| dc.contributor.advisor | Doğan , A. Lale | |
| dc.contributor.author | Haznedaroğlu , Elif | |
| dc.date.accessioned | 2018-07-18T13:52:44Z | |
| dc.date.available | 2018-07-18T13:52:44Z | |
| dc.date.issued | 2018-07-18 | |
| dc.date.submitted | 2018-06-25 | |
| dc.identifier.citation | Haznedaroğlu, E., Inhibition of Serum and Glucocorticoid Inducible Kinase 1 (SGK1) in Triple Negative Breast Cancer, Hacettepe University Institute of Health Sciences Tumor Biology and Immunology Doctor
of Philosophy Thesis, Ankara, 2018. | tr_TR |
| dc.identifier.uri | http://hdl.handle.net/11655/4691 | |
| dc.description.abstract | Glucocorticoid receptor overexpression leads to poor prognosis in breast cancer, particularly in triple negative phenotype. This poor prognosis has been shown to be due to the activation of SGK1 (serum and glucocorticoid inducible kinase 1). In this study, we analyzed SGK1 levels and sensitivity of a panel of TNBC (triple negative breast cancer) cell lines towards SGK1 inhibitor GSK650394. Among these cell lines, MDA-MB-436 cells, displaying markedly elevated SGK1 and showing high phosphorylation of the SGK1 substrate NDRG1 (N-Myc downstream regulated gene 1), was unresponsive to the SGK1 inhibitor. The other cell lines with varying SGK1 levels (MDA MB 231, HCC1937 and BT549) showed marked decrease of NDGR1 phosphorylation due to kinase activity inhibition. Intriguingly, despite GSK650394 sensitivity in these cells, pharmacological SGK1 inhibition did not decrease GSK3β phosphorylation, exhibiting no effect on GSK3β reactivation. In addition, SGK1 inhibition did not change E-cadherin and vimentin expression showing that epithelial-mesenchymal transition (EMT) phenotype was not suppressed. Accordingly, Slug, Snail and Twist mRNA levels were not affected from SGK1 inhibition. GSK650394 treatment suppressed proliferation in MDA-MB-231 cells and led to a slight decrease in S-phase. The results of this study support the idea that SGK1 inhibition strategies may have therapeutic potential in triple negative breast cancer. | en |
| dc.description.sponsorship | Hacettepe University Scientific Research Projects Coordination Unit (Project No: THD-2016-12969). | tr_TR |
| dc.description.tableofcontents | PAGE OF APPROVAL iii
YAYINLAMA VE FİKRİ MÜLKİYET HAKLARI BEYANI iv
ETHICAL DECLARATION v
ACKNOWLEDGEMENTS vi
ABSTRACT vii
ÖZET viii
CONTENTS ix
SYMBOLS AND ABBREVATIONS xiii
INDEX OF FIGURES xv
INDEX OF TABLES xix
1. INTRODUCTION 1
2. REVIEW OF LITERATURE 3
2.1. Development of The Mammary Tissue 3
2.2. Breast Cancer 4
2.2.1. Important Signaling Pathways in Breast Cancer 5
2.2.2. Triple Negative Breast Cancer 9
2.3 Breast Cancer and Stress Receptors 12
2.3.1. Breast Cancer and SGK1 14
2.4. EMT 18
3. MATERIAL AND METHODS 22
3.1. Substances Used for This Study 22
3.2. Tools and Equipment 23
3.3. Study Plan 25
3.4. Cell Culture 26
3.4.1. Passaging and Counting of Cells with Trypsin-EDTA solution 26
3.4.2. Freezing of Cells 27
3.5. Drug Incubation 28
3.6. MTT Assay 28
3.7. mRNA Expression Analysis 28
3.7.1. RNA Isolation from Cells 29
3.7.2. One-step Real Time PCR (Polymerase Chain Reaction) 29
3.8. Western Blot and Protein Analysis 30
3.8.1. Preparation of Protein Lysates and Protein Quantification 30
3.8.2. Polyacrylamide Gel Electrophoresis 32
3.8.3. Semi-dry Transfer and Blotting with Trans-Blot Turbo system 33
3.8.4. Chemiluminescent Imaging 33
3.8.5. Reblotting for Loading Control Proteins 34
3.8.6. Densitometry Analysis 34
3.9. Crystal Violet Assay 34
3.10. Cell Cycle Analysis 35
3.11. Statistical Analysis 35
4. RESULTS 36
4.1. SGK1 mRNA and protein expressions in triple negative breast cancer 36
cells
4.1.1. SGK1 protein expressions in triple negative breast cancer cells 37
4.1.2. SGK1 mRNA expressions in triple negative breast cancer cells 37
4.2. Determination of subtoxic doses of GSK650394 with MTT Assay 38
4.2.1. Determination of subtoxic doses of GSK650394 with MTT Assay 39
in MDA-MB-231 cells
4.2.2. Determination of subtoxic doses of GSK650394 with MTT Assay 41
in MDA-MB-436 cells
4.2.3. Determination of subtoxic doses of GSK650394 with MTT Assay 42
in HCC1937 cells
4.2.4. Determination of subtoxic doses of GSK650394 with MTT Assay 44
in BT549 cells
4.2.5. Determination of subtoxic doses of GSK650394 with MTT Assay 45
in MDA-MB-468 cells
4.3. Determination of SGK1 inhibition through phospho-NDRG1 45
protein expression levels in triple negative breast cancer cell lines
4.3.1. Determination of SGK1 inhibition through phospho-NDRG1 45
protein expression levels in MDA-MB-231 cells
4.3.2. Determination of SGK1 inhibition through phospho-NDRG1 46
protein expression levels in BT549 cells
4.3.3. Determination of SGK1 inhibition through phospho-NDRG1 47
protein expression levels in HCC1937 cells
4.3.4. Determination of SGK1 inhibition through phospho-NDRG1 48
protein expression levels in MDA-MB-436 cells
4.3.5. Determination of SGK1 inhibition through phospho-NDRG1 51
protein expression levels in MDA-MB-468 cells
4.4. Effect of SGK1 inhibition via GSK650394 on the proliferation 52
of MDA-MB-231 cells
4.4.1. Effect of SGK1 inhibition via GSK650394 on the proliferation 52
of MDA-MB-231 cells macroscopically
4.4.2. Effect of SGK1 inhibition via GSK650394 on the proliferation 53
of MDA-MB-231 cells microscopically
4.5. Effect of SGK1 inhibition via GSK650394 on the cell cycle 54
phases distribution in MDA-MB-231 cells
4.6. Effect of SGK1 inhibition on phospho-GSK3 beta expression 55
in triple negative breast cancer cell lines
4.6.1. Effect of SGK1 inhibition on phospho-GSK3 beta expression 56
in MDA-MB-231 cell line
4.6.2. Effect of SGK1 inhibition on phospho-GSK3 beta expression 57
in HCC1937 cell line
4.6.3. Effect of SGK1 inhibition on phospho-GSK3 beta expression 58
in BT549 cell line
4.6.4. Effect of SGK1 inhibition on phospho-GSK3 beta expression 59
in MDA-MB-436 cell line
4.7. Effect of SGK1 inhibition via GSK650394 on EMT related proteins 59
E-cadherin and vimentin protein expressions in triple negative breast
cancer cell lines
4.7.1. Effect of SGK1 inhibition via GSK650394 on E-cadherin protein 60
expressions in triple negative breast cancer cell lines
4.7.2. Effect of SGK1 inhibition via GSK650394 on vimentin protein 61
expressions in triple negative breast cancer cell lines
4.8. Effect of SGK1 inhibition via GSK650394 on EMT related 64
transcription factors in MDA-MB-231 and BT549 cell lines
4.8.1. Effect of SGK1 inhibition via GSK650394 on EMT related 65
transcription factors in MDA-MB-231 cells
4.8.2. Effect of SGK1 inhibition via GSK650394 on EMT related 67
transcription factors in BT549 cells
5. DISCUSSION 70
6. CONCLUSION AND RECOMMENDATIONS 75
7. REFERENCES 76
8. SUPPLEMENTS 83
Supp.1. Approval of the Ethics Committee 83
Supp.2. Abstracts Derived from This Thesis 84
9. CURRICULUM VITAE 86 | tr_TR |
| dc.language.iso | en | tr_TR |
| dc.publisher | Kanser Enstitüsü | tr_TR |
| dc.rights | info:eu-repo/semantics/restrictedAccess | tr_TR |
| dc.subject | Breast cancer | tr_TR |
| dc.subject | Triple negative breast cancer | tr_TR |
| dc.subject | Triple negative breast cancer | tr_TR |
| dc.subject | SGK1 | tr_TR |
| dc.subject | GSK650394 | |
| dc.title | Inhibition of Serum and Glucocortıcoıd Inducible Kinase 1 (Sgk1) in Triple Negative Breast Cancer | tr_eng |
| dc.type | info:eu-repo/semantics/doctoralThesis | tr_TR |
| dc.description.ozet | Glukokortikoid reseptörünün ekspresyonunda artış özellikle triple negatif meme kanserinde kötü prognoza yol açmaktadır. Bu kötü prognozun SGK1 (Serum ve Glukokortikoid ile İndüklenebilen Kinaz 1) aktivasyonuna bağlı olduğu ortaya konulmuştur. Bu çalışmada, TNBC (triple negatif meme kanseri) hücre hatlarında SGK1 düzeyi ve SGK1 inhibitörü olan GSK650394’e duyarlılıkları incelendi. Bu hücreler arasında, SGK1 ekspresyonu belirgin olarak yüksek bulunan MDA-MB-436 hücreleri, SGK1 inhibisyonuna yanıt vermedi ve SGK1’in substratı olan NDRG1’in (N-Myc downstream regulated gene 1) fosforilasyonu yüksek düzeyde bulundu. SGK1 düzeyleri farklılık gösteren diğer hücre hatlarında (MDA-MB-231, HCC1937 ve BT549) ise kinaz aktivitesinin inhibisyonuna bağlı olarak NDRG1’in fosforilasyonunda belirgin azalma gözlendi. İlginç olarak, bu hücreler GSK650394’e duyarlı olmalarına karşın SGK1’in farmakolojik inhibisyonu GSK3β’nın fosforilasyonunda azalmaya yol açmadı ve GSK3β ‘nın yeniden aktivasyonu gözlenmedi. Buna ilave olarak, SGK1 inhibisyonu E-kaderin ve vimentin ekspresyonlarını değiştirmedi ve epitelyal-mezenkimal dönüşüm (EMT) ile ilgili baskılanma saptanmadı. Slug, Snail ve Twist mRNA düzeyleri de SGK1 inhibisyonundan etkilenmedi. GSK650394 ile inkübasyon sonucu, MDA-MB-231 hücrelerinin proliferasyonunda baskılanma ve S- fazında hafif azalma gözlendi. Bu çalışmanın sonuçları, SGK1 inhibisyonuna yönelik stratejilerin triple negatif meme kanserinde terapötik potansiyeli olabileceği görüşünü desteklemektedir. | tr_TR |
| dc.contributor.department | Temel Onkoloji | tr_TR |
