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dc.contributor.advisorDoğan, Ayşe Lale
dc.contributor.authorEbrahimi, Mohammad Azim
dc.date.accessioned2018-06-05T13:19:20Z
dc.date.available2018-06-05T13:19:20Z
dc.date.issued2018
dc.date.submitted2018-05-29
dc.identifier.urihttp://hdl.handle.net/11655/4521
dc.description.abstractMetadherin gene (MTDH/ AEG-1/ Lyric) encodes a 582- aminoacid protein, metadherin (MTDH), which is almost undetectable by immunohistochemistry in normal breast tissue. MTDH expression is markedly increased in breast cancer cell lines as well as breast cancer patients which is found to be positively correlated with poor prognosis. The constitutive activation of PI3K/Akt/mTOR pathway triggers MTDH expression via GSK3β inactivation. In this thesis, the effect of PI-103 (dual PI3K/mTOR inhibitor) on MTDH expression in HER2+ breast cancer cell lines was investigated using qPCR and Western Blot. The results indicated different levels of inhibiton for MTDH expression in HER2 overexpressing cells. In MDA-MB-453 cells, incubation with PI-103 decreased MTDH mRNA and protein level significantly (p<0.05) which was considered in quite accordance with Akt dependency of these cells. MTDH mRNA expression decreased in SKBR-3 cells after 8 hours following a slight increase in 24 hours. The protein expression level was also affected moderately from PI-103. Taking into consideration the remarkable PTEN expression in these cells, we may speculate that decreased response to drug inhibitory effect of PI-103 may be attributed to the inverse corrrelation between MTDH protein level and PTEN activity. Finally, BT474 cells did not show significant change for MTDH at mRNA and protein level. In addition to cell lines, serum MTDH level of breast cancer patients (n=80) was evaluated by ELISA. Among molecular subtypes, HER2+ patients (n=9) had significantly high level of MTDH compared to patients diagnosed as Luminal A (n=20), and TNBC (n=32) (p<0.05) . There was no significant difference between HER2+ and Luminal B (n=19) subtypes. MTDH level was found significantly high in metastatic breast cancer (n=13) compared to nonmetastatic group (n=66) (p<0.01). In conclusion, PI3K/Akt targeting therapeutic strategies in HER2+ breast cancer may be involved in suppression of invasion and metastasis through regulation of MTDH expression.en
dc.description.tableofcontentsAPPROVAL PAGE iii YAYIMLAMA VE FİKRİ MÜLKİYET HAKLARI BEYANI iv ETHICAL DECLARATION .v ACKNOWLEDGEMENTS vi ABSTRACT vii ÖZET viii TABLE OF CONTENTS ix LIST OF ABBREVIATIONS xii LIST OF FIGURES xvi LIST OF TABLES xviii 1. INTRODUCTION 1 2. GENERAL INFORMATION 4 2.1 Breast Cancer 4 2.2 Receptor Tyrosine Kinase (RTK) 6 2.3 Epidermal Growth Factor Receptors (EGFR) and Regulation 8 2.4 Epidermal Growth Factor (EGFR) and Its Relation to Cancer 10 2.5 PI3K/Akt Signaling Pathway 12 2.6 PI3K/Akt Signaling Pathway and Breast Cancer 14 2.7 Metadherin 15 2.8 Therapeutic Approach to PI3K/Akt/mTOR Pathway 19 2.9 Aim of the Study 20 3. MATERIAL AND METHODS 21 3.1 Chemicals, Kits and Buffer Solutions 21 3.2 Equipments and Devices 25 3.3 Cell Culture 29 3.4 Trypsinization 29 3.5 Cell Passage 30 3.6 Cell Freezing 30 3.7 Mycoplasma Test For The Culture 31 3.8 Drug Administration 32 3.9 RNA Isolation 33 3.10 cDNA Synthesis from Obtained RNAs 35 3.11 Preparation of Drug Treatment and Lysate with PI-103 for Protein Assay 36 3.12 Lyric/Metadherin Protein Detection and Analysis by Western Blot Method in All Cells 37 3.13 Patient Specimens and ELISA Assay 38 3.14 Statistical Analysis 39 4. RESULTS 40 4.1 Effect of pharmacological inhibition of PI3K pathway with PI-103 dual inhibitor on protein expression of metadherin (MTDH) in breast cancer cell lines 40 4.2 Effect of pharmacological inhibition of PI3K pathway with PI-103 on metadherin mRNA expressions in MDA-MB-453, BT-474 and SKBR-3 cell lines 47 4.2.1 Effect of time-dependent incubation of PI-103 on metadherin mRNA expression in the MDA-MB-453 cell line 48 4.2.2. Effect of time-dependent incubation of PI-103 on metadherin mRNA expression in the BT-474 cell line 48 4.2.3. Effect of time-dependent incubation of PI-103 on metadherin mRNA expression in the SKBR-3 cell line 49 4.3 Evaluation of metadherin level in breast cancer patients 51 4.3.1 Evaluation of metadherin level in different molecular subtypes of breast cancer 52 4.3.2 Evaluation of metadherin level according to the immunostaining score of HER2 in breast cancer patients 53 4.3.3 Comparison of metadherin level between metastatic or non-metastatic breast cancer patients 54 4.3.4 Evaluation of metadherin level according to tumor grade of breast cancer patients 55 4.3.5 Comparison of metadherin level in node positive or node negative in breast cancer patients 55 5. DISCUSSION 57 6. CONCLUSIONS AND FUTURE DIRECTIONS 60 7.REFERENCES 61 8.APPENDIX Appendix-1: Ethics committee approval of cell lines Appendix-2: Ethics committee approval of patient materials Appendix-3: Poster presentation related to this thesis CURRICILUM VITAEtr_TR
dc.language.isoengtr_TR
dc.publisherSağlık Bilimleri Enstitüsütr_TR
dc.rightsinfo:eu-repo/semantics/closedAccesstr_TR
dc.subjectMetadherintr_TR
dc.subjectbreast cancer
dc.subjectPI3K
dc.subjectmTOR
dc.subjectPI-103
dc.titleEvaluation of Metadherin Expression in Breast Cancertr_TR
dc.typedoctoralThesistr_TR
dc.description.ozetMetadherin geni (MTDH/ AEG-1/ Lyric) 582 aminoasit uzunluğunda bir protein olan metadherini (MTDH) kodlamaktadır. Normal meme dokusunda immünohistokimya yöntemiyle ölçülemeyecek kadar az olan MTDH ekspresyonu meme kanseri hücre hatlarında ve meme kanseri hastalarında belirgin olarak artmaktadır ve MTDH düzeyi kötü prognoz ile doğru orantılıdır. Bu tezde, HER2+ meme kanseri hücre hatlarında dual PI3K ve mTOR inhibitörü olan PI-103’ün MTDH ekspresyonuna etkisi qPCR ve Western Blot yöntemleri ile incelendi. Sonuçlarımız, HER2 ekspresyonu artmış olan meme kanseri hücrelerinde farklı düzeylerde MTDH inhibisyonu olduğunu ortaya koydu. MDA-MB-453 hücrelerinde, PI-103 inkübasyonu MTDH mRNA ve protein düzeyini anlamlı olarak azalttı (p<0.05). Bu sonuç, hücrelerin Akt bağımlılığı ile oldukça uyumlu olarak değerlendirildi. SKBR-3 hücrelerinde, MTDH mRNA ekspresyonunda 8 saat sonra gözlenen azalmayı 24 saat sonunda hafif bir artış takip etti. Protein ekspresyonu da PI-103’ten orta düzeyde etkilendi. Bu hücrelerdeki belirgin PTEN ekspresyonunu dikkate aldığımızda, PI-103’ün inhibitör etkisine karşı düşük yanıt alınmasının, MTDH protein düzeyi ile PTEN aktivitesi arasında ters yönde korelasyonun bulunması ile bağlantılı olabileceğini öne sürebiliriz. Son olarak, BT474 hücreleri, mRNA ve protein düzeyinde anlamlı bir değişiklik göstermedi. Hücre hatlarına ilave olarak, meme kanseri hastalarının serumlarındaki (n=80) MTDH düzeyi ELISA yöntemiyle incelendi. Moleküler alttipler arasında, HER2+ hastaların (n=9) MTDH düzeyi, diğer alttipler olan Luminal A (n=20), ve TNBC (n=32) tanısı alan hastalarla kıyaslandığında anlamlı olarak yüksek bulundu (p<0.05). HER2+ ve Luminal B (n=19) alttipleri arasında anlamlı fark bulunmadı. MTDH düzeyi, metastatik meme kanseri grubunda (n=13), nonmetastatik grup (n=66) ile karşılaştırıldığında, anlamlı olarak yüksek saptandı (p<0.01). Sonuç olarak, HER2+ meme kanserinde PI3K/Akt yolağını hedefleyen terapötik stratejiler MTDH ekspresyonunun regülasyonu aracılığıyla invazyon ve metastazın baskılanmasında rol oynayabilirler.tr_TR
dc.contributor.departmentTemel Onkolojitr_TR


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