Targeted Inhibition of Fascin Function Blocks Tumour Invasion and Metastatic Colonization

ARTICLE Received 30 Jun 2014 | Accepted 12 May 2015 | Published 17 Jun 2015 DOI: 10.1038/ncomms8465 Targeted inhibition of fascin function blocks tumour invasion and metastatic colonization Fang-Ke Huang1, Shaoqin Han1, Bowen Xing1, Jianyun Huang1, Bingqian Liu1, Francois Bordeleau2, Cynthia A. Reinhart-King2, J Jillian Zhang1 & Xin-Yun Huang1 One of the key steps during tumour metastasis is tumour cell migration and invasion, which require actin cytoskeletal reorganization. Among the critical actin cytoskeletal protrusion structures are the filopodia, which act like cell sensory organs to communicate with the extracellular microenvironment and participate in fundamental cell functions such as cell adhesion, spreading and migration in the three-dimensional environment. Fascin is the main actin-bundling protein in filopodia. Using high-throughput screening, here we identify and characterize small molecules that inhibit the actin-bundling activity of fascin. Focusing on one such inhibitor, we demonstrate that it specifically blocks filopodial formation, tumour cell migration and invasion in vitro, and metastasis in vivo. Hence, target-specific anti-fascin agents have a therapeutic potential for cancer treatment. 1 Department of Physiology and Biophysics, Cornell University Weill Medical College, New York, New York 10065, USA. 2 Department of Biomedical Engineering, Cornell University, Ithaca, New York 14853, USA. Correspondence and requests for materials should be addressed to X.-Y.H. (email: [email protected]). NATURE COMMUNICATIONS | 6:7465 | DOI: 10.1038/ncomms8465 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8465 n recent years, significant progress has been made in the tomography and analysed the in vitro reconstituted treatment of cancer, particularly with the development of unconstrained three-dimensional (3D) bundles formed by fascin Itargeted therapy. However, there is very little advancement in and actin filaments. These bundles consisted of a hexagonal the area of blocking tumour metastasis, which remains the lattice of parallel actin filaments where fascin formed the cross- primary cause of mortality of cancer patients1,2. Metastasis is bridges, and these bundles were similar to the filopodial bundles comprised of a series of events that a primary tumour spreads isolated from native tissues/cells44. from its initial site to secondary tissues/organs3. This invasion- Here we screened chemical libraries and identified small- metastasis cascade includes cell migration/invasion, intravasation, molecule compounds that specifically inhibit the biochemical survival in the circulation, arrest in a distant capillary bed function of fascin to bundle actin filaments. These inhibitors also and extravasation into and colonization within the organ impair the cellular function of fascin of inducing the formation of parenchyma. Inhibition of any one of these steps could block filopodia in two-dimensional and 3D environment. Furthermore, the entire metastatic process. the small-molecule inhibitor blocks breast tumour cell migration, Tumour cell migration and invasion are critical steps in invasion and metastasis. Our data demonstrate that fascin metastasis4–10. Migration provides tumour cells the ability to inhibitors could be developed to inhibit metastatic dissemination. leave the primary tumour bed (local invasion), enter into blood vessels, then exit the circulation and infiltrate the distant tissues/ organs. For cell migration and invasion to proceed, actin Results cytoskeleton must be reorganized by forming actin polymers Identification of small-molecule inhibitors of fascin. Previously, and bundles to dynamically change cell shapes11. Individual actin using protein affinity purification, we had identified fascin as a filaments are flexible and insufficient for membrane protrusion. protein target of some migrastatin (a natural product) analogues, Bundling of actin filaments provides rigidity against the which inhibit tumour cell migration, invasion and metastasis35. compressive force from the plasma membrane12. Owing to the unexpected nature of potential toxicity of One of the main actin filament-based membrane protrusions is small-molecule drugs, it is the best and usual practice of drug filopodia13. Filopodia are finger-like plasma membrane protrusions development to have more than one class of chemical compounds that are formed upon remodelling of the actin cytoskeleton beneath for a given protein target. The migrastatin analogues that we have the plasma membrane13. They can be viewed as a sensory organ of previously investigated belong to a class of chemical compounds the cells that are used to detect and assimilate signals as well as to with similar core chemical scaffold. Therefore, we screened explore and move into the surrounding microenvironment14–16. chemical libraries to identify new compounds that inhibit the Filopodia are also involved in transporting signalling proteins actin-bundling activity of fascin and are structurally different within tissues over a long range16. Metastatic tumour cells are rich from migrastatin analogues. in filopodia, and the number of which correlates with their To identify small-molecule inhibitors of fascin for future invasiveness17,18. Recently, filopodium-like protrusions have also clinical applications and for in vivo biological function studies of been implicated to be critical for metastatic tumour cells to fascin, we have screened chemical libraries to identify compounds colonize in the secondary tissues or organs19. that inhibit the actin-bundling activity of fascin. Instead of Fascin is the primary actin cross-linker in filopodia and has identifying small-molecule compounds that bind to fascin, we no amino-acid sequence homology with other actin-binding have chosen to identify small molecules that inhibit the function proteins20–25. It has a molecular mass of B55 kDa, functions as a of fascin to bundle actin. The rationales were: (i) It is the actin- monomer and is required to maximally cross-link the actin bundling activity of fascin that is important for tumour cell filaments into straight, compact and rigid bundles, to impart migration and invasion. (ii) Based on the X-ray crystal structure distinct mechanical stiffness to actin bundles26,27. Fascin holds of fascin, there are many potential binding sites on fascin for 10–30 parallel actin filaments together to form filopodia small molecules, in addition to the two main actin-binding 60–200 nm in diameter13. Elevated levels of fascin have been pockets44. Some fascin-binding compounds might not inhibit the found in many types of metastatic tumours and are correlated actin-bundling function of fascin. with clinically aggressive phenotypes, poor prognosis and shorter We developed an imaging-based assay where we could observe survival28–30. Human fascin expression is low or absent in normal the fascin-mediated actin bundles (Fig. 1). This method was an adult epithelial cells, but highly expressed in embryonic stages31,32. adaptation of a method that we used previously35,44. Because the Fascin gene-knockout mice are normal, likely due to the F-actin filaments or F-actin bundles bear negative charges, we functional compensation of other actin-bundling proteins during reasoned that a positively charged surface would be able to embryonic development33. Fascin point mutations in Drosophila capture the F-actin filaments and bundles (Fig. 1a,b). By labelling (the singed mutants) cause defective actin bundling and lead to F-actin with phalloidin-conjugated with fluorescent dye, we could curled bristles, waved body hairs and impaired oogenesis34. visualize the F-actin structures using fluorescent microscopy Furthermore, when ectopically expressed, fascin promotes tumour (Fig. 1b,c). Actin bundles formed by fascin were long and thick in cell migration, invasion and metastasis28. Moreover, it has been shape. In contrast, in the absence of fascin, actin filaments suggested that upregulation of fascin is part of the programme of organized into a network of short and thin curved threads epithelial-to-mesenchymal transition that confers motility and (Fig. 1b). We then titrated the concentrations of both F-actin and invasion properties on tumour cells29. Therefore, fascin has been fascin, and found that fascin-mediated actin bundles were most suggested as a therapeutic target for blocking tumour cell distinct when the final concentrations of fascin and F-actin were migration, invasion and metastasis31,32,35–43. close to 0.2 mM, with a molar ratio of 1:1. The assay displayed Previously, we solved the X-ray crystal structures of the wild- striking differences between images with and without fascin, type fascin and four fascin mutants to define the active and showing its potential to be developed into a high-content imaging inactive configurations of fascin, and revealed the structural basis assay (Fig. 1c,d). for the conformational changes of fascin during the actin-binding During the screening, four images were taken for each well. process35,44. From a systematic mutagenesis study of 100 fascin The shading correction (processed) and background subtraction mutants, we identified at least two major actin-binding sites on (thresholded) produced images with homogenous illumination fascin, and that each of these actin-binding sites is essential for (Fig. 1c). The microscopic actin-bundling assay was then the filopodial formation in cells44. We used cryo-electron optimized and

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