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857: Aggravation of ER stress by combination of proteasome inhibitors and HIV protease inhibitors results in preferencial killing of triple-negative breast cancer cells in vitro
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 2. Gender differences in ER beta expression could be one of the reasons of gender differences in NSCLC disease progression. 3. We believe that at least half of the NSCLC pts (especially women) with ER beta high and moderate expression could benefit from adjuvant antiestrogen treatment. Thereby of ERbeta have to be obligate index among the other tumor markers studying in NSCLC pts. Supported by RFBR grants (13-04-01004-a, 12-04-00028-a, 14-04-31734mol-a) and scholarship of the President of Russian Federation (376.2012.4.). No conflict of interest. 856 Molecular mechanisms of sorafenib-induced apoptosis in cancer cells C. Kuznia1 , B. Klinger2 , J. Keil3 , B. Seliger4 , C. Falk3 , N. Rohwer5 , T. Cramer5 , 1 R. Schafer ¨ 1 , N. Bluthgen ¨ , C. Sers1 . 1 Charite´ Berlin, Institute of Pathology, Berlin, Germany, 2 Humboldt University of Berlin, Institute for Theoretical Biology, Berlin, Germany, 3 Hannover Medical School, Integrated Research and Treatment Center Transplantation, Hannover, Germany, 4 Martin Luther University, Institute for Medical Immunology, Halle, Germany, 5 Charite´ Berlin, Medical Department Division of Hepatology and Gastroenterology, Berlin, Germany Introduction: The Raf kinase inhibitor Sorafenib and the MEK inhibitor U0126 target the same pathway; yet, Sorafenib treatment of HRAS transformed human embryonic kidney cells induces apoptosis while U0126 treatment does not. A time-resolved analysis of this effect by protein and mRNA arrays revealed an involvement of the stress-related protein DNA damageinducible transcript 4 (DDIT4, also known as Redd1) and mTOR signalling. Most interestingly, the pattern of Sorafenib-increased DDIT4 expression and apoptosis induction was additionally seen in renal carcinoma cell lines, which represent the clinical application of this inhibitor. The present study aims to further improve our understanding of the underlying molecular mechanisms resulting in Sorafenib-induced apoptosis (or resistance), focusing on the role of DDIT4 and altered mTOR signalling. Materials and Methods: To identify possible variations in cancer-associated genes, three different renal carcinoma cell lines were analysed by exome sequencing. The activation of mTOR and associated components of the signaling network were examined using Bio-Plex assays and Western Blot. To functionally investigate the link between DDIT4 and apoptosis we performed a shRNA-mediated knock-down of DDIT4. The activating transcription factor 4 (ATF4) and the hypoxia-inducible factors (HIF)-1alpha and HIF-2alpha, that are described in the literature as regulators of DDIT4, were also analyzed using Real-Time-PCR, Western Blot and shRNA-mediated knock-down. Results and Discussion: Exome sequencing revealed no mutations in genes coding for proteins of the investigated signalling pathways except for one cell line with variations in the mTOR and VHL coding region. A closer examination of the mTOR pathway by Bio-Plex assay and Western Blot revealed no differences in mTOR inhibition between cells with and without DDIT4-mediated apoptosis. But the shRNA-mediated knock-down of both ATF4 and DDIT4 resulted in an inhibition of caspase-3 cleavage and abrogated apoptosis, whereas the HIF knockdown was not sufficient. Conclusion: These results encourage the hypothesis that Sorafenib activates ATF4 and subsequently induces DDIT4 in human renal cancer, which plays an important role for apoptosis. So far, the mechanisms leading towards a successful induction of apoptosis are still not clear, but our results strongly indicate an involvement of stress signalling. No conflict of interest. 857 Aggravation of ER stress by combination of proteasome inhibitors and HIV protease inhibitors results in preferencial killing of triple-negative breast cancer cells in vitro 1 J. Gibbons Marsico1 , T. Heger2 , M. Kraus3 , J. Bader3 , M. Jorger ¨ , C.H. Driessen1 . 1 Kantonsspital St. Gallen, Oncology and Haematology, 2 St. Gallen, Switzerland, EMPA, Department for Biomaterials, St. Gallen, Switzerland, 3 Kantonsspital St. Gallen, Laboratory of Experimental Oncology, St. Gallen, Switzerland
Background: Breast cancer (BC) is the most frequent cancer and the principal cause of cancer death for females worldwide. The expressions of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) serve as important diagnostic markers for breast cancer aggressiveness and invasiveness. Nevertheless, several molecular therapies that have been developed in the past decade show promising effects against these subtypes of BC (i.e. tamoxifen, trastuzumab, lapatinib). Triple-negative breast cancer (TNBC), however, carries poor prognosis due to its aggressive biology, fast development of drug resistance, and lack of molecular targets such as hormone receptors and HER2. Until now, chemotherapy remains the standard care for advanced TNBC, with a poor median overall survival. Recently, pharmacological aggravation of endoplasmic reticulum stress (ERS) has become an attractive strategy not only for multiple myeloma, but also for
solid malignancies. In our work we present evidence that pharmacological aggravation of ERS may lead to efficient killing of breast cancer preclinical models in vitro. Methods: We determined the cytotoxic activity of proteasome inhibitors (bortezomib, bzb; carfilzomib, cfb) alone and in combination with the two HIV protease inhibitors nelfinavir (nel) and lopinavir (lop) in five different breast cancer cell lines (HER2+: BT 474 and SK-BR 3, and HER2-: BT 549, MDAMB 231, and MDA-MB 468). To assess cytotoxicity of each drug and their combinations, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay was applied. Results: Both proteasome inhibitors moderately reduced cell survival in all BC cell lines at clinically relevant concentrations (125 nM) when applied alone (8−36%). At higher concentrations (1 mM) the irreversible proteasome inhibitor cfb exhibited higher cytotoxicity (>60%) than bzb (>80%) in all tested cell lines. Importantly, combinations with HIV protease inhibitors yielded significantly enhanced cytotoxicity and mostly synergistic responses in all cell lines compared with either proteasome (see table) or HIV protease inhibitors alone. Moreover, preliminary data shows that these combinations cause strong cytotoxic effects in TNBC cell lines. Conclusions: Drug combinations of the irreversible proteasome inhibitor carfilzomib and an HIV protease inhibitor such as lopinavir or nelfinavir resulted in substantial cytotoxicity in preclinical models of TNBC. The concept of pharmacological aggravation of ERS is promising in patients with TNBC, and a respective phase IB-II clinical study is in early planning. Table: Cytotoxic effects of mono and combination experiments in five BC cell lines Cell line
Cell death (%), mean±SD Bzb (125 nM)
MDA-MB 468 MDA-MB 231 BT 549 BT 474 SK-BR-3
Cfb (125 nM)
−
Nel
Lop
−
Nel
Lop
27±8 36±8 23±3 8±6 nd
57±0.1 56±0.1 90±1 28±6 nd
80±1 57±2 56±0.2 32±3 nd
21±7 41±2 44±3 33±6 30±2
65±2 74±4 91±1 82±6 68±3
79±4 78±8 69±4 61±1 21±4
nd, not determined. No conflict of interest. 858 Simvastatin modifies the expression of stemness and epithelial– mesenchymal transition (EMT) markers in ovarian cancer-initiating cells resulting in decreased cell migration S. Kato1 , L. Abarzua1 , K. Hormazabal1 , A. Delpiano1 , M.L. Bravo2 , R. Orellana2 , J. Branes1 , E. Castellon3 , M. Cuello-Fredes1 , G.I. Owen2 . 1 Faculty of Medicine P. Universidad Catolica de Chile, Obstetrics and Gynecology, Santiago, Chile, 2 Faculty of Biological Sciences P. Universidad Catolica de Chile, Physiology, Santiago, Chile, 3 Faculty of Medicine University of Chile, Laboratory of Cellular and Molecular Andrology, Santiago, Chile Introduction: Worldwide ovarian cancer is the most deadly gynecologic malignancy. A major determinant of its lethality is the late diagnosis when the disease has spread out to the abdominal cavity. To metastasize ovarian cancer cells must experience epithelial mesenchymal transition (EMT) allowing detaching from the primary tumor, adapting and surviving in ascites until settling in a new supporting microenvironment. Only a small subset of cells known as cancer-initiating cells (CICs) and expressing EMT and stemness markers are able to migrate and metastasize. Previously, we have demonstrated that ovarian cancer cells have a metabolic dependence in mevalonate synthesis. These cells overexpress HMGCoA reductase and experience cell death when exposed to statins, a selective inhibitor of the enzyme. In the present work we explore if statins could inhibit cell migration in cancer-initiating cells and study the explanatory mechanisms. Material and Methods: Ovarian CICs were isolated from cell lines (SKOV3, HEY, UCI101) using stemness-selecting media in low attachment culture plates. Real-Time PCR, immunoblotting and immunocytochemistry were used to characterize CICs and non-CICs with stemness (Oct-4, nanog, CD44) and EMT (N-cadherin, Snail, Zeb2) markers. CICs and non-CICs were exposed to vehicle or statin (Simvastatin 0.1−10 uM) for up to 48 h. Cell death and cell migration were analyzed by PARP cleavage immunoblotting and Boyden chamber migration assay, respectively. Results: Ovarian cancer-initiating cells (CICs) grew forming ‘ovospheres’ (small floating cell aggregates) in selection culturing conditions. CICs expressed higher levels of Oct-4, nanog, and CDD44 compared to nonCICs. Statin exposure resulted in reduced detection of the cleaved form of PARP in CICs compared to non-CICs reflecting decreased cell death and less sensitivity to the drug of this subpopulation. However, despite not inducing cell death, simvastatin significantly reduced the percentage of migrating CIC cells.
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 2. Gender differences in ER beta expression could be one of the reasons of gender differences in NSCLC disease progression. 3. We believe that at least half of the NSCLC pts (especially women) with ER beta high and moderate expression could benefit from adjuvant antiestrogen treatment. Thereby of ERbeta have to be obligate index among the other tumor markers studying in NSCLC pts. Supported by RFBR grants (13-04-01004-a, 12-04-00028-a, 14-04-31734mol-a) and scholarship of the President of Russian Federation (376.2012.4.). No conflict of interest. 856 Molecular mechanisms of sorafenib-induced apoptosis in cancer cells C. Kuznia1 , B. Klinger2 , J. Keil3 , B. Seliger4 , C. Falk3 , N. Rohwer5 , T. Cramer5 , 1 R. Schafer ¨ 1 , N. Bluthgen ¨ , C. Sers1 . 1 Charite´ Berlin, Institute of Pathology, Berlin, Germany, 2 Humboldt University of Berlin, Institute for Theoretical Biology, Berlin, Germany, 3 Hannover Medical School, Integrated Research and Treatment Center Transplantation, Hannover, Germany, 4 Martin Luther University, Institute for Medical Immunology, Halle, Germany, 5 Charite´ Berlin, Medical Department Division of Hepatology and Gastroenterology, Berlin, Germany Introduction: The Raf kinase inhibitor Sorafenib and the MEK inhibitor U0126 target the same pathway; yet, Sorafenib treatment of HRAS transformed human embryonic kidney cells induces apoptosis while U0126 treatment does not. A time-resolved analysis of this effect by protein and mRNA arrays revealed an involvement of the stress-related protein DNA damageinducible transcript 4 (DDIT4, also known as Redd1) and mTOR signalling. Most interestingly, the pattern of Sorafenib-increased DDIT4 expression and apoptosis induction was additionally seen in renal carcinoma cell lines, which represent the clinical application of this inhibitor. The present study aims to further improve our understanding of the underlying molecular mechanisms resulting in Sorafenib-induced apoptosis (or resistance), focusing on the role of DDIT4 and altered mTOR signalling. Materials and Methods: To identify possible variations in cancer-associated genes, three different renal carcinoma cell lines were analysed by exome sequencing. The activation of mTOR and associated components of the signaling network were examined using Bio-Plex assays and Western Blot. To functionally investigate the link between DDIT4 and apoptosis we performed a shRNA-mediated knock-down of DDIT4. The activating transcription factor 4 (ATF4) and the hypoxia-inducible factors (HIF)-1alpha and HIF-2alpha, that are described in the literature as regulators of DDIT4, were also analyzed using Real-Time-PCR, Western Blot and shRNA-mediated knock-down. Results and Discussion: Exome sequencing revealed no mutations in genes coding for proteins of the investigated signalling pathways except for one cell line with variations in the mTOR and VHL coding region. A closer examination of the mTOR pathway by Bio-Plex assay and Western Blot revealed no differences in mTOR inhibition between cells with and without DDIT4-mediated apoptosis. But the shRNA-mediated knock-down of both ATF4 and DDIT4 resulted in an inhibition of caspase-3 cleavage and abrogated apoptosis, whereas the HIF knockdown was not sufficient. Conclusion: These results encourage the hypothesis that Sorafenib activates ATF4 and subsequently induces DDIT4 in human renal cancer, which plays an important role for apoptosis. So far, the mechanisms leading towards a successful induction of apoptosis are still not clear, but our results strongly indicate an involvement of stress signalling. No conflict of interest. 857 Aggravation of ER stress by combination of proteasome inhibitors and HIV protease inhibitors results in preferencial killing of triple-negative breast cancer cells in vitro 1 J. Gibbons Marsico1 , T. Heger2 , M. Kraus3 , J. Bader3 , M. Jorger ¨ , C.H. Driessen1 . 1 Kantonsspital St. Gallen, Oncology and Haematology, 2 St. Gallen, Switzerland, EMPA, Department for Biomaterials, St. Gallen, Switzerland, 3 Kantonsspital St. Gallen, Laboratory of Experimental Oncology, St. Gallen, Switzerland
Background: Breast cancer (BC) is the most frequent cancer and the principal cause of cancer death for females worldwide. The expressions of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) serve as important diagnostic markers for breast cancer aggressiveness and invasiveness. Nevertheless, several molecular therapies that have been developed in the past decade show promising effects against these subtypes of BC (i.e. tamoxifen, trastuzumab, lapatinib). Triple-negative breast cancer (TNBC), however, carries poor prognosis due to its aggressive biology, fast development of drug resistance, and lack of molecular targets such as hormone receptors and HER2. Until now, chemotherapy remains the standard care for advanced TNBC, with a poor median overall survival. Recently, pharmacological aggravation of endoplasmic reticulum stress (ERS) has become an attractive strategy not only for multiple myeloma, but also for
solid malignancies. In our work we present evidence that pharmacological aggravation of ERS may lead to efficient killing of breast cancer preclinical models in vitro. Methods: We determined the cytotoxic activity of proteasome inhibitors (bortezomib, bzb; carfilzomib, cfb) alone and in combination with the two HIV protease inhibitors nelfinavir (nel) and lopinavir (lop) in five different breast cancer cell lines (HER2+: BT 474 and SK-BR 3, and HER2-: BT 549, MDAMB 231, and MDA-MB 468). To assess cytotoxicity of each drug and their combinations, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay was applied. Results: Both proteasome inhibitors moderately reduced cell survival in all BC cell lines at clinically relevant concentrations (125 nM) when applied alone (8−36%). At higher concentrations (1 mM) the irreversible proteasome inhibitor cfb exhibited higher cytotoxicity (>60%) than bzb (>80%) in all tested cell lines. Importantly, combinations with HIV protease inhibitors yielded significantly enhanced cytotoxicity and mostly synergistic responses in all cell lines compared with either proteasome (see table) or HIV protease inhibitors alone. Moreover, preliminary data shows that these combinations cause strong cytotoxic effects in TNBC cell lines. Conclusions: Drug combinations of the irreversible proteasome inhibitor carfilzomib and an HIV protease inhibitor such as lopinavir or nelfinavir resulted in substantial cytotoxicity in preclinical models of TNBC. The concept of pharmacological aggravation of ERS is promising in patients with TNBC, and a respective phase IB-II clinical study is in early planning. Table: Cytotoxic effects of mono and combination experiments in five BC cell lines Cell line
Cell death (%), mean±SD Bzb (125 nM)
MDA-MB 468 MDA-MB 231 BT 549 BT 474 SK-BR-3
Cfb (125 nM)
−
Nel
Lop
−
Nel
Lop
27±8 36±8 23±3 8±6 nd
57±0.1 56±0.1 90±1 28±6 nd
80±1 57±2 56±0.2 32±3 nd
21±7 41±2 44±3 33±6 30±2
65±2 74±4 91±1 82±6 68±3
79±4 78±8 69±4 61±1 21±4
nd, not determined. No conflict of interest. 858 Simvastatin modifies the expression of stemness and epithelial– mesenchymal transition (EMT) markers in ovarian cancer-initiating cells resulting in decreased cell migration S. Kato1 , L. Abarzua1 , K. Hormazabal1 , A. Delpiano1 , M.L. Bravo2 , R. Orellana2 , J. Branes1 , E. Castellon3 , M. Cuello-Fredes1 , G.I. Owen2 . 1 Faculty of Medicine P. Universidad Catolica de Chile, Obstetrics and Gynecology, Santiago, Chile, 2 Faculty of Biological Sciences P. Universidad Catolica de Chile, Physiology, Santiago, Chile, 3 Faculty of Medicine University of Chile, Laboratory of Cellular and Molecular Andrology, Santiago, Chile Introduction: Worldwide ovarian cancer is the most deadly gynecologic malignancy. A major determinant of its lethality is the late diagnosis when the disease has spread out to the abdominal cavity. To metastasize ovarian cancer cells must experience epithelial mesenchymal transition (EMT) allowing detaching from the primary tumor, adapting and surviving in ascites until settling in a new supporting microenvironment. Only a small subset of cells known as cancer-initiating cells (CICs) and expressing EMT and stemness markers are able to migrate and metastasize. Previously, we have demonstrated that ovarian cancer cells have a metabolic dependence in mevalonate synthesis. These cells overexpress HMGCoA reductase and experience cell death when exposed to statins, a selective inhibitor of the enzyme. In the present work we explore if statins could inhibit cell migration in cancer-initiating cells and study the explanatory mechanisms. Material and Methods: Ovarian CICs were isolated from cell lines (SKOV3, HEY, UCI101) using stemness-selecting media in low attachment culture plates. Real-Time PCR, immunoblotting and immunocytochemistry were used to characterize CICs and non-CICs with stemness (Oct-4, nanog, CD44) and EMT (N-cadherin, Snail, Zeb2) markers. CICs and non-CICs were exposed to vehicle or statin (Simvastatin 0.1−10 uM) for up to 48 h. Cell death and cell migration were analyzed by PARP cleavage immunoblotting and Boyden chamber migration assay, respectively. Results: Ovarian cancer-initiating cells (CICs) grew forming ‘ovospheres’ (small floating cell aggregates) in selection culturing conditions. CICs expressed higher levels of Oct-4, nanog, and CDD44 compared to nonCICs. Statin exposure resulted in reduced detection of the cleaved form of PARP in CICs compared to non-CICs reflecting decreased cell death and less sensitivity to the drug of this subpopulation. However, despite not inducing cell death, simvastatin significantly reduced the percentage of migrating CIC cells.