Clinical Relevance and Functional Role of Nuclear Met in Hepatocellular Carcinoma

( Sze Keong Tey ),( Edith Yuk Ting Tse ),( Frankie Chi Fat Ko ),( Xiao Wen Mao ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: Met is a receptor tyrosine kinase which triggers a wide range of normal physiological signaling cascades. However, a perturbation of the Met pathway is commonly found in human cancers. Emerging evidence has shown the presence of nuclear Met in some cancerous tissues and cell lines, suggesting that nuclear Met could have unexplored functions in the nucleus. The present study aimed to assess the expression and functions of nuclear Met in hepatocellular carcinoma (HCC). Methods: Nuclear Met expression of 103 clinicopathologically characterized HCC paired samples was examined by immunohistochemistry using an antibody against the carboxyl terminus of Met. Statistical analyses were applied to evaluate the association of nMet with different clinical parameters. Nuclear localization of Met was determined by western blot analysis and immunofluorescence microscopy. Met cytoplasmic fragments were characterized by in vitro functional assay such as migration, invasion and proliferation in HCC cells. Nude mice model was employed to investigate the in vivo functional impact of nuclear Met. Results: Nuclear Met is overexpressed in nearly 90% of HCC paired samples and its expression is progressively increased along HCC development from non-tumorous liver tissue to advanced HCC. Nonetheless, nuclear Met overexpression is significantly associated with venous invasion and poorer overall survival. We found that nuclear Met, which has a lower molecular weight than Met, could only be detected using an antibody against the carboxyl terminus of Met (C28) in tumorous tissues. This finding strongly suggests that nuclear Met only comprises of the carboxyl cytoplasmic region of full length Met. Moreover, both western blot analysis of nuclear fraction of HCC cells and immunofluorescence confirmed the nuclear localization of Met. We designed construct J1, J3 and T2 that encode Met fragment truncated after tyrosine residues D972 and P1027 in the juxtamembrane region and after tyrosine kinase domain beginning at L1157, respectively. Immunofluorescence microscopy showed both J1 and J3 constructs are dominantly expressed in the nucleus whereas T2 construct is expressed in the cytoplasm. These observations indicated the region in between J1 and T2 as the important region that facilitates the nuclear localization of Met. In vitro functional assay showed that nMet significantly promoted HCC cell proliferation and anchorage independent growth. It also significantly augmented HCC cell migration and invasiveness. Besides that, nMet also enhanced HCC tumor formation in animal model. Furthermore, we showed that nMet promoted tumor invasiveness and aggressiveness through NF-κ B/MMP2 pathway. Conclusions: Nuclear Met is overexpressed and associated with venous invasion and poorer overall survival in HCC. We found that nuclear Met is actually the carboxyl terminal fragment of Met and translocates into nucleus to promote invasiveness in HCC cells.

Deubiquitinase BRCC3 promotes the migration, invasion and EMT progression of colon adenocarcinoma by stabilizing MET expression

Feng Xiu,He Shengnan,Chen Ying,Zhang Liang 한국유전학회 2024 Genes & Genomics Vol.46 No.5

Background Breast cancer type 1 susceptibility protein/breast cancer type 2 susceptibility protein-containing complex subunit 3 (BRCC3), a deubiquitinase (DUBs), is overexpressed in various cancers. However, the underlying biological roles of BRCC3 in adenocarcinoma colon (COAD) have yet to be decrypted. Objective In this work, we explored the potential biological function of BRCC3 in the natural process of COAD cells. Methods The expression levels of BRCC3 in COAD tissues and cell lines were investigated via quantitative real time polymerase chain reaction and western blotting analyses. Meanwhile, short hairpin RNAs targeting BRCC3 (sh-BRCC3) or mesenchymal-epithelial transition factor (MET) (sh-MET) were used to investigate the biological function, including proliferation, apoptosis, migration, invasion, and epithelial–mesenchymal transition (EMT) progression in COAD cells. Furthermore, the expression levels of EMT-related biomarkers were detected with western blotting analysis. Furthermore, we also performed Co-IP assay to identify the correlation between BRCC3 and MET. Results BRCC3 expression was increased in COAD tissues and cell lines. ShRNA-mediated downmodulation of BRCC3 in COAD cell lines induced EMT progression. BRCC3 knockdown resulted in decreased migration as well as invasion and increased apoptosis of SW480 and Lovo cells. Besides, MET was regulated by BRCC3 and involved in the migration, invasion, and EMT in SW480 and Lovo cells. Finally, we uncovered that the overexpressed MET reversed the effects of BRCC3 knockdown in COAD cell development. Conclusions BRCC3 acted as a critical factor in the development of COAD by deubiquitinating and stabilizing MET, which might provide an emerging biomarker for the therapeutic and diagnosis strategy of COAD. Background Breast cancer type 1 susceptibility protein/breast cancer type 2 susceptibility protein-containing complex subunit 3 (BRCC3), a deubiquitinase (DUBs), is overexpressed in various cancers. However, the underlying biological roles of BRCC3 in adenocarcinoma colon (COAD) have yet to be decrypted. Objective In this work, we explored the potential biological function of BRCC3 in the natural process of COAD cells. Methods The expression levels of BRCC3 in COAD tissues and cell lines were investigated via quantitative real time polymerase chain reaction and western blotting analyses. Meanwhile, short hairpin RNAs targeting BRCC3 (sh-BRCC3) or mesenchymal-epithelial transition factor (MET) (sh-MET) were used to investigate the biological function, including proliferation, apoptosis, migration, invasion, and epithelial–mesenchymal transition (EMT) progression in COAD cells. Furthermore, the expression levels of EMT-related biomarkers were detected with western blotting analysis. Furthermore, we also performed Co-IP assay to identify the correlation between BRCC3 and MET. Results BRCC3 expression was increased in COAD tissues and cell lines. ShRNA-mediated downmodulation of BRCC3 in COAD cell lines induced EMT progression. BRCC3 knockdown resulted in decreased migration as well as invasion and increased apoptosis of SW480 and Lovo cells. Besides, MET was regulated by BRCC3 and involved in the migration, invasion, and EMT in SW480 and Lovo cells. Finally, we uncovered that the overexpressed MET reversed the effects of BRCC3 knockdown in COAD cell development. Conclusions BRCC3 acted as a critical factor in the development of COAD by deubiquitinating and stabilizing MET, which might provide an emerging biomarker for the therapeutic and diagnosis strategy of COAD.

Apo(a) Pentanucleotide (TTTTA) Repeat Polymorphism의 대립인자 분포

황상현,김우양,전사일,민원기 대한임상병리학회 2000 대한임상병리학회지 Vol.20 No.3

Immunohistochemical Study to Evaluate the Prognostic Significance of Four Biomolecular Markers in Radiotherapy of Nasopharyngeal Carcinoma

김연주(Yeon-Joo Kim)ㆍ이승희(Seung Hee Lee)ㆍ우홍균(Hong-Gyun Wu)ㆍ고현정(Heounjeong Go)ㆍ전윤경(Yoon Kyung Jeon) 대한방사선종양학회 2010 Radiation Oncology Journal Vol.28 No.2

목 적: 방사선 치료를 받은 코인두암 환자의 치료 전 조직을 면역조직화학염색하여 생체분자적 예후인자를 찾고자하였다.대상 및 방법: 1998년부터 2006년까지 방사선 치료를 받은 코인두암환자는 68명이었다. 이중 38명의 환자에서 면 역조직화학염색을 위한 파라핀 블록을 찾을 수 있었다. 전체 환자 중 31명은 미분화암종이었고, 7명은 편평세포암종이었다. 전체 환자의 84%가 2002 American Joint Committee on Cancer Stage III or IV 환자였다. 전체 환자의파라핀 블록을 이용하여 Met, COX-2, epidermal growth factor receptor (EGFR), nm23-H1에 대해 면역조직화학염색을 시행하였다.결 과: 전체 환자의 중앙 추적 기간은 30개월이었고 생존 환자의 중앙 추적 기간은 39개월이었다. 높은(≥50%)Met 발현을 보인 환자들의 5년 생존율은 48%, 낮은 Met 발현을 보인 환자들의 5년 생존율은 84%로 이는 통계적으로 유의한 차이를 보였다(p=0.02). Met 발현 정도는 다변량 분석에서도 유의한 인자로 분석되었다(p=0.01). Met발현은 종양의 병기, 성별, 나이, 항암제나 방사선 치료에 대한 반응 정도와 상관관계를 보이지 않았다. Met 발현정도는 COX-2 발현과 중등도의 상관관계를 보였으나(Pearson coefficient 0.496, p<0.01), COX-2 발현은 전체생존율에 영향을 미치지 않았다. 본 연구에서는 nm23-H1이나 EGFR의 발현은 예후인자가 아닌 것으로 분석되었다.결 론: 방사선치료를 받은 코인두암에서, 높은(≥ 50%) Met 발현은 전체생존율에 영향을 미치는 독립적인 예후인자일 가능성이 있다. Purpose: We performed an immunohistochemical study with pre-treatment biopsy specimens to evaluate the prognostic significance of four biomolecular markers which can be used as a predictive assay for radiotherapy (RT) treatment of nasopharyngeal carcinoma (NPC). Materials and Methods: From January 1998 through December 2006, 68 patients were histologically diagnosed as non-metastatic NPC and treated by RT. Only 38 patients had the paraffin block for the immunohistochemical study. Thirty-one patients had undifferentiated carcinoma and 7 patients had squamous cell carcinoma. Thirtytwo patients (84%) had advanced stage NPC (2002 AJCC Stage III∼IV). Immunohistochemical staining was performed for Met, COX-2, nm23-H1, and epidermal growth factor receptor (EGFR) expression using routine methods. Results: The median follow-up time was 30 months (range, 11 to 83 months) for all patients, and 39 months (range, 19 to 83 months) for surviving patients. The 5-year overall survival (OS) rate of the patients with high Met extent (≥50%) was significantly lower than that of the patients with low Met extent (48% vs. 84%, p=0.02). In addition, Met extent was also a significant prognostic factor in multivariate analysis (p=0.01). No correlation was observed between Met extent and T stage, N stage, stage group, gender, age, and the response to chemotherapy or RT. Met extent showed moderate correlation with COX-2 expression (Pearson coefficient 0.496, p<0.01), but COX-2 expression did not affect OS. Neither nm23-H1 or EGFR expression was a prognostic factor for OS in this study. Conclusion: High Met extent (≥50%) might be an independent prognostic factor that predicts poor OS in NPC treated with RT.

미생물 전기화학 기술이 설치된 단일 혐기성소화조에서 유기성폐기물로부터 메탄생성

박준규(Jungyu Park),전동걸(Dongjie Tian),이범(Beom Lee),전항배(Hangbae Jun) 大韓環境工學會 2016 대한환경공학회지 Vol.38 No.4

Glucose (C6H12O6)의 이론적인 최대 메탄수율은 표준상태(1 atm, 0℃)를 기준으로 0.35 LCH₄/gCOD이지만, 전통적인 혐기성소화조에서 유기물이 메탄으로 전환되는 양은 연구의 방법이나 유기물의 종류에 따라 매우 다양하게 보고되고 있으며, 대부분의 연구실 규모 실험에서 안정화 후 메탄 수율은 0.35 LCH₄/gCOD 이하로 나타난다. 최근, 미생물 전기화학 기술(Microbial Electrochemical Technology, MET)은 지속가능한 신재생에너지 생산 기술로서 큰 주목을 받고 있으며, MET를 혐기성소화조에 적용할 경우 고농도의 유기성폐기물의 빠른 분해가 가능할 뿐만 아니라 전기화학적인 반응에 의해 휘발성지방산(VFAs)이나 독성물질, 생분해 불가능한 물질까지도 분해가 가능하며, 소화조 내 미생물의 활성을 높이고 바이오가스의 생산량을 극대화 할 수 있다고 알려져 있다. 본 연구에서는 MET가 혐기성소화의 메탄발생에 미치는 영향에 대하여 연구하기 위해 음식물 탈리액과 하수슬러지의 원소조성에 따른 이론적인 최대 메탄수율을 분석하였으며, BMP (Biochemical Methane Potential) 실험과 연속식 실험을 통한 메탄수율의 특성을 평가하였다. 그 결과, MET가 적용된 혐기성소화에서의 메탄수율은 일반적인 혐기성소화조에 비하여 기질에 따라 2-3배 정도 높았으며, 이론적인 최대 메탄수율에 미치지는 못하였으나 일부는 거의 근접한 결과가 도출되었다. 또한, 일반적인 혐기성소화조와 MET가 적용된 혐기성소화조의 안정화 후 바이오가스의 조성은 거의 유사하게 나타났다. 결과적으로, MET가 혐기성소화조의 유기물 제거효율을 향상시켜 메탄발생량을 증가시킨 것으로 나타났으며, 향후 추가적인 연구를 통하여 MET에서 메탄발생 메카니즘이 명확히 규명되어야 할 것이다. Theoretical maximum methane yield of glucose at STP (1 atm, 0℃) is 0.35LCH₄/gCOD. However, most researched actual methane yields of anaerobic digester (AD) on lab scale is lower than theoretical ones. A wide range of them have been reported according to experiments methods and types of organic matters. Recent year, a MET (Microbial electrochemical technology) is a promising technology for producing sustainable bio energies from AD via rapid degradation of high concentration organic wastes, VFAs (Volatile Fatty Acids), toxic materials and non-degradable organic matters with electrochemical reactions. In this study, methane yields of food waste leachate and sewage waste sludge were evaluated by using BMP (Biochemical Methane Potential) and con-tinuous AD tests. As the results, methane production volume from the anaerobic digester equipped with MET (AD+MET) was higher than conventional AD in the ratio of 2 to 3 times. The actual methane yields from all experiments were lower than those of theoretical value of glucose. The methane yield, however, from the AD+MET occurred similar to the theoretical one. Moreover, biogas compositions of AD and AD+MET were similar. Consequently, methane production from anaerobic digester with MET increased from the result of higher organic removal efficiency, while, further researches should be required for investigating methane production mechanisms in the anaerobic digester with MET.

기초 : 뇌하수체 선종에서 Met Tyrosine Kinase 수용체의 발현과 의미

박정언 ( Jung Eon Park ),김화정 ( Hwa Jung Kim ),이복순 ( Bok Soon Lee ),이재호 ( Jae Ho Lee ),김세혁 ( Se Hyuk Kim ) 대한뇌종양학회 2008 대한뇌종양학회지 Vol.7 No.2

Objective:Met tyrosine kinase receptor is known as the receptor of hepatocyte growth factor(HGF) and met-HGF signaling has been shown to affect a wide range of biological activities, including angiogenesis, cellular motility, growth, and morphogenetic differentiation. However, little is known about its significance in pituitary adenomas(PAs). The goal of this study is to verify Met-expression and its implication in PAs. Material & Methods:We collected fresh specimens from 29 PAs(11 functioning, 18 nonfunctioning) during surgery. The samples were subjected to RT-PCR using human wild type Met(wtMet) primer to assess Met expression in RNA level, and western blotting to assess its expression in protein level. We analyzed the relationship between Met expression in mRNA or protein level and clinico-radiological factors. Results:wtMet mRNA was expressed in 65.5%(19/29) and Met protein in 17.9%(5/28). Among 29 PAs, the incidence of wtMet mRNA expression was significantly higher in functioning PAs(90.9%) compared to nonfunctioning PAs (50%). And patients` age was significantly younger in wtMet expressed group(mean:42.8 years) compared to nonexpressed group(mean:55.1 years). The expression of wtMet mRNA or Met protein was not correlated with the other clinico-radiological factors including the invasiveness. Conclusion:Our results showed a positive relationship between wild type Met mRNA expression and endocrine activity. We suggest that wild type Met(wtMet) mRNA expression might be one of the markers of functioning adenomas and its clinical implication should be studied further.

RON and MET Co-overexpression Are Significant Pathological Characteristics of Poor Survival and Therapeutic Targets of Tyrosine Kinase Inhibitors in Triple-Negative Breast Cancer

Tian-Hao Weng,Min-Ya Yao,Xiang-Ming Xu,Chen-Yu Hu,Shu-Hao Yao,Yi-Zhi Liu,Zhi-Gang Wu,Tao-Ming Tang,Pei-Fen Fu,Ming-Hai Wang,Hang-Ping Yao 대한암학회 2020 Cancer Research and Treatment Vol.52 No.3

Purpose Triple-negative breast cancer (TNBC) is highly malignant and has poor prognosis and a high mortality rate. The lack of effective therapy has spurred our investigation of new targets for treating this malignant cancer. Here, we identified RON (macrophage-stimulating 1 receptor) and MET (MET proto-oncogene, receptor tyrosine kinase) as a prognostic biomarker and therapeutic targets for potential TNBC treatment. Materials and Methods We analyzed RON and MET expression in 187 primary TNBC clinical samples with immunohistochemistry. We validated the targeted therapeutic effects of RON and MET in TNBC using three tyrosine kinase inhibitors (TKIs): BMS-777607, INCB28060, and tivantinib. The preclinical therapeutic efficacy of the TKIs was mainly estimated using a TNBC xenograft model. Results Patients with TNBC had widespread, abnormal expression of RON and MET. There was RON overexpression, MET overexpression, and RON and MET co-overexpression in 63 (33.7%), 63 (33.7%), and 43 cases (23.0%), respectively, which had poor prognosis and short survival. In vivo, the TKI targeting RON ant MET inhibited the activation of the downstream signaling molecules, inhibited TNBC cell migration and proliferation, and increased TNBC cell apoptosis; in the xenograft model, they significantly inhibited tumor growth and shrank tumor volumes. The TKI targeting RON and Met, such as BMS-777607 and tivantinib, yielded stronger anti-tumor effects than INCB28060. Conclusion RON and MET co-overexpression can be significant pathological characteristics in TNBC for poor prognosis. TKIs targeting RON and MET have stronger drug development potential for treating TNBC.

MET1-Dependent DNA Methylation Represses Light Signaling and Influences Plant Regeneration in Arabidopsis

Shim, Sangrea,Lee, Hong Gil,Seo, Pil Joon Korean Society for Molecular and Cellular Biology 2021 Molecules and cells Vol.44 No.10

Plant somatic cells can be reprogrammed into a pluripotent cell mass, called callus, which can be subsequently used for de novo shoot regeneration through a two-step in vitro tissue culture method. MET1-dependent CG methylation has been implicated in plant regeneration in Arabidopsis, because the met1-3 mutant exhibits increased shoot regeneration compared with the wild-type. To understand the role of MET1 in de novo shoot regeneration, we compared the genome-wide DNA methylomes and transcriptomes of wildtype and met1-3 callus and leaf. The CG methylation patterns were largely unchanged during leaf-to-callus transition, suggesting that the altered regeneration phenotype of met1-3 was caused by the constitutively hypomethylated genes, independent of the tissue type. In particular, MET1-dependent CG methylation was observed at the blue light receptor genes, CRYPTOCHROME 1 (CRY1) and CRY2, which reduced their expression. Coexpression network analysis revealed that the CRY1 gene was closely linked to cytokinin signaling genes. Consistently, functional enrichment analysis of differentially expressed genes in met1-3 showed that gene ontology terms related to light and hormone signaling were overrepresented. Overall, our findings indicate that MET1-dependent repression of light and cytokinin signaling influences plant regeneration capacity and shoot identity establishment.

Acquired Resistance of MET-Amplified Non-small Cell Lung Cancer Cells to the MET Inhibitor Capmatinib

김슬기,김태민,김동완,김소연,김미소,안용운,김범석,허대석 대한암학회 2019 Cancer Research and Treatment Vol.51 No.3

Purpose Amplified mesenchymal-epithelial transition factor, MET, is a receptor tyrosine kinase (RTK) that has been considered a druggable target in non-small cell lung cancer (NSCLC). Although multiple MET tyrosine kinase inhibitors (TKIs) are being actively developed for MET-driven NSCLC, the mechanisms of acquired resistance to MET-TKIs have not been well elucidated. To understand the mechanisms of resistance and establish therapeutic strategies, we developed an in vitro model using the MET-amplified NSCLC cell line EBC-1. Materials and Methods We established capmatinib-resistant NSCLC cell lines and identified alternative signaling pathways using 3! mRNA sequencing and human phospho-RTK arrays. Copy number alterations were evaluated by quantitative polymerase chain reaction and cell proliferation assay; activation of RTKs and downstream effectors were compared between the parental cell line EBC-1 and the resistant cell lines. Results We found that EBC-CR1 showed an epidermal growth factor receptor (EGFR)–dependent growth and sensitivity to afatinib, an irreversible EGFR TKI. EBC-CR2 cells that had overexpression of EGFR-MET heterodimer dramatically responded to combined capmatinib with afatinib. In addition, EBC-CR3 cells derived from EBC-CR1 cells that activated EGFR with amplified phosphoinositide-3 kinase catalytic subunit " (PIK3CA) were sensitive to combined afatinib with BYL719, a phosphoinositide 3-kinase " (PI3K") inhibitor. Conclusion Our in vitro studies suggested that activation of EGFR signaling and/or genetic alteration of downstream effectors like PIK3CA were alternative resistance mechanisms used by capmatinib- resistant NSCLC cell lines. In addition, combined treatments with MET, EGFR, and PI3K" inhibitors may be effective therapeutic strategies in capmatinib-resistant NSCLC patients.

Association between the Catechol-O-Methyltransferase (COMT) Val158Met Polymorphism and Alexithymia in Patients with Obsessive-Compulsive Disorder

김세주,고민정,강지인,남궁기,이수영 연세대학교의과대학 2016 Yonsei medical journal Vol.57 No.3

Purpose: Alexithymia, defined as a deficit in the ability to recognize and describe one’s own feelings, may be related to the developmentand maintenance of obsessive-compulsive symptoms. The aim of this study was to evaluate the association between the catechol-O-methyltransferase (COMT) Val158Met polymorphism and alexithymia in patients with obsessive-compulsive disorder (OCD). Materials and Methods: We recruited 244 patients with OCD (169 males, 75 females). Alexithymia was assessed using the 20-item Toronto Alexithymia Scale (TAS-20), and genotyping of the COMT Val158Met polymorphism was evaluated. Results: Patients with the COMT Val/Val genotype had significantly higher total and “difficulty identifying feelings” (DIF) subdimensionscores than those with the Val/Met or Met/Met genotypes. Patients with the COMT Val/Val genotype had significantly higher “difficulty describing feelings” (DDF) subdimension scores than those with the COMT Val/Met genotype. However, there were no differences in the scores for the “externally oriented thinking” (EOT) subdimension among the three genotypes. Conclusion: These results indicate that the high-activity Val allele of the COMT Val158Met polymorphism is associated with increasedalexithymic traits in patients with OCD. The present finding suggests that alexithymia is an endophenotype of OCD that is mediated by the COMT Val158Met polymorphism.