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| Project Details |
| Funding Scheme : | General Research Fund | ||||||||||||||||
| Project Number : | 783310 | ||||||||||||||||
| Project Title(English) : | Interplay of caveolin-1 and met receptor in liver cancer metastasis | ||||||||||||||||
| Project Title(Chinese) : | Cav-1和Met受體在肝癌轉移的相互作用 | ||||||||||||||||
| 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) : |
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| Panel : | Biology & Medicine | ||||||||||||||||
| Subject Area : | Biological Sciences | ||||||||||||||||
| Exercise Year : | 2010 / 11 | ||||||||||||||||
| Fund Approved : | 887,400 | ||||||||||||||||
| Project Status : | Completed | ||||||||||||||||
| Completion Date : | 30-6-2014 | ||||||||||||||||
| Project Objectives : |
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| Abstract as per original application (English/Chinese): |
肝細胞癌（HCC）是一種在東南亞特別普遍的癌病，在香港HCC所引致的死亡率更位列第二。由於HCC的復發率和癌細胞轉移較高，預後比較困難。因此，鑑定有效的生物標記不單能提昇臨床管理的效果，也可協助發展新的療法。小窩蛋白-1（Cav1）的命名是基於它作為細胞膜小窩的基本結構及功能元件。在焦點粘聯中能發現非細胞膜小窩Cav1，在其中參與焦點粘聯動力和細胞遷移。新的證據顯示Cav1與人類癌細胞的轉移有極強的關係。事實上，我們的研究結果顯示Cav1在轉移的HCC細胞株中的表達顯著增高。在生理方面，我們觀察到Cav1在人類臨床樣本中的非腫瘤肝組織，原位腫瘤和轉移的肝癌組織中的表達逐步增加。Cav1在轉移癌細胞中的顯著表達說明Cav1與肝癌轉移有關。從功能上來看，提高Cav1在HCC細胞株中的表達能促進細胞的生長，遷移和侵襲，也能促進腫瘤在小鼠模型中的生長，而抑制HCC細胞株中Cav1的表達則能妨礙腫瘤在裸鼠體內形成。我們的初步證據表示，Cav1調節肝細胞生長因子（HGF）誘導的遷移和入侵，並增強肝細胞生長因子受體 (Met) 在肝癌細胞中的表達。異常的HGF-Met信號通路在癌細胞轉移中扮演著關鍵角色，過度Met的表達更是一個肝癌轉移的預後指標。我們猜測Cav1在肝癌轉移中激活Met和其下游信號通路有著極其重要的作用。在這項研究中，我們目的是闡明Cav1在肝癌轉移中介導Met活化的分子學基礎及功能。我們建議 1）在肝癌細胞中劃定Cav1和Met信號通路的相互作用，2）利用體外活化實驗和原位移植模型，描繪Cav1介導Met活化的功能，以及 3）評估在臨床肝癌組織中的Cav1和Met表達的臨床及預後意義。據我們了解，這研究是首次探討Cav1在肝癌轉移中調節HGF-Met信號的作用。這項研究也將為Cav1在肝癌轉移的功能提供有力的論證。此外，臨床病理分析Cav1介導Met激活有可能產生深遠的預後價值以供臨床剖析之用。 |
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| Realisation of objectives: | 1. To delineate the molecular interaction of Cav1 and Met signaling pathways in HCC Both Cav1 and Met were profoundly expressed in metastatic HCC cells. Using PLC cells as a working model, the protein and mRNA expressions of the Met were upregulated in PLC Cav1 overexpressing (PLC/Cav1) cells. With hepatocyte growth factor (HGF) stimulation, Met-ERK1/2 signaling cascade was transactivated. A notable increase and higher level of pMet was detected in PLC/Cav1 cells as compared to the control. The capacity for Cav1 to interact with Met was illustrated by their colocalization at perinuclear and cytoplasmic regions. Their colocalization in the early endosomes suggested that Cav1 might affect the endosomal dynamics and the subcellular localization of Met. 2. To characterize the functional effect of Cav1-mediated Met activation in HCC Cav1 overexpressing and knockdown stable clones were established and subjected to different functional assays. Collectively, our data demonstrated the salient contribution of Cav1 in promoting in vitro cell growth, anchorage independent growth, motility and invasiveness, as well as the in vivo tumorgenicity and metastasis of HCC cells. Extensive cell scattering was observed in PLC/Cav1 overexpressing cells upon HGF stimulation. The number of scattered colonies under HGF stimulation was increased remarkably. Such de-compacted colonies were not observed in control. To manifest the involvement of HGF/Met signaling in Cav1 promoted motility and invasiveness, we perturbed the HGF/Met signaling pathway by suppressing the Met expression and activity. Suppression of Met expression by siRNA against Met or inhibition of Met activity using Met kinase inhibitor SU11274 in PLC/Cav1 overexpressing cells largely suppressed HGF-induced migration and invasiveness of PLC/Cav1 cells. The number of scattered colonies formed under HGF stimulation was also substantially reduced. 3. To evaluate the clinicopathological relevance and prognostic significance of Cav1 and Met expressions in HCC clinical tissues Immunohistochemistry was performed in 106 paired non-tumorous and tumorous liver tissues and 31 sets of samples comprising non-tumorous livers, primary HCCs and extrahepatic metastatic tumors from the same patients. Cav1 was not detected in non-tumorous liver but exclusively expressed in tumorous liver. Cav1 overexpression was observed in 36% of primary tumors and significantly associated with venous invasion. Increase in cav1 expression was also detected in 35.5% of metastatic tumors when compared to the primary tumors. These observations suggested the promoting role of Cav1 in HCC tumorigenesis and metastasis. Met was also found to be overexpressed in HCC. However, significant association between Cav1 and Met was not found. | ||||||||||||||||
| Summary of objectives addressed: |
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| Research Outcome | |||||||||||||||||
| Major findings and research outcome: | Clinical significance of Cav1 in HCC Cav1 expression was upregulated in non-metastatic HCC cell lines as compared to the non-tumourigenic liver cell line, and dramatically overexpressed in metastatic cell lines. The observation in human HCC tissues was in accordance with that observed in cell line panel. Cav1 was not expressed in non-tumourous liver but overexpressed in primary and extrahepatic tumours. We demonstrated for the first time the progressive increase of Cav1 along with cancer progression. Clinicopathological analysis further revealed a significant association of Cav1 expression with venous invasion. These findings collectively suggest the significance of Cav1 as a prognostic marker in HCC. (J Pathology 2012, AACR annual meeting abstract 2009) Oncogenic properties of Cav1 in HCC tumorigenesis and metastasis Overexpression and knockdown approaches were performed to characterize the functions of Cav1 in HCC. The promoting role of Cav1 in in vitro cell growth, motility and invasiveness was demonstrated. Moreover, Cav1 overexpression dramatically augmented the in vivo tumorgenicity and metastasis of HCC cells. Collectively, our data demonstrated the undisputable role of Cav1 as a promoter in HCC tumorigenesis and metastasis. (J Pathology 2012). Mechanisms underlying the oncogenic capacity of Cav1 To provide mechanistic insights, we examined the involvement of the HGF/Met signaling pathway in Cav1 mediated tumourigenesis. Our data demonstrated clearly the transactivation of HGF/Met-ERK1/2 signaling cascade in Cav1 overexpressing cells. Ectopic expression of Cav1 upregulated the mRNA and protein expression of Met receptor, thus sensitizing the HCC cells to HGF-induced cell growth, motility and invasiveness. We further pinpointed the ERK1/2 signaling to be the downstream of Cav1-mediated HGF/Met transactivation. Such unprecedented observation explained in part the oncogenic function of Cav1 in HCC and elucidated the signaling pathways involved. (Asian Pacific Association for the Study of the Liver conference abstract 2010, Days of Molecular Medicine conference abstract 2010) Role of hypoxia-induced Cav1 expression in HCC metastasis Similar to Met surfact receptor, Cav1 was found to be induced by hypoxia. It was found that Cav1 was upregulated at both the transcriptional and translational levels. Its expression was also enhanced in the hypoxic region of tumor mass. The hypoxia-induced upregulation of Cav1 was found to be regulated by HIF1alpha. Functionally, upregulation of Cav1 promoted HCC cell migration and invasiveness. Using cDNA expression profiling, S100P was found to be the downstream target of Cav1. Hypoxia upregulated the expressions of Cav1 and S100P. Silencing of Cav1 abolished the upregulation of S100P under hypoxia indicating that Cav1 is the upstream regulator of S100P. Functional study also revealed that Cav1 augmented cell migration and invasiveness through S100P. (AACR annual meeting abstract 2011) | ||||||||||||||||
| Potential for further development of the research and the proposed course of action: |
The novel findings of our study provided a comprehensive understanding regarding the functional implications and mechanistic nature of Cav1 in HCC tumorigenesis and metastasis. Mechanistic study also provided evidence about the Cav1-mediated activation of Met pathway as the underlying basis of the oncogenic property of Cav1. Our data also suggested the capacity for Cav1 to interact with Met as illustrated by their colocalization at perinuclear and cytoplasmic regions. In addition, we identified the perinuclear region, where Cav1 and Met colocalize, to be the early endosomes. Colocalization of Met and EEA1 endosomal marker was revealed in our study and this finding has implicated the endosomal dynamics of Met regulated by Cav1. Endocytosis and endosomal sorting have been shown to be involved in the nuclear entry of surface receptor. The role of Cav1 in the internalization of Met thus warrants further investigation. Indeed, our data also revealed the presence of nuclear Met (nMet) in the nucleus of HCC cells. Emerging evidence has shown that the localization of Met is not restricted to the cell membrane and cytoplasm. Nuclear Met has been detected in different cell types. The functional role and clinical implications of nMet are awaited to be further investigated. | ||||||||||||||||
| Layman's Summary of Completion Report: | In this study, we elucidated the functions and molecular mechanism of Cav1-mediated Met activation in HCC metastasis. Metastasis is a key event at the advanced stage of hepatocarcinogenesis. In light of the emergence of Cav1 as a new master of cancer metastasis in various cancer types and its undefined role in HCC metastasis, it is important to unveil the role of Cav1 in HCC. The current understanding about the molecular mechanisms of Cav1 in cancer metastasis is conflicting. Also, the underlying basis of Cav1 in HCC metastasis has never been addressed. Our work on dissecting the interplay of Cav1 and Met signaling pathways in HCC metastasis is original. To our knowledge, this is the first study to investigate the effect of Cav1 overexpression on Met signaling cascades in HCC metastasis. This study has deciphered novel molecular pathways contributing to the invasiveness and metastasis of HCC. The functional data obtained from animal work has provided the first in vivo evidence about the functional effect of Cav1 in HCC metastasis. In addition, the clinicopathological data obtained in this study has determined the significance of Cav1 as a prognostic marker and a potential therapeutic target for advanced HCC. | ||||||||||||||||
| Research Output | |||||||||||||||||
| Peer-reviewed journal publication(s) arising directly from this research project : (* denotes the corresponding author) |
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| Recognized international conference(s) in which paper(s) related to this research project was/were delivered : |
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| Other impact (e.g. award of patents or prizes, collaboration with other research institutions, technology transfer, etc.): |
Awards: Tse Yuk Ting, a PhD student who involved in this project was awarded the following awards and scholarships for her oral presentation on her project and academic excellence. 1. Young Investigator Award, 15th Hong Kong International Cancer Congress 2. Best Presentation Award, 8th, Department of Pathology, February 2010 3. Best Presentation Award, 9th, Department of Pathology, July 2010 4. YS and Christabel Lung Postgraduate Scholarship Her PhD thesis was marked as outstanding (top 5%) by the examiners and she was nominated for thesis prize. Collaboration: Dr. Yam (PI) and Dr. Alice Wong (co-I) has established research collaboration not only on this funded GRF, but also on research work related to caveolin-1 and Met in different cancer models. Apart from their published paper on this funded project (as listed in research output), they also have another joint paper entitled “c-Met overexpression contributes to the acquired apoptotic resistance of non-adherent ovarian cancer cells through a cross-talk mediated by phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2” published in Neoplasia 12(2):128-138, 2010 by Tang MKS, Zhou HY, Yam JWP and Wong AST. Their collaboration has been continued in joint application for RGC GRF in the subsequent application exercise. They successfully secured three RGC GRF in year 2011 and 2013. Technology transfer: Dr. Leda Raptis (Department of Biomedical and Molecular Sciences, School of Medicine, Queen’s University, Canada) requested a series of 5’deleted Caveolin-1 promoter constructs. | ||||||||||||||||
| SCREEN ID: SCRRM00542 |