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Project Details
Funding Scheme : General Research Fund
Project Number : 17113116
Project Title(English) : Exosomes released from nuclear Met expressing cells promote liver cancer metastasis and formation of lung premetastatic niche  
Project Title(Chinese) : 核Met表達細胞源性外泌體促使肝癌轉移及肺轉移前微環境的形成 
Principal Investigator(English) : Prof YAM, Judy Wai Ping  
Principal Investigator(Chinese) :  
Department : Department of Pathology
Institution : The University of Hong Kong
E-mail Address : judyyam@pathology.hku.hk 
Tel : 22552681 
Co - Investigator(s) :
Prof Wong, Alice Sze-Tsai
Panel : Biology & Medicine
Subject Area : Medicine, Dentistry & Health
Exercise Year : 2016 / 17
Fund Approved : 933,936
Project Status : Completed
Completion Date : 30-6-2020
Project Objectives :
To characterize the functional effect of exosomes in HCC cell migration and invasiveness and distant metastasis
To delineate the role of TGF-beta1 in HCC cell aggressiveness
To evaluate the mechanistic basis of exosomes in the formation of pre-metastatic niche in lung tissues
Abstract as per original application
(English/Chinese):
Met受體酪氨酸激酶可以激發活一系列生理性信號通路。然而Met自身的激活則常見於癌症，因此Met也成為了有前景的治療靶點。最新證據表明核Met（nMet）在癌症組織及在癌細胞系中表達，提示nMet在細胞核里有不為人知的功能。這對目前針對Met表面受體的治療方式提出了挑戰。我們之前的研究第一次證明了nMet在人類肝癌中的臨床意義。nMet過表達與肝癌靜脈侵襲及較差的預後顯著相關。相應的，nMet過表達促進了肝癌的生長和轉移。我們隨後的研究闡明了nMet驅使肝癌轉移的機制并發現了外泌體在這之中的重要性。近年來關於外泌體的研究主要集中在細胞間交流，特別是外泌體參與癌症轉移及轉移前微環境的形成。外泌體分泌具有功能性的分子傳遞至身體內特定的其他遠端組織，促使其形成適合癌細胞轉移的微環境。我們的研究表明表達nMet的肝癌細胞釋放的外泌體增加了肝癌早期細胞的轉移和侵襲能力。動物實驗顯示靜脈注射nMet-外泌體增加了肝癌得轉移的機率。經蛋白組學分析，TGF-1參與調節肝癌侵襲性。同時表達nMet的肝癌細胞顯示了比對照組細胞更高的TGF-1含量。因此我們假設表達nMet的肝癌細胞源性外泌體促使肝癌轉移及促進遠處器官轉移前微環境的形成的功能與產生TGF-1有關。在此次研究中，我們將探討nMet表達細胞源外泌體的功能及機制。我們將1）功能性的探討外泌體在肝癌細胞遷徙，侵襲及遠處轉移的作用，2）闡明TGF-1在肝癌侵襲性的作用及3）評價外泌體促使轉移前微環境建立的機制。此次的研究將會給肝癌轉移的研究提供新的看點及給與腫瘤源性外泌體對腫瘤轉移提供了新的機制。
Realisation of objectives: The project objectives as per original application has been thoroughly assessed and revised for better research improvement and outcome. Generally, the term “extracellular vesicles” (EVs) was used to replace “exosome” in whole study as suggested by the nomenclature criteria reported by the International Society of Extracellular Vesicles (ISEV). We have successfully accomplished objective 1 by characterization of the functional effect of EVs derive from nuclear Met (nMet) cells in HCC cell colony formation, migration and invasion ability. Nonetheless, we also demonstrated the effect of nMet-EVs in promoting distant metastasis in mice. For objective 2 and 3, we have revised the pre-proposed protein target TGF-beta 1. Our further study of TGF-beta 1 failed to deliver favorable outcome that warrants further investigation. In order to find the new potential candidates, we performed protein profiling using Orbitrap Fusion Lumos Tribrid mass spectrometer, which is more powerful in detecting minute protein inputs, on our EV samples. We identified a panel of proteins that belong to three family clusters that are important in catalyzing protein synthesis. We validated these candidates by quantitative PCR and immunoblotting. We performed both in vitro and in vivo functional assay to comprehensively elucidate the role of these candidates within EVs in promoting cancer metastasis. Our study has revealed the new evidences on how cancer cells utilized EVs to deliver key components that enhance cancer aggressiveness.
Summary of objectives addressed:
Objectives Addressed Percentage achieved
1.To characterize the functional effect of exosomes in HCC cell migration and invasiveness and distant metastasisYes100%
2.To delineate the role of TGF-beta1 in HCC cell aggressiveness Yes60%
3.To evaluate the mechanistic basis of exosomes in the formation of pre-metastatic niche in lung tissuesYes90%
Research Outcome
Major findings and research outcome: Extracellular vesicles (EVs) were isolated from the conditioned medium of MHCC97L cells that express nuclear Met (nMet) by ultracentrifugation. The integrity of isolated EVs was validated by transmission electron microscopy, western blotting and nanoparticle tracking analyzer. nMet-EVs exhibited an augmented potency to promote colony formation, migration and invasion ability of naïve parental MIHA, Huh7 and PLC/PRF/5 cells when compared to vector control EVs (Vec-EVs). The effect of nMet-EVs in promoting metastasis was also observed in the in vivo experimental metastasis assay by the coinjection of EVs and murine p53-/-;Myc-transduced hepatoblasts. These findings suggest that nMet-EVs could facilitate the extravasation and colonization of tumors cells in lungs. To ascertain the differential biological activities between Vec- and nMet-EVs, we compared their proteomic profiles using mass spectrometry. We identified a panel of proteins that belong to three protein complexes, namely eukaryotic initiation factors (eIFs) that are involved in the initiation of eukaryotic translation, ribosomal protein L (RPL) and ribosomal protein S (RPS) that are responsible for the assembly of 60S and 40S subunits, respectively. Both 60S and 40S are founding subunits of eukaryotic 80S ribosome that catalyzes the synthesis of proteins. A total of 13 protein candidates that are upregulated by more than 2-fold in nMet-EVs were selected for further validation by quantitative PCR and immunoblotting. The data revealed the increased expression of these proteins in nMet cells and established the association between cellular overexpression and enrichment in the EVs. To elucidate the functional role of these protein candidates in nMet-EVs, EIF3I, EIF4G2, RPS3A, and RPL10 were selected for further study. We employed lentiviral based knock-down approach where sh-EIF3I, shEIF4G2, shRPS3A and shRPL10 clones were established in nMet cells. EVs were collected from these knock-down clones and subjected to in vitro functional study. The effect of these EVs on colony formation, migration and invasion was diminished when compared to control nMet-EVs. In vivo experimental metastasis model also provided similar evidence. To investigate whether nMet expressing cells could evade from the effect of rapamycin, which actions on mTOR signaling pathway that regulate protein synthesis, naïve PLC cells were co-treated with rapamycin and nMet-EVs. Such combination treatment could rescue the expression of c-Myc protein, where it is normally suppressed by the effect of rapamycin. However, co-treatment of rapamycin with EVs of knock-down clones failed to rescue the c-Myc expression, suggesting those proteins within nMet-EVs are involved in protein translation that are important in driving cancer aggressiveness.
Potential for further development of the research
and the proposed course of action:		 Our data has revealed nuclear Met (nMet) expressing cells secrete extracellular vesicles (EVs) enriched with a panel of proteins involved in the regulation of protein translation to promote cancer aggressiveness. However, the detailed regulatory mechanism of how these proteins being packaged into EVs remained to be unanswered. Further study focuses on molecular sorting machinery and biogenesis of EVs could unravel the mechanistic basis underlying the oncogenic activities of tumor cell derived EVs. Also, our data provided new evidences on how cancer cells could evade from the effect of rapamycin, an inhibitor for mTOR signaling pathway that regulates cell growth and metabolism, through the action of EVs. Our findings provide insights into the innovative treatment leading to the blockage of EVs biogenesis or EVs activity, alone or in combination with rapamycin may serve as a therapeutic option beyond the current limited treatments for HCC.
Layman's Summary of
Completion Report:		 Hepatocellular carcinoma (HCC) accounts for most primary liver cancers. World Health Organization (WHO) estimates that more than 1 million patients will die from liver cancer in 2030. Understanding the underlying mechanism of how HCC acquires cancer aggressiveness is crucial to improve the current diagnosis and treatment for HCC patients. Our previous study provided the first evidence about the presence of truncated Met receptor, a well-known oncogene that was frequently deregulated in many types of cancer, in the nucleus of HCC which has profound prognostic value. These observations have led to current study in which we demonstrated that extracellular vesicles (EVs), in nanometer-sized, released by nuclear Met expressing cells, promote cancer metastasis. Within these oncogenic vesicles, we identified a panel of proteins belong to the same family cluster that are mainly involved in protein translation and functioning as the potent modulators of cancer aggressiveness. By reducing the expression of these protein targets in the EVs, cancer metastasis was profoundly attenuated in mice. The outcome of study suggests an unreported underlying mechanism that involves nuclear Met for the enhancement of cancer metastasis and provides important insights into the development of therapeutic strategies against aberrant Met in human cancers.
Research Output
Peer-reviewed journal publication(s)
arising directly from this research project :
(* denotes the corresponding author)
Year of
Publication		 Author(s) Title and Journal/Book Accessible from Institution Repository
Tey SK, Chung CYS, Yam JWP*  Nuclear Met expressing cells derived extracellular vesicles promote liver cancer metastasis via enrichment of proteins that regulate translation  No 
Recognized international conference(s)
in which paper(s) related to this research
project was/were delivered :
Month/Year/City Title Conference Name
Korea Exosome released from nuclear Met expressing cells promote liver cancer metastasis and formation of lung premetastatic niche.  The Liver Week 2017 
Barcelona Role of exosomes in liver cancer metastasis.  ISEV (The International Society for Extracellular Vesicles) 2018 Annual meeting 
Other impact
(e.g. award of patents or prizes,
collaboration with other research institutions,
technology transfer, etc.):
  SCREEN ID: SCRRM00542