Rebecca D. Dodd

Cross Species Genomic Analysis Identifies a Mouse Model as Undifferentiated Pleomorphic Sarcoma/Malignant Fibrous Histiocytoma

Undifferentiated pleomorphic sarcoma/Malignant Fibrous Histiocytoma (MFH) is one of the most common subtypes of human soft tissue sarcoma. Using cross species genomic analysis, we define a geneset from the LSL-KrasG12D; Trp53Flox/Flox mouse model of soft tissue sarcoma that is highly enriched in human MFH. With this mouse geneset as a filter, we identify expression of the RAS target FOXM1 in human MFH. Expression of Foxm1 is elevated in mouse sarcomas that metastasize to the lung and tissue microarray analysis of human MFH correlates overexpression of FOXM1 with metastasis. These results suggest that genomic alterations present in human MFH are conserved in the LSL-KrasG12D; p53Flox/Flox mouse model of soft tissue sarcoma and demonstrate the utility of this pre-clinical model.

MicroRNA-182 drives metastasis of primary sarcomas by targeting multiple genes

Metastasis causes most cancer deaths, but is incompletely understood. MicroRNAs can regulate metastasis, but it is not known whether a single miRNA can regulate metastasis in primary cancer models in vivo. We compared the expression of miRNAs in metastatic and nonmetastatic primary mouse sarcomas and found that microRNA-182 (miR-182) was markedly overexpressed in some tumors that metastasized to the lungs. By utilizing genetically engineered mice with either deletion of or overexpression of miR-182 in primary sarcomas, we discovered that deletion of miR-182 substantially decreased, while overexpression of miR-182 considerably increased, the rate of lung metastasis after amputation of the tumor-bearing limb. Additionally, deletion of miR-182 decreased circulating tumor cells (CTCs), while overexpression of miR-182 increased CTCs, suggesting that miR-182 regulates intravasation of cancer cells into the circulation. We identified 4 miR-182 targets that inhibit either the migration of tumor cells or the degradation of the extracellular matrix. Notably, restoration of any of these targets in isolation did not alter the metastatic potential of sarcoma cells injected orthotopically, but the simultaneous restoration of all 4 targets together substantially decreased the number of metastases. These results demonstrate that a single miRNA can regulate metastasis of primary tumors in vivo by coordinated regulation of multiple genes.

Intraoperative detection and removal of microscopic residual sarcoma using wide-field imaging

The goal of limb‐sparing surgery for a soft tissue sarcoma of the extremity is to remove all malignant cells while preserving limb function. After initial surgery, microscopic residual disease in the tumor bed will cause a local recurrence in approximately 33% of patients with sarcoma. To help identify these patients, the authors developed an in vivo imaging system to investigate the suitability of molecular imaging for intraoperative visualization.

Efficacy of sunitinib and radiotherapy in genetically engineered mouse model of soft-tissue sarcoma.

PURPOSE Sunitinib (SU) is a multitargeted receptor tyrosine kinase inhibitor of the vascular endothelial growth factor and platelet-derived growth factor receptors. The present study examined SU and radiotherapy (RT) in a genetically engineered mouse model of soft tissue sarcoma (STS). METHODS AND MATERIALS Primary extremity STSs were generated in genetically engineered mice. The mice were randomized to treatment with SU, RT (10 Gy x 2), or both (SU+RT). Changes in the tumor vasculature before and after treatment were assessed in vivo using fluorescence-mediated tomography. The control and treated tumors were harvested and extensively analyzed. RESULTS The mean fluorescence in the tumors was not decreased by RT but decreased 38-44% in tumors treated with SU or SU+RT. The control tumors grew to a mean of 1378 mm(3) after 12 days. SU alone or RT alone delayed tumor growth by 56% and 41%, respectively, but maximal growth inhibition (71%) was observed with the combination therapy. SU target effects were confirmed by loss of target receptor phosphorylation and alterations in SU-related gene expression. Cancer cell proliferation was decreased and apoptosis increased in the SU and RT groups, with a synergistic effect on apoptosis observed in the SU+RT group. RT had a minimal effect on the tumor microvessel density and endothelial cell-specific apoptosis, but SU alone or SU+RT decreased the microvessel density by >66% and induced significant endothelial cell apoptosis. CONCLUSION SU inhibited STS growth by effects on both cancer cells and tumor vasculature. SU also augmented the efficacy of RT, suggesting that this combination strategy could improve local control of STS.

NF1 deletion generates multiple subtypes of soft-tissue sarcoma that respond to MEK inhibition

Soft-tissue sarcomas are a heterogeneous group of tumors arising from connective tissue. Recently, mutations in the neurofibromin 1 (NF1) tumor suppressor gene were identified in multiple subtypes of human soft-tissue sarcomas. To study the effect of NF1 inactivation in the initiation and progression of distinct sarcoma subtypes, we have developed a novel mouse model of temporally and spatially restricted NF1-deleted sarcoma. To generate primary sarcomas, we inject adenovirus containing Cre recombinase into NF1flox/flox; Ink4a/Arfflox/flox mice at two distinct orthotopic sites: intramuscularly or in the sciatic nerve. The mice develop either high-grade myogenic sarcomas or malignant peripheral nerve sheath tumor (MPNST)-like tumors, respectively. These tumors reflect the histologic properties and spectrum of sarcomas found in patients. To explore the use of this model for preclinical studies, we conducted a study of mitogen-activated protein kinase (MAPK) pathway inhibition with the MEK inhibitor PD325901. Treatment with PD325901 delays tumor growth through decreased cyclin D1 mRNA and cell proliferation. We also examined the effects of MEK inhibition on the native tumor stroma and find that PD325901 decreases VEGFα expression in tumor cells with a corresponding decrease in microvessel density. Taken together, our results use a primary tumor model to show that sarcomas can be generated by loss of NF1 and Ink4a/Arf, and that these tumors are sensitive to MEK inhibition by direct effects on tumor cells and the surrounding microenvironment. These studies suggest that MEK inhibitors should be further explored as potential sarcoma therapies in patients with tumors containing NF1 deletion. Mol Cancer Ther; 12(9); 1906–17. ©2013 AACR.

Analysis of mRNA Partitioning Between the Cytosol and Endoplasmic Reticulum Compartments of Mammalian Cells

All eukaryotic cells display a dramatic partitioning of mRNAs between the cytosol and endoplasmic reticulum (ER) compartments-mRNAs encoding secretory and integral membrane proteins are highly enriched on ER-bound ribosomes and mRNAs encoding cytoplasmic/nucleoplasmic proteins are enriched on cytosolic ribosomes. In current views, this partitioning phenomenon occurs through positive selection-mRNAs encoding signal sequence-bearing proteins are directed into the signal recognition particle pathway early in translation and trafficked as mRNA/ribosome/nascent polypeptide chain complexes to the ER. In the absence of an encoded signal sequence, mRNAs undergo continued translation on cytosolic ribosomes. Recent genome-wide analyses of mRNA partitioning between the cytosol and the ER compartments have identified subsets of mRNAs that are non-canonically partitioned to the ER-although lacking an encoded signal sequence, they are translated on ER-bound ribosomes. These findings suggest that multiple, and as yet unidentified, pathways exist for directing mRNA partitioning in the cell. In this contribution, we briefly review the literature describing the subcellular partitioning patterns of mRNAs and present a detailed methodology for studying this fundamental, yet poorly understood process.

Induction of the unfolded protein response drives enhanced metabolism and chemoresistance in glioma cells.

The unfolded protein response (UPR) is an endoplasmic reticulum (ER)-based cytoprotective mechanism acting to prevent pathologies accompanying protein aggregation. It is frequently active in tumors, but relatively unstudied in gliomas. We hypothesized that UPR stress effects on glioma cells might protect tumors from additional exogenous stress (ie, chemotherapeutics), postulating that protection was concurrent with altered tumor cell metabolism. Using human brain tumor cell lines, xenograft tumors, human samples and gene expression databases, we determined molecular features of glioma cell UPR induction/activation, and here report a detailed analysis of UPR transcriptional/translational/metabolic responses. Immunohistochemistry, Western and Northern blots identified elevated levels of UPR transcription factors and downstream ER chaperone targets in gliomas. Microarray profiling revealed distinct regulation of stress responses between xenograft tumors and parent cell lines, with gene ontology and network analyses linking gene expression to cell survival and metabolic processes. Human glioma samples were examined for levels of the ER chaperone GRP94 by immunohistochemistry and for other UPR components by Western blotting. Gene and protein expression data from patient gliomas correlated poor patient prognoses with increased expression of ER chaperones, UPR target genes, and metabolic enzymes (glycolysis and lipogenesis). NMR-based metabolomic studies revealed increased metabolic outputs in glucose uptake with elevated glycolytic activity as well as increased phospholipid turnover. Elevated levels of amino acids, antioxidants, and cholesterol were also evident upon UPR stress; in particular, recurrent tumors had overall higher lipid outputs and elevated specific UPR arms. Clonogenicity studies following temozolomide treatment of stressed or unstressed cells demonstrated UPR-induced chemoresistance. Our data characterize the UPR in glioma cells and human tumors, and link the UPR to chemoresistance possibly via enhanced metabolism. Given the role of the UPR in the balance between cell survival and apoptosis, targeting the UPR and/or controlling metabolic activity may prove beneficial for malignant glioma therapeutics.

Distinct and Overlapping Sarcoma Subtypes Initiated from Muscle Stem and Progenitor Cells

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, whereas undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue sarcomas diagnosed in adults. To investigate the myogenic cell(s) of origin of these sarcomas, we used Pax7-CreER and MyoD-CreER mice to transform Pax7(+) and MyoD(+) myogenic progenitors by expressing oncogenic Kras(G12D) and deleting Trp53 in vivo. Pax7-CreER mice developed RMS and UPS, whereas MyoD-CreER mice developed UPS. Using gene set enrichment analysis, RMS and UPS each clustered specifically within their human counterparts. These results suggest that RMS and UPS have distinct and overlapping cells of origin within the muscle lineage. Taking them together, we have established mouse models of soft tissue sarcoma from muscle stem and progenitor cells.

Animal models of soft-tissue sarcoma

Soft-tissue sarcomas (STSs) are rare mesenchymal tumors that arise from muscle, fat and connective tissue. Currently, over 75 subtypes of STS are recognized. The rarity and heterogeneity of patient samples complicate clinical investigations into sarcoma biology. Model organisms might provide traction to our understanding and treatment of the disease. Over the past 10 years, many successful animal models of STS have been developed, primarily genetically engineered mice and zebrafish. These models are useful for studying the relevant oncogenes, signaling pathways and other cell changes involved in generating STSs. Recently, these model systems have become preclinical platforms in which to evaluate new drugs and treatment regimens. Thus, animal models are useful surrogates for understanding STS disease susceptibility and pathogenesis as well as for testing potential therapeutic strategies.

Myogenic transcription factors regulate pro-metastatic MIR-182

Approximately 30% of patients with soft-tissue sarcoma die from pulmonary metastases. The mechanisms that drive sarcoma metastasis are not well understood. Recently, we identified miR-182 as a driver of sarcoma metastasis in a primary mouse model of soft-tissue sarcoma. We also observed elevated miR-182 in a subset of primary human sarcomas that metastasized to the lungs. Here, we show that myogenic differentiation factors regulate miR-182 levels to contribute to metastasis in mouse models. We find that MyoD directly binds the miR-182 promoter to increase miR-182 expression. Furthermore, mechanistic studies revealed that Pax7 can promote sarcoma metastasis in vivo through MyoD-dependent regulation of pro-metastatic miR-182. Taken together, these results suggest that sarcoma metastasis can be partially controlled through Pax7/MyoD-dependent activation of miR-182 and provide insight into the role that myogenic transcription factors have in sarcoma progression.