Loss of ATRX promotes aggressive features of osteosarcoma with increased NF-κB signaling and integrin binding.

Osteosarcoma (OS) is a lethal disease with few known targeted therapies. Here, we show that decreased ATRX expression is associated with more aggressive tumor cell phenotypes, including increased growth, migration, invasion, and metastasis. These phenotypic changes correspond with activation of NF-κB signaling, extracellular matrix remodeling, increased integrin αvß3 expression, and ETS family transcription factor binding. Here, we characterize these changes in vitro, in vivo, and in a data set of human OS patients. This increased aggression substantially sensitizes ATRX-deficient OS cells to integrin signaling inhibition. Thus, ATRX plays an important tumor-suppression role in OS, and loss of function of this gene may underlie new therapeutic vulnerabilities. The relationship between ATRX expression and integrin binding, NF-κB activation, and ETS family transcription factor binding has not been described in previous studies and may impact the pathophysiology of other diseases with ATRX loss, including other cancers and the ATR-X α thalassemia intellectual disability syndrome.

Coronin 1C inhibits melanoma metastasis through regulation of MT1-MMP-containing extracellular vesicle secretion.

Coronin 1C is overexpressed in multiple tumors, leading to the widely held view that this gene drives tumor progression, but this hypothesis has not been rigorously tested in melanoma. Here, we combined a conditional knockout of Coronin 1C with a genetically engineered mouse model of PTEN/BRAF-driven melanoma. Loss of Coronin 1C in this model increases both primary tumor growth rates and distant metastases. Coronin 1C-null cells isolated from this model are more invasive in vitro and produce more metastatic lesions in orthotopic transplants than Coronin 1C-reexpressing cells due to the shedding of extracellular vesicles (EVs) containing MT1-MMP. Interestingly, these vesicles contain melanosome markers suggesting a melanoma-specific mechanism of EV release, regulated by Coronin 1C, that contributes to the high rates of metastasis in melanoma.

The ARP2/3 complex prevents excessive formin activity during cytokinesis.

Cytokinesis completes cell division by constriction of an actomyosin contractile ring that separates the two daughter cells. Here we use the early Caenorhabditis elegans embryo to explore how the actin filament network in the ring and the surrounding cortex is regulated by the single cytokinesis formin CYK-1 and the ARP2/3 complex, which nucleate nonbranched and branched filaments, respectively. We show that CYK-1 and the ARP2/3 complex are the predominant F-actin nucleators responsible for generating distinct cortical F-actin architectures and that depletion of either nucleator affects the kinetics of cytokinesis. CYK-1 is critical for normal F-actin levels in the contractile ring, and acute inhibition of CYK-1 after furrow ingression slows ring constriction rate, suggesting that CYK-1 activity is required throughout ring constriction. Surprisingly, although the ARP2/3 complex does not localize in the contractile ring, depletion of the ARP2 subunit or treatment with ARP2/3 complex inhibitor delays contractile ring formation and constriction. We present evidence that the delays are due to an excess in formin-nucleated cortical F-actin, suggesting that the ARP2/3 complex negatively regulates CYK-1 activity. We conclude that the kinetics of cytokinesis are modulated by interplay between the two major actin filament nucleators.

New Mechanisms of Resistance to MEK Inhibitors in Melanoma Revealed by Intravital Imaging.

Targeted therapeutics that are initially effective in cancer patients nearly invariably engender resistance at some stage, an inherent challenge in the use of any molecular-targeted drug in cancer settings. In this study, we evaluated resistance mechanisms arising in metastatic melanoma to MAPK pathway kinase inhibitors as a strategy to identify candidate strategies to limit risks of resistance. To investigate longitudinal responses, we developed an intravital serial imaging approach that can directly visualize drug response in an inducible RAF-driven, autochthonous murine model of melanoma incorporating a fluorescent reporter allele (tdTomatoLSL). Using this system, we visualized formation and progression of tumors in situ, starting from the single-cell level longitudinally over time. Reliable reporting of the status of primary murine tumors treated with the selective MEK1/2 inhibitor (MEKi) trametinib illustrated a time-course of initial drug response and persistence, followed by the development of drug resistance. We found that tumor cells adjacent to bundled collagen had a preferential persistence in response to MEKi. Unbiased transcriptional and kinome reprogramming analyses from selected treatment time points suggested increased c-Kit and PI3K/AKT pathway activation in resistant tumors, along with enhanced expression of epithelial genes and epithelial-mesenchymal transition downregulation signatures with development of MEKi resistance. Similar trends were observed following simultaneous treatment with BRAF and MEK inhibitors aligned to standard-of-care combination therapy, suggesting these reprogramming events were not specific to MEKi alone. Overall, our results illuminate the integration of tumor-stroma dynamics with tissue plasticity in melanoma progression and provide new insights into the basis for drug response, persistence, and resistance.Significance: A longitudinal study tracks the course of MEKi treatment in an autochthonous imageable murine model of melanoma from initial response to therapeutic resistance, offering new insights into the basis for drug response, persistence, and resistance. Cancer Res; 78(2); 542-57. ©2017 AACR.

Arp2/3 Complex Is Required for Macrophage Integrin Functions but Is Dispensable for FcR Phagocytosis and In Vivo Motility.

The Arp2/3 complex nucleates branched actin, forming networks involved in lamellipodial protrusion, phagocytosis, and cell adhesion. We derived primary bone marrow macrophages lacking Arp2/3 complex (Arpc2-/-) and directly tested its role in macrophage functions. Despite protrusion and actin assembly defects, Arpc2-/- macrophages competently phagocytose via FcR and chemotax toward CSF and CX3CL1. However, CR3 phagocytosis and fibronectin haptotaxis, both integrin-dependent processes, are disrupted. Integrin-responsive actin assembly and αM/ß2 integrin localization are compromised in Arpc2-/- cells. Using an in vivo system to observe endogenous monocytes migrating toward full-thickness ear wounds we found that Arpc2-/- monocytes maintain cell speeds and directionality similar to control. Our work reveals that the Arp2/3 complex is not a general requirement for phagocytosis or chemotaxis but is a critical driver of integrin-dependent processes. We demonstrate further that cells lacking Arp2/3 complex function in vivo remain capable of executing important physiological responses that require rapid directional motility.

Tortuous Microvessels Contribute to Wound Healing via Sprouting Angiogenesis.

OBJECTIVE: Wound healing is accompanied by neoangiogenesis, and new vessels are thought to originate primarily from the microcirculation; however, how these vessels form and resolve during wound healing is poorly understood. Here, we investigated properties of the smallest capillaries during wound healing to determine their spatial organization and the kinetics of formation and resolution. APPROACH AND RESULTS: We used intravital imaging and high-resolution microscopy to identify a new type of vessel in wounds, called tortuous microvessels. Longitudinal studies showed that tortuous microvessels increased in frequency after injury, normalized as the wound healed, and were closely associated with the wound site. Tortuous microvessels had aberrant cell shapes, increased permeability, and distinct interactions with circulating microspheres, suggesting altered flow dynamics. Moreover, tortuous microvessels disproportionately contributed to wound angiogenesis by sprouting exuberantly and significantly more frequently than nearby normal capillaries. CONCLUSIONS: A new type of transient wound vessel, tortuous microvessels, sprout dynamically and disproportionately contribute to wound-healing neoangiogenesis, likely as a result of altered properties downstream of flow disturbances. These new findings suggest entry points for therapeutic intervention.

Tumor Presence Induces Global Immune Changes and Enhances Nanoparticle Clearance.

Long-circulating nanoparticles are essential for increasing tumor accumulation to provide therapeutic efficacy. While it is known that tumor presence can alter the immune system, very few studies have explored this impact on nanoparticle circulation. In this report, we demonstrate how the presence of a tumor can change the local and global immune system, which dramatically increases particle clearance. We found that tumor presence significantly increased clearance of PRINT hydrogel nanoparticles from the circulation, resulting in increased accumulation in the liver and spleen, due to an increase in M2-like macrophages. Our findings highlight the need to better understand interactions between immune status and nanoparticle clearance, and suggest that further consideration of immune function is required for success in preclinical and clinical nanoparticle studies.

Intravital imaging of a spheroid-based orthotopic model of melanoma in the mouse ear skin.

Multiphoton microscopy is a powerful tool that enables the visualization of fluorescently tagged tumor cells and their stromal interactions within tissues in vivo. We have developed an orthotopic model of implanting multicellular melanoma tumor spheroids into the dermis of the mouse ear skin without the requirement for invasive surgery. Here, we demonstrate the utility of this approach to observe the primary tumor, single cell actin dynamics, and tumor-associated vasculature. These methods can be broadly applied to investigate an array of biological questions regarding tumor cell behavior in vivo.