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Graduate School of Ajou University Department of Biomedical Sciences 4. Theses(Ph.D)

Differential regulation of mTORc1 and mTORc2 by expression of BTG2/TIS21/PC3 inhibits breast cancer cell growth and malignancy

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dc.contributor.advisorIn Kyoung Lim-
dc.contributor.authorSanthoshkumar Sundaramoorthy-
dc.date.accessioned2022-11-29T03:01:30Z-
dc.date.available2022-11-29T03:01:30Z-
dc.date.issued2018-02-
dc.identifier.other27469-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/21243-
dc.description학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2018. 2-
dc.description.tableofcontentsPart I 1 I. INTRODUCTION 1 II. MATERIALS AND METHODS 5 A. MATERIALS 5 B. METHODS 5 1. Cell culture 5 2. Adenoviral transduction of HeLa cells with BTG2 gene 5 3. Cloning of κB-response element (κB-RE) into pGL3 basic vector 5 4. Transfection analyses 6 5. Chromatin immunoprecipitation (ChIP) assay 6 6. Measurement of the level of reactive oxygen species 7 7. Small interfering RNAs (siRNAs) and preparation of lentivirus with IκBα mutant 7 8. RT-PCR 7 9. Immunoblot analyses 8 10. Cell synchronization and FACS analysis 8 III. RESULTS 9 A. BTG2 upregulates MnSOD expression in HeLa cells 9 B. BTG2 activates NFκB-response element of MnSOD gene in the 2nd intron 11 C. BTG2 mediatedNFκB activation is regulated by IκBα degradation 14 D. BTG2-enhanced IκBα degradation is regulated by p-Akt1 17 E. BTG2 enhances G2/M arrest along with reduction of H2O2 level 20 IV. DISCUSSION 25 V. CONCLUSION 27 Part II 28 I. INTRODUCTION 28 II. MATERIALS AND METHODS 31 A. MATERIALS 31 B. METHODS 31 1. Cell Culture, Antibodies and Reagents 31 2. Preparation of Ad-BTG2 virus and Reconstitution of BTG2 gene Expression 31 3. mTOR Kinase Assays 32 4. In vivo Analyses and Preparation of Mouse Tissues 32 5. Chromatin Immunoprecipitation Analysis (ChIP) 32 6. Immunohistochemistry and Survival Analysis 32 7. Immunoprecipitation 33 8. MTT assay 33 9. Colony Formation assay 33 10. FACS analysis 33 11. Transfections of siRNA and Plasmid DNAs 33 12. Reverse transcriptional PCR and RTqPCR 34 13. Subcellular fractionation 34 14. Live cell imaging of GFP-AKT PH localization 34 15. GEO data analysis 34 16. In-vitro Protein Purifications 35 17. Statistical analysis 35 III. RESULTS 36 A. BTG2 differentially regulates the mTOR interaction with Raptor and Rictor 36 B. Interaction of Raptor with BTG2 inhibits mTORc1 activity in breast cancer cells 42 C. BTG2 upregulates mTORc2 activity in both normal and cancer cells 49 D. Rictor activates AKT1, but not AKT2, in response to BTG2 expression 54 E. BTG2-induced pAKT1S473 downregulates NFAT1 60 F. In vivo evidence of BTG2 expression that blocks breast cancer invasion 68 IV. DISCUSSION 78 V. CONCLUSION 81 REFERENCES 83 국문요약 94-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.titleDifferential regulation of mTORc1 and mTORc2 by expression of BTG2/TIS21/PC3 inhibits breast cancer cell growth and malignancy-
dc.title.alternativemTORc1의 억제와 mTORc2 활성을 통한 BTG2/TIS21/PC3의 유방암 악성화 억제 및 세포 분열 억제 기전 연구-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.department일반대학원 의생명과학과-
dc.date.awarded2018. 2-
dc.description.degreeDoctoral-
dc.identifier.localId800821-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000027469-
dc.description.alternativeAbstract12-O-tetradecanoyl phorbol-13-acetate (TPA)-inducible sequences 21 (TIS21)-an ortholog of B-cell translocation gene 2 (BTG2) in humans and pheochromocytoma cell-3 (PC3) in rats- is an antiproliferative and early growth response gene and belongs to a family of six proteins which includes BTG1-4 and Tob1, Tob2. All these proteins contain APRO domain at the N-terminus with highly conserved sequences termed Box A and Box B. However the C-terminus is less conserved except for the presence of PAM2 motif (Poly-A binding protein Motif 2) in Tob 1 and 2. BTG2 is induced following DNA damage in a p53-dependent and -independent manner by protein kinase C-δ. BTG2 induces cell cycle arrest by inhibiting cyclin D1 and E, as well as CDK4, depending on pRB activity, and by interacting with cyclin B1/CDK1. BTG2 is constitutively expressed in all human tissues but lost during the early stage of carcinogenesis and loss of BTG2 expression is associated with the size and grade of ER-positive breast cancers along with breast tumor progression. We previously reported that BTG2 inhibits invadopodia formation by downregulating reactive oxygen species (ROS) through targeting of the mDia genes and regulated AKT phosphorylation at S473 . We therefore tried to explore the mechanism of AKT activation by the BTG2 gene and its outcome in breast carcinogenesis. It has been reported that AKT1 inhibits cancer progression, but maintains cancer cell growth; however, here, we proposed novel mechanisms of AKT1 for inhibiting both cancer progression and cancer growth following expression of the tumor suppressor, BTG2, in triple negative breast cancer cells via differential regulation of mTORc1 and mTORc2. Expression of BTG2 inhibited the mTOR-Raptor interaction, and increased mTOR binding to Rictor. BTG2 selectively induced phosphorylation of AKT1, not AKT2, by mTORc2 and reduced expression of PHLPP2, an AKT1-specific phosphatase. BTG2-induced pAKT1S473 degraded NFAT1, which regulates transcription of cytokines, chemokine receptors and possibly PHLPP2, resulting in modification of the tumor microenvironment. In human breast cancer tissues, expression of NFAT1 and PHLPP2 was significantly higher compared to the low expression of BTG2. In tsc-null and p53-null mouse embryonic fibroblasts (MEFs), BTG2 was substituted for the tsc gene, -a negative regulator of mTORc1 activation- , and was also evident in -null mice. Constitutive expression of BTG2 in the basal cells of normal mammary ducts was maintained in ductal-carcinoma in situ (DCIS) of human breast cancer but absent in infiltrating breast cancer, as examined by immunohistochemistry. Indeed, the relative risk of lymph node invasion in BTG2+ breast cancer patients was approximately 1/10th that of BTG2- patients, and overall survival of ER-LN+ breast cancer patients was significantly higher among BTG2 high expressers than low expressers. Indeed, BTG2 expression significantly inhibited the in vitro colony forming ability of triple negative breast cancer cells. We present here the signaling pathways that regulate cancer growth and progression by expression of BTG2, which inhibits cancer growth via the BTG2-tsc1/2-mTORc1-p70S6K axis and downregulates cancer progression via the BTG2-mTORc2-AKT1-NFAT1-PHLPP2 axis. Therefore, we suggest the BTG2 gene is a promising candidate to combat intractable breast cancer.-
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