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ENQUIRE PROJECT DETAILS BY GENERAL PUBLIC |
| Project Details |
| Funding Scheme : | General Research Fund | ||||||||||||||||||||
| Project Number : | 17122014 | ||||||||||||||||||||
| Project Title(English) : | Sialyl Lewis x antigen: a critical molecule for early stage tumor metastasis? | ||||||||||||||||||||
| Project Title(Chinese) : | 唾液酸化Lewis x抗原： 腫瘤轉移早期關鍵因子？ | ||||||||||||||||||||
| 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 : | 2014 / 15 | ||||||||||||||||||||
| Fund Approved : | 1,051,688 | ||||||||||||||||||||
| Project Status : | Completed | ||||||||||||||||||||
| Completion Date : | 31-12-2017 | ||||||||||||||||||||
| Project Objectives : |
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| Abstract as per original application (English/Chinese): |
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| Realisation of objectives: | Objective 1: Peritoneal metastasis is an important factor affecting the prognosis of patients with ovarian cancer. The attachment of ascitic ovarian tumor sphere forming cells to the peritoneal mesothelium is a key and pivotal step to the onset of the metastatic spread. This tumor cell lodgment requires the presence of specialized adhesion molecules capable of binding under shear stress, but not static, conditions. Thus, it is important to develop 3D tumor model that recapitulates shear-induced changes and, consequently, natural tumor progression in real time. Here, we developed a clinically relevant 3D model generated under controlled physiological shear stress conditions, which could serve as a precise experimental model to study molecular mechanisms involved in ovarian tumor progression. Using this 3D-microfluidic platform and flow cytometry, we identified sLex glycoprotein as a ligand that played potential roles during rolling and firm adhesion events in the metastatic cascade. Glycosyltransferases/glycosidases in glycosylation machinery involved in the synthesis of sLex such as a1,4-galactosyltransferase 1, a2,3-sialytransferase 3, and a1,3-fucosyltransferase 5 (FUT5) were significantly increased in highly metastatic (M-) compared to non-metastatic (NM)-CSCs. While the sLex glycan displayed canonical biochemical features such as sialofucosylation and nonsulfation, our data also unveiled novel features of sLex different from other sLex ligands (2, 3). Knocking down FUT5, a key enzyme in the synthesis of sLex, suppressed adhesion of M-CSCs and metastatic spread in mice. Percentage achieved: 100% Objective 2: We show for the first time that M-CSCs and NM-CSCs differed in their adhesion efficiency to the peritoneal mesothelium under physiological flow conditions. M-CSCs, as compared to NM-CSCs, exhibited slower rolling velocity, with an average rolling velocity of 56.7±0.4 um/s (at least 2-fold less than required for NM-CSCs), higher tethering frequency (1.8-fold at 0.05 dynes/cm2), and bound more firmly to the peritoneal mesothelium. The maximum adhesion percentage was found at 0.04 to 0.07 dynes/cm2, which matches with ascitic shear stress. In addition to E-selectin, we showed that P-selectin expressed by the peritoneal mesothelium also played an important role in this interaction. P-selectin had a greater strength than E-selectin in adhesion. Treatment with anti-E-, P- or L-selectin increased the rolling velocities of M-CSCs when compared with that of isotype control (24.8% increase by anti-E-selectin, 63.3% increase by anti-P-selectin, and 22.5% increase by anti-L-selectin in median rolling velocities). Notably, blockade of P-selectin almost completely abrogated (~88%) tumor-mesothelial adhesion, whereas inhibition of E-selectin or L-selectin had partial effects (48% by anti-E-selectin; 28% by anti-L-selectin). Consistently, significant adherence to P-selectin-Fc (59%), but not E-selectin- or L-selectin-Fc, was observed with M-CSCs. Percentage achieved: 100% Objective 3: We show for the first time that the sLex-P-selectin interaction activates P-cadherin, but not other cadherins or b1 integrin, for the firm adhesion to the peritoneal mesothelium. Moreover, this adhesion activates specific downstream signaling paths to confer metastatic success. We found that P-cadherin could activate the accumulation of p120 catenin in the cytoplasm and the subsequent tumor cell migration and invasion. The use of P-cadherin siRNA or neutralizing antibody to inhibit P-cadherin expression and function resulted in diminished p120 catenin activation, confirming that the effect was P-cadherin specific. Moreover, we showed an essential role for insulin-like growth factor-1 receptor (IGF-1R) as a novel sLex-bearing ligand in the peritoneal milieu, which in turn activated mesothelial cells. P-cadherin was associated with complex formation with IGF-1R, which could induce the ligand-independent activation of IGF-1R. Inhibition of IGF-1R expression or its activity significantly inhibited the P-cadherin-dependent p120ctn activation. Furthermore, we uncovered a new link of microRNA-199a-3p, phosphatidylinositol 3-kinase/Akt, and multidrug transporter activation in shear stress-induced CSC enrichment. Percentage achieved: 100% These findings not only reveal a novel class of sLex ligand but also unveil a previously unrecognized role for P-selectin in mediating tumor-mesothelial adhesive interactions under shear stress, which provide new perspectives on shear stress-based biology in the peritoneum, and that its targeting may represent a potential therapeutic advantage for metastatic ovarian cancer. Publications (*, corresponding author) (Note: ALL as corresponding author): Journal article: 1. Li SS, Ip CK, Tang MY, Tong Y, Zhang JZ, Hassan AA, Mak AS, Yung S, Chan TM, Ip PP, Lee CL, Chiu PC, Lee LT, Zeng JZ, Shum HC, and Wong AS* Sialyl Lewis x-P-selectin cascade mediates tumor-mesothelial adhesion in ascitic fluid shear flow. Nat. Commun. (in revision) (impact factor: 12.353). 2. Neoh KH, Hassan AA, Chen A, Sun Y, Liu P, Xu KF*, Wong AS*, and Han R* (2017) Rethinking liquid biopsy: microfluidic assays of mobile tumor cells in human body fluids. Biomaterials 150: 112-124. (impact factor: 8.402) 3. Li S, Ip CK, Tang MY, Sy SK, Yung S, Chan TM, Yang M, Shum HC, and Wong AS* (2017) Modeling ovarian cancer multicellular spheroid behaviors in a dynamic 3D peritoneal microdevice. J. Vis. Exp. 120: doi:10.3791/55337 (impact factor: 1.232). 4. Ip CK, Li SS, Tang MY, Sy SK, Ren Y, Shum HC, and Wong AS* (2016) Stemness and chemoresistance in epithelial ovarian carcinoma cells under shear stress. Sci. Rep. 6: 26788 (impact factor: 5.578). Conference proceedings: 5. Li SS, Ip CK, Shum HC, and Wong AS* (2018) Shear stress downregulation of miR-199a-3p drives chemoresistance in ovarian cancer cells. 12th Biennial Ovarian Cancer Research Symposium, Seattle, Washington (Abstract No. GMM-059). 6. Li S, Ip CK, Hassan AA, Tang MY, Yung S, Chan TM, Shum HC, and Wong AS* (2017) Sialyl Lewis x-P-selectin connection between ovarian tumor-mesothelium in early stage metastasis. AACR Annual Meeting 2017, Washington, DC (Abstract No. 5885). 7. Li S, Ip CK, Hassan AA, Tang MY, Yung S, Chan TM, Chiu PC, Shum HC, and Wong AS* (2016) Sialyl Lewis x drives early stage ovarian cancer metastasis through modulation of tumor-mesothelial adhesion. Annual Scientific Meeting of the Society of Endocrinology, Metabolism and Reproduction, Hong Kong (Abstract No. P-11) [awarded Best Basic Science Poster Presentation]. 8. Ip CK, Li S, Tang MY, Sy SK, Shum HC, and Wong AS* (2015) Sialyl Lewis x is essential for early stage ovarian tumor metastasis through modulation of tumor-mesothelial adhesion. ASCB Annual Meeting 2015, San Diego, CA (Abstract No. P1086). | ||||||||||||||||||||
| Summary of objectives addressed: |
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| Research Outcome | |||||||||||||||||||||
| Major findings and research outcome: | Objective 1: We have developed a customizable microfluidic platform to overcome the technical limitations of the conventional approaches and to recapitulate peritoneal dissemination in dynamic flow. Using this microfluidic platform, we have uncovered a novel sLex-bearing P-selectin ligand in ovarian cancer cells, which is distinct from those previously defined P-selectin ligands. Very little is known about the selectin ligands on ovarian carcinoma cells. The sLex-bearing glycan present on M-CSCs binds P-selectin in a rigid fucosylation-dependent and relatively loose sialyation-dependent manner, instead of a simple sLex structure that relies equally on fucose and sialic acid moieties. Such biochemistry is of biological significance, as fucosylation has been shown with increased affinity and bind selectin more efficiently. Whereas sulfation is a common feature of P-selectin ligands, our results suggest that the sLex-bearing glycan on M-CSCs does not require sulfation for its binding to P-selectin. There is evidence that sulfated polysaccharides of P-selectin ligands tend to be more resilient to stress which is required for binding under high shear conditions. Publications: Journal articles: #1, #3 Conference proceeding: #6-8 Objective 2: We show for the first time that CSCs were effectively captured to HPMCs at 0.03-0.15 dynes/cm2 (a physiologically relevant range of shear stress of the ascites) with maximal activity at a shear stress of 0.05 dynes/cm2, consistent with a catch bond that requires a clear threshold shear to initiate rolling. Strikingly, both the kinetic and mechanical properties that govern tumor-mesothelial interaction were significantly different between the two CSCs populations. Compared with NM-CSC, M-CSCs had a higher tethering frequency and lower rolling velocity on HPMCs, resulting in a higher percentage of firm adhesion of M-CSCs to HPMCs. Mechanistically, this interaction is mediated by P-selectin expressed by the peritoneal mesothelium. Tumor xenograft models and clinical samples corroborate the relevance of these findings. Publications: Journal articles: #1 Conference proceeding: #6-8 Objective 3: On exploring how the sLex-P-selectin interaction mediates firm adhesion, we show that this is through a critical member, P-cadherin. Moreover, we show that sLex-P-selectin can transduce an outside-in p120 catenin metastatic signaling cascade through the crosstalk of IGF-1R and P-cadherin. This may provide an attractive mechanism whereby sLex-P-selectin can affect tumor progression in ovarian cancer and may explain the highly aggressive behavior of ovarian tumors expressing these two molecules. Furthermore, we uncovered a new link of microRNA-199a-3p, phosphatidylinositol 3-kinase/Akt, and multidrug transporter activation in shear stress-induced CSC enrichment. These findings not only shed new light on the significance of hydrodynamics in cancer progression, but also emphasize the need of a flow-informed framework in the development of therapeutics. Publication: Journal article: #2, #4 Conference proceeding: #5 Taken together, our data strongly support the important role for a sLex-P-selectin cascade in ovarian cancer metastasis and provide a mechanistic rationale for targeting the sLex-P-selectin cascade 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 sLex-P-selectin cascade on tumor-mesothelial adhesion under ascitic fluid shear flow that has a pivotal role in the metastatic progression of ovarian cancer. This reveals a new direction for understanding the oncogenic role of sLex-P-selectin in ovarian cancer metastasis and implicate that sLex-P-selectin could be a promising therapeutic target. Our findings underscore a critical role for FUT5 during the development and maintenance of the metastasis. While most of the glycosyltransferases genes involved in sLex synthesis are constitutively expressed to produce sLex direct precursor, FUT5 catalyzing the last and rate-limiting step of sLex synthesis by adding fucose to the precursor are normally switched off. We are actively pursuing in this direction to document such effect using more extensive in vitro, in vivo, and clinicopathological studies. Given that glycans can be rationally selected based on the targeted glycosyltransferase, it is of great interest to identify the key enzyme in this process. Peritoneal metastasis has also been detected in patients with breast, gastric, and colon cancer. To expand our understanding of the sLex-P-selectin cascade, we plan to examine if this sLex-P-selectin cascade is also seen in other tumor types in which peritoneal metastasis is an important pathological process. We will also examine the level and effect of sLex-P-selectin interaction in healthy donors or non-cancerous patients. These findings should highlight the functional importance of sLex-P-selectin 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 critical, rate-limiting step of metastasis is the ability of tumor cells to colonize specific target organs, which involves specific cell-cell recognition. Moreover, tumor cell lodgement requires binding under shear stress, but not static, conditions. Therefore, unraveling the intricate details of this particular adhesion is of obvious importance. Several of our new findings of this project indicate that the sLex-P-selectin cascade is a pivotal regulator of tumor-mesothelium interaction under ascitic fluid shear flow and that it could be a promising target. (i) We show for the first time a preferential binding of the metastatic populations of cancer stem/tumor-initiating cells than the non-metastatic populations of cancer stem/tumor-initiating cells to the peritoneal mesothelium. (ii) We also present evidence that this interaction is mediated by a sLex-P-selectin cascade. (iii) We uncover a new uncommon non-sulfated sLex epitope serves as a distinct P-selectin binding determinant. These results reveal a new direction for understanding the oncogenic roles of a sLex-P-selectin cascade and highlight its importance as a viable therapeutic target. Targeting sLex-P-selectin may thus be a useful approach to decrease metastatic colonization, 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 |