초록 ▼

본 연구과제는 난치성 전이암 치료를 위한 표적 분자 발굴 및 진단 마커 개발을 목표로 수행되었다.
1. 암 전이 조절 신규유전자의 발굴 및 기능 규명
① 암전이 억제 신규타겟 2종 (SL-15, SL-772) 발굴 및 암전이 관련 기전 규명
- 신규 타겟 SL-15 (콜레스테롤합성 관련 효소) 저해에 의한 대장암 전이 기능 (anoikis escape 현상 유도) 및 기전 (GSK3β/β-catenin/ZEB1/2 & p53 degradation 경로) 규명함
- 신규 타겟 SL-772(분비 단백질) 저해에 의

본 연구과제는 난치성 전이암 치료를 위한 표적 분자 발굴 및 진단 마커 개발을 목표로 수행되었다.
1. 암 전이 조절 신규유전자의 발굴 및 기능 규명
① 암전이 억제 신규타겟 2종 (SL-15, SL-772) 발굴 및 암전이 관련 기전 규명
- 신규 타겟 SL-15 (콜레스테롤합성 관련 효소) 저해에 의한 대장암 전이 기능 (anoikis escape 현상 유도) 및 기전 (GSK3β/β-catenin/ZEB1/2 & p53 degradation 경로) 규명함
- 신규 타겟 SL-772(분비 단백질) 저해에 의한 간암 전이 기능 및 기전 (DKK4/wnt signaling 경로) 규명함
② 신규 유전자 VGLL1의 발굴 및 기능 규명
- cancer stem cell 분화과정에서 발굴된 유전자들의 암세포 전이 관련 스크리닝을 통하여 신규타겟 VGLL1을 발굴하고 위암 세포/조직에서 발현량이 증가되어 있음을 확인함
- 신규타겟 VGLL1의 발암/전이 관련 기능/기전을 검증함 (in vitro/in vivo)
2. 난치성 전이암 조기/예후/치료진단의 바이오 마커 규명 및 진단기술 개발
① 난치성 전이암 조기/예후/치료 진단의 바이오 마커 발굴 및 검증을 수행함
- 전이관련 진단 바이오마커 (CCBM 201, 207, 301, 356)를 전이성 임상시료에서 규명/검증함
② 전이암 조기/예후/치료진단의 바이오 마커 규명 및 진단법을 확립함 (ELISA, LUMINEX)
- 전이관련 진단 바이오마커의 항원/항체의 확보 및 생산과 임상시료에의 적용 및 유의성을 검증함
③ CCBM 205, 213, 356의 암세포 성장 및 전이과정에 미치는 영향 및 기능을 분석함
④ CCBM 201 유전자의 항암제 내성 조절이 오토페이지와의 관련 기전을 규명함
⑤ CCBM 205, 213, 356의 전이/암화 관련 기능 분석 및 진단 마커로의 임상적 활용성 재검증함 (100 case 이상 임상시료)
3. 전이성 암 치료 저해 물질 발굴 및 표적 검증
① 대장암 및 췌장암 전이를 억제하는 항암활성물질 MD-164 (=Benproperine)을 도출하여 동물에서 암전이 억제활성 및 항암활성을 검증함. (MD-164는 현재 진해거담제로 사용 중인 약물)
② biotin-benproperine 화합물을 합성하여 affinity chromatography 방법으로 벤프로페린의 표적분자 ARPC2를 확인함
③ HSF1의 활성을 저해하여 항암효능을 나타내는 활성물질 KRIBB31 (=Cantharidin)을 도출하여, 동물에서 항암활성을 확인함
(Cantharidin은 지난 2천년동안 중국에서 항암약제로 사용한 곤충유래 화합물로서 현재 QininⓇ (Cantharidin sodium) 약물명으로 중국 Guizhou magic Pharmaceutical Co. 제조)
본 연구로부터 IF>10 3편 (ANGEWANDTE CHEMIE INT. ED., GUT, GASTROENTEROLOGY), IF>5 27편 등을 포함 SCI(E)/SCI 66편을 출판하였고, 국제특허 등록 10건, 국내특허 등록 19건을 완료하였음

Abstract ▼

Results
1. Regulation of cancer metastasis by SL-15 and 772.
We found that SL-15 and SL-772 were highly expressed in colon and liver cancers.
However, the roles of SL-15 and 772 dysfunction in cancer largely remain unclear. Here, we demonstrated the involvement of SL-15 and 772 in cancer me

Results
1. Regulation of cancer metastasis by SL-15 and 772.
We found that SL-15 and SL-772 were highly expressed in colon and liver cancers.
However, the roles of SL-15 and 772 dysfunction in cancer largely remain unclear. Here, we demonstrated the involvement of SL-15 and 772 in cancer metastasis. SL-15 and 772 specific siRNA significantly knocked-downed its expression and resulted in increase of cell migration and invasion. In addition, the knockdown of SL-15 and 772 could prohibit degradation of β-catenin via inactivation of GSK3β, and finally induced epithelial-to-mesenchymal transition (EMT), causing anoikis escape. Therefore, our present study suggests that SL-15 and 772 play a critical role in regulation of the cancer metastasis in colon and liver cancer, and this proteins may assist to development of novel therapeutic strategies for cancer metastasis.
2. Regulation of cancer progression and metastasis by VGLL1.
Vestigial-like 1 (VGLL1) is a poorly characterized gene encoding a transcriptional co-activator structurally homologous to TAZ and YAP that modulates the Hippo pathway in Drosophila. VGLL1 identified as a novel cancer-associated gene by RNA seq analysis of differentiated cancer stem cell samples. We confirmed the expression of VGLL1 in human normal/cancer cell lines and found that the level of VGLL1 increased in stomach cancer cell lines. Moreover VGLL1 expression was observed in 15% of normal tissues, and while found in 55.3% stomach adenocarcinoma tissues.
We investigated the potential biological function of VGLL1 in stomach cancer. VGLL1 overexpression induced cell growth, migration and invasion in human stomach cancer cell, NUGC3. VGLL1 knockdown inhibited the binding to extracellular matrix components such as collagen, fibronectin, cell migration and invasion in NUGC3 and AGS cells. We profiled whole-genome gene expression in VGLL1 overexpression/knockdown NUGC3 cells by microarray. One potential candidate gene that came out of microarray data is MMP-9 (Matrix metallopeptidase-9). We confirmed that TGF-β-induced MMP9 expression was inhibited by treatment with siVGLL1 and siTEAD4.
Nude mice inoculated with parent HEK293T or VGLL1 overexpressing cells. After 4 weeks, the tumor volume of VGLL1 overexpressing cells showed 3-fold increase than those established by parent cells. Thus, VGLL1 contributes to stomach cancer progression and metastasis and may provide as a novel target for human cancer treatment.
3. Identification of colorectal cancer related biomarkers and diagnosis kit.
In order to identify diagnostic/prognostic/theragnostic markers, we searched overexpressed genes in metastatic colorectal cancer by using triple-paired tissues DNA microarray analysis.
From this, we Identified colorectal cancer related biomarkers (CCBM 201, 205, 213, 356) for the early detection of metastatic colorectal cancer. And then, we confirmed their expression (CCBM 201, 205, 213, 356) in serum and tissues. For the development of diagnosis technology, we produced poly/monoclonal antibody for CCBM201, CCBM205, CCMB213, and CCBM356.
Finally, we developed early diagnosis kit for colorectal cancer using serum diagnostic markers.
4. Identification of MD-164 as an inhibitor of DLD1/PRL-3 migration
To search for migration inhibitors, we used transwell migration assay for PRL-3-overexpressing DLD-1 cells that had treated with 783 medicinal drugs and 57 herbal medicines. From this, MD-164 was discovered as a migration inhibitor. MD-164 is widely used as a cough suppressant in non-productive cough. In order to identify target protein for MD-164, we synthesized biotin-MD-164 compound and affinity chromatography was carried out with cell lysates. From this, we identified ARPC2 as a target of MD-164. Oral administration of MD-164 efficiently inhibited metastasis as well as primary tumor growth.
These results suggest that MD-164 inhibits the migration and invasion of PRL-3-overexpressing DLD-1 cells and can be good candidate for the therapeutic control of cancer cell invasion or metastasis.
5. KRIBB31(=Cantharidin) inhibits HSF1 activity and induces cancer cell apoptosis.
To identify inhibitors of HSF1 activity, we performed cell-based screening with a library of marketed and experimental drugs and identified KRIBB31 as an HSF1 inhibior. KRIBB31 is a potent antitumor agent from traditional Chinese medicine. KRIBB31 inhibited heat shock-induced luciferase activity with an IC50 of 4.2μM. In contrast, KRIBB31 did not inhibit NF-κB luciferase reporter activity, demonstrating that KRIBB31 is not a general transcription inhibitor. When the HCT-116 colorectal cancer cells were exposed to heat shock in the presence of KRIBB31, the induction of HSF1 downstream target proteins, such as HSP70 and BAG3 (Bcl-2-associated athanogene domain 3), was suppressed. HSP70 and its co-chaperone BAG3 have been reported to protect cells from apoptosis by stabilizing anti-apoptotic Bcl-2 family proteins. As expected, treating HCT-116 cancer cells with KRIBB31 significantly decreased the amounts of BCL-2, BCL-xL, and MCL-1 protein and induced apoptotic cell death. Chromatin immunoprecipitation analysis showed that KRIBB31 inhibited the binding of HSF1 to the HSP70 promoter and subsequently blocked HSF1-dependent p-TEFb recruitment.
Therefore, the p-TEFb-dependent phosphorylation of the C-terminal domain of RNA polymerase II was blocked, arresting transcription at the elongation step. PP2A inhibition with PP2CA siRNA or okadaic acid did not block HSF1 activity, suggesting that KRIBB31 inhibits HSF1 in a PP2A-independent manner. We show for the first time that KRIBB31 inhibits HSF1 transcriptional activity.
Recently we screened and identified another HSF1 inhibitor, KRIBB41 from natural compound library. KRIBB41 is component from edible fruit and inhibits HSF1 activity in a concentration-dependent manner. KRIBB41 blocked HSPs mRNA and protein expression and induced apoptosis. KRIBB41 inhibited HSF1 binding to the hsp70 promoter, blocking HSF1-dependent RNA polymerase II activity. Intraperitonial treatment of nude mice with KRIBB41 at 30 mg/kg resulted in a 35.7% (P<0.001) inhibition of tumor growth.

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보고서 요약서(보고서 초록) ... 4
• 요 약 문 ... 5
• SUMMARY ... 12
• CONTENTS ... 15
• 목차 ... 16
• 제 1 장 연구개발과제의 개요 ... 17
• 제1절 연구개발의 목적 ... 17
• 제2절 연구개발의 필요성 ... 17
• 제 2 장 국내외 기술개발 현황 ... 25
• 제 1절 연구개발대상 기술의 국내·외 수준 ... 25
• 제 2절 국내·외의 연구 현황 ... 25
• 제 3 장 연구개발 수행 내용 및 결과 ... 28
• 제 1 절 암 전이 조절 신규유전자의 발굴 및 기능규명 ... 28
• 제2절 전이암 조기/예후/치료 진단의 바이오 마커 발굴 및 검증 ... 47
• 제3절 전이성 암 치료 표적 검증 및 저해 물질 발굴 ... 66
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 79
• 제1절 연도별 연구목표 ... 79
• 제2절 평가 착안점에 입각한 연구개발 목표 ... 82
• 제3절 세부연구목표 달성도 ... 84
• 제4절 관련분야에의 기여도 ... 85
• 제 5 장 연구개발결과의 활용계획 ... 86
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 87
• 제 7 장 연구시설·장비 현황 ... 91
• 제 8 장 참고문헌 ... 92
• 끝페이지 ... 94