhibitors, used to reduce cholesterol synthesis, are also