[보고서]암 대사에서 Autophagy의 역할 규명을 통한 바이오 마커 및 항암제 개발

손재경

암 대사에서 Autophagy의 역할 규명을 통한 바이오 마커 및 항암제 개발
Understanding the Role of Autophagy in Cancer Metabolism 원문보기

보고서 정보
주관연구기관	울산대학교 University of Ulsan
연구책임자	손재경
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2018-05
과제시작연도	2017
주관부처	보건복지부 [Ministry of Health & Welfare(MW)(MW)
등록번호	TRKO202000008414
과제고유번호	1465023408
사업명	암연구소및국가암관리사업본부연구운영비지원
DB 구축일자	2020-07-29
키워드	Autophagy.Metabolomics.Glutamine metabolism.BCAA metabolism.Matrine.

초록 ▼

◉ 연구개발사업의 목적
본 연구개발의 최종목표는 대사체 흐름에서 autophagy에 의해 조절되는 대사물질을 발굴하고, 또한 autophagy에 의해 조절되는 대사관련 신호전달경로를 규명하여 그 기작을 밝힌 뒤 동물 모델에서도 이와 같은 조절이 일어나는지를 확인함으로써 암 대사에서 autophagy의 중요성을 이해하고 중요 대사물질 및 대사 관련 신호전달 조절물질을 난치성 암 진단 및 치료제의 표적으로 사용하는 것임.

◉ 연구방법
- Autophagy에 의하여 조절되는 대사물질을 찾기 위하여 autophagy가 결핍된 세포주를 확보하고 untargeted-metabolomics study를 수행하여 후보 대사물질을 확보하고, 다시 targeted-metabolomics study를 수행하여 autophagy에 의하여 조절되는 대사물질인지 증명함.
- 어떤 기작으로 autophagy에 의해 후보 대사물질이 조절되는지 규명하기 위하여 micro array를 수행, 후보 대사 유전자를 확보함. 다시 후보 대사 유전자가 결핍된 세포주를 확보하고, 대사체 분석을 수행하여 실제로 autophagy에 의해 변화하는 대사물질과 동일한지 확인하여 후보 대사 유전자를 증명함.
- 이렇게 발견된 후보 유전자가 항암제 개발을 위한 타겟 가능성을 in vitro와 in vivo에서 확인함,

◉ 연구결과
1. Autophagy는 췌장암 glutamine metabolism에서 중요한 역할을 함.
- Untargeted, targeted metabolomics를 통하여 autophagy가 glutamine level를 조절하여 glutamine metabolism에서 중요한 역할을 한다는 것을 확인하였음. autophagy가 glutamine transporter gene을 조절하여 glutamine metabolism을 조절하는 기작을 규명하였음. 췌장암 세포내에 intracellular glutamine level을 조절하는데 다양한 기작이 관여하는 것을 확인하였음. 췌장암 세포내에 intracellular glutamine level은 두 개의 parallel pathway를 통해 유지됨. 하나는 전형적인 glutamine transporter를 통한 것이며, 다른 하나는 macropinocytosis-mediated glutamine 공급으로 이 대사 경로에는 autophagy가 반드시 필요함. 그러므로 intracellular glutamine level를 조절하는 두 개의 대사 경로를 동시에 억제할 경우 TCA cycle의 영양분 공급이 저해되어 췌장암 세포 사멸을 유도한다는 것을 규명하였음.

2. Autophagy는 Branched-chain amino acids (BCAA) metabolism에서 중요한 역할을 함.
- Untargeted, targeted metabolomics를 통하여 autophagy가 BCAA metabolism을 조절하여 췌장암 세포 성장에서 중요한 역할을 한다는 것을 확인하였음. 대사체 분석을 통하여 BCAA metabolism 억제하였을 경우 TCA cycle의 영양분 공급에는 별다른 영향을 주지 않지만, fatty acid 합성이 현저하게 감소하는 것을 확인하였음. 그러므로 autophagy에 의해 조절되는 BCAA metabolism은 fatty acid 합성에 중요한 영양분을 공급함으로써, 췌장암 세포 성장에 중요한 역할을 한다는 것을 규명하였음.

3. Matrine은 autophagy에 조절되는 energy metabolism을 억제하여 췌장암 세포 성장의 억제를 유도함.
- Matrine이 새로운 autophagy 억제제로써의 가능성을 확인하였음. Matrine은 췌장암 세포에서 전사인자인 stat3의 발현을 억제하고, 억제된 stat3는 cathepsin B와 D의 활성화를 억제하여 lysosomal protease의 기능을 감소시킴으로써 결국 autolysosome의 형성을 방해하여 autophagy의 억제를 유도한다는 기작을 규명하였음. 이와 같이 matrine에 의해서 억제된 autophagy는 췌장암 세포 성장에 필요한 energy metabolism을 억제함으로써 췌장암 세포의 성장을 현저하게 감소하는 것을 확인하였음. chloroquine은 현재 autophagy 억제제로써 임상 실험중이지만, 여러 가지 문제점을 보여주고 있음. 그러므로 matrine이 새로운 autophagy의 억제제로써 가능성을 제시하였음.

(출처 : 요약문 4p)

Abstract ▼

◉ The goal of this project
The final goal of this project is to understand the role of autophagy in pancreatic cancer metabolism by exploring autophagy-regulated metabolites in metabolomics flux and investigating the molecular mechanism by which autophagy regulates cancer metabolism.
Thus, this project will be able to identify novel metabolic targets by assessing the affect of metabolism-focused therapies.

◉ Experimental design and methods
- In order to identify the therapeutic potential of targeting autophagy-regulated metabolism in pancreatic cancer, we will first make a stable cell line expressing shATG5 or ATG7 and perform untargeted and targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify autophagy-regulated metabolites.
- In order to examine the molecular mechanism by which autophagy regulates cancer metabolism, we will perform micro array. Based on the data of micro array, we will make a stable cell line expressing shRNA gene which is regulated by autophagy and perform targeted LC-MS/MS.
- We will explore the therapeutic potential of targeting autophagy-regulated metabolism in vitro and in vivo.

◉ The results of this project
1. Autophagy is required for PDAC glutamine metabolism
- The implication of autophagy in regulating cancer metabolism recently started to be uncovered. However, the precise roles of autophagy in cancer metabolism are still unclear.
Here, we show that autophagy plays a critical role in glutamine metabolism, which is required for tumor survival. Pancreatic ductal adenocarcinoma (PDAC) cells require both autophagy and typical glutamine transporters to maintain intracellular glutamine levels. Glutamine deprivation, but not that of glucose, led to the activation of macropinocytosis-associated autophagy through TFEB induction and translocation into the nucleus. In contrast, glutamine uptake increased as a compensatory response to decreased intracellular glutamine levels upon autophagy inhibition. Moreover, autophagy inhibition and glutamine deprivation did not induce cell death, while glutamine deprivation dramatically activated apoptotic cell death upon autophagy inhibition. Interestingly, the addition of α-ketoglutarate significantly rescued the apoptotic cell death caused by the combination of the inhibition of autophagy with glutamine deprivation. Our data suggest that macropinocytosis-associated autophagy is a critical process providing glutamine for anaplerosis of the TCA cycle in PDAC. Thus, targeting both autophagy and glutamine metabolism to completely block glutamine supply may provide new therapeutic approaches to treat refractory tumors.

2. Autophagy is critical for PDAC BCAA metabolism
- Elevations of the three branched chain amino acids (BCAAs) isoleucine, leucine and valine are associated with a greater than two-fold increased risk of future PDAC diagnosis.
However, the precise roles of BCAA metabolism in cancer are still unclear. Here, we find that autophagy plays an important role in BCAA metabolism, which is required for PDAC growth. Branched-chain alpha-keto acid dehydrogenase (BCKDH) knockdown has no significant effect on TCA cycle intermediates, whereas BCKDH knockdown dramatically decreases fatty acids synthesis. Thus, our data indicate that autophagy-mediated BCAA metabolism is an essential process providing carbon sources for fatty acids synthesis.

3. Matrine suppresses KRAS-driven pancreatic cancer growth by inhibiting autophagy-mediated energy metabolism
- Matrine is a natural compound extracted from the herb Sophora flavescens Ait which is widely used in traditional Chinese medicine for treating various diseases. Recently, matrine was reported to have antitumor effects against a variety of cancers without any obvious side effects; however, the molecular mechanisms of its antiproliferative effects on cancer are unclear. Here, we report that matrine inhibits autophagy-mediated energy metabolism, which is necessary for pancreatic cancer growth. We found that matrine significantly reduces pancreatic cancer growth in vitro and in vivo by insufficiently maintaining mitochondrial metabolic function and energy level. We also found that either pyruvate or α-ketoglutarate supplementation markedly rescues pancreatic cancer cell growth following matrine treatment.
Inhibition of mitochondrial energy production results from matrine-mediated autophagy inhibition by impairing the function of lysosomal protease. Matrine-mediated autophagy inhibition requires stat3 downregulation. Furthermore, we found that the antitumor effect of matrine on pancreatic cancer growth depends on the mutation of the KRAS oncogene.
Together, our data suggest that matrine can suppress the growth of KRAS-mutant pancreatic cancer by inhibiting autophagy-mediated energy metabolism.

(출처 : Project Summery 6p)

목차 Contents

표지 ... 1
제출문 ... 2
목차 ... 3
요약문 ... 4
Project Summery ... 6
1. 연구과제의 최종 연구개발 목표 ... 8
2. 연구과제의 연구대상 및 방법 ... 10
3. 연구과제의 연구개발결과 ... 13
4. 연구과제의 연구결과 고찰 및 결론 ... 30
5. 연구과제의 연구성과 및 목표달성도 ... 31
(1) 연구성과 총괄 ... 31
(2) 연구성과 상세내역 ... 33
(3) 연구개발과제의 목표달성도 ... 44
6. 연구과제의 활용계획 ... 45
(1) 연구종료 3년까지 예상 연구성과 ... 45
(2) 연구성과의 활용계획 ... 45
7. 참고문헌 ... 47
8. 첨부서류 ... 47
끝페이지 ... 56

표/그림 (18)

표 Autophagy inhibitors
표 Glutamine, but not glucose, deprivation induces autophagy. (A) PDAC cells were plated in complete medium, which was replaced the following day with glucose or glutamine-free medium and then incubated for another 24 h. Cell lysates were immunoblotted for LC3 and p62. (B) 8988T cells were cultured in complete or glutamine free medium with or without CQ (10 μM) for 24 h and immunoblotted for LC3. (C) 8988T cells were infected with a lentivirus expressing GFP-LC3, grown in complete, glucose-free, or glutamine-free medium for 24 h and analyzed for LC3 dots. (D) PDAC cells were treated with 2DG (10 mM) or BPTES (10 μM) for 24 h and immunoblotted for LC3. (E) Effect of GLS1 knockdown on LC3 levels in 8988T cells expressing a control shRNA (shGFP) or glutaminase 1 shRNAs (shGLS1s)
표 Autophagy is required to maintain intracellular glutamine levels. (A) PDAC cells were treated with CQ (10 μM) for 24 h. (B) Relative glutamine consumption rate of 8988T cells expressing a control shRNA (shGFP) or a shRNA targeting ATG7. Western blot confirmed the knockdown of ATG7 expression. (C) WT MEFs and atg5-/-MEFsexpressingKRasG12Vwereculturedincompletemediumfor24h. Western blot confirmed the knockdown of ATG5 expression. (A-C) Glutamine levels were measured in the medium using a metabolite analyzer (BioProfile FLEX analyzer) to monitor glutamine consumption. The glutamine consumption rate was presented after normalization by the number of cells. (D) 8988T cells were plated in complete medium, which was replaced by glutamine-free medium the following day and then incubated for another 24 h with or without CQ (10 μM). Glutamine levels were monitored by using LC-MS/MS. (E) 8988T cells expressing a control (shGFP) or ATG7 shRNA were cultured in complete or glutamine-free medium for 24 h and glutamine levels were monitored by using LC-MS/MS. Error bars represent the s.d. of triplicate wells from a representative experiment. **, p < 0.01
표 TFEB transcription factor regulates autophagy upon glutamine deprivation. (A and B) The expression of TFEB was determined by quantitative RT-PCR (A) or western blot (B) in PDAC cells 24 h after supplementing with glucose or glutamine-free medium. (C) PDAC cells expressing a control (shGFP) or TFEB shRNAs (shTFEBs) were plated in the complete medium, which was replaced with glucose or glutamine-free medium the following day and then incubated for another 24 h. Cell lysates were immunoblotted for LC3 and p62. (D) Subcellular localization of GFP-TFEB in MIAPaCa2 was monitored by fluorescent microscopy analysis after incubating for 24 hr under either glutamine-replete or glutamine-free conditions, 20 μm. Error bars represent the s.d. of triplicate wells from a representative experiment. **, p < 0.01
표 Glutamine deprivation and autophagy inhibition exacerbate Ras mutant cell survival. (A and B) 8988T cells were plated in the complete medium, which was replaced with glucose or glutamine-free medium the following day and then incubated for another 24 h with or without CQ (10 μM). Cell death was assessed by using the annexin V/PI assay (A) and cleaved caspase-3 and -9 expression was assessed by immunoblotting (B). Error bars represent the s.d. of three separate experiments. (C) 8988T cells were treated with CQ (10 μ M) and BPTES (10 μM) alone, or in combination for 24 h and cell death was determined by using the annexin V/PI assay. Error bars represent the s.d. of three separate experiments. (D and E) 8988T cells expressing a control (shGFP) or ATG7 shRNAs (shATGs) were plated in the complete medium, which was replaced with glucose or glutamine-free medium the following day and then incubated for another 24 h. Cell death was assessed by using the annexin V/PI assay (D) and cleaved caspase-3 and -9 expression was assessed by immunoblotting. ATG7 knockdown was confirmed by western blot (E). Error bars represent the s.d. of three separate experiments. (F and G) WT MEFs and atg5-/- MEFs expressing KRas G12V were plated in the complete medium, which was replaced with glutamine-free medium the following day and then incubated for another 24 h. Cell death was assessed by using the annexin V/PI assay (F) and immunoblotted for PARP and cleaved caspase-3 expression was assessed by immunoblotting (G). Error bars represent the s.d. of three separate experiments. (H) Subcutaneous MIAPaCa2-driven tumors were established in 6-week old male mice. CQ (20 mg kg−1 per day), BPTES (4 mg kg−1 per day) alone, or in combination, were administered daily via intraperitoneal injection. Tumor growth was assessed once the tumor volume reached 150 mm3.Dataareshownasthemeanoffivemiceineachgroup± SEM. NS, not significant. **, p < 0.01
표 Glutamine deprivation increases macropinocytosis (A) Macropinocytosis was assessed in BxPC3 and MIAPaCa2 cells by monitoring the uptake of FITC-Dextran under either glutamine-replete or glutamine-free conditions. Scale bars, 20 μm. (B) The relative macropinocytic uptake of BxPC3 and MIAPaCa2. MIAPaCa2 was quantified by image-based determination of the total macropinocytic vesicle area compared with the DAPI-stained area of cells. Data are expressed relative to the values of BxPC3 observed in the glutamine-free conditions. Data are shown as the mean of five images in each experiment ± SEM. (Error bars represent the SEM of values from five representative images per experiment) (C) Macropinocytic uptake of MIAPaCa2 cells induced by glutamine deprivation was inhibited by the treatment with EIPA (50 μM). (D) The levels of macropinocytic uptake of MIAPaCa2. MIAPaCa2 were quantified by image-based determination as shown in (B). Data are expressed relative to the values of MIAPaCa2 in the glutamine-free conditions. (E) MIAPaCa2 cells were cultured in glutamine deprivation medium for 6 days, either with or without 2% BSA and further with EIPA treatment. Growth levels were measured by cell counting. Data are expressed relative to the values observed in the glutamine-free conditions. (F) MIAPaCa2 cells expressing ATG7 shRNA and control shRNA were cultured in glutamine-free medium for 16 h after treatment with FITC-Dextran and macropinocytic degradation was determined by monitoring intracellular FITC-Dextran. Scale bars, 20 m. (G) Levels of macropinocytic uptake of MIAPaCa2 cells were quantified by image-based determination as shown in (B). Data are relative to the values of MIAPaCa2 cells cultured in glutamine-free conditions. Error bars represent the SEM of triplicates from a representative experiment. *, p < 0.05
표 Inhibition of metabolic pathways providing substrates for the TCA cycle induces apoptotic cell death in PDAC. (A) 8988Tcells were plated in the complete medium, which was replaced by glutamine-free medium the following day and then incubated for another 24 h with or without EIPA (25 μM). Glutamine levels were monitored by using LC-MS/MS. Error bars represent the s.d. of triplicate wells from a representative experiment. (B and C) PDAC cells were plated in the complete medium which was replaced by glutamine-free medium the following day and then incubated for another 24 h with or without EIPA (25 μM) for 24 h. Cell death was assessed by using the annexin V/PI assay (B) and cleaved-3, -9 and PARP expression was determined by immunoblotting (C). Error bars represent the s.d. of three separate experiments. (D) 8988T cells expressing a control shRNA (shGFP) or TFEB shRNAs (shTFEBs) were plated in the complete medium, which was replaced by glucose or glutamine-free medium the following day and then incubated for another 24 h. Cell lysates were immunoblotted for cleaved caspase-3 and PARP. TFEB knockdown was confirmed by western blot. (E) TCA metabolite pools were analyzed by using LC-MS/MS in 8988T cells expressing a control (shGFP) or ATG7 shRNA. Error bars represent the s.d. of triplicate wells from a representative experiment. (F) 8988T cells were plated in the complete medium, which was replaced by glutamine-free medium supplemented with NAC (2 mM), GSH (2 mM), or MAKG (2 mM) in the absence or presence of CQ (10 μM) the following day and then incubated for another 24 h. NAC, N-acetylcysteine; GSH, glutathione; MAKG, dimethyl α -ketoglutarate. Error bars represent the s.d. of triplicate wells from a representative experiment. *, p < 0.05; **, p < 0.01
표 Model of maintaining intracellular levels of glutamine via two parallel pathways, including macropinocytosis-associated autophagy and a canonical glutamine transportation pathway
표 Autophagy가 억제 되였을 때 BCAAs metabolism이 감소됨
표 BCKDH 억제는 췌장암 세포의 성장을 저해함
표 BCAA metabolism은 fatty acid 합성에 중요함. CIT: citrate, AKG: alpha-ketoglutarate, FUM: fumarate, SUC: Succinate, MAL: malate
표 BCAA-mediated fatty acids production
표 Effect of matrine on pancreatic ductal adenocarcinoma (PDAC) growth. (A) PDAC cells were treated with the indicated concentration of matrine and assayed for cell growth. (B) PDAC cells were treated with 2-mM matrine for 24 h and assayed for cell viability. (C) Colony-forming assays were performed to assess the ability of matrine to inhibit PDAC growth. Error bars represent the s.d. of triplicate wells from a representative experiment. (D) Subcutaneous 8988T-driven tumors were established in severe combined immunodeficiency (SCID) mice. Matrine (50 mg/kg or 100 mg/kg) was administered daily by intraperitoneal injection. Data are shown as the mean of five mice in each group ± SEM
표 Matrine reduces levels of tricarboxylic acid cycle (TCA)–cycle intermediates. (A) 8988T cells were treated with 2-mM matrine for 24 h and assayed for intracellular adenosine triphosphate (ATP). (B) Oxygen consumption rates were measured with an extracellular flux analyzer. 8988T cells were treated with 2-mM matrine. Cells were sequentially treated with 2-μM oligomycin, 5-μM FCCP, and 2-μM rotenone. (C) TCA metabolite pools in 8988T cells treated with 2-mM matrine were analyzed using LC-MS/MS. Error bars represent the s.d. of three independently prepared samples. (D and E) 8988T cells were treated with 1-mM matrine in the presence of either 7-mM methyl pyruvate or 7-mM dimethyl α-ketoglutarate and assayed for cell growth (D) and assayed for colony formation (E). Error bars represent the s.d. of triplicate wells from a representative experiment. p < .05; **p < .01
표 Effect of matrine on autophagy. (A) PDAC cells were treated with the indicated concentration of matrine for 24 h and immunoblotted with the indicated antibodies. (B) 8988T cells expressing GFP-LC3 were treated with 2-mM matrine for 24 h and analyzed for LC3 dots. (C) Autophagic vacuoles in 8988T cells treated with 2-mM matrine for 24 h were analyzed by transmission electron microscopy. Notes: N, nucleus; L, lipid; A, autophagic vacuoles
표 Matrine inhibits the maturation of cathepsins. (A and B) Expression levels of Beclin1, ATG3, ATG5, and ATG7 were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting in PDAC cells treated with 2-mM matrine for 24 h. Error bars represent the s.d. of triplicate wells from a representative experiment. (C) PDAC cells were treated with 2-mM matrine for 24 h in the absence or presence of 10-μM chloroquine (CQ) and immunoblotted with the indicated antibodies. (D) PDAC cells were treated with the indicated concentration of matrine for 24 h and immunoblotted with p62. (E) LysoTracker staining was performed on PDAC cells treated with either 10-μM CQ or 2-mM matrine for 24 h. (F) PDAC cells were treated with 2-mM matrine for 24 h and immunoblotted with the indicated antibodies. (G) 8988T cells were treated with 2-mM matrine for 24 h and assayed for DQ-BSA cleavage activity
표 Matrine inhibits the maturation of cathepsins by reducing Stat3. (A) PDAC cells were treated with the indicated concentration of matrine for 24 h and immunoblotted with Stat3. (B and C) PDAC cells were treated with the indicated concentration of stattic for 24 h and immunoblotted with the indicated antibodies. (D) PDAC cells were treated with the indicated concentration of stattic and assayed for cell growth. (E) 8988T cells expressing a vector or constitutively active form of Stat3 (Stat3-c) were treated with 1-mM matrine and assayed for cell growth. Western blotting confirmed the overexpression of Stat3-c. Error bars represent the s.d. of triplicate wells from a representative experiment. p < .05; **
표 Matrine does not inhibit growth of KRAS-wt PDAC. (A) BXPC3 cells were treated with the indicated concentration of matrine for 24 h and immunoblotted with LC3. (B) BXPC3 cells were treated with 2-mM matrine for 24 h and assayed for intracellular ATP. (C) Oxygen consumption rates were measured using an extracellular flux analyzer. BXPC3 cells were treated with 2-mM matrine. Cells were sequentially treated with 2-μM oligomycin, 5-μ M FCCP, and 2-μM rotenone. (D) TCA metabolite pools in BXPC3 cells treated with 2-mM matrine were analyzed LC-MS/MS. Error bars represent the s.d. of three independently prepared samples. (E) Colony-forming assays were performed to assess the ability of matrine to inhibit BXPC3 growth. (F) BXPC3 cells were treated with the indicated concentration of matrine and assayed for cell growth. Error bars represent the s.d. of triplicate wells from a representative experiment

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안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

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