MiR-130b modulates the invasive, migratory, and metastatic behavior of leiomyosarcoma.

Leiomyosarcoma (LMS) is an aggressive, often poorly differentiated cancer of the smooth muscle (SM) lineage for which the molecular drivers of transformation and progression are poorly understood. In microRNA (miRNA) profiling studies, miR-130b was previously found to be upregulated in LMS vs. normal SM, and down-regulated during the differentiation of mesenchymal stem cells (MSCs) into SM, suggesting a role in LMS tumor progression. In the present study, the effects of miR-130b on human LMS tumorigenesis were investigated. Stable miR-130b overexpression enhanced invasion of LMS cells in vitro, and led to the formation of undifferentiated, pleomorphic tumors in vivo, with increased growth and metastatic potential compared to control LMS cells. TSC1 was identified as a direct miR-130b target in luciferase-3'UTR assays, and shRNA-mediated knockdown of TSC1 replicated miR-130b effects. Loss-of-function and gain-of-function studies showed that miR-130b levels regulate cell morphology and motility. Following miR-130b suppression, LMS cells adopted a rounded morphology, amoeboid mode of cell movement and enhanced invasive capacity that was Rho/ROCK dependent. Conversely, miR-130b-overexpressing LMS cells exhibited Rho-independent invasion, accompanied by down-regulation of Rho-pathway effectors. In mesenchymal stem cells, both miR-130b overexpression and TSC1 silencing independently impaired SM differentiation in vitro. Together, the data reveal miR-130b as a pro-oncogenic miRNA in LMS and support a miR-130b-TSC1 regulatory network that enhances tumor progression via inhibition of SM differentiation.

Tsc1 Regulates the Proliferation Capacity of Bone-Marrow Derived Mesenchymal Stem Cells.

TSC1 is a tumor suppressor that inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). TSC1 mutations are associated with Tuberous Sclerosis Complex (TSC), characterized by multiple benign tumors of mesenchymal and epithelial origin. TSC1 modulates self-renewal and differentiation in hematopoietic stem cells; however, its effects on mesenchymal stem cells (MSCs) are unknown. We investigated the impact of Tsc1 inactivation in murine bone marrow (BM)-MSCs, using tissue-specific, transgelin (Tagln)-mediated cre-recombination, targeting both BM-MSCs and smooth muscle cells. Tsc1 mutants were viable, but homozygous inactivation led to a dwarfed appearance with TSC-like pathologies in multiple organs and reduced survival. In young (28 day old) mice, Tsc1 deficiency-induced significant cell expansion of non-hematopoietic BM in vivo, and MSC colony-forming potential in vitro, that was normalized upon treatment with the mTOR inhibitor, everolimus. The hyperproliferative BM-MSC phenotype was lost in aged (1.5 yr) mice, and Tsc1 inactivation was also accompanied by elevated ROS and increased senescence. ShRNA-mediated knockdown of Tsc1 in BM-MSCs replicated the hyperproliferative BM-MSC phenotype and led to impaired adipogenic and myogenic differentiation. Our data show that Tsc1 is a negative regulator of BM-MSC proliferation and support a pivotal role for the Tsc1-mTOR axis in the maintenance of the mesenchymal progenitor pool.

In Vivo Modeling of Chemoresistant Neuroblastoma Provides New Insights into Chemorefractory Disease and Metastasis.

Neuroblastoma is a pediatric cancer that is frequently metastatic and resistant to conventional treatment. In part, a lack of natively metastatic, chemoresistant in vivo models has limited our insight into the development of aggressive disease. The Th-MYCN genetically engineered mouse model develops rapidly progressive chemosensitive neuroblastoma and lacks clinically relevant metastases. To study tumor progression in a context more reflective of clinical therapy, we delivered multicycle treatment with cyclophosphamide to Th-MYCN mice, individualizing therapy using MRI, to generate the Th-MYCN CPM32 model. These mice developed chemoresistance and spontaneous bone marrow metastases. Tumors exhibited an altered immune microenvironment with increased stroma and tumor-associated fibroblasts. Analysis of copy number aberrations revealed genomic changes characteristic of human MYCN-amplified neuroblastoma, specifically copy number gains at mouse chromosome 11, syntenic with gains on human chromosome 17q. RNA sequencing revealed enriched expression of genes associated with 17q gain and upregulation of genes associated with high-risk neuroblastoma, such as the cell-cycle regulator cyclin B1-interacting protein 1 (Ccnb1ip1) and thymidine kinase (TK1). The antiapoptotic, prometastatic JAK-STAT3 pathway was activated in chemoresistant tumors, and treatment with the JAK1/JAK2 inhibitor CYT387 reduced progression of chemoresistant tumors and increased survival. Our results highlight that under treatment conditions that mimic chemotherapy in human patients, Th-MYCN mice develop genomic, microenvironmental, and clinical features reminiscent of human chemorefractory disease. The Th-MYCN CPM32 model therefore is a useful tool to dissect in detail mechanisms that drive metastasis and chemoresistance, and highlights dysregulation of signaling pathways such as JAK-STAT3 that could be targeted to improve treatment of aggressive disease. SIGNIFICANCE: An in vivo mouse model of high-risk treatment-resistant neuroblastoma exhibits changes in the tumor microenvironment, widespread metastases, and sensitivity to JAK1/2 inhibition.

Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-ß-OTX2-SNAIL via PTEN inhibition.

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-ß signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-ß activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-ß/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.

Preclinical transgenic and patient-derived xenograft models recapitulate the radiological features of human adamantinomatous craniopharyngioma.

To assess the clinical relevance of transgenic and patient-derived xenograft models of adamantinomatous craniopharyngioma (ACP) using serial magnetic resonance imaging (MRI) and high resolution post-mortem microcomputed tomography (µ-CT), with correlation with histology and human ACP imaging. The growth patterns and radiological features of tumors arising in Hesx1Cre/+ ;Ctnnb1lox(ex3)/+ transgenic mice, and of patient-derived ACP xenografts implanted in the cerebral cortex, were monitored longitudinally in vivo with anatomical and functional MRI, and by ex vivo µ-CT at study end. Pathological correlates with hematoxylin and eosin stained sections were investigated. Early enlargement and heterogeneity of Hesx1Cre/+ ;Ctnnb1lox(ex3)/+ mouse pituitaries was evident at initial imaging at 8 weeks, which was followed by enlargement of a solid tumor, and development of cysts and hemorrhage. Tumors demonstrated MRI features that recapitulated those of human ACP, specifically, T1 -weighted signal enhancement in the solid tumor component following Gd-DTPA administration, and in some animals, hyperintense cysts on FLAIR and T1 -weighted images. Ex vivo µ-CT correlated with MRI findings and identified smaller cysts, which were confirmed by histology. Characteristic histological features, including wet keratin and calcification, were visible on µ-CT and verified by histological sections of patient-derived ACP xenografts. The Hesx1Cre/+ ;Ctnnb1lox(ex3)/+ transgenic mouse model and cerebral patient-derived ACP xenografts recapitulate a number of the key radiological features of the human disease and provide promising foundations for in vivo trials of novel therapeutics for the treatment of these tumors.

Pilot Study to Determine Safety and Efficacy of Paclitaxel Infusion in De Novo Peripheral Lesions Using the Atrium ClearWay Balloon.

OBJECTIVE: To evaluate the safety and efficacy of a one-time infusion of paclitaxel through an Atrium ClearWay balloon in infra inguinal de novo peripheral lesions. METHODS: This is a single-center prospective study looking at treatment of 50 limbs. Treatment includes standard infra inguinal endovascular revascularization followed by a pre-prescribed infusion of paclitaxel. Control is standard reintervention without subsequent paclitaxel infusion. Patients were followed at one, four, and 10 months with ankle-brachial index (ABI)s, arterial duplex of the treated limb, and Rutherford classification stage measured before and after procedures and at each follow-up. Freedom from binary restenosis was tracked with duplex ultrasound, and freedom from target lesion revascularization (TLR) was also tracked in the treatment group. Binary restenosis and TLR data was harvested from the patient record for the control group. RESULTS: Average ABI and Rutherford classification stage improved as expected. The treatment group had a freedom from TLR rate of 86 percent and a freedom from binary restenosis rate of 80 percent at 10 months. Average ABI improved from 0.65 at baseline to 0.94 at 10 months in the treatment group. The control group had a 72 percent freedom from TLR and a 58 percent freedom from binary restenosis at 10 months. Average ABI of the control group improved from 0.67 at baseline to 0.85 at 10 months in the control group. There were no amputations, open bypass revascularizations, or hypersensitivity reactions observed in the treatment group. CONCLUSIONS: Infusion of paclitaxel in de novo lesions appears to be a safe and efficacious treatment in the peripheral vasculature when compared to a historical control group. While it is early, it appears that the patients do receive some benefit from this one time infusion, and this approach should be studied further.

Immunoassays for the quantification of ALK and phosphorylated ALK support the evaluation of on-target ALK inhibitors in neuroblastoma.

Targeted inhibition of anaplastic lymphoma kinase (ALK) is a successful approach for the treatment of many ALK-aberrant malignancies; however, the presence of resistant mutations necessitates both the development of more potent compounds and pharmacodynamic methods with which to determine their efficacy. We describe immunoassays designed to quantitate phosphorylation of ALK, and their use in preclinical models of neuroblastoma, a pediatric malignancy in which gain-of-function ALK mutations predict a poor overall outcome to conventional treatment. Validation of the immunoassays is presented using a panel of neuroblastoma cell lines and evidence of on-target ALK inhibition provided by treatment of a genetically engineered murine model of neuroblastoma with two clinical ALK inhibitors, crizotinib and ceritinib, highlighting the superior efficacy of ceritinib.

Examination of near-wall hemodynamic parameters in the renal bridging stent of various stent graft configurations for repairing visceral branched aortic aneurysms.

OBJECTIVE: This study examined the flow behavior of four stent graft configurations for endovascular repair of complex aneurysms of the descending aorta. METHODS: Computational fluid dynamics models with transient boundary conditions and rigid wall simplifying assumptions were developed and used with four distinct geometries to compare various near-wall hemodynamic parameters. RESULTS: Graphic plots for time-averaged wall shear stress, oscillating shear index, and relative residence time were presented and compared among the four stent graft configurations of interest. CONCLUSIONS: Abrupt 90° and 180° changes in stent geometry (particularly in the side branches) cause a high momentum change and thus increased flow separation and mixing, which has significant implications in blood flow characteristics near the wall. By comparison, longer bridging stents provide more gradual changes in momentum, thus allowing blood flow to develop before reaching the target vessel.

Osteoporosis: A Review.

Osteoporosis is a silent disease which can significantly increase an individual's risk of fracture. Fractures can be physically debilitating and consequently cause a financial burden on the patient, so it is key to treat osteoporosis before a fracture occurs. Osteoporosis is defined based on the patient's bone mineral density (BMD) as compared to a young adult's BMD using standard deviations (SD). Normal BMD is within 1 SD of young adult mean, osteopenia is between 1 and 2.5 SD less, and osteoporosis is greater than 2.5 SD less than the mean. Severe osteoporosis is defined similar to osteoporosis but includes the presence of fragility fractures.

Tackling Crizotinib Resistance: The Pathway from Drug Discovery to the Pediatric Clinic.

Neuroblastoma is a childhood malignancy that has not yet benefitted from the rapid progress in the development of small-molecule therapeutics for cancer. An opportunity to take advantage of pharmaceutical innovation in this area arose when the identification of ALK fusion proteins in non-small cell lung cancer (NSCLC) occurred in parallel to the discovery of point mutations of ALK in neuroblastomas. ALK is now known to be a marker of poor outcome in neuroblastoma, and therefore, urgent development of specific ALK inhibitors to treat this devastating disease is a necessity. However, the translation of small molecules from adult directly into pediatric practice has thus far been challenging, due to mutation-specific structural variances in the ALK kinase domain. We discuss how the most recent structural and biological characterizations of ALK are directing preclinical and clinical studies of ALK inhibitors for both NSCLC and neuroblastoma.

Limited miR-17-92 overexpression drives hematologic malignancies.

The overexpression of microRNA cluster miR-17-92 has been implicated in development of solid tumors and hematological malignancies. The role of miR-17-92 in lymphomagenesis has been extensively investigated; however, because of the developmental defects caused by miR-17-92 dysregulation, its ability to drive tumorigenesis has remained undetermined until recently. Here we demonstrate that overexpression of miR-17-92 in a limited number of hematopoietic cells is sufficient to cause B cell malignancies. In sum, our study provides a novel and physiologically relevant model that exposes the potent ability of miR-17-92 to act as a driver of tumorigenesis.

The kinase ALK stimulates the kinase ERK5 to promote the expression of the oncogene MYCN in neuroblastoma.

Anaplastic lymphoma kinase (ALK) is an important molecular target in neuroblastoma. Although tyrosine kinase inhibitors abrogating ALK activity are currently in clinical use for the treatment of ALK-positive (ALK(+)) disease, monotherapy with ALK tyrosine kinase inhibitors may not be an adequate solution for ALK(+) neuroblastoma patients. Increased expression of the gene encoding the transcription factor MYCN is common in neuroblastomas and correlates with poor prognosis. We found that the kinase ERK5 [also known as big mitogen-activated protein kinase (MAPK) 1 (BMK1)] is activated by ALK through a pathway mediated by phosphoinositide 3-kinase (PI3K), AKT, MAPK kinase kinase 3 (MEKK3), and MAPK kinase 5 (MEK5). ALK-induced transcription of MYCN and stimulation of cell proliferation required ERK5. Pharmacological or RNA interference-mediated inhibition of ERK5 suppressed the proliferation of neuroblastoma cells in culture and enhanced the antitumor efficacy of the ALK inhibitor crizotinib in both cells and xenograft models. Together, our results indicate that ERK5 mediates ALK-induced transcription of MYCN and proliferation of neuroblastoma, suggesting that targeting both ERK5 and ALK may be beneficial in neuroblastoma patients.

A novel endovascular debranching technique using physician-assembled endografts for repair of thoracoabdominal aneurysms.

OBJECTIVE: The objective of this study was to demonstrate a technique that uses physician-assembled endografts to make use of the benefits of parallel grafts while also providing for circumferential seal and fixation in repair of thoracoabdominal aneurysms in inoperable patients. METHODS: A single-center all-comers retrospective analysis of 14 patients was performed that looked at the early outcomes of patients treated for thoracoabdominal aneurysms. Three Crawford type II, four type III, four type IV, and three type V thoracoabdominal aneurysms were treated. Contrast material, fluoroscopy time, length of stay, clinical success, and technical success were measured. RESULTS: There was no in-hospital, 30-day, or 6-month mortality. We found two type III endoleaks in the early design. One required coil embolization. Average volume of contrast material and average fluoroscopy time were 76.9 mL and 119.1 minutes, respectively. Average length of stay was 10.5 days, and average procedure time was 251.2 minutes. Clinical success was observed in 78.6% of patients to date, and technical success was observed in 85.7% of patients. CONCLUSIONS: Short-term results show that this approach is safe. The device can be safely implanted, is off-the-shelf, and can treat each of the Crawford thoracoabdominal aneurysm types. Finally, the assembly of off-the-shelf components may shorten the regulatory path for this physician-assembled endograft.

Treatment of a massive left femoral arteriovenous malformation using an innovative modular hybrid bifurcated stent graft system.

BACKGROUND: Arteriovenous malformations (AVMs) are difficult to treat and manage because of their high recurrence and complication rates. In particular, peripheral AVMs pose multiple clinical challenges because of their high flow rates and the frequent presence of multifocal nidi. METHODS: A 37-year-old man with a massive AVM involving the left common, deep, and superficial femoral arteries and veins is discussed herein. After initially being treated at another facility with coil embolization in 2005, he went untreated until he presented to us in April 2012 with swelling, tissue breakdown, leg ulcers, pain, and difficulty walking. When our extensive packed coil embolization proved ineffective, we knew that other standard treatments would be impractical, given the size of the AVM. Because the patient was in significant danger of bleeding, we treated him endovascularly with a system of modified stent grafts to exclude the arterial branches feeding multiple nidi. RESULTS: Postoperative computed tomography angiography scans revealed exclusion of the AVM and excellent flow to the deep and superficial femoral arteries. At 6 months postoperatively, the patient had no complications, and the leg continued to decompress. At 8 months postoperatively, we started additional treatment using percutaneous sclerotherapy to treat residual areas. CONCLUSION: A modular hybrid bifurcated stent graft system is a viable option to treat or manage complex peripheral arteriovenous malformations.

Dual Pten/Tp53 suppression promotes sarcoma progression by activating Notch signaling.

Soft tissue sarcomas are a heterogeneous group of tumors associated with poor clinical outcome. Although a subset of soft tissue sarcomas is characterized by simple karyotypes and recurrent chromosomal translocations, the mechanisms driving cytogenetically complex sarcomas are largely unknown. Clinical evidence led us to partially inactivate Pten and Tp53 in the smooth muscle lineage of mice, which developed high-grade undifferentiated pleomorphic sarcomas, leiomyosarcomas, and carcinosarcomas that widely recapitulate the human disease, including the aberrant karyotype and metastatic behavior. Pten was found haploinsufficient, whereas the wild-type allele of Tp53 invariably gained point mutations. Gene expression profiles showed up-regulated Notch signaling in Pten(Δ/+)Tp53(Δ/+) tumors compared with Pten(+/+)Tp53(Δ/+) tumors. Consistently, Pten silencing exacerbated the clonogenic and invasive potential of Tp53-deficient bone marrow-derived mouse mesenchymal stem cells and tumor cells and activated the Notch pathway. Moreover, the increased oncogenic behavior of Pten(Δ/+)Tp53(Δ/+) and shPten-transduced Pten(+/+)Tp53(Δ/+) tumor cells was counteracted by treatment with a γ-secretase inhibitor, suggesting that the aggressiveness of those tumors can be attributed, at least in part, to enhanced Notch signaling. This study demonstrates a cooperative role for Pten and Tp53 suppression in complex karyotype sarcomas while establishing Notch as an important functional player in the cross talk of these pathways during tumor progression. Our results highlight the importance of molecularly subclassifying patients with high-grade sarcoma for targeted treatments.

Cardiovascular dysregulation of miR-17-92 causes a lethal hypertrophic cardiomyopathy and arrhythmogenesis.

MicroRNA cluster miR-17-92 has been implicated in cardiovascular development and function, yet its precise mechanisms of action in these contexts are uncertain. This study aimed to investigate the role of miR-17-92 in morphogenesis and function of cardiac and smooth muscle tissues. To do so, a mouse model of conditional overexpression of miR-17-92 in cardiac and smooth muscle tissues was generated. Extensive cardiac functional studies identified a dose-dependent induction of dilated, hypertrophic cardiomyopathy, and arrhythmia inducibility in transgenic animals, which correlated with premature mortality (98.3 ± 42.5 d, P<0.0001). Expression analyses revealed the abundance of Pten transcript, a known miR-17-92 target, to be inversely correlated with miR-17-92 expression levels and heart size. In addition, we demonstrated through 3'-UTR luciferase assays and expression analyses that Connexin43 (Cx43) is a novel direct target of miR-19a/b and its expression is suppressed in transgenic hearts. Taken together, these data demonstrate that dysregulated expression of miR-17-92 during cardiovascular morphogenesis results in a lethal cardiomyopathy, possibly in part through direct repression of Pten and Cx43. This study highlights the importance of miR-17-92 in both normal and pathological functions of the heart, and provides a model that may serve as a useful platform to test novel antiarrhythmic therapeutics.

A differentiation-based microRNA signature identifies leiomyosarcoma as a mesenchymal stem cell-related malignancy.

Smooth muscle (SM) is a spontaneously contractile tissue that provides physical support and function to organs such as the uterus. Uterine smooth muscle-related neoplasia comprise common well-differentiated benign lesions called leiomyomas (ULM), and rare, highly aggressive and pleomorphic tumors named leiomyosarcomas (ULMS). MicroRNAs (miRNAs) are small non-coding RNAs that play essential roles in normal cellular development and tissue homeostasis that can be used to accurately subclassify different tumor types. Here, we demonstrate that miRNAs are required for full smooth muscle cell (SMC) differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). We also report a miRNA signature associated with this process. Moreover, we show that this signature, along with miRNA profiles for ULMS and ULM, are able to subclassify tumors of smooth muscle origin along SM differentiation. Using multiple computational analyses, we determined that ULMS are more similar to hMSCs as opposed to ULM, which are linked with more mature SMCs and myometrium. Furthermore, a comparison of the SM differentiation and ULMS miRNA signatures identified miRNAs strictly associated with SM maturation or transformation, as well as those modulated in both processes indicating a possible dual role. These results support separate origins and/or divergent transformation pathways for ULM and ULMS, resulting in drastically different states of differentiation. In summary, this work expands on our knowledge of the regulation of SM differentiation and sarcoma pathogenesis.

The temporal program of chromosome replication: genomewide replication in clb5{Delta} Saccharomyces cerevisiae.

Temporal regulation of origin activation is widely thought to explain the pattern of early- and late-replicating domains in the Saccharomyces cerevisiae genome. Recently, single-molecule analysis of replication suggested that stochastic processes acting on origins with different probabilities of activation could generate the observed kinetics of replication without requiring an underlying temporal order. To distinguish between these possibilities, we examined a clb5Delta strain, where origin firing is largely limited to the first half of S phase, to ask whether all origins nonspecifically show decreased firing (as expected for disordered firing) or if only some origins ("late" origins) are affected. Approximately half the origins in the mutant genome show delayed replication while the remainder replicate largely on time. The delayed regions can encompass hundreds of kilobases and generally correspond to regions that replicate late in wild-type cells. Kinetic analysis of replication in wild-type cells reveals broad windows of origin firing for both early and late origins. Our results are consistent with a temporal model in which origins can show some heterogeneity in both time and probability of origin firing, but clustering of temporally like origins nevertheless yields a genome that is organized into blocks showing different replication times.