Nuclear fascin regulates cancer cell survival.

Fascin is an important regulator of F-actin bundling leading to enhanced filopodia assembly. Fascin is also overexpressed in most solid tumours where it supports invasion through control of F-actin structures at the periphery and nuclear envelope. Recently, fascin has been identified in the nucleus of a broad range of cell types but the contributions of nuclear fascin to cancer cell behaviour remain unknown. Here, we demonstrate that fascin bundles F-actin within the nucleus to support chromatin organisation and efficient DDR. Fascin associates directly with phosphorylated Histone H3 leading to regulated levels of nuclear fascin to support these phenotypes. Forcing nuclear fascin accumulation through the expression of nuclear-targeted fascin-specific nanobodies or inhibition of Histone H3 kinases results in enhanced and sustained nuclear F-actin bundling leading to reduced invasion, viability, and nuclear fascin-specific/driven apoptosis. These findings represent an additional important route through which fascin can support tumourigenesis and provide insight into potential pathways for targeted fascin-dependent cancer cell killing.

VCA nanobodies target N-WASp to reduce invadopodium formation and functioning.

Invasive cancer cells develop small actin-based protrusions called invadopodia, which perform a primordial role in metastasis and extracellular matrix remodelling. Neural Wiskott-Aldrich syndrome protein (N-WASp) is a scaffold protein which can directly bind to actin monomers and Arp2/3 and is a crucial player in the formation of an invadopodium precursor. Expression modulation has pointed to an important role for N-WASp in invadopodium formation but the role of its C-terminal VCA domain in this process remains unknown. In this study, we generated alpaca nanobodies against the N-WASp VCA domain and investigated if these nanobodies affect invadopodium formation. By using this approach, we were able to study functions of a selected functional/structural N-WASp protein domain in living cells, without requiring overexpression, dominant negative mutants or siRNAs which target the gene, and hence the entire protein. When expressed as intrabodies, the VCA nanobodies significantly reduced invadopodium formation in both MDA-MB-231 breast cancer and HNSCC61 head and neck squamous cancer cells. Furthermore, expression of distinct VCA Nbs (VCA Nb7 and VCA Nb14) in PC-3 prostate cancer cells resulted in reduced overall matrix degradation without affecting MMP9 secretion/activation or MT1-MMP localisation at invadopodial membranes. From these results, we conclude that we have generated nanobodies targeting N-WASp which reduce invadopodium formation and functioning, most likely via regulation of N-WASp-Arp2/3 complex interaction, indicating that this region of N-WASp plays an important role in these processes.

Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion.

Cancer cells exploit different strategies to escape from the primary tumor, gain access to the circulation, disseminate throughout the body, and form metastases, the leading cause of death by cancer. Invadopodia, proteolytically active plasma membrane extensions, are essential in this escape mechanism. Cortactin is involved in every phase of invadopodia formation, and its overexpression is associated with increased invadopodia formation, extracellular matrix degradation, and cancer cell invasion. To analyze endogenous cortactin domain function in these processes, we characterized the effects of nanobodies that are specific for the N-terminal acidic domain of cortactin and expected to target small epitopes within this domain. These nanobodies inhibit cortactin-mediated actin-related protein (Arp)2/3 activation, and, after their intracellular expression in cancer cells, decrease invadopodia formation, extracellular matrix degradation, and cancer cell invasion. In addition, one of the nanobodies affects Arp2/3 interaction and invadopodium stability, and a nanobody targeting the Src homology 3 domain of cortactin enabled comparison of 2 functional regions in invadopodium formation or stability. Given their common and distinct effects, we validate cortactin nanobodies as an instrument to selectively block and study distinct domains within a protein with unprecedented precision, aiding rational future generation of protein domain-selective therapeutic compounds.-Bertier, L., Boucherie, C., Zwaenepoel, O., Vanloo, B., Van Troys, M., Van Audenhove, I., Gettemans, J. Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion.

A new survivin tracer tracks, delocalizes and captures endogenous survivin at different subcellular locations and in distinct organelles.

Survivin, the smallest member of the inhibitor of apoptosis protein family, plays a central role during mitosis and exerts a cytoprotective function. Survivin is highly expressed in most cancer types and contributes to multiple facets of carcinogenesis. The molecular mechanisms underlying its highly diverse functions need to be extensively explored, which is crucial for rational design of future personalized therapeutics. In this study, we have generated an alpaca survivin nanobody (SVVNb8) that binds with low nanomolar affinity to its target. When expressed as an intrabody in HeLa cells, SVVNb8 faithfully tracks survivin during different phases of mitosis without interfering with survivin function. Furthermore, coupling SVVNb8 with a subcellular delocalization tag efficiently redirects endogenous survivin towards the nucleus, the cytoplasm, peroxisomes and even to the intermembrane space of mitochondria where it presumably interacts with resident mitochondrial survivin. Based on our findings, we believe that SVVNb8 is an excellent instrument to further elucidate survivin biology and topography, and can serve as a model system to investigate mitochondrial and peroxisomal (survivin) protein import.

Nanobodies as Versatile Tools to Understand, Diagnose, Visualize and Treat Cancer.

Since their discovery, nanobodies have been used extensively in the fields of research, diagnostics and therapy. These antigen binding fragments, originating from Camelid heavy-chain antibodies, possess unusual hallmarks in terms of (small) size, stability, solubility and specificity, hence allowing cost-effective production and sometimes outperforming monoclonal antibodies. In this review, we evaluate the current status of nanobodies to study, diagnose, visualize or inhibit cancer-specific proteins and processes. Nanobodies are highly adaptable tools for cancer research as they enable specific modulation of targets, enzymatic and non-enzymatic proteins alike. Molecular imaging studies benefit from the rapid, homogeneous tumor accumulation of nanobodies and their fast blood clearance, permitting previously unattainable fast tumor visualization. Moreover, they are endowed with considerable therapeutic potential as inhibitors of receptor-ligand pairs and deliverers of drugs or drug-loaded nanoparticles towards tumors. More in vivo and clinical studies are however eagerly awaited to unleash their full potential.

Fascin Rigidity and L-plastin Flexibility Cooperate in Cancer Cell Invadopodia and Filopodia.

Invadopodia and filopodia are dynamic, actin-based protrusions contributing to cancer cell migration, invasion, and metastasis. The force of actin bundles is essential for their protrusive activity. The bundling protein fascin is known to play a role in both invadopodia and filopodia. As it is more and more acknowledged that functionally related proteins cooperate, it is unlikely that only fascin bundles actin in these protrusions. Another interesting candidate is L-plastin, normally expressed in hematopoietic cells, but considered a common marker of many cancer types. We identified L-plastin as a new component of invadopodia, where it contributes to degradation and invasiveness. By means of specific, high-affinity nanobodies inhibiting bundling of fascin or L-plastin, we further unraveled their cooperative mode of action. We show that the bundlers cannot compensate for each other due to strikingly different bundling characteristics: L-plastin bundles are much thinner and less tightly packed. Composite bundles adopt an intermediate phenotype, with fascin delivering the rigidity and strength for protrusive force and structural stability, whereas L-plastin accounts for the flexibility needed for elongation. Consistent with this, elevated L-plastin expression promotes elongation and reduces protrusion density in cells with relatively lower L-plastin than fascin levels.

Use of Nanobodies to Localize Endogenous Cytoskeletal Proteins and to Determine Their Contribution to Cancer Cell Invasion by Using an ECM Degradation Assay.

There are numerous ways to study actin cytoskeletal structures, and thereby identify the underlying mechanisms of organization and their regulating proteins. Traditional approaches make use of protein overexpression or siRNA. However to study or modulate resident endogenous proteins, complementary methods are required. Since the discovery of nanobodies in 1993, they have proven to represent interesting tools in a variety of applications due to their high affinity, solubility, and stability. Especially their intracellular functionality makes them ideally suited for the study of actin cytoskeletal regulation. Here we provide a protocol to clone nanobody cDNAs in frame with an EGFP or mCherry fluorescent tag. We explain how to transfect this fusion protein in eukaryotic (cancer) cells and how to perform immunofluorescence. This allows microscopic analysis of endogenous (cytoskeletal) proteins and gives insight into their endogenous localization. Moreover, we outline an extracellular matrix (ECM) degradation assay as an application of the general protocol. By seeding cells onto a fluorescently labeled gelatin matrix, degradation can be quantified by means of a matrix degradation index. This assay demonstrates the contribution of a protein during cancer cell invasiveness in vitro and the potential of a nanobody to inhibit this degradation through modulation of its target.

Fascin actin bundling controls podosome turnover and disassembly while cortactin is involved in podosome assembly by its SH3 domain in THP-1 macrophages and dendritic cells.

Podosomes are dynamic degrading devices present in myeloid cells among other cell types. They consist of an actin core with associated regulators, surrounded by an adhesive ring. Both fascin and cortactin are known constituents but the role of fascin actin bundling is still unclear and cortactin research rather focuses on its homologue hematopoietic lineage cell-specific protein-1 (HS1). A fascin nanobody (FASNb5) that inhibits actin bundling and a cortactin nanobody (CORNb2) specifically targeting its Src-homology 3 (SH3) domain were used as unique tools to study the function of these regulators in podosome dynamics in both THP-1 macrophages and dendritic cells (DC). Upon intracellular FASNb5 expression, the few podosomes present were aberrantly stable, long-living and large, suggesting a role for fascin actin bundling in podosome turnover and disassembly. Fascin modulates this by balancing the equilibrium between branched and bundled actin networks. In the presence of CORNb2, the few podosomes formed show disrupted structures but their dynamics were unaffected. This suggests a role of the cortactin SH3 domain in podosome assembly. Remarkably, both nanobody-induced podosome-losses were compensated for by focal adhesion structures. Furthermore, matrix degradation capacities were altered and migratory phenotypes were lost. In conclusion, the cortactin SH3 domain contributes to podosome assembly while fascin actin bundling is a master regulator of podosome disassembly in THP-1 macrophages and DC.

Combination of interleukin-12 gene therapy, metronomic cyclophosphamide and DNA cancer vaccination directs all arms of the immune system towards tumor eradication.

In this work a combination therapy that acts upon the immune suppressive, the innate and specific arms of the immune system is proposed. This combination therapy, which consists of intratumoral interleukin-12 (IL-12) gene therapy, human tyrosinase (hTyr) DNA vaccination and metronomic cyclophosphamide (CPX), was evaluated in a B16-F10 mouse model. The following groups were compared: (1) no treatment, (2) control vector, (3) intratumoral IL-12 gene therapy, (4) intratumoral IL-12 gene therapy+metronomic CPX, (5) intratumoral IL-12 gene therapy+metronomic CPX+hTyr DNA vaccination. Next to clinical efficacy and safety, we characterized acute effects of IL-12 and anti-tumor immune response after a second tumor challenge. All treatment groups showed increased survival and higher cure rates than control groups. Survival of non-cured mice was increased when metronomic CPX was combined with IL-12 gene therapy. Furthermore, mice that received metronomic CPX had significantly lower percentages of regulatory T cells. Addition of the hTyr DNA vaccine increased cure rate and resulted in increased survival compared to other treatment groups. We also demonstrated that the manifest necrosis within days after IL-12 gene therapy is at least partly due to IL-12 mediated activation of NK cells. All cured mice were resistant to a second challenge. A humoral memory response against the tumor cells was observed in all groups that received IL-12 gene therapy, while a cellular memory response was observed only in the vaccinated mice. In conclusion, every component of this combination treatment contributed a unique immunologic trait with associated clinical benefits.

Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization.

Invadopodia are actin-rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen-binding domains of Camelid heavy-chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA-MB-231 breast and PC-3 prostate cancer cells. A nanobody (K(d)~35 nM, 1:1 stoichiometry) that disrupts fascin F-actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion. Cortactin-SH3 dependent WIP recruitment toward the plasma membrane was specifically inhibited by a cortactin nanobody (K(d)~75 nM, 1:1 stoichiometry). This functional domain is shown to be important for formation of properly organized invadopodia, MMP-9 secretion, matrix degradation, and cancer cell invasion. Notably, using a subcellular delocalization strategy to trigger protein loss of function, we uncovered a fascin-bundling-independent role in MMP-9 secretion. Hence, we demonstrate that nanobodies enable high resolution protein function mapping in cells.

Mapping cytoskeletal protein function in cells by means of nanobodies.

Nanobodies or VHHs are single domain antigen binding fragments derived from heavy-chain antibodies naturally occurring in species of the Camelidae. Due to their ease of cloning, high solubility and intrinsic stability, they can be produced at low cost. Their small size, combined with high affinity and antigen specificity, enables recognition of a broad range of structural (undruggable) proteins and enzymes alike. Focusing on two actin binding proteins, gelsolin and CapG, we summarize a general protocol for the generation, cloning and production of nanobodies. Furthermore, we describe multiple ways to characterize antigen-nanobody binding in more detail and we shed light on some applications with recombinant nanobodies. The use of nanobodies as intrabodies is clarified through several case studies revealing new cytoskeletal protein properties and testifying to the utility of nanobodies as intracellular bona fide protein inhibitors. Moreover, as nanobodies can traverse the plasma membrane of eukaryotic cells by means of the enteropathogenic E. coli type III protein secretion system, we show that in this promising way of nanobody delivery, actin pedestal formation can be affected following nanobody injection.