In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding secretory/membrane proteins are translated on endoplasmic reticulum (ER)-bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on free ribosomes. mRNA partitioning between the two compartments occurs via positive selection: free ribosomes engaged in the translation of signal sequence-encoding mRNAs are trafficked from the cytosol to the ER. After translation termination, ER-bound ribosomes are thought to dissociate, thereby completing a cycle of mRNA partitioning. At present, the physiological basis for termination-coupled ribosome release is unknown. To gain insight into this process, we examined ribosome and mRNA partitioning during the unfolded protein response, key elements of which include suppression of the initiation stage of protein synthesis and polyribosome breakdown. We report that unfolded protein response (UPR)-elicited polyribosome breakdown resulted in the continued association, rather than release, of ER-bound ribosomes. Under these conditions, mRNA translation in the cytosol was suppressed, whereas mRNA translation on the ER was sustained. Furthermore, mRNAs encoding key soluble stress proteins (XBP-1 and ATF-4) were translated primarily on ER-bound ribosomes. These studies demonstrate that ribosome release from the ER is termination independent and identify new and unexpected roles for the ER compartment in the translational response to induction of the unfolded protein response.

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 NF1^flox/flox; Ink4a/Arf^flox/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.

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.

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.

Genetically engineered mouse models that employ site-specific recombinase technology are important tools for cancer research but can be costly and time-consuming. The CRISPR-Cas9 system has been adapted to generate autochthonous tumours in mice, but how these tumours compare to tumours generated by conventional recombinase technology remains to be fully explored. Here we use CRISPR-Cas9 to generate multiple subtypes of primary sarcomas efficiently in wild type and genetically engineered mice. These data demonstrate that CRISPR-Cas9 can be used to generate multiple subtypes of soft tissue sarcomas in mice. Primary sarcomas generated with CRISPR-Cas9 and Cre recombinase technology had similar histology, growth kinetics, copy number variation and mutational load as assessed by whole exome sequencing. These results show that sarcomas generated with CRISPR-Cas9 technology are similar to sarcomas generated with conventional modelling techniques and suggest that CRISPR-Cas9 can be used to more rapidly generate genotypically and phenotypically similar cancers.

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.