The Role of Bcl-xL and EGFR in the Malignant Progression of Neurofibromatosis type 1
신경섬유종증 제1형의 악성화에 관여하는 Bcl-xL과 EGFR의 작용기전 연구
- 주제(키워드) "Neurofibromatosis type 1" "Bcl-xL" "EGFR"
- 발행기관 아주대학교
- 지도교수 정선용
- 발행년도 2013
- 학위수여년월 2013. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000013530
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Neurofibromatosis Type 1 (NF1) is one of the most commonly inherited autosomal dominant human genetic disorders, with an incidence of approximately 1 in 3000~3500 individuals worldwide. NF1 is caused by loss-of-function mutations in the NF1 gene encoding neurofibromin, a GTPase-activating protein that regulates the small GTPase Ras signaling pathway by converting the active GTP-Ras to an inactive GDP-Ras. The transformation of benign plexiform neurofibromas (PNs) to malignant peripheral nerve sheet tumors (MPNSTs) in patients with NF1 is notable and is of far greater concern because MPNSTs are a type of aggressive sarcoma and are a major cause of mortality in patients with NF1. Yet the pathogenesis is poorly understood. In addition to the NF1 mutation, it has been suggested that other genetic or epigenetic changes may participate in the malignant change of benign tumor tissues to MPNSTs. Despite robust histological and molecular analyses that have been conducted to compare PNs and MPNSTs, the exact molecular pathogenesis of the malignant transformation in NF1 patients has not yet been elucidated. Long-term investigations using a multi-model therapeutic strategy revealed that there are still many hurdles to overcome in chemotherapy for the NF1-associated MPNSTs. In this study, I aimed to clarify the molecular mechanisms of NF1’s malignant progression, which may lead to finding new target molecules and/or drugs for improved chemotherapeutic approaches that treat NF1-associated MPNSTs effectively. Since hyperexpression of epidermal growth factor receptor (EGFR) in Schwann cells (SCs) is frequently observed in NF1-associated MPNST tissues, I first examined whether the basal EGFR expression levels were different between SCs derived from normal tissues or MPNSTs. By performing a comparison analysis between normal and MPNST SCs, I found that EGFR expression levels were significantly higher in the MPNST SCs. Interestingly, I found that the expression level of the EGFR protein was inversely related to the expression level of neurofibromin in all the SCs tested, suggesting that upregulated EGFR in the NF1-associated MPNSTs may be caused by neurofibromin deficiency. Manipulation of NF1 gene expression by RNA interference (RNAi) and overexpression demonstrated that neurofibromin plays a role in regulating the transcriptional expression of the EGFR. The results of site-specific chromatin immunoprecipitation (ChIP) targeting the phosphorylated Sp1 (pSp1)-binding site of the EGFR gene revealed that the knockdown of NF1 stimulated the binding of pSp1 to the EGFR gene. In addition, inhibition of extracellular signal-regulated kinase 1 and 2 (Erk1/2), a Ras downstream protein regulated by neurofibromin and an upstream regulator of Sp1, resulted in a weak increase in the EGFR level in neurofibromin-depleted cells. These results demonstrated that neurofibromin regulates EGFR expression by modulating the binding of the Sp1 transcription factor to the EGFR gene promoter. The results also demonstrate that the Ras/Erk/Sp1-signaling pathway mediates the EGFR upregulation in NF1-deficient MPNST cells. Crucially, I found that the antiapoptotic Bcl-xL protein was upregulated in MPNST cells and the increased Bcl-xL expression caused an increase in an MPNST cell’s resistance to anticancer drugs. These results suggest that the alteration of the Bcl-xL expression level by somatic expression changes in the NF1 locus may contribute to the development of MPNSTs in otherwise benign tumors. The manipulation of NF1 gene expression levels demonstrated that the decreased transcriptional expression of the NF1 gene, as in the neurofibromin-mediated EGFR expression, caused the high expression of Bcl-xL in MPNST cells. Because Bcl-xL is also one of the downstream proteins of the Ras/Erk/Sp1 pathway, I tested the effect of Erk1/2 in regulation of the neurofibromin-mediated Bcl-xL expression. Treatment with the Erk1/2 inhibitor PD98059 resulted in a weak increase in the Bcl-xL level in the neurofibromin-depleted cells, indicating that Erk1/2 activation caused the Bcl-xL upregulation in MPNST cells. In addition, knockdown of SP1 and NF1 genes demonstrated that the Ras/Erk/Sp1-signaling pathway mediated the Bcl-xL upregulation in the MPNST cells. I unexpectedly found that Bcl-xL modulated neurofibromin levels in the opposite direction. The knockdown of Bcl-xL caused an increase in the neurofibromin level, whereas Bcl-xL overexpression resulted in a decreased neurofibromin level. I discovered that Bcl-xL regulates neurofibromin levels by modulating the ubiquitin-mediated proteolysis of neurofibromin. In particular, Bcl-xL negatively regulated the neurofibromin level by increasing the binding of ETEA, a ubiquitin-related protein, to neurofibromin. These results suggest that Bcl-xL plays a crucial role in the regulation of EGFR-mediated Ras signaling in a positive feedback manner by modulating cellular neurofibromin levels. I further demonstrated that either the depletion of Bcl-xL expression by RNAi or the inactivation of Bcl-xL by ABT-737, a mimetic of the BH3-only protein BAD, was very effective in sensitizing the MPNST cells to apoptotic cell death when combined with the tested anticancer drug Doxorubicin. Because immunohistological staining results showed that both EGFR and Bcl-xL proteins were upregulated in MPNST tissues (when compared to that in PN tissues from patients with NF1), I tested the effect of the combined treatment of the EGFR inhibitor Erlotinib, the Bcl-xL inhibitor ABT-737, and the apoptosis inducer Doxorubicin in MPNST cells. A low concentration of Erlotinib, ABT-737, and Doxorubicin could effectively induce synergistic cytotoxicity in the MPNST cells with minimal side effects. These results suggest that pharmacological inhibition of both EGFR and Bcl-xL in combination with anticancer drug-inducing apoptosis may be a potential therapeutic strategy for the treatment of NF1-associated MPNSTs. Collectively, the results suggest that an NF1 deficiency-mediated elevation in the Ras/Erk/Sp1-signaling pathway may cause the upregulation of EGFR and Bcl-xL in MPNST cells and may provide a new chemotherapeutic target in patients with NF1 and MPNSTs.
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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS v
LIST OF FIGURES viii
LIST OF TABLES xi
I. INTRODUCTION 1
II. MATERIALS AND METHODS 12
1. Antibodies and reagents 12
2. Patients tissue samples and primary culture 12
3. Cell lines 15
4. Hematoxylin and eosin staining 15
5. Immunohistochemistry 15
6. Cell viability assay 16
7. Plasmids, small interfering RNAs (siRNAs) and transfection 16
8. Real-time reverse transcription-polymerase chain reaction (RT-PCR) 17
9. Chromatin immunoprecipitation (ChIP) assay 18
10. Immunoprecipitation and immunoblotting 19
11. Cytoplasmic and nuclear fractionation 20
12. Ras activation assay 20
13. Immunofluorescence cell staining 21
III. RESULTS 22
1. The EGFR expression level was closely related to the neurofibromin expression level in both normal and MPNST cells 22
2. Manipulation of NF1 gene expression caused alterations in the transcriptional
expression of the EGFR gene 25
3. Neurofibromin-regulated EGFR expression occurred by modulating the binding of
the Sp1 transcription factor to the EGFR gene promoter 28
4. Neurofibromin-regulated EGFR expression occurred by modulating the Erk/Sp1-signaling pathway 35
5. MPNST cells were more resistant than normal cells to anticancer drugs inducing apoptosis 37
6. The downregulation of NF1 expression reduced the apoptosis sensitivity to Doxorubicin in normal cells 39
7. The NF1 gene dose mediated the Bcl-xL expression level in normal and MPNST cells 41
8. The endogenous Bcl-xL was upregulated in MPNST cells 44
9. The Bcl-xL expression level was closely related to the apoptosis sensitivity to Doxorubicin in both normal and MPNST cells 46
10. NF1 deficiency-induced Bcl-xL expression occurred by activating the Ras/Erk/Sp1 signaling pathway 49
11. Both the phosphorylated EGFR and Bcl-xL were expressed more in MPNST than in PN tissues form NF1 patients 53
12. Bcl-xL regulated the neurofibromin expression level 56
13. Bcl-xL regulated the ubiquitin-mediated proteolysis of neurofibromin
by modulating the ETEA binding to neurofibromin 58
14. Bcl-xL negatively regulated neurofibromin by enhancing the ubiquitin-mediated proteolysis of neurofibromin in MPNST cells. 62
15. Bcl-xL positively regulated EGFR by reducing the neurofibromin level 64
16. The Bcl-xL inhibitor ABT-737 enhanced chemotherapeutic sensitivity to anticancer drugs in MPNST cells 66
17. The combined treatment of Erlotinib, ABT-737, and Doxorubicin synergistically enhanced apoptotic cell death in NF1-deficient MPNST cells 69
IV. DISCUSSION 72
V. CONCLUSION 83
REFERENCES 84
국문요약 101
