갑상선암에서 TERT promoter 변이의 의의와 BRAF, RAS 변이와의 시너지 상호작용

학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의학과, 2018. 2. 박영주.Recent reports suggest that mutations in the promoter of the gene encoding telomerase reverse transcriptase (TERT) affect thyroid cancer outcomes. I aimed to investigate the clinical significance of TERT promoter mutation in thyroid cancer and its synergistic interaction with BRAF and RAS mutations. Furthermore, molecular mechanisms of the oncogene interaction by genomic analysis using next-generation sequencing database were explored. TERT promoter mutations were detected in 4.5% of all differentiated thyroid cancers and associated with poor prognosis. These mutations were more frequent in tumors also harboring either BRAF (4.8%) or RAS mutations (11.3%). The prevalence of TERT promoter mutations was higher in high-risk patients: 9.1% and 12.9% in the ATA high-risk and advanced TNM stage groups, respectively. Among high-risk patients, the presence of TERT promoter mutations additively increased the risk of both recurrence and disease-specific mortality. The coexistence of BRAF and TERT promoter mutations had a synergistic effect on the clinicopathological characteristics and long-term prognosis of papillary thyroid cancer (PTC) and I firstly confirmed this by meta-analysis. From the analyses of RNA sequencing data and in vitro experiments, I could confirm that TERT mRNA expression was increased by adding the BRAF mutation to the TERT promoter mutation (fold change, 17.00q-value = 1.36×10-13). Furthermore, this increase was due to, at least in part, the upregulated expression of E-twenty-six (ETS), especially ETV1, ETV4, and ETV5 by BRAF mutation. The coexisting mutations showed changes in the almost same intracellular signaling pathways as BRAF mutation alone, however, amplified the changes of the expression level of genes associated with altered pathways. Moreover, the inflammation and adhesion-related pathways were activated by adding TERT expression in BRAF-mutated PTCs. Notably, I firstly reported that the coexistence of RAS and TERT promoter mutations was associated with a higher rate of recurrence, suggesting that they had additive effects on the prognosis, similarly to BRAF and TERT promoter mutations. As for the mechanism, I could confirm that this genetic duet significantly increased TERT expression (fold change, 5.58q-value = 0.004) compared with the expression in tumors harboring RAS or TERT promoter mutation alone. Moreover, adding the TERT promoter mutation or expression to the RAS mutation, there were significant changes in transcriptional profile, which activated the aggressive intracellular pathways including MAPK pathways. In conclusion, genetic screening for TERT promoter mutations in high-risk patients with thyroid cancer might bolster the prediction of mortality and recurrence. In addition, molecular testing of TERT promoter mutation with BRAF or RAS mutation together may be useful in assisting with risk stratification in clinical settings. Furthermore, I can suggest that the mechanism of synergistic oncogene interaction between TERT and BRAF or RAS be explained by increased TERT expression, which may result from the BRAF or RAS-induced upregulation of several ETS transcription factors. Pathways related to aggressive behaviors of tumors are activated by the genetic duetBRAF and TERT or RAS and TERT.Introduction 1 1. Increase in the prevalence of thyroid cancer and importance of predicting prognosis 1 2. Prevalence of genetic alterations in thyroid cancer 2 3. Telomerase reverse transcriptase (TERT) and the promoter mutations 4 4. Effects of the coexistence of BRAF and TERT promoter mutations on clinical outcomes in thyroid cancer 6 5. Effects of the coexistence of RAS and TERT promoter mutations on clinical outcomes in thyroid cancer 7 6. Potential molecular mechanisms of synergistic oncogene interaction between TERT and BRAF or RAS 8 7. Hypothesis 10 8. Aims of study 10 Chapter I. Prevalence and clinical significance of TERT mutation in thyroid cancer 12 Materials and methods 13 Results 18 Discussion 33 Chapter II. TERT promoter and BRAF mutations in papillary thyroid cancer 37 II-1. Clinical significance of TERT and BRAF mutations in papillary thyroid cancer 38 Materials and methods 38 Results 42 II-2. Meta-analysis of synergistic effects of coexisting TERT and BRAF mutations on clinical outcomes 48 Materials and methods 48 Results 51 II-3. Molecular genetic mechanisms of synergistic interaction between TERT promoter and BRAF mutations 81 Materials and methods 81 Results 89 Discussion 123 Chapter III. TERT promoter and RAS mutations in follicular thyroid cancer 132 III-1. Clinical significance of TERT and RAS mutations in papillary thyroid cancer 133 Materials and methods 133 Results 137 III-2. Molecular genetic mechanisms of synergistic interaction between TERT promoter and RAS mutations 149 Materials and methods 149 Results 151 Discussion 163 References 170 Summary and conclusions 186 Abstract in Korean 188Docto

Effects of Thyroid Stimulating Hormone on Tumor Growth by Modulating Tumor Microenvironment in Thyroid Cancer

학위논문 (석사)-- 서울대학교 대학원 : 의학과 중개의학 전공, 2016. 2. 박영주.Introduction: The stimulatory effect of thyroid stimulating hormone (TSH) through TSH receptor signal pathways on the growth of thyrocytes is well-demonstrated. Differentiated thyroid cancer (DTC) expresses TSH receptors and retains responsiveness to TSH. Thus, TSH suppression has been used as an important and effective treatment in patients with DTC. Since tumor microenvironment including angiogenesis has a crucial role in cancer progression and metastasis, we investigated whether the effects of TSH on tumor growth are also mediated by tumor microenvironment using a mouse model of DTC. Materials and Methods: BHP10-3SC DTC cells, which express TSH receptors, were subcutaneously implanted on 7-week-old BALB/c nu/nu mice. When the greater diameter of tumor became 5 mm or larger, recombinant human TSH (rhTSH, 1.5 μg/g) or vehicle was started to be injected intraperitoneally. Tumor size was measured every 3 days. After 15 days, tumor histology was examined and vascular endothelial growth factor (VEGF) mRNA expression was analyzed using real time-PCR. For supporting in vivo results, in vitro experiments to demonstrate TSH effects on angiogenesis were performed in BHP10-3SC and human endothelial cells. Results: Tumors in rhTSH group grew more rapidly than controls, and there was a significant difference in the tumor volume (on day 15, 1733.4 ± 793.5 mm3 vs. 1148.8 ± 471.1 mm3, respectively, P = 0.010). The vascular density in tumors was significantly increased in rhTSH group (13.8 ± 0.8% vs. 5.7 ± 0.8% in control, P = 0.021). Moreover, more tortuous and dilated vessels were observed in tumors of rhTSH group compared with controls (23.0 ± 1.7 μm vs. 7.4 ± 0.5 μm in vascular diameter, respectively, P <0.001). In addition, the macrophage infiltration in tumors was significantly increased in rhTSH group (27.6 ± 11.6% vs. 12.1 ± 4.3% in control, P = 0.004). In vitro experiments showed that TSH induced a significant up-regulation of VEGF-A mRNA expression in BHP10-3SC cells. Conditioned medium of TSH-treated BHP10-3SC cells (TSH-CM) contained higher concentration of VEGF-A than saline-treated CM (control-CM). Finally, treatment of TSH-CM significantly enhance potentials of cell migration and tube formation in human endothelial cells, HMVEC or HUVEC. Conclusions: TSH supports the growth of thyroid cancer via enhancing abnormal vasculature and subsequent recruitment of macrophages in tumor microenvironments.Introduction 1 1. Positive correlation between serum TSH level and tumor progression in thyroid cancer patients 1 2. Regulatory mechanism of TSH on growth of differentiated thyroid tumor cells 1 3. Vascular endothelial growth factor (VEGF)-regulated angiogenesis in thyroid cancer 2 4. Tumor-associated macrophage (TAM) in thyroid cancer 4 5. Aims of Study 4 Materials and Methods 6 1. Mice 6 2. Cell cultures 6 3. Experimental protocols of TSH treatment on mouse tumor model 8 4. Measurement of tumor size 13 5. Measurement of serum thyroid hormone 14 6. Immunofluorescence, H&E, and immunohistochemistry 14 7. Electron microscopy 15 8. RNA extraction and real time-PCR analysis 16 9. Conditioned medium of TSH-treated BHP10-SC cells 17 10. Measurement of VEGF-A protein levels 17 11. Cell migration and tube formation assay 17 12. Morphometric analysis 18 13. Statistical analysis 19 Results 20 1. Time effects of TSH on angiogenesis in normal thyroid and thyroid cancer 20 1) Effects of TSH on thyrocytes and angiogenesis in thyroid gland 20 2) Effects of TSH on angiogenesis in thyroid cancer 21 2. Effects of TSH on tumor growth 26 1) Effects of cell passages on tumorigenesis of xenografts 26 2) Effects of TSH on tumor growth 29 3. In vivo effects of TSH on angiogenesis 34 1) Angiogenesis and vascular permeability 34 2) Endothelial fenestration 34 3) VEGF expression 35 4. In vivo effects of TSH on TAM 39 5. In vitro effects of TSH on angiogenesis 41 Discussion 48 References 52 Abstract in Korean 58Maste