Melissa Desouza

The actin cytoskeleton as a sensor and mediator of apoptosis

Apoptosis is an important biological process required for the removal of unwanted or damaged cells. Mounting evidence implicates the actin cytoskeleton as both a sensor and mediator of apoptosis. Studies also suggest that actin binding proteins (ABPs) significantly contribute to apoptosis and that actin dynamics play a key role in regulating apoptosis signaling. Changes in the organization of the actin cytoskeleton has been attributed to the process of malignant transformation and it is hypothesized that remodeling of the actin cytoskeleton may enable tumor cells to evade normal apoptotic signaling. This review aims to illuminate the role of the actin cytoskeleton in apoptosis by systematically analyzing how actin and ABPs regulate different apoptosis pathways and to also highlight the potential for developing novel compounds that target tumor-specific actin filaments.

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.

Tropomyosin isoform 3 promotes the formation of filopodia by regulating the recruitment of actin-binding proteins to actin filaments.

Tropomyosins are believed to function in part by stabilizing actin filaments. However, accumulating evidence suggests that fundamental differences in function exist between tropomyosin isoforms, which contributes to the formation of functionally distinct filament populations. We investigated the functions of the high-molecular-weight isoform Tm3 and examined the molecular properties of Tm3-containing actin filament populations. Overexpression of the Tm3 isoform specifically induced the formation of filopodia and changes in actin solubility. We observed alterations in actin-binding protein recruitment to filaments, co-incident with changes in expression levels, which can account for this functional outcome. Tm3-associated filaments recruit active actin depolymerizing factor and are bundled into filopodia by fascin, which is both up-regulated and preferentially associated with Tm3-containing filaments in the Tm3 overexpressing cells. This study provides further insight into the isoform-specific roles of different tropomyosin isoforms. We conclude that variation in the tropomyosin isoform composition of microfilaments provides a mechanism to generate functionally distinct filament populations.

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

Tropomyosins induce neuritogenesis and determine neurite branching patterns in B35 neuroblastoma cells.

BACKGROUND The actin cytoskeleton is critically involved in the regulation of neurite outgrowth. RESULTS The actin cytoskeleton-associated protein tropomyosin induces neurite outgrowth in B35 neuroblastoma cells and regulates neurite branching in an isoform-dependent manner. CONCLUSIONS Our data indicate that tropomyosins are key regulators of the actin cytoskeleton during neurite outgrowth. SIGNIFICANCE Revealing the molecular machinery that regulates the actin cytoskeleton during neurite outgrowth may provide new therapeutic strategies to promote neurite regeneration after nerve injury. SUMMARY The formation of a branched network of neurites between communicating neurons is required for all higher functions in the nervous system. The dynamics of the actin cytoskeleton is fundamental to morphological changes in cell shape and the establishment of these branched networks. The actin-associated proteins tropomyosins have previously been shown to impact on different aspects of neurite formation. Here we demonstrate that an increased expression of tropomyosins is sufficient to induce the formation of neurites in B35 neuroblastoma cells. Furthermore, our data highlight the functional diversity of different tropomyosin isoforms during neuritogenesis. Tropomyosins differentially impact on the expression levels of the actin filament bundling protein fascin and increase the formation of filopodia along the length of neurites. Our data suggest that tropomyosins are central regulators of actin filament populations which drive distinct aspects of neuronal morphogenesis.

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

Abstract B50: Synergistic anti-neuroblastoma efficacy using combinatorial cytoskeletal inhibitors

Children with high risk neuroblastoma still have a poor response rate to existing chemotherapeutics making it imperative that new classes of compounds are developed to treat this disease. The actin cytoskeleton is an ideal chemotherapeutic target due to its role in numerous biological processes essential for tumor cell growth and survival. Targeting actin however has been problematic due to unacceptable levels of toxicity associated with impacting actin containing structures essential for normal cell function. We have developed a novel class of compounds which target tropomyosin, the second core component of an actin microfilament. By targeting the cancer associated tropomyosin, Tm5NM1, we are able to discriminate between the actin filament populations in normal and transformed cells. We have demonstrated that our first in class anti-tropomyosin compound, TR100, impacts actin filament integrity leading to tumor cell death in vitro and in vivo in neuroblastoma models. In this study we elucidate the molecular mechanisms by which disruption of the actin cytoskeleton by TR100 induces tumor cell death. We also investigate the efficacy of this novel class of compound in combination with existing chemotherapeutics. Preliminary studies have demonstrated that low exposure of neuroblastoma cells to TR100 results in a G0G1 arrest. Increased exposure leads to the activation of the mitochondrial apoptotic pathway as measured by increases in both caspase activity and mitochondrial permeability. To delineate the signaling pathways involved in anti-tropomyosin compound induced apoptosis, we have conducted a kinexus phospho-array. Using this approach we have identified the activation of key stress response pathways. Treatment of the SH-EP neuroblastoma cell line with anti-tropomyosin compounds for 8h results in reduced phospho-activity of key intermediates of the MEK/ERK pathway, in particular a significant decrease in the phosphorylation of the RSK1/2 family of kinases. Preliminary data suggest that the compounds mediate their effect through the downregulation of the MEK/ERK and p38/JNK cell survival pathways. We have now extended this study to investigate the impact of the drugs in combination with other established chemotherapeutic agents. In particular compounds which target the microtubules, another key cytoskeletal structure within the cells. In CHLA20 neuroblastoma cells, TR100 showed significant synergy when used in combination with both paclitaxel (microtubule stabilizing) and vincristine (microtubule destabilizing) with a combinatorial index Citation Format: Justine R. Stehn, Duo Chen, Mark A. Currier, David Eaves, Melissa Desouza, Matthew Moosavian, Timothy P. Cripe, Peter W. Gunning. Synergistic anti-neuroblastoma efficacy using combinatorial cytoskeletal inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B50.

Abstract C208: Targeting the actin cytoskeleton of neuroblastoma: Elucidating the mechanism by which a novel class of anti-cancer compounds induces tumor cell death.

The actin cytoskeleton is an ideal chemotherapeutic target due to its role in numerous biological processes essential for tumor cell growth and survival. Targeting actin however has been problematic due to unacceptable levels of toxicity associated with impacting actin containing structures essential for normal cell function. We have developed a novel class of compounds which target tropomyosin, the second core component of an actin microfilament. By targeting the cancer associated tropomyosin, Tm5NM1 we are able to discriminate between the actin filament populations in normal and transformed cells. We have demonstrated that our first in class anti-Tm compound, TR100, impacts actin filament integrity leading to tumor cell death in vitro and in vivo in neuroblastoma and melanoma models. In this study we investigate the molecular mechanisms by which disruption of the actin cytoskeleton with the next generation anti-Tm compounds leads to tumor cell death. Preliminary studies have indicated that these compounds initiate cell death via the intrinsic mitochondrial apoptotic pathway. SH-EP neuroblastoma cells treated with TR200 showed a dose dependent increase in both caspase activation and annexin V staining as measured by Western Blotting and FACS analysis respectively. Significant changes in mitochondrial membrane integrity were also observed using TMRE staining in SH-EP cells treated with 5 µM TR200 for 24h. Interestingly, mitochondrial permeabilization was independent of the presence of proapoptotic factors Bax and Bak as Bax/Bak null MEFs still displayed sensitivity to TR200. To delineate the signaling pathways leading to apoptosis, a kinexus phospho-antibody array was conducted using SH-EP cells treated with 10 µM DTR200 for 8 and 24h. DTR200 treatment for 8h reduced the phospho-activity of key intermediates of the MEK/ERK pathway, in particular a significant decrease in the phosphorylation of the RSK1/2 family of kinases. Preliminary data would suggest that the compounds at 24h are mediating their effect through the downregulation of the MEK/ERK and p38/JNK cell survival pathways. We are now validating these findings to elucidate which signaling pathways mediate the apoptotic response to the anti-Tm compounds. Understanding the molecular mechanisms involved in anti-Tm mediated cell death will allow us to determine how these compounds can be used in combination with existing therapies to lower the apoptotic threshold of a tumor cell and ultimately improve the outcome of patients with neuroblastoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C208. Citation Format: Melissa Desouza, Margaret Nguyen, Peter W. Gunning, Justine R. Stehn. Targeting the actin cytoskeleton of neuroblastoma: Elucidating the mechanism by which a novel class of anti-cancer compounds induces tumor cell death. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C208.

Abstract 1827: Developing chemotherapeutics which selectively disable the actin cytoskeleton of tumor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The actin cytoskeleton is an important regulator of a variety of cellular functions making it an ideal chemotherapeutic target. Despite this promise no anti-actin compounds are used in current chemotherapy, primarily due to the inability of existing anti-actin agents to discriminate between the actin cytoskeleton of tumor cells and that of the muscle sarcomere. We have previously shown that tropomyosin, an integral component of the actin cytoskeleton, defines functionally distinct actin filament populations. We have designed a new class of compounds which target a specific cancer associated tropomyosin isoform, Tm5NM1. Anti-tropomyosin (Tm) compounds were selected based on ability to target the actin cytoskeleton and efficacy against a panel of neuroblastoma and melanoma cell lines. The lead compound, TR100, was shown to be effective against a panel of tumor cell lines (average EC50 3µM). Preliminary studies demonstrate that TR100 induces cell death via the intrinsic (mitochondrial) apoptotic pathway and the mechanism appears to be independent of the pro apoptotic factors BAX and BAK. In 3D melanoma spheroid models, which more accurately mimic the tumor microenvironment, TR100 inhibited cell growth and motility. This translated to a reduction in tumor cell growth in neuroblastoma and melanoma xenograft models. Analysis of drug treated animals showed no evidence of cardiac damage as measured by blood Troponin I levels and the intraventricular septum thickness of isolated hearts. These results demonstrate the possibility of targeting distinct actin filament populations based on tropomyosin composition. Next generation anti-Tm compounds with improved efficacy and specificity have now been developed. Preliminary data demonstrate that these compounds exhibit increased selectivity for transformed cells. Taken together, our findings suggest that the anti-Tm compounds show a significant improvement in the therapeutic window compared to existing anti-actin agents. This novel approach and the development of the new class of anti-Tm compounds may be the key for disabling a long sought after target, the actin cytoskeleton, and may lead to a new class of chemotherapeutics active against a broad range of cancer types. 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 1827. doi:1538-7445.AM2012-1827

Abstract A178: Developing chemotherapeutics which selectively disable the actin cytoskeleton of tumor cells.

Background: The actin cytoskeleton is an important regulator of a variety of cellular functions including cell motility, adhesion, and proliferation, making it an ideal chemotherapeutic target. Despite this promise there are still no anti-actin compounds used in current chemotherapy, primarily due to the inability of existing anti-actin agents to discriminate between the actin cytoskeleton of tumor cells and the actin filaments of the muscle sarcomere. We have previously shown that tropomyosin, an integral component of the actin cytoskeleton, defines functionally distinct populations of actin filaments. We have identified a specific tropomyosin isoform common to all tumor cells tested to date which regulates cell proliferation and have designed a new class of compounds to target this filament population. Summary of Results: Anti-tropomyosin (Tm) compounds were selected based their ability to target the actin cytoskeleton and their efficacy against a panel of neuroblastoma and melanoma cell lines. The lead compound, TR100, was shown to be effective against a panel of tumor cell lines with an average EC50 of 3μM. When tested in 3D melanoma spheroid models, which more accurately mimic the tumor microenvironment, TR100 inhibited melanoma cell growth and motility. This effect translated to a reduction in tumor cell growth in vivo in both neuroblastoma and melanoma xenograft models. In vitro data using isolated rat cardiomyocytes demonstrated that TR100 had minimal impact on contractile function. In vivo data from the drug treated animals also showed no evidence of cardiac damage as measured by blood Troponin I levels and no changes in the intraventricular septum thickness of isolated hearts. These results demonstrate that it is possible to target distinct actin filament populations based on the tropomyosin composition. Next generation anti-Tm compounds with improved efficacy and specificity have now been developed. Preliminary data demonstrate that these compounds exhibit increased selectivity for transformed cells. Conclusions: Taken together, our findings suggest that the anti-Tm compounds show a significant improvement in the therapeutic window compared to existing anti-actin agents. This novel approach and the development of the new class of anti-Tm compounds may be the key for disabling a long sought after target, the actin cytoskeleton, and may lead to a new class of chemotherapeutics active against a broad range of cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A178.