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| Project Details |
| Funding Scheme : | General Research Fund | ||||||||||||||||
| Project Number : | 781013 | ||||||||||||||||
| Project Title(English) : | Soluble E-cadherin: a novel molecule regulating angiogenesis? | ||||||||||||||||
| Project Title(Chinese) : | 可溶性E-cadherin：一種新的血管生成調控因子? | ||||||||||||||||
| Principal Investigator(English) : | Prof Wong, Alice Sze-Tsai | ||||||||||||||||
| Principal Investigator(Chinese) : | |||||||||||||||||
| Department : | School of Biological Sciences | ||||||||||||||||
| Institution : | The University of Hong Kong | ||||||||||||||||
| E-mail Address : | awong1@hku.hk | ||||||||||||||||
| Tel : | 22990865 | ||||||||||||||||
| Co - Investigator(s) : |
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| Panel : | Biology & Medicine | ||||||||||||||||
| Subject Area : | Biological Sciences | ||||||||||||||||
| Exercise Year : | 2013 / 14 | ||||||||||||||||
| Fund Approved : | 1,039,239 | ||||||||||||||||
| Project Status : | Completed | ||||||||||||||||
| Completion Date : | 31-12-2016 | ||||||||||||||||
| Project Objectives : |
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| Abstract as per original application (English/Chinese): |
腫瘤血管生成是癌症侵襲轉移中的重要步驟，相關血管生成調控因子及其調控機制的研究受到廣泛關注。E-cadherin是一種細胞間黏連蛋白，它的缺失是癌症侵襲轉移的標志物。我們已於早前發現了卵巢癌中E-cadherin的失調機制及其與癌症發展的關係。E-cadherin合成后以跨膜蛋白的形式發揮作用，其胞外區被剪切后釋放出可溶性E-cadherin (sE-cad)，進而使細胞表面功能性E-cadherin迅速減少。sE-cad在卵巢癌病人的血清和癌性腹水中的含量增高，但是其相關的腫瘤侵襲調控機制仍不明確。許多研究只關注E-cadherin缺失在減弱細胞間黏附中的作用，但是sE-cad本身是否具有生物活性尚不清楚。而於最近，我們首次發現sE-cad能夠介導血管生成的相關信號，並一步証實腫瘤來源囊泡與sE-cad剪切及釋放的激活密切相關。根據以上結果我們推測sE-cad對血管形成至關重要，腫瘤來源囊泡是激活sE-cad級聯信號的關鍵機制之一。 我們希望通過體內外實驗繼續深入的研究：(1)揭示sE-cad在血管生成不同階段中的作用；(2)剖析sE-cad在內皮細胞中的功能及調控機制，及其可能涉及一種新的sE-cad-VE-cadherin信號軸；(3)闡明腫瘤血管形成過程中sE-cad剪切及釋放的機制。這個研究將揭示腫瘤血管生成與腫瘤侵襲的關係，為血管生成調控提供理論基礎，並為新型靶向治療藥物的開發提供思路。 |
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| Realisation of objectives: | Objective 1: Angiogenesis is a multistep process which involves cell adhesion, migration, differentiation, and growth. As such, we assayed for these activities to define a mechanism of action. In this report, we have successfully shown for the first time that soluble E-cadherin (sE-cad) is a key regulator of tumor angiogenesis, which is involved in one or more rate-limiting steps of the angiogenic cascade in vitro and in vivo. Besides ectodomain shedding, we also present evidence for a novel paradigm of sE-cad production in the form of exosomes. Using a refined analysis, we showed that sE-cad was predominantly expressed in Golgi/Trans-Golgi network-enriched fractions. By employing various tests on morphology (electron microscope), density (sucrose gradient), protein composition (Western blotting), and size (nanoparticle tracking), we showed exosomes as a vehicle for sE-cad. Using immunoelectron microscopy and BODIPY-TR ceramide fluorescent dye, we further showed the surface localization and uptake of exogenous purified sE-cad-positives exosomes by endothelial cells. sE-cad-positive exosomes were a potent inducer of angiogenesis. Furthermore, sE-cad-positive exosomes could also be found in the ascites of ovarian cancer patients and promoted angiogenesis in vitro and in vivo, confirming that sE-cad involved in it is clinically relevant. Percentage achieved: 100% Objective 2: In search of mechanisms underlying sE-cad regulation of angiogenesis, we found that sE-cad mediated heterophilic transconnection with VE-cadherin. This provides a novel and attractive mechanism whereby endothelial cells, which lack E-cadherin, can lead to angiogenic signaling. This heterophilic interaction may serve as a new paradigm of cell-cell communication. We further showed that a Wnt/b-catenin signaling cascade was critically involved in this novel sE-cad/VE-cadherin heterotypic transconnection that contributed to angiogenesis. Although the mechanism of b-catenin activation could be mediated through MAP kinase, we ruled out this possibility and showed that these events were mediated via a PI3K/Akt-dependent signaling. We also showed that sE-cad-VE-cadherin transduced a novel sequential activation of b-catenin and NFkB signaling, which should provide further new insights into the mechanisms of sE-cad functions. Percentage achieved: 100% Objective 3: In search of how the sE-cad on exosomes is cleaved, we show that this is mediated through activation of the membrane-bound metalloproteinases ADAM10 and MMP25. Moreover, we show that Rab25, which is highly expressed in ovarian cancer cells but not normal ovarian surface epithelium, is required for exosome release. Interestingly, we found that HGF, EGF, and hypoxic stimulus augmented the generation and release of exosomes containing ADAM10, MMP-25, Rab25 and E-cadherin, suggesting that metastatic factors in the tumor microenvironment may be responsible for sE-cad release. These studies provide the first evidence for the regulation of a novel form of tumor-endothelial interaction in the molecular control of angiogenesis. Percentage achieved: 100% Publications: Journal articles: 1. Tang MK, Yue PY, Ip PP, Huang RL, Lai HC, Cheung AN, Tse KY, Ngan HY, and Wong AST* Soluble E-cadherin promotes tumor angiogenesis and localizes to exosome surface. Nat. Commun. (in second revision). 2. Tang MK and Wong AST* (2015) Exosomes: Emerging biomarkers and targets for ovarian cancer. Cancer Lett. 367: 26-33. 3. Tang MK and Wong AST* ADAM10 and MMP25 in the tumor microenvironment-induced ectodomain cleavage of E-cadherin (in preparation). Conference proceedings: 4. Tang MK and Wong AST* (2014) An angiogenic function for E-cadherin in ovarian cancer. AACR Annual Meeting 2014, San Diego, CA, USA (Abstract No. 9). 5. Tang MK and Wong AST* (2015) An angiogenic role of E-cadherin-positive exosomes in ovarian cancer. AACR Annual Meeting 2015, Philadelphia, PA, USA (Abstract No. 1035). | ||||||||||||||||
| Summary of objectives addressed: |
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| Research Outcome | |||||||||||||||||
| Major findings and research outcome: | Objective 1: Here, we show for the first time that sE-cad (an 80-kDa soluble form), which is highly expressed in the malignant ascites of ovarian cancer patients, is a potent inducer of angiogenesis. We demonstrated that sE-cad stimulates (a) endothelial cell migration, (b) permeability, (c) endothelial cell tube formation, and (d) neovascularization in Matrigel implants. In addition to ectodomain shedding, we provide further evidence that sE-cad is abundantly released in the form of exosomes. In vivo and clinical data prove the relevance of sE-cad-positive exosomes for malignant ascites formation and widespread peritoneal dissemination. We confirm the localization of sE-cad on the surface and its uptake by the endothelial cells. Publications: Journal articles: #1, #2 Conference proceeding: #4 Objective 2: We show for the first time that sE-cad mediates heterophilic transconnection with VE-cadherin. This provides a novel and attractive mechanism whereby endothelial cells, which lack E-cadherin, can lead to angiogenic signaling. We also show that sE-cad-VE-cadherin transduces a novel sequential activation of b-catenin and NFkB signaling, which should provide further new insights into the mechanisms of sE-cad functions. Whereas crossregulation between b-catenin and NFkB has been generally reported, we found that they were two parallel pathways, which may allow continuous input from the signaling pathway for sustaining transcriptional and subsequent cellular response. The combined operation of different mechanisms suggests that the interaction of sE-cad/VE-cadherin is a crucial event and emphasizes the important role of sE-cad/VE-cadherin that has in contributing to the angiogenic process. Publications: Journal articles: #1, #2 Conference proceeding: #5 Objective 3: On exploring how the sE-cad on exosomes is cleaved, we show that this is through two critical members of the membrane-bound metalloproteinases, ADAM10 and MMP-25. Moreover, we show that Rab25, which is highly expressed in ovarian cancer cells but not normal ovarian surface epithelium, is required for exosome release. Interestingly, we found that this cleavage and release of sE-cad could be regulated by metastatic factors in the malignant ascites, in particular HGF, EGF and hypoxic stimulus, suggesting a role for the tumor microenvironment. To date, while exosomes may help create a tumor microenvironment is well defined, the underlying mechanisms of how the tumor microenvironment may regulate the cleavage and release of exosomes are largely unknown. These studies provide the first evidence for the regulation of a novel form of tumor-endothelial interaction in the molecular control of angiogenesis. Publication: Journal article: #3 Taken together, our data strongly support the important role for sE-cad in ovarian cancer metastasis and provide a mechanistic rationale for targeting sE-cad or its associated signaling in the treatment of metastatic ovarian cancer. | ||||||||||||||||
| Potential for further development of the research and the proposed course of action: |
We reveal the novel identification of sE-cad regulation on angiogenesis that has a pivotal role in the metastatic progression of ovarian cancer. This reveals a new direction for understanding the oncogenic role of sE-cad in ovarian cancer metastasis and implicate that sE-cad could be a promising therapeutic target. We found that sE-cad possessed similar VEGF efficiency in angiogenesis, but in a VEGF-independent manner. We are actively pursuing in this direction to document such effect using more extensive in vitro and in vivo studies with and without anti-VEGF agents. Given the development of resistance in targeting VEGF and that VEGF inhibitors also interfere with the normal VEGF pathway leading to numerous adverse side effects, it is of great interest to identify other angiogenic regulators with complimentary mechanisms. High serum sE-cad level has also been detected in patients with breast, gastric, and colon cancer and its expression positively correlates with the aggressive behavior. To expand our understanding of the angiogenic properties of sE-cad, we plan to examine if the sE-cad release is also seen in peritoneal cancers or other cancer types. We will also examine the level and effect of sE-cad in healthy donors or non-cancerous patients. These findings should highlight the functional importance of sE-cad in impacting malignant cell behavior and implies important clinical implications. | ||||||||||||||||
| Layman's Summary of Completion Report: | One of the greatest unmet needs compromising the successful treatment of cancer is metastasis. A key to successful metastasis is the formation of new vasculature, known as angiogenesis. Therefore, unraveling molecular mechanisms of angiogenesis is of obvious importance. Several of our new findings of this project indicate that sE-cad, which is highly expressed in the serum and malignant ascites of ovarian carcinoma patients, is a pivotal regulator of angiogenesis and that it could be a promising target. (i) We show for the first time a new role for sE-cad on angiogenesis. (ii) We also present evidence that exosomes are a novel major platform for the cleavage and release of sE-cad. (iii) We uncover a new heterophiliic interaction between sE-cad and VE-cadherin that transduces a novel sequential activation of b-catenin and NFkB signaling that may explain its significant angiogenesis promoting effects. These results reveal a new direction for understanding the oncogenic roles of sE-cad and highlight its importance as a viable therapeutic target. Targeting sE-cad may thus be a useful approach to decrease ascites formation, the most distressing complication for ovarian cancer. | ||||||||||||||||
| 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.): |
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| SCREEN ID: SCRRM00542 |