Teresa Bonello

A Novel Class of Anticancer Compounds Targets the Actin Cytoskeleton in Tumor Cells

The actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers.

New approaches to targeting the actin cytoskeleton for chemotherapy

The actin cytoskeleton is indispensable for normal cellular function. In particular, several actin-based structures coordinate cellular motility, a process hijacked by tumor cells in order to facilitate their propagation to distant sites. The actin cytoskeleton, therefore, represents a point for chemotherapeutic intervention. The challenge in disrupting the actin cytoskeleton is in preserving actin-driven contraction of cardiac and skeletal muscle. By targeting actin-binding proteins with altered expression in malignancy, it may be possible to achieve tumor-specific toxicity. A number of actin-binding proteins act cooperatively and synergistically to regulate actin structures required for motility. The actin cytoskeleton is characterized by a significant degree of plasticity. Targeting specific actin-binding proteins for chemotherapy will only be successful if no other compensatory mechanisms exist.

A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development.

The impact of tropomyosins on actin filament assembly is isoform specific

Abstract Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actins interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1–5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27–85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules.

Stable incorporation of α‐smooth muscle actin into stress fibers is dependent on specific tropomyosin isoforms

α‐Smooth Muscle Actin (α‐SMA), a widely characterized cytoskeletal protein, represents the hallmark of myofibroblast differentiation. Transforming growth factorβ1 (TGFβ1) stimulates α‐SMA expression and incorporation into stress fibers, thus providing an increased myofibroblast contractile force that participates in tissue remodeling. We have addressed the molecular mechanism by which α‐SMA is stably incorporated into stress fibers in human myofibroblasts following exposure to TGFβ1. The unique N‐terminal sequence AcEEED, which is critical for α‐SMA incorporation into stress fibers, was used to screen for AcEEED binding proteins. Tropomyosins were identified as candidate binding proteins. We find that after TGFβ1 treatment elevated levels of the Tpm1.6/7 isoforms, and to a lesser extent Tpm2.1, precede the increase in α‐SMA. RNA interference experiments demonstrate that α‐SMA fails to stably incorporate into stress fibers of TGFβ1 treated fibroblasts depleted of Tpm1.6/7, but not other tropomyosins. This does not appear to be due to exclusive interactions between α‐SMA and just the Tpm1.6/7 isoforms. We propose that an additional AcEEED binding factor may be required to generate α‐SMA filaments containing just Tpm1.6/7 which result in stable incorporation of the resulting filaments into stress fibers.

Therapeutic Targeting of the Actin Cytoskeleton in Cancer

In cancer, actin filament populations and associated remodelling proteins are involved in driving proliferation, apoptosis and motility. Furthermore, a web of signalling pathways converge with the actin cytoskeleton to regulate these functions. Importantly, the actin cytoskeleton is a heterogeneous assembly of filament populations, each contributing to shared and unique cellular functions. The current range of actin-disrupting compounds are limited in their therapeutic use as they cannot discriminate between functionally specific populations of actin. Universal disruption of actin is likely to be intolerable in a clinical setting. Dissecting the regulation and composition of these filament populations will allow for treatments tailored to target the unique cytoskeletal repertoire of tumour cells. Identifying specific actin filament populations which are indispensible for tumour cell function is the focus of current work.

Rap1 acts via multiple mechanisms to position Canoe and adherens junctions and mediate apical-basal polarity establishment

ABSTRACT Epithelial apical-basal polarity drives assembly and function of most animal tissues. Polarity initiation requires cell-cell adherens junction assembly at the apical-basolateral boundary. Defining the mechanisms underlying polarity establishment remains a key issue. Drosophila embryos provide an ideal model, as 6000 polarized cells assemble simultaneously. Current data place the actin-junctional linker Canoe (fly homolog of Afadin) at the top of the polarity hierarchy, where it directs Bazooka/Par3 and adherens junction positioning. Here we define mechanisms regulating Canoe localization/function. Spatial organization of Canoe is multifaceted, involving membrane localization, recruitment to nascent junctions and macromolecular assembly at tricellular junctions. Our data suggest apical activation of the small GTPase Rap1 regulates all three events, but support multiple modes of regulation. The Rap1GEF Dizzy (PDZ-GEF) is crucial for Canoe tricellular junction enrichment but not apical retention. The Rap1-interacting RA domains of Canoe mediate adherens junction and tricellular junction recruitment but are dispensable for membrane localization. Our data also support a role for Canoe multimerization. These multifactorial inputs shape Canoe localization, correct Bazooka and adherens junction positioning, and thus apical-basal polarity. We integrate the existing data into a new polarity establishment model. Highlighted Article: Correct apical clustering of Cno, an essential cue for setting up the apical-basal polarity axis, is regulated by active Rap1 acting both through, and independently of, the Cno Rap1-binding domain.

Phosphomimetic S3D cofilin binds but only weakly severs actin filaments

Many biological processes, including cell division, growth, and motility, rely on rapid remodeling of the actin cytoskeleton and on actin filament severing by the regulatory protein cofilin. Phosphorylation of vertebrate cofilin at Ser-3 regulates both actin binding and severing. Substitution of serine with aspartate at position 3 (S3D) is widely used to mimic cofilin phosphorylation in cells and in vitro. The S3D substitution weakens cofilin binding to filaments, and it is presumed that subsequent reduction in cofilin occupancy inhibits filament severing, but this hypothesis has remained untested. Here, using time-resolved phosphorescence anisotropy, electron cryomicroscopy, and all-atom molecular dynamics simulations, we show that S3D cofilin indeed binds filaments with lower affinity, but also with a higher cooperativity than wild-type cofilin, and severs actin weakly across a broad range of occupancies. We found that three factors contribute to the severing deficiency of S3D cofilin. First, the high cooperativity of S3D cofilin generates fewer boundaries between bare and decorated actin segments where severing occurs preferentially. Second, S3D cofilin only weakly alters filament bending and twisting dynamics and therefore does not introduce the mechanical discontinuities required for efficient filament severing at boundaries. Third, Ser-3 modification (i.e. substitution with Asp or phosphorylation) “undocks” and repositions the cofilin N terminus away from the filament axis, which compromises S3D cofilins ability to weaken longitudinal filament subunit interactions. Collectively, our results demonstrate that, in addition to inhibiting actin binding, Ser-3 modification favors formation of a cofilin-binding mode that is unable to sufficiently alter filament mechanical properties and promote severing.

Abstract 5230: Improving the specificity of drugs which target the actin cytoskeleton for cancer therapy

The actin cytoskeleton is fundamental in the regulation of cellular processes involved in tumourigenesis and is therefore a highly desirable chemotherapeutic target. However, the non-specific nature of actin-targeting drugs makes them ineffective due to their toxic action on the heart. We have designed a class of drugs that target the second core component of actin filaments, tropomyosin. Tm5NM1, a cytoskeletal tropomyosin isoform, is upregulated in a variety of tumour cells and patient samples, and is known to promote tumour cell growth. Our lead drug, TR100, targets Tm5NM1 and potentially other isoforms, reducing cell proliferation in vitro in a panel of melanoma and neuroblastoma cell lines and decreases the rate of tumour growth in vivo in melanoma and neuroblastoma mouse models without affecting heart integrity. The difficulty in improving the specificity of these anti-tropomyosin compounds for Tm5NM1 was the lack of a suitable cell-based system to test the drugs. NIH3T3 stable cells overexpressing fluorophore-tagged Tm5NM1 or the muscle-specific isoform αfastTm now allows us to determine the impact of our drugs on functionally distinct actin populations. Since tropomyosin is a structural protein with no known measurable catalytic activity it has previously been challenging to design a high throughput assay to quantitate specific changes in actin filament architecture. High content imaging provides a system to image actin filament integrity which can be quantified using an algorithm designed in collaboration with the CSIRO. Using this algorithm we can calculate the impact of the anti-tropomyosin compounds on actin filaments in co-cultured stable cells. Development of these assays will contribute to the design of more specific drugs which target defined tropomyosin containing actin populations highly expressed in cancer without showing toxicity in muscle, thereby improving the therapeutic index of anti-tropomyosin drugs for the clinical treatment of a wide array of cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5230. doi:1538-7445.AM2012-5230

Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.