Stochastic Model of Tsc1 Lesions in Mouse Brain

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder due to mutations in either TSC1 or TSC2 that affects many organs with hamartomas and tumors. TSC-associated brain lesions include subependymal nodules, subependymal giant cell astrocytomas and tubers. Neurologic manifestations in TSC comprise a high frequency of mental retardation and developmental disorders including autism, as well as epilepsy. Here, we describe a new mouse model of TSC brain lesions in which complete loss of Tsc1 is achieved in multiple brain cell types in a stochastic pattern. Injection of an adeno-associated virus vector encoding Cre recombinase into the cerebral ventricles of mice homozygous for a Tsc1 conditional allele on the day of birth led to reduced survival, and pathologic findings of enlarged neurons, cortical heterotopias, subependymal nodules, and hydrocephalus. The severity of clinical and pathologic findings as well as survival was shown to be dependent upon the dose and serotype of Cre virus injected. Although several other models of TSC brain disease exist, this model is unique in that the pathology reflects a variety of TSC-associated lesions involving different numbers and types of cells. This model provides a valuable and unique addition for therapeutic assessment

Cross-Species Array Comparative Genomic Hybridization Identifies Novel Oncogenic Events in Zebrafish and Human Embryonal Rhabdomyosarcoma

Human cancer genomes are highly complex, making it challenging to identify specific drivers of cancer growth, progression, and tumor maintenance. To bypass this obstacle, we have applied array comparative genomic hybridization (array CGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to rapidly identify genomic copy number aberrations and novel candidate oncogenes in human disease. Zebrafish ERMS contain small, focal regions of low-copy amplification. These same regions were commonly amplified in human disease. For example, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, low-copy gains in human disease. Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS – identifying critical genes associated with tumor maintenance. Knockdown studies identified important roles for Cyclin D2 (CCND2), Homeobox Protein C6 (HOXC6) and PlexinA1 (PLXNA1) in human ERMS cell proliferation. PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth. By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish. Importantly, VEGFA expression correlated with poor clinical outcome in patients with ERMS, implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. Our results demonstrate the utility of array CGH and cross-species comparisons to identify candidate oncogenes essential for the pathogenesis of human cancer

Knockdown of PLXNA1 impairs migration of human ERMS cells.

<p>Representative images of ERMS cells transfected with gene-specific siRNAs at 0 hr (A, control siRNA; C, <i>CCND2</i> siRNA; E, <i>HOXC6</i> siRNA; G, <i>PLXNA1</i> siRNA) and 22 hrs (B, control siRNA; D, <i>CCND2</i> siRNA; F, <i>HOXC6</i> siRNA; H, <i>PLXNA1</i> siRNA) following gap creation. Scale bar indicates 100 µm. (I) Quantification of data from wound healing assay. Each error bar indicates standard deviation across 5–6 independent replicates. (J) A Transwell migration assay was performed in RD cells that stably express either a control shRNA or two independent <i>PLXNA1</i> shRNAs. Migration was assessed after 24 hours. Each error bar indicates standard deviation across six fields at 200× magnification. Asterisks denote p<0. 05.</p

Chemical inhibition of VEGF signaling by cediranib reduces ERMS growth <i>in vivo</i>.

<p>Syngeneic CG1 fish were transplanted with ERMS cells that co-expressed <i>rag2-KRASG12D</i> and <i>rag2-dsRED</i>. Fish with engrafted tumors were treated with DMSO vehicle (A–F) or 100 nM of cediranib for 7 days (G–L). Pre-treatment (A–C and G–I) and post-treatment images (D–F and J–L) of representative fish. Bright field (A,D,G,J), dsRED fluorescence (B,E,H,K) and merged image planes (C,F,I,L). Scale bar is 3 mm. (M) Quantification of relative volume change for individual animals. (N–O) <i>fli1-GFP</i> transgenic zebrafish were transplanted with dsRED-labeled ERMS and treated with DMSO (N) and cediranib (O). Scale bar equals 50 µm. (P) Microvessel density quantification. Asterisk indicates statistically significant difference between DMSO and cediranib-treated groups based on student t-test. Each error bar indicates standard deviation from 3 fields of microvessels for each animal. EDU incorporation analysis in DMSO (Q) or cediranib (R) treated fish. Scale bar is 50 µm. (S) Quantification of EDU analysis across each cohort of animals. Each error bar indicates standard deviation of percent EDU+ cells found within 3 fields for each animal.</p

Comparison of array CGH analyses in zebrafish and human ERMS.

<p>Zebrafish gene-containing, CNAs were compared to those identified in 26 primary human ERMS samples by Paulson et al. (2011). Genes selected for characterization in this study are in bold.</p

Knockdown of PLXNA1 induced differentiation and impaired anchorage-independent growth of human ERMS cells.

<p>RD cells stained with myosin heavy chain (MF20) and DAPI following culture under differentiation conditions for 72 hrs. (A) Control siRNA. (B) <i>PLXNA1</i> smart-pool siRNA. (C) Control scrambled shRNA. (D) <i>PLXNA1</i> shRNA-1. DAPI, blue; MF20-positive cell, green. (E) Quantification of MF-20 immunofluorescence in siRNA and shRNA-knockdown RD cells. Asterisk indicates significant differences between gene knock- down and control cells (p<0.05). Error bars denote standard deviation. (F) Western analysis of <i>PLXNA1</i> shRNA stable knockdown; sc, scrambled control shRNA; 1, <i>PLXNA1</i> shRNA-1; 2, <i>PLXNA1</i> shRNA-2. A soft agar colony formation assay to assess PLXNA1 knockdown effects on anchorage-independent growth (G–I). (G) Control scrambled shRNA. (H) <i>PLXNA1</i> shRNA. (I) Quantification of colony formation assay results. Error bar indicates standard deviation from triplicate experiments.</p

Array CGH reveals cancer-specific chromosomal abnormalities in zebrafish ERMS.

<p>(A) Summary of common gene-containing CNA gains (green) and losses (red) in 20 animals examined. Only recurrent CNAs found in ≥3 samples are shown. The height of each bar correlates with the frequency of each aberration. Detailed view of regional gains for <i>vegfa</i> on chromosome 4 (B), <i>ccnd2a</i> on chromosome 25 (C), <i>hoxc6a</i> on chromosome 23 (D), and <i>plxna1</i> on chromosome 6 (E). Y-axis denotes log2 ratio of the probes and X-axis denotes genomic coordinates.</p