S-Space Graduate School of Convergence Science and Technology (융합과학기술대학원) Dept. of Molecular and Biopharmaceutical Sciences (분자의학 및 바이오제약학과) Theses (Master's Degree_분자의학 및 바이오제약학과)
Genistein Inhibits Proliferation of BRCA1 Deficient Breast Cancer Cells: the GPR30-Akt Axis as a Potential Target
- 융합과학기술대학원 분자의학 및 바이오제약학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 : 융합과학기술대학원 분자의학 및 바이오제약학과, 2018. 2. 서영준.
- Absence or mutation of BRCA1 has been associated with development and progression of breast cancer. In this study, I investigated the proliferation signaling pathway in triple negative breast cancer (TNBC) cells with respect to the presence of functional BRCA1 and whether this signaling can be inhibited by genistein. Two human breast cancer cell lines, MDA-MB-231 (wild-type BRCA1) and HCC1937 (mutated BRCA1), were compared to ascertain the difference in growth signaling depending on the presence of functional BRCA1. Additionally, MDA-MB-231 cells were transfected with BRCA1-siRNA for gene silencing to verify whether the difference was truly due to BRCA1.
BRCA1 deficient breast cancer cells showed increased cell proliferation compared to breast cancer cells harboring wild-type BRCA1. In addition, BRCA1 silenced MDA-MB-231 cells, when injected into nude mice, were capable of forming larger tumors. These results indicate that deficiency in BRCA1 promotes aggressive proliferation of breast cancer. Elevated expression of cell proliferation targets P-Akt and GPR30 was observed in BRCA1 mutated HCC1937 cells. Diminished phosphorylation of Akt in GPR30-siRNA transfected HCC1937 cells suggests regulation of Akt signaling by GPR30. Furthermore, BRCA1 deficient breast cancer cells had greater accumulation of intracellular reactive oxygen species (ROS), which was associated with a low Nrf2 mRNA level.
Genistein, an isoflavone derived from soybeans, has been reported to have chemotherapeutic as well as chemopreventive properties. Genistein was chosen to be tested as a representative phytochemical that might inhibit aggressive proliferation of BRCA1 deficient TNBC cells by inactivating GPR30. I examined the effects of genistein on proliferation and growth of TNBC cells, and how these effects differ depending on the presence or absence of functionally active BRCA1. Anti-proliferative effects of genistein were assessed by the MTT, migration and clonogenic assays. Genistein treatment reduced viability, migration and colony formation of TNBC cells to a greater extent in HCC1937 cells than in MDA-MB-231 cells. Likewise, MDA-MB-231 cells that were transfected with BRCA1-siRNA were more susceptible to genistein-mediated growth inhibition. Effects of genistein on cell cycle progression were examined by flow cytometry. Genistein caused cell cycle arrest at the G2/M phase in BRCA1 mutant cells, leading to down-regulation of Cyclin B1. Genistein treatment also decreased the level of phosphorylated Akt, an upstream kinase of Cyclin B1, more prominently in BRCA1 mutated breast cancer cells. In addition, the expression of GPR30 was reduced upon genistein treatment. Moreover, up-regulation of Nrf2 and a decrease in the ROS level were observed after genistein treatment.
Taken together, lack of functional BRCA1 activates GPR30 signaling, thereby phosphorylating Akt and promoting cell proliferation. Genistein induces G2/M phase arrest by down-regulating Cyclin B1 expression through suppression of Akt phosphorylation and GPR30 activation in BRCA1 deficient breast cancer cells.