- Email: [email protected]
Anti-cancer stem cell activity of the hedgehog inhibitor GANT61 in ER-positive breast cancer cells
15th St.Gallen International Breast Cancer Conference / The Breast 32S1 (2017) S22–S77
administered radio- and/or chemotherapy, to avoid excessive cytotoxic harm due to cumulative effect of all applied therapies. Disclosure of Interest: No significant relationships. P048 Tumour-preventive effects of clove buds in mammary carcinoma model P. Kubatka1 *, S. Uramová1, M. Kello2, K. Kajo3, P. Žúbor1, J. Mojžiš2, Z. Lasabová1, M. Péč1, M. Adamkov1, J. Danko1. 1Comenius University in Bratislava, Jessenius Faculty of Medicine, Martin, Slovakia, 2University of Pavol Jozef Šafárik, Medical Faculty, Košice, Slovakia, 3Department of Pathology, Slovak Medical University and St. Elisabeth Oncology Institute, Bratislava, Slovakia Aims: There has been a considerable interest in the identification of natural plant foods for developing effective agents against cancer. The anti-tumour effects of cloves in the in vivo and in vitro breast cancer model were evaluated in this study. Methods: Dried flower buds of cloves (CLO) were used at two concentrations of 0.1% and 1% through diet during 13 weeks after the administration of chemocarcinogen. After autopsy, histopathological and immunohistochemical analyses of rat mammary carcinomas were performed. Moreover, in vitro evaluation using MCF-7 cells was carried out. Results: Dietary administered CLO caused the dose-dependent decrease in tumour frequency by 47.5% and 58.5% when compared to control. Analysis of carcinoma cells in animals showed bcl-2, Ki67, VEGF, CD24, and CD44 expression decrease and Bax, caspase3, and ALDH1 expression increase after high dose CLO administration. MDA levels were substantially decreased in rat carcinomas in both CLO groups. The evaluation of histone modifications revealed increase in lysine trimethylations and acetylations (H4K20me3, H4K16ac) in carcinomas after CLO administration. TIMP3 promoter methylation levels of CpG3, CpG4, CpG5 islands were altered in treated cancer cells. An increase in total RASSF1A promoter methylation (three CpG sites) in CLO 1 group was found. In vitro studies showed antiproliferative and proapoptotic effects of CLO extract in MCF-7 cells (analyses of cytotoxicity, Brdu, cell cycle, annexin V/PI, caspase-7, Bcl-2, and mitochondrial membrane potential). Conclusions: Our results demonstrate, for the first time, a distinct tumour-preventive effect of cloves in the breast cancer model. Study was supported by the grant VEGA 1/0108/16. Disclosure of Interest: No significant relationships. P49 Tamoxifen resistance in breast cancer is correlated with poor prognosis through CD24 and CD44 mesenchymal ability up-regulation Y.-l. Kuo1 *, T.-I. Hsu2, W.-C. Wang3, M.-J. Chen3. 1Department of Surgery, National Cheng Kung University, Tainan, Taiwan, 2Department of Orthopedics, E-Da Hospital, Kaohsiung, Taiwan, 3Department of General Surgery, Chi Mei Medical Center, Tainan, Taiwan Aim: Breast cancer is clinically defined as four intrinsic subtypes according to expression of estrogen receptor (ER), progesterone receptor (PR), and Her-2/neu. In the recent studies showed that breast cancer patients with Tamoxifen resistance have poor response to chemotherapy. Furthermore, these patients develop recurrence earlier after treatment. From the clinical observation, the ER status and the related signaling pathway seems important for the therapy. However, the role of ER/PR status is still unclearly with therapy progression and prognosis. Our first step is to clarify the ER status and the correlation of disease prognosis. Methods: In our experiment, the tamoxifen resistant MCF-7 cell line was generated with a 12.5 μM concentration. This cell line was
S39
verified with HER2 negative status. The morphology of MCF-7 with tamoxifen resistance showed spindle-like shape. Results: The MTT assay data showed that the IC50 of tamoxifen in MCF7 and resistance is 139.99 and 309.98 mM. The IC50 of taxol in tamoxifen is lower than parental (38.99 v.s 103.15 μM). The Western blotting data showed that the ER status is up-regulated and CD24 and CD44 are up-regulated. The in vivo animal model demonstrated that cells with tamoxifen resistance have the aggressive disease progression. Conclusions: Our data demonstrated that breast cancer cells with HER2 negative, ER/PR positive status with tamoxifen resistance is strongly correlated with poor prognosis through CD-24 and CD-44 up-regulated with mesenchymal ability. Disclosure of Interest: No significant relationships. P050 Anti-cancer stem cell activity of the hedgehog inhibitor GANT61 in ER-positive breast cancer cells J. Kurebayashi1 *, Y. Koike1, Y. Ohta1, W. Saitoh1, T. Yamashita1, N. Kanomata2, T. Moriya2. 1Department of Breast and Thyroid Surgery, Kawasaki Medical School, Kurashiki, Japan, 2Department of Pathology 2, Kawasaki Medical School, Kurashiki, Japan Aims: Estradiol (E2) increases not only the growth of, but also the cancer stem cell (CSC) proportion in estrogen receptor (ER)-positive breast cancer cells. The molecular mechanisms responsible for the regulation of CSCs have not yet been elucidated. A recent study has suggested that the non-canonical hedgehog (Hh) pathway activated by E2 plays an important role in regulating the CSC proportion in ERpositive breast cancer cells. Therefore, we investigated the anti-CSC activity of the non-canonical Hh inhibitor GANT61 in ER-positive breast cancer cells. The combined anti-cell growth and anti-CSC activities of GANT61 with the antiestrogens, 4-hydroxy-tamoxifen and fulvestrant were also examined. Methods: The effects of GANT61 on cell growth, cell cycle progression, apoptosis, and the CSC proportion were investigated in four ERpositive breast cancer cell lines: MCF-7, T-47D, KPL-1, and KPL-3C. The CSC proportion was measured using either the mammosphere assay or CD44/CD24 assay. The expression levels of pivotal molecules in the Hh pathway, sonic Hh (SHH), glioma-associated oncogene (GLI) 1, GLI2, and GLI3, were also measured. Furthermore, the combined effects of GANT61 with antiestrogens on anti-cell growth and antiCSC activities were investigated. Results: E2 significantly increased cell growth and the CSC proportion in all ER-positive breast cancer cell lines tested. E2 also increased the expression levels of GLI1, GLI2, and SHH, but not GLI3. GANT61 dose-dependently decreased cell growth in association with G1-S cell cycle retardation and enhanced apoptosis. GANT61 decreased the E2-induced CSC proportion, as measured by the mammosphere assay, in all cell lines. Antiestrogens also decreased E2-induced cell growth and the CSC proportion. The combined treatments of GANT61 with antiestrogens additively enhanced anticell growth and/or anti-CSC activities in some ER-positive breast cancer cell lines. Conclusions: The non-canonical Hh inhibitor GANT61 inhibited not only growth, but also the CSC proportion increased by E2 in ER-positive breast cancer cells. GANT61 enhanced the anti-cell growth and/or anti-CSC activities of antiestrogens in some ERpositive breast cancer cell lines. These results suggest for the first time that GANT61 is an active therapeutic agent in the treatment of patients with ER-positive breast cancer. Disclosure of Interest: J. Kurebayashi received advisory/consultation fees and research funding from Takeda Pharmaceutical Co., Limited. J. Kurebayashi received research funding from Takeda Pharmaceutical Co., Esai Co., Chugai Co. and AstraZeneca Pharmaceuticals. The other authors declare that they have no conflict of interest.
administered radio- and/or chemotherapy, to avoid excessive cytotoxic harm due to cumulative effect of all applied therapies. Disclosure of Interest: No significant relationships. P048 Tumour-preventive effects of clove buds in mammary carcinoma model P. Kubatka1 *, S. Uramová1, M. Kello2, K. Kajo3, P. Žúbor1, J. Mojžiš2, Z. Lasabová1, M. Péč1, M. Adamkov1, J. Danko1. 1Comenius University in Bratislava, Jessenius Faculty of Medicine, Martin, Slovakia, 2University of Pavol Jozef Šafárik, Medical Faculty, Košice, Slovakia, 3Department of Pathology, Slovak Medical University and St. Elisabeth Oncology Institute, Bratislava, Slovakia Aims: There has been a considerable interest in the identification of natural plant foods for developing effective agents against cancer. The anti-tumour effects of cloves in the in vivo and in vitro breast cancer model were evaluated in this study. Methods: Dried flower buds of cloves (CLO) were used at two concentrations of 0.1% and 1% through diet during 13 weeks after the administration of chemocarcinogen. After autopsy, histopathological and immunohistochemical analyses of rat mammary carcinomas were performed. Moreover, in vitro evaluation using MCF-7 cells was carried out. Results: Dietary administered CLO caused the dose-dependent decrease in tumour frequency by 47.5% and 58.5% when compared to control. Analysis of carcinoma cells in animals showed bcl-2, Ki67, VEGF, CD24, and CD44 expression decrease and Bax, caspase3, and ALDH1 expression increase after high dose CLO administration. MDA levels were substantially decreased in rat carcinomas in both CLO groups. The evaluation of histone modifications revealed increase in lysine trimethylations and acetylations (H4K20me3, H4K16ac) in carcinomas after CLO administration. TIMP3 promoter methylation levels of CpG3, CpG4, CpG5 islands were altered in treated cancer cells. An increase in total RASSF1A promoter methylation (three CpG sites) in CLO 1 group was found. In vitro studies showed antiproliferative and proapoptotic effects of CLO extract in MCF-7 cells (analyses of cytotoxicity, Brdu, cell cycle, annexin V/PI, caspase-7, Bcl-2, and mitochondrial membrane potential). Conclusions: Our results demonstrate, for the first time, a distinct tumour-preventive effect of cloves in the breast cancer model. Study was supported by the grant VEGA 1/0108/16. Disclosure of Interest: No significant relationships. P49 Tamoxifen resistance in breast cancer is correlated with poor prognosis through CD24 and CD44 mesenchymal ability up-regulation Y.-l. Kuo1 *, T.-I. Hsu2, W.-C. Wang3, M.-J. Chen3. 1Department of Surgery, National Cheng Kung University, Tainan, Taiwan, 2Department of Orthopedics, E-Da Hospital, Kaohsiung, Taiwan, 3Department of General Surgery, Chi Mei Medical Center, Tainan, Taiwan Aim: Breast cancer is clinically defined as four intrinsic subtypes according to expression of estrogen receptor (ER), progesterone receptor (PR), and Her-2/neu. In the recent studies showed that breast cancer patients with Tamoxifen resistance have poor response to chemotherapy. Furthermore, these patients develop recurrence earlier after treatment. From the clinical observation, the ER status and the related signaling pathway seems important for the therapy. However, the role of ER/PR status is still unclearly with therapy progression and prognosis. Our first step is to clarify the ER status and the correlation of disease prognosis. Methods: In our experiment, the tamoxifen resistant MCF-7 cell line was generated with a 12.5 μM concentration. This cell line was
S39
verified with HER2 negative status. The morphology of MCF-7 with tamoxifen resistance showed spindle-like shape. Results: The MTT assay data showed that the IC50 of tamoxifen in MCF7 and resistance is 139.99 and 309.98 mM. The IC50 of taxol in tamoxifen is lower than parental (38.99 v.s 103.15 μM). The Western blotting data showed that the ER status is up-regulated and CD24 and CD44 are up-regulated. The in vivo animal model demonstrated that cells with tamoxifen resistance have the aggressive disease progression. Conclusions: Our data demonstrated that breast cancer cells with HER2 negative, ER/PR positive status with tamoxifen resistance is strongly correlated with poor prognosis through CD-24 and CD-44 up-regulated with mesenchymal ability. Disclosure of Interest: No significant relationships. P050 Anti-cancer stem cell activity of the hedgehog inhibitor GANT61 in ER-positive breast cancer cells J. Kurebayashi1 *, Y. Koike1, Y. Ohta1, W. Saitoh1, T. Yamashita1, N. Kanomata2, T. Moriya2. 1Department of Breast and Thyroid Surgery, Kawasaki Medical School, Kurashiki, Japan, 2Department of Pathology 2, Kawasaki Medical School, Kurashiki, Japan Aims: Estradiol (E2) increases not only the growth of, but also the cancer stem cell (CSC) proportion in estrogen receptor (ER)-positive breast cancer cells. The molecular mechanisms responsible for the regulation of CSCs have not yet been elucidated. A recent study has suggested that the non-canonical hedgehog (Hh) pathway activated by E2 plays an important role in regulating the CSC proportion in ERpositive breast cancer cells. Therefore, we investigated the anti-CSC activity of the non-canonical Hh inhibitor GANT61 in ER-positive breast cancer cells. The combined anti-cell growth and anti-CSC activities of GANT61 with the antiestrogens, 4-hydroxy-tamoxifen and fulvestrant were also examined. Methods: The effects of GANT61 on cell growth, cell cycle progression, apoptosis, and the CSC proportion were investigated in four ERpositive breast cancer cell lines: MCF-7, T-47D, KPL-1, and KPL-3C. The CSC proportion was measured using either the mammosphere assay or CD44/CD24 assay. The expression levels of pivotal molecules in the Hh pathway, sonic Hh (SHH), glioma-associated oncogene (GLI) 1, GLI2, and GLI3, were also measured. Furthermore, the combined effects of GANT61 with antiestrogens on anti-cell growth and antiCSC activities were investigated. Results: E2 significantly increased cell growth and the CSC proportion in all ER-positive breast cancer cell lines tested. E2 also increased the expression levels of GLI1, GLI2, and SHH, but not GLI3. GANT61 dose-dependently decreased cell growth in association with G1-S cell cycle retardation and enhanced apoptosis. GANT61 decreased the E2-induced CSC proportion, as measured by the mammosphere assay, in all cell lines. Antiestrogens also decreased E2-induced cell growth and the CSC proportion. The combined treatments of GANT61 with antiestrogens additively enhanced anticell growth and/or anti-CSC activities in some ER-positive breast cancer cell lines. Conclusions: The non-canonical Hh inhibitor GANT61 inhibited not only growth, but also the CSC proportion increased by E2 in ER-positive breast cancer cells. GANT61 enhanced the anti-cell growth and/or anti-CSC activities of antiestrogens in some ERpositive breast cancer cell lines. These results suggest for the first time that GANT61 is an active therapeutic agent in the treatment of patients with ER-positive breast cancer. Disclosure of Interest: J. Kurebayashi received advisory/consultation fees and research funding from Takeda Pharmaceutical Co., Limited. J. Kurebayashi received research funding from Takeda Pharmaceutical Co., Esai Co., Chugai Co. and AstraZeneca Pharmaceuticals. The other authors declare that they have no conflict of interest.