보고서 정보
주관연구기관 |
한국생명공학연구원 Korea Research Institute of Bioscience and Biotechnology |
연구책임자 |
원미선
|
참여연구자 |
김보경
,
반현승
,
임주영
,
원경재
,
정영진
,
홍예슬
|
보고서유형 | 1단계보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2013-10 |
과제시작연도 |
2012 |
주관부처 |
미래창조과학부 KA |
사업 관리 기관 |
한국연구재단 |
등록번호 |
TRKO201400006441 |
과제고유번호 |
1345167482 |
DB 구축일자 |
2014-05-31
|
키워드 |
암치료타겟,타겟 검증,맞춤의료,폐암,유전자 복구,세포사멸cancer therapeutic target,target validation,lung cancer,DNA repair,apoptosis
|
초록
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1. NUAAP의 전사조절
- 페암세포주/HEK293세포주: NUAAP 전사발현에 프로모터 P1이 주요부위, P2/P4는 조절 부위임
- 전사인자 NFAT2가 P1에 결합하며 NFAT2 signaling이 NUAAP 발현 조절에 중요함.
- P4에 특이적으로 결합하는 hnRNPK를 발굴하였고 NUAAP 전사 조절 관련성을 확인하였음.
- NUAAP 발현 저해 약물 스크리닝 시스템을 구축, 신규 약물 1400 종 스크리닝 수행, 시스템 검증함.
2. NUAAP alternative splicing
-
1. NUAAP의 전사조절
- 페암세포주/HEK293세포주: NUAAP 전사발현에 프로모터 P1이 주요부위, P2/P4는 조절 부위임
- 전사인자 NFAT2가 P1에 결합하며 NFAT2 signaling이 NUAAP 발현 조절에 중요함.
- P4에 특이적으로 결합하는 hnRNPK를 발굴하였고 NUAAP 전사 조절 관련성을 확인하였음.
- NUAAP 발현 저해 약물 스크리닝 시스템을 구축, 신규 약물 1400 종 스크리닝 수행, 시스템 검증함.
2. NUAAP alternative splicing
- NUAAP의 alternative splicing에 의해 다양한 구조의 transcript variants 생성됨.
- DNA damage를 유도하는 UV나 CPT가 NUAAP mRNA의 alternative splicing을 촉진하고 동시에 짧은 단백질 단편이 생성되어 총 NUAAP 단백질 감소를 야기함.
3. NUAAP의 DNA damage 억제 효능
- NUAAP knockdown은 암세포주에 따라 p38/p53 경로 활성화로 apoptosis/mitotic cell death 유도.
- CPT에 의한 DNA damage 가 NUAAP 과발현에 의해 억제되고, NUAAP knockdown과 동시 DNA damage 유도 약물 처리는 암세포주 성장억제에 시너지 효능을 나타냄.
4. NUAAP의 기능
- NUAAP knockdown /과발현에 의해 STAT3 단백질 양적 변화: NUAAP과 STAT3와 직접 결합하여 NUAAP가 STAT3를 stabilize에 관여함.
- NUAAP과 상호작용하는 단백질 ß-catenin 전사 관련된 CBY1, apoptosis 관련 APIP 및 Ubiquitination 분해관련 CHIP이 발굴되었고 ChIP-seq에서 apoptosis 유도 CAPN8을 발굴하였음.
5. NUAAP 저해에 의한 항암 효능
- 다양한 폐암 세포주에 대해 NUAAP knockdown으로 in vitro 세포 성장 억제 효능
- in vivo NUAAP knockdowni에 의한 항종양 효능, NUAAP knockdown과 γ-radition의 병용효능
- 폐암 모델 쥐에서 in vivo toxicity 조사하였음.
Abstract
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NUAAP (FLJ25416, human Noxin, hNoxin, C11Orf82) shares 56% homology with mouse noxin. NUAAP was identified as a cancer-related protein which is highly expressed in colorectal and lung cancer tissues. The hNoxin gene (Genbank NM_145018) has six exons, including a translation start site in the third e
NUAAP (FLJ25416, human Noxin, hNoxin, C11Orf82) shares 56% homology with mouse noxin. NUAAP was identified as a cancer-related protein which is highly expressed in colorectal and lung cancer tissues. The hNoxin gene (Genbank NM_145018) has six exons, including a translation start site in the third exon, and encodes a 998-amino acid polypeptide. NUAAP contains a DNA-binding C-domain in RPA1, which mediates DNA metabolic processes, such as DNA replication and DNA repair. We characterized that NUAAP expression is induced by UV and that NUAAP is involved in anti-apoptotic function. Therefore, we named it NUAAP, Noxin-like UV-inducible anti-apoptotic protein.
Expression of NUAAP was examined in normal human tissues. The strong expression of NUAAP was detected in human testis, pancreas, and prostate, with weaker expression in the lung, stomach, thymus, colon, and heart, which was similar to that of Noxin in normal mouse tissues. In situ mRNA hybridization assay demonstratdd a large amount of NUAAP mRNAs were observed in both colorectal and lung cancer tissues compared with normal tissues. RT-PCR also showed NUAAP mRNA levels were higher in colorectal cancer and lung cancer cells, indicating that NUAAP was strongly expressed in colorectal and lung cancer tissues. Cell cycle analysis by cell synchronization through starvation revealed that NUAAP mRNA levels increased at early S phase and remained high until late S phase in both cells despite 4h difference due to slower growth of WI-38 cells. Expression of NUAAP was induced by ultraviolet (UV) irradiation.
At first, transcriptional start site of NUAAP was examined by 5'-Race experiment and found 32 bp downstram of the site reported as a transcription start site. To study transcriptional regulation of NUAAP gene, promoter analysis was carried out by examining transcription factor binding sites and cloning the region in front of exon 1, exon 2 and exon 4 to construct reporter assay system using luciferase. Reporter assay revealed P1 containing -350 ~ +158 region was crucial to express NUAAP transcripts and both P2 and P4 played a role in suppressing transcription of NUAAP gene. Transcription factors E2F1 and NFAT2 were involved in the regulation of NUAAP transcription. The site-directed mutagenesis of NFAT2 binding site, ChIP analysis and reporter assay showed NFAT2 bound to NFAT-binding site and the region of -199~-79 was important to regulate NUAAP expression. It was clear that NFAT2 knockdown caused growth inhibition of lung cancer cells, H1703 and A549 cells, and that overexpression of NUAAP rescued cells from death induced by NFAT2 knockdown, indicating NFAT2 regulates NUAAP expression. We also found that EGF, TNFa, IL-6 increased promoter activity of NUAAP. The gene expressions regulated by EGF signaling also relevant to NUAAP expression. Next, in the attempt to isolate DNA binding proteins to suppress P1 activity, 5 different 35 mer biotin-labeled oligonucleltides in P4 were synthesized and used for pull-down assay with nuclear lysates. The specific protein bound to P4 was isolated and analyzed by MALDI-TOF. The knockdown of hnRNPK, which was identified as a protein bound to P4, increased NUAAP expression suggesting hnRNPK functions as suppressor of NUAAP expression even though the exact role of hnRNPK is not clear at this moment.
NUAAP gene was reported to generate various transcripts by alternative splicing. However, we identified more transcripts in addition to transcripts showed in database (UCSC Genome Browser) Major transcript contains exon 1 through exon6. Small amounts of various transcripts generated by alternative splicing included deletion of exon 4, deletion of both exon4 and 5, addition of 214, or 75 bp between exon 2 and exon3. The insertion of 134 bp between exon3 and exon 4 caused frameshift mutations resulted in generation of a short polypeptide. Interestingly, DNA damage agents such as UV, and camptothecin treatment enhanced alternative splicing and resulted in reduction of functional NUAAP due to early stop with deletions of exon4 and both exon4 and 5 leading polypeptides of 120 aa and 66 aa respectively.
Interestingly, we found that NUAAP knockdown decreased STAT3 protein level without effect on STAT3 mRNA level. On the contrary, overexpression of NUAAP increased STAT3 protein level without change in the amount of STAT3 mRNA, suggesting NUAAP affects the stability of STAT3 protein. Immunoprecipitation assay of Flag-NUAAP with antibody against Flag showed NUAAP interacted with STAT3. Then, STAT3 protein stability was determined in the presence of cycloheximide, protein synthesis inhibitor. It was demonstrated that the stability of STAT3 protein decreased in the NUAAP-knockdowned cells suggesting NUAAP plays a role in stabilizing STAT3 protin. The mechanism of stabilization of STAT3 by NUAAP is under investigation.
Knockdown of NUAAP caused growth inhibition of colorectal and lung cancer cells. The comet assay and Western blot analysis revealed that NUAAP knockdown induced apoptosis through activation of p38 mitogen-activated protein kinase (MAPK)/p53 in A549 cells. In A549 cells, inhibition of p38 MAPK during NUAAP knockdown-mediated apoptosis suppressed phosphorylation of p53, rescuing cells from death. It is clear that NUAAP knockdown induces apoptosis of A549 cells by activating phosphorylation of p38 MAPK/p53 and expressing p53 target genes Bax and Puma. Furthermore, simultaneous NUAAP knockdown and treatment with DNA-damaging agents, such as camptothecin (CPT) and UV irradiation, enhanced apoptosis, whereas (TSA) did not. However, transient overexpression of NUAAP rescued cells from DNA damage-induced apoptosis but did not block apoptosis in the absence of DNA damage. These results suggest that NUAAP may be associated with inhibition of a poptosis in response to DNA damage. Transient overexpression of NUAAP rescued A549 cells from CPT-induced DNA damage and apoptosis but not from TSA-induced growth inhibition. Furthermore, the combination of NUAAP knockdown and treatment with the DNA-damage agents, CPT or UV irradiation, resulted in synergistic effects on the apoptosis of A549 cells. The observation that NUAAP knockdown sensitized cells to apoptosis following DNA damage supports the hypothesis that NUAAP may be an anti-apoptotic protein involved in DNA repair or cell survival.
To understand function of NUAAP, we attempt to isolate NUAAP-interacting proteins by yeast two-hybrid analysis. In preparation of a bait of NUAAP, it was shown most region of NUAAP (1-783) bound to DNA. We used small polypeptide of NUAAP (784-998 aa) as a bait, and obtained 81 clones of total 21 candidate proteins, including CHIP, PLSCR1 and Cby1. We demonstrated in vitro binding of NUAAP with CHIP, PLSCR1 or Cby1. Furthermore, in vivo binding of CHIP, PLSCR1 or Cby1 with Flag-NUAAP by coimmunoprecipitation using Flag antibody was also demonstrated. The functional relevance of NUAAP with these proteins are also under investigation.
To validate NUAAP as a therapeutic target of lung cancer, in vivo xenograft assay was carried out using NUAAP siRNA in lung cancer A549 and H1703 model. NUAAP knockdown using F7 (0.5 mg/kg, 1.0 mg/kg) induced tumor regression compared to control scrambled siRNA. Then, combination effect of siRNA knockdown and x-ray irradiation on tumor growth was examined in C33A ovarian cancer cells. The combination of NUAAP knockdown and x-ray irradiation caused significant regression of tumor (67%), showing DNA damage in the tissues of mouse treated with both NUAAP knockdown and x-ray irradiation. This result suggests that NUAAP is involved in DNA damage repair during DNA synthesis of S phase and apoptosis. Then, liver toxicity caused by NUAAP knockdown was investigated in A549 mouse model. In NUAAP siRNA treated mouse, changes in BUN, AST, ALT, LDH enzymatic activities were not significant, suggesting NUAAP knockdown may not cause adverse effect on normal organ or cells.
To develop combination therapeutics in NUAAP knockdowned cells, we examined growth inhibition of 7 cancer cells by combination of anti-tumor agents and NUAAP siRNA. NUAAP knockdown demonstrated more stronger effect on growth inhibition of H1703, A549 and H1299 cells, which were insensitive to tytrosine kinase gefitinib and erlotinib compared to Calu3 and H827 cells. Even though H1703 cells showed higher GI50 (>10 μM) of erlotinib than H827 (sub nM) or Calu3 (4.3 μM), combination treatment of NUAAP knockdown and erlotinib or gefitinib on H1703 cells resulted in synergistic effect on growth inhibition. In addition, PKC inhibitor Go6076 also induced synergistic effect on growth inhibition by combination with NUAAP knockdown.
Because we validated NUAAP as an therapeutic cancer target of lung cancer in this project, we constructed drug screening system to develop novel global anti-cancer drugs using NUAAP promoter. The stable cells line expressing P1 promoter was generated and tested using chemical library of novel 1500 compounds.
Taken together, the transcription of NUAAP, which is strongly expressed in cancer tissues, is mainly regulated at P1 promoter by NFAT2 signaling. NUAAP may stabilize STAT3 protein by direct binding. In vivo xenograft assay also demonstrated that NUAAP is an potential therapeutic target of lung cancer.
목차 Contents
- 표 지 ... 1
- 제 출 문 ... 2
- 보고서 요약서 ... 3
- 요 약 문 ... 4
- SUMMARY ... 18
- CONTENTS ... 23
- 목 차 ... 24
- 제1장 연구개발 과제의 개요 ... 25
- 제1절 맞춤의료 및 표적 항암제 개발의 중요성 ... 25
- 제2절 항암 치료 타겟 발굴의 중요성 ... 26
- 제3절 본 연구 개발의 필요성 ... 29
- 제2장 국내외 기술개발 현황 ... 32
- 제1절 항암 타겟 발굴 및 표적항암제의 시장 동향 ... 32
- 제3장 연구개발 수행 내용 및 결과 ... 38
- 제1절 실험재료 ... 38
- 제2절 실험방법 ... 39
- 제3절 결과 및 고찰 ... 45
- 제4장 목표달성도 및 관련분야에의 기여도 ... 101
- 제1절 연구개발의 최종목표 ... 101
- 제2절 연차별 연구개발 목표 및 내용 ... 102
- 제4절 연구수행 내용 및 결과 ... 104
- 제4절 계획대비 달성도 ... 112
- 제5장 연구개발 결과의 활용계획 ... 116
- 제1절 연구개발결과의 활용방안 ... 116
- 제2절 기대 성과 ... 116
- 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 118
- 제1절 NFAT2의 암관련성 ... 118
- 제2절 암유전자 STAT3 및 NFAT2/STAT3 관련성 ... 119
- 제7장 연구시설ᆞ장비 현황 ... 121
- 제8장 참고문헌 ... 122
- 끝페이지 ... 132
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