Clinical Impact of Comprehensive Molecular Profiling in Adolescents and Young Adults with Sarcoma.

Sarcomas are a heterogenous group of tumours that commonly carry poor prognosis with limited therapeutic options. Adolescents and young adults (AYAs) with sarcoma are a unique and understudied patient population that have only achieved modest survival gains compared to other groups. We present our institutional experience of AYAs with sarcoma who underwent comprehensive molecular profiling (CMP) via either large-panel targeted DNA sequencing or whole genome and transcriptome sequencing and evaluated the feasibility and clinical impact of this approach. Genomic variants detected were determined to be clinically relevant and actionable following evaluation by the Molecular Tumour Board. Clinicians provided feedback regarding the utility of testing three months after reporting. Twenty-five patients who were recruited for CMP are included in this analysis. The median time from consent to final molecular report was 45 days (interquartile range: 37-57). Potentially actionable variants were detected for 14 patients (56%), and new treatment recommendations were identified for 12 patients (48%). Pathogenic germline variants were identified in three patients (12%), and one patient had a change in diagnosis. The implementation of CMP for AYAs with sarcoma is clinically valuable, feasible, and should be increasingly integrated into routine clinical practice as technologies and turnaround times continue to improve.

Mouse Models for Exploring the Biological Consequences and Clinical Significance of PIK3CA Mutations.

The phosphatidylinositol 3-kinase (PI3K) pathway is involved in a myriad of cellular signalling pathways that regulate cell growth, metabolism, proliferation and survival. As a result, alterations in the PI3K pathway are frequently associated with human cancers. Indeed, PIK3CA-the gene encoding the p110α catalytic subunit of PI3K-is one of the most commonly mutated human oncogenes. PIK3CA mutations have also been implicated in non-malignant conditions including congenital overgrowth syndromes and vascular malformations. In order to study the role of PIK3CA mutations in driving tumorigenesis and tissue overgrowth and to test potential therapeutic interventions for these conditions, model systems are essential. In this review we discuss the various mouse models currently available for preclinical studies into the biological consequences and clinical significance of PIK3CA mutations.

STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones.

The spatial and temporal regulation of calcium signaling in neuronal growth cones is essential for axon guidance. In growth cones, the endoplasmic reticulum (ER) is a significant source of calcium signals. However, it is not clear whether the ER is remodeled during motile events to localize calcium signals in steering growth cones. The expression of the ER-calcium sensor, stromal interacting molecule 1 (STIM1) is necessary for growth cone steering toward the calcium-dependent guidance cue BDNF, with STIM1 functioning to sustain calcium signals through store-operated calcium entry. However, STIM1 is also required for growth cone steering away from semaphorin-3a, a guidance cue that does not activate ER-calcium release, suggesting multiple functions of STIM1 within growth cones (Mitchell et al., 2012). STIM1 also interacts with microtubule plus-end binding proteins EB1/EB3 (Grigoriev et al., 2008). Here, we show that STIM1 associates with EB1/EB3 in growth cones and that STIM1 expression is critical for microtubule recruitment and subsequent ER remodeling to the motile side of steering growth cones. Furthermore, we extend our data in vivo, demonstrating that zSTIM1 is required for axon guidance in actively navigating zebrafish motor neurons, regulating calcium signaling and filopodial formation. These data demonstrate that, in response to multiple guidance cues, STIM1 couples microtubule organization and ER-derived calcium signals, thereby providing a mechanism where STIM1-mediated ER remodeling, particularly in filopodia, regulates spatiotemporal calcium signals during axon guidance.SIGNIFICANCE STATEMENT Defects in both axon guidance and endoplasmic reticulum (ER) function are implicated in a range of developmental disorders. During neuronal circuit development, the spatial localization of calcium signals controls the growth cone cytoskeleton to direct motility. We demonstrate a novel role for stromal interacting molecule 1 (STIM1) in regulating microtubule and subsequent ER remodeling in navigating growth cones. We show that STIM1, an activator of store-operated calcium entry, regulates the dynamics of microtubule-binding proteins EB1/EB3, coupling ER to microtubules, within filopodia, thereby steering growth cones. The STIM1-microtubule-ER interaction provides a new model for spatial localization of calcium signals in navigating growth cones in the nascent nervous system.

Tropomyosin Tpm 2.1 loss induces glioblastoma spreading in soft brain-like environments.

INTRODUCTION: The brain is a very soft tissue. Glioblastoma (GBM) brain tumours are highly infiltrative into the surrounding healthy brain tissue and invasion mechanisms that have been defined using rigid substrates therefore may not apply to GBM dissemination. GBMs characteristically lose expression of the high molecular weight tropomyosins, a class of actin-associating proteins and essential regulators of the actin stress fibres and focal adhesions that underpin cell migration on rigid substrates. METHODS: Here, we investigated how loss of the high molecular weight tropomyosins affects GBM on soft matrices that recapitulate the biomechanical architecture of the brain. RESULTS: We find that Tpm 2.1 is down-regulated in GBM grown on soft substrates. We demonstrate that Tpm 2.1 depletion by siRNA induces cell spreading and elongation in soft 3D hydrogels, irrespective of matrix composition. Tpm 1.7, a second high molecular weight tropomyosin is also down-regulated when cells are cultured on soft brain-like surfaces and we show that effects of this isoform are matrix dependent, with Tpm 1.7 inducing cell rounding in 3D collagen gels. Finally, we show that the absence of Tpm 2.1 from primary patient-derived GBMs correlates with elongated, mesenchymal invasion. CONCLUSIONS: We propose that Tpm 2.1 down-regulation facilitates GBM colonisation of the soft brain environment. This specialisation of the GBM actin cytoskeleton organisation that is highly suited to the soft brain-like environment may provide novel therapeutic targets for arresting GBM invasion.

Small molecule targeting of the actin associating protein tropomyosin Tpm3.1 increases neuroblastoma cell response to inhibition of Rac-mediated multicellular invasion.

The migration and invasion of cells through tissues in the body is facilitated by a dynamic actin cytoskeleton. The actin-associating protein, tropomyosin Tpm3.1 has emerged to play important roles in cell migration and invasion. To date, investigations have focused on single cell migration and invasion where Tpm3.1 expression is inversely associated with Rac GTPase-mediated cell invasion. While single cell and collective cell invasion have many features in common, collective invasion is additionally impacted by cell-cell adhesion, and the role of Tpm3.1 in collective invasion has not been established. In the present study we have modelled multicellular invasion using neuroblastoma spheroids embedded in 3D collagen and analysed the function of Tpm3.1 using recently established compounds that target the Tpm3.1 C-terminus. The major findings from our study reveal that combined Rac inhibition and Tpm3.1 targeting result in greater inhibition of multicellular invasion than either treatment alone. Together, the data suggest that Tpm3.1 disruption sensitises neuroblastoma cells to inhibition of Rac-mediated multicellular invasion.

How does calcium interact with the cytoskeleton to regulate growth cone motility during axon pathfinding?

The precision with which neurons form connections is crucial for the normal development and function of the nervous system. The development of neuronal circuitry in the nervous system is accomplished by axon pathfinding: a process where growth cones guide axons through the embryonic environment to connect with their appropriate synaptic partners to form functional circuits. Despite intense efforts over many years to understand how this process is regulated, the complete repertoire of molecular mechanisms that govern the growth cone cytoskeleton and hence motility, remain unresolved. A central tenet in the axon guidance field is that calcium signals regulate growth cone behaviours such as extension, turning and pausing by regulating rearrangements of the growth cone cytoskeleton. Here, we provide evidence that not only the amplitude of a calcium signal is critical for growth cone motility but also the source of calcium mobilisation. We provide an example of this idea by demonstrating that manipulation of calcium signalling via L-type voltage gated calcium channels can perturb sensory neuron motility towards a source of netrin-1. Understanding how calcium signals can be transduced to initiate cytoskeletal changes represents a significant gap in our current knowledge of the mechanisms that govern axon guidance, and consequently the formation of functional neural circuits in the developing nervous system.

Rac GTPase regulation of 3D invasion in neuroblastomas lacking MYCN amplification.

Neuroblastomas are highly invasive tumors that occur in pediatric patients and treatment of invasive disease remains a challenge. The study of cells invading in 3-dimensional (3D) hydrogels has revealed morphologically distinct modes of invasion by which cancer cells adapt to the local tissue environment in order to invade local tissue. Specifically, the small G protein Rac GTPase has been implicated as regulating the elongated/mesenchymal mode of cell invasion. In the present study we demonstrate an inverse association between Rac expression and amplification of MYCN, a well-established prognostic indicator in neuroblastoma. Moreover, the association further tracks with previously described morphological variants of neuroblastoma. Importantly, while MYCN amplification is associated with universally poor prognosis, the clinical course of patients whose tumors lack MYCN amplification are more difficult to predict. Therefore, we analyzed the role that Rac plays in regulating the invasive behavior of neuroblastoma cells lacking MYCN amplification. Using siRNA targeting Rac in single cell suspensions in 3D collagen gels and Rac inhibition of multicellular spheroids (MCS) embedded in collagen gels, we find that the high Rac-expressing lines differ in their morphological response to Rac depletion and inhibition. Live cell imaging of embedded MCS reveals distinct individual and collective modes of invasion between the cell lines. Critically, Rac inhibition blocked both individual and collective invasion in 2 of the 3 high Rac expressing cell lines. Our study suggests that Rac activity may be an important determinant of metastatic capability in subsets of neuroblastoma cells lacking MYCN amplification.

Cooperative cell invasion: matrix metalloproteinase-mediated incorporation between cells.

Progression to metastatic disease is a leading cause of cancer death. Tumors are a complex mixture of cell types, both genetically heterogeneous malignant cells and associated nonmalignant cells. Models mimicking this heterogeneous cell environment have revealed that invasive cell populations can induce dissemination by otherwise poorly/noninvasive tumor cells, known as cooperative invasion. Neuroblastoma tumors arise in children and are characterized by mixed cellular populations in vivo, consisting chiefly of neuronal (N)-type and substrate (S)-type cells. The S-type cells have all the hallmarks of invasive leader cell populations and have been coisolated with N-type cells from metastatic bone lesions, but to date their ability to induce cooperative invasion has not been investigated. Therefore, in the present study, we analyzed the invasive behavior of mixed N-type and S-type multicellular spheroids embedded in three-dimensional collagen gels. Our analyses show that S-type cells induce invasion of either single cells or small cell clusters of N-type cells. In contrast to other reports of cooperative invasion in which mixed cultures exhibit a follow-the-leader mechanism, we show coincident emergence of S- and N-type cells from mixed spheroids. Our data suggest mutual effects between the two cell types. Thus, whereas coculture with S-type cells induces N-type invasion, coculture with N-type cells slows S-type invasion. Using matrix metalloproteinase (MMP) inhibitors and cell incorporation assays, we demonstrate that MMP activity is required for S-type cells to insert into layers of N-type cells. Our study therefore highlights an important role for S-type neuroblastoma cells in the invasion process and reveals a new mechanism of cooperative invasion.

STIM1 is necessary for store-operated calcium entry in turning growth cones.

Coordinated calcium signalling is vital for neuronal growth cone function and axon pathfinding. Although store-operated calcium entry (SOCE) has been suggested to be an important source of calcium in growth cone navigation, the mechanisms that regulate calcium signalling, particularly the regulation of internal calcium stores within growth cones, are yet to be fully determined. Stromal Interaction Molecule 1 (STIM1) is a calcium-sensing protein localized in the endoplasmic reticulum membrane that interacts with Orai proteins in the plasma membrane to initiate SOCE and refilling of intracellular calcium stores. We hypothesize that STIM1- and Orai1/2-mediated SOCE are necessary for growth cone turning responses to extracellular guidance cues. We show that STIM1 and Orai reorganize into puncta upon store depletion and during growth cone turning with STIM1 localization biased towards the turning side (high calcium side) of the growth cone. Importantly, STIM1 knock-down perturbed growth cone turning responses to the guidance cues brain-derived neurotrophic factor and semaphorin-3a (Sema-3a), as well as abolishing Sema-3a-induced growth cone collapse. Furthermore, STIM1 knock-down abolished SOCE induced by brain-derived neurotrophic factor, but not Sema-3a. Our data suggest that STIM1 is essential for correct growth cone navigation, playing multiple roles in growth cone motility, including the activation of SOCE.

Homer regulates calcium signalling in growth cone turning.

BACKGROUND: Homer proteins are post-synaptic density proteins with known functions in receptor trafficking and calcium homeostasis. While they are key mediators of synaptic plasticity, they are also known to function in axon guidance, albeit by mechanisms that are yet to be elucidated. Homer proteins couple extracellular receptors - such as metabotropic glutamate receptors and the transient receptor potential canonical family of cation channels - to intracellular receptors such as inositol triphosphate and ryanodine receptors on intracellular calcium stores and, therefore, are well placed to regulate calcium dynamics within the neural growth cone. Here we used growth cones from dorsal root ganglia, a well established model in the field of axon guidance, and a growth cone turning assay to examine Homer1 function in axon guidance. RESULTS: Homer1 knockdown reversed growth cone turning from attraction to repulsion in response to the calcium-dependent guidance cues brain derived neurotrophic factor and netrin-1. Conversely, Homer1 knockdown had no effect on repulsion to the calcium-independent guidance cue Semaphorin-3A. This reversal of attractive turning suggested a requirement for Homer1 in a molecular switch. Pharmacological experiments confirmed that the operational state of a calcium-calmodulin dependent protein kinase II/calcineurin phosphatase molecular switch was dependent on Homer1 expression. Calcium imaging of motile growth cones revealed that Homer1 is required for guidance-cue-induced rise of cytosolic calcium and the attenuation of spontaneous cytosolic calcium transients. Homer1 knockdown-induced calcium transients and turning were inhibited by antagonists of store-operated channels. In addition, immunocytochemistry revealed the close association of Homer1 with the store-operated proteins TRPC1 and STIM1 within dorsal root ganglia growth cones. CONCLUSION: These experiments provide evidence that Homer1 is an essential component of the calcium signalling repertoire within motile growth cones, regulating guidance-cue-induced calcium release and maintaining basal cytosolic calcium.