- PDF Free Download

Size: px

Start display at page:

Download ""

현빈 한
6 years ago
Views:

5 Δ

7 Epidermal growth factor receptor (EGFR) is well established target for cancer treatment and EGFR tyrosine kinase (TK) inhibitors such as gefinitib and elotinib have been developed as anti-cancer drugs. Although non-small cell lung carcinoma (NSCLC) with activating EGFR mutant L858R responded well to gefinitib and elotinib, tumors with double mutated EGFR T709M-L858R developed acquired resistance to those drugs. The C.elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from worms to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants we expressed three LET-23 chimeras where its tyrosine kinase (TK) domain was replaced with either human wild-type TK domain (LET-23::hEGFR-TK), TK domain with L858R mutation:let-23::hegfr-tk[l858r] (jgis6), or TK domain with T790M-L858R mutations:let-23::hegfr-tk[t790m-l858r] (jgis25) in C.elegans vulval cells. The wild-type EGFR-TK chimeric receptors rescued let-23 mutant phenotypes and the activating mutant TK chimeras induced a multivulva (Muv) phenotype in a wild-type background. The anticancer drugs gefitinib and erlotinib can suppress the Muv phenotype in jgis6 expressing transgenic animals, but not in jgis25 transgenic animals. We screened inhibitors suppressing the Muv phenotype of jgis6 using 400 natural componds extracted from plants. We treated 50ug/ml concentration of plant extracts to the synchronized L1 larvae using 96 well plates. As a result, 12 plant extracts inhibited the Muv phenotype of jgis6 more than 20%. Among 12 plants, Eurycoma longifolia is known to induce apoptosis of the breast cancer cell line and have cytotoxicity for the lung cancer cell line. We further tested 12 plant extracts using two breast and one prostate cancer cell lines, and 8 plant extacts inhibited cell proliferation. Therefore, jgis6 is proven to be acceptable for screening anti-cancer agent.

8 Next, we also screened using 7,680 chemical library, of which target and mechanism are not known. Finally, 7 chemicals inhibited the Muv phenotype of jgis6 well, and they have the same basic structures. We continue to test using 244 derivatives of these 7 chemicals, and found that 15 chemicals are more effective than gefitinib. However, no chemicals suppressed the Muv phenotype of the EGFR-TKI resistant model jgis25. We tried to screen using 1,280 small chemicals of which target and mechanism are known. AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors for EGFR-mediated biological function. Both chemicals inhibited the Muv phenotype of jgis6, but only U0126 could inhibit the Muv phenotype of jgis25. In conclusion, transgenic C.elegans expressing chimeric LET-23::EGFR-TK proteins provide a model system that can be utilized in mutation-specific screens for new anticancer drugs. We also tried to apply jgis6 which is developed for screening the anticancer drug to reveal the interaction and relationship between the cell proliferation from the activated EGFR and extracellular matrix(ecm) molecules. We planed to screen mutants which are involved in the components or organization of the cuticle, then tested the change of hyperplasia in that mutants by mating with jgis6. For this purpose, we screened suppressors of transgenic worms expressing dominant ROL-6 collagen proteins. We named these Rol suppressor mutants as suro mutant. Most mutants enhance the Muv phenotype of jgis6, but only one mutant inhibited the Muv phenotype of jgis6. We first cloned the suro-4 mutant, and found a mutation in the sqt-1 gene. SQT-1 is a famous collagen protein, and it is closely related to ROL-6. We further tested the suro-4 mutant by mating with jgis25 and jgis26 which express EGFR-TKI resistant LET-23::EGFR[T790M L858R]. The suro-4 mutation also increased the Muv phenotype of jgis25 and jgis26. In particular, suro-4 increased the Muv phenotype of jgis26 dramatically. The suro-1 mutant inhibited the Muv phenotype of jgis6 and exhibited a different ROL-6::GFP localization pattern compared to other Dpy mutants. We identified mutations in R11A5.7, which encodes a zinc-carboxypeptidase homologue. The expression pattern in the hypodermis and genetic interaction with other collagen modifying enzyme mutants imply that the regulatory role of this novel carboxypeptidase for collagen processing and cuticle organization. This result helps further understanding of the cuticle formation during larval development and the relationship between epithelial cell proliferation and ECM. In summary, we made in vivo screening model targeting the EGFR pathway using C. elegans vulval development pathway and the activating mutant form of human EGFR. We characterized and evaluated this model by the molecular genetic analysis and pilot screens with 400 natural compounds and approximately 9,000 small chemicals. In addition, we tried to elucidate the relationship of the epithelial cell proliferation from the activated EGFR signal and ECM using this model. As a result, we found a novel zinc-carboxypeptidase A homolog and reported the function of this CPA.

9 인간의돌연변이형 EGFR 의발현에의한꼬마선충 (C. elegans) 과형성 (Hyperplasia) 모델을제작하고, 이모델을통해세포증식의신호전달을조절하는유전자를발굴하고, 변이형 EGFR 신호전달계의저해제탐색가능성검증.

37 FEBS letters (3.2) PLoS one (4.7) 2010; Molecules and Cell (1.8) 2010; FEBS Journal (3.1) 2009;

38 Molecules and Cell (1.9) Molecules and Cell (1.9)

39 인간의돌연변이형 EGFR의발현에의 한꼬마선충 (C. elegans) 과형성 (Hyperplasia) 모델 을제작하고, 이모 델을통해세포증 식의신호전달을 조절하는유전자를 발굴하고, 변이형 EGFR 신호전달계 의저해제탐색가 능성검증

2016 학년도약학대학면접문제해설 문제 2 아래의질문에 3-4분이내로답하시오. 표피성장인자수용체 (epidermal growth factor receptor, EGFR) 는수용체티로신인산화효소군 (receptor tyrosine kinases, RTKs) 의일종으로서세

2016 학년도약학대학면접문제해설 문제 2 아래의질문에 3-4분이내로답하시오. 표피성장인자수용체 (epidermal growth factor receptor, EGFR) 는수용체티로신인산화효소군 (receptor tyrosine kinases, RTKs) 의일종으로서세 본문제에대한지적소유권은동국대학교에있습니다. 본교의서면허락없이무단으로출판, 게재, 사용할수없습니다. 문제 2 2016 학년도약학대학면접문제 아래의질문에 3-4 분이내로답하시오. 표피성장인자수용체 (epidermal growth factor receptor, EGFR) 는수용체티로신 인산화효소군 (receptor tyrosine kinases, RTKs) 의일종으로서세포의생존과증식