The Oncolytic Activity of Myxoma Virus against Soft Tissue Sarcoma Is Mediated by the Overexpression of Ribonucleotide Reductase.

BACKGROUND: Myxoma virus (MYXV) is an oncolytic poxvirus that lacks the gene for 1 of the subunits of ribonucleotide reductase (RR), a crucial DNA synthesis and repair enzyme. The overexpression of RR has been implicated in the invasiveness of several cancers, including soft tissue sarcomas (STS). The purpose of the study was to investigate the oncolytic efficacy of MYXV in STS with different levels of RR expression. METHODS: The oncolytic effect of recombinant MYXV was evaluated in 4 human STS cell lines, LS141 (a dedifferentiated liposarcoma), DDLS8817 (a dedifferentiated liposarcoma), RDD2213 (recurrent dedifferentiated liposarcoma), and HSSYII (a synovial sarcoma) using infectivity and cytotoxicity assays. Following the overexpression of RRM2 by cDNA transfection and silencing of RRM2 by siRRM2 in these STS cell lines, the RRM2 expression levels were analyzed by Western blot. RESULTS: We observed a direct correlation between viral oncolysis and RRM2 mRNA levels (R = 0.96) in STS. Higher RRM2 expression was associated with a more robust cell kill. Silencing the RRM2 gene led to significantly greater cell survival (80%) compared with the control group (P = .003), whereas overexpression of the RRM2 increased viral oncolysis by 33% (P < .001). CONCLUSIONS: Our results show that the oncolytic effects of MYXV correlate directly with RR expression levels and are enhanced in STS cell lines with naturally occurring or artificially induced high expression levels of RR. Myxoma virus holds promise in the treatment of advanced soft tissue cancer, especially in tumors overexpressing RR.

Copy number losses define subgroups of dedifferentiated liposarcoma with poor prognosis and genomic instability.

PURPOSE: Molecular events underlying progression of well-differentiated liposarcoma (WDLS) to dedifferentiated liposarcoma (DDLS) are poorly defined. This study sought to identify copy number alterations (CNA) associated with dedifferentiation of WDLS, with DDLS morphology, and with patient outcomes. EXPERIMENTAL DESIGN: Fifty-five WDLS and 52 DDLS were analyzed using Agilent 244K comparative genomic hybridization and Affymetrix U133A expression arrays. CNAs were identified by RAE analysis. Thirty-nine of the DDLS specimens were categorized morphologically by a single pathologist. RESULTS: Nine regions of CNA were identified as recurrent in DDLS but not WDLS; 79% of DDLS had at least one of these CNAs. Loss of the chromosome segment 11q23-24, the most common event, was observed only in DDLS that morphologically resembled the genomically complex sarcomas, undifferentiated pleomorphic sarcoma and myxofibrosarcoma. 11q23-24 loss was itself associated with increased genomic complexity in DDLS. Loss of 19q13, but not 11q23-24, was associated with poor prognosis. Median disease-specific survival was shorter for patients with19q13 loss (27 months) than for patients with diploid 19q13 (>90 months; P < 0.0025), and 19q13 loss was associated with local recurrence (HR, 2.86; P = 0.013). Common copy number losses were associated with transcriptional downregulation of potential tumor suppressors and adipogenesis-related genes (e.g., EI24 and CEBPA). CONCLUSIONS: Dedifferentiation of WDLS is associated with recurrent CNAs in 79% of tumors. In DDLS, loss of 11q23-24 is associated with genomic complexity and distinct morphology whereas loss of 19q13 predicts poor prognosis. CNAs in liposarcoma improve risk stratification for patients and will help identify potential tumor suppressors driving liposarcoma progression.

Restoration of C/EBPα in dedifferentiated liposarcoma induces G2/M cell cycle arrest and apoptosis.

Well-differentiated liposarcoma (WDLS) and dedifferentiated liposarcoma (DDLS) represent the most common biological group of liposarcoma, and there is a pressing need to develop targeted therapies for patients with advanced disease. To identify potential therapeutic targets, we sought to identify differences in the adipogenic pathways between DDLS, WDLS, and normal adipose tissue. In a microarray analysis of DDLS (n = 84), WDLS (n = 79), and normal fat (n = 23), C/EBPα, a transcription factor involved in cell cycle regulation and differentiation, was underexpressed in DDLS when compared to both WDLS and normal fat (15.2- and 27.8-fold, respectively). In normal adipose-derived stem cells, C/EBPα expression was strongly induced when cells were cultured in differentiation media, but in three DDLS cell lines, this induction was nearly absent. We restored C/EBPα expression in one of the cell lines (DDLS8817) by transfection of an inducible C/EBPα expression vector. Inducing C/EBPα expression reduced proliferation and caused cells to accumulate in G2/M. Under differentiation conditions, the cell proliferation was reduced further, and 66% of the DDLS cells containing the inducible C/EBPα expression vector underwent apoptosis as demonstrated by annexin V staining. These cells in differentiation conditions expressed early adipocyte-specific mRNAs such as LPL and FABP4, but they failed to accumulate intracellular lipid droplets, a characteristic of mature adipocytes. These results demonstrate that loss of C/EBPα is an important factor in suppressing apoptosis and maintaining the dedifferentiated state in DDLS. Restoring C/EBPα may be a useful therapeutic approach for DDLS.

Small RNA sequencing and functional characterization reveals MicroRNA-143 tumor suppressor activity in liposarcoma.

Liposarcoma remains the most common mesenchymal cancer, with a mortality rate of 60% among patients with this disease. To address the present lack of therapeutic options, we embarked upon a study of microRNA (miRNA) expression alterations associated with liposarcomagenesis with the goal of exploiting differentially expressed miRNAs and the gene products they regulate as potential therapeutic targets. MicroRNA expression was profiled in samples of normal adipose tissue, well-differentiated liposarcoma, and dedifferentiated liposarcoma by both deep sequencing of small RNA libraries and hybridization-based Agilent microarrays. The expression profiles discriminated liposarcoma from normal adipose tissue and well differentiated from dedifferentiated disease. We defined over 40 miRNAs that were dysregulated in dedifferentiated liposarcomas in both the sequencing and the microarray analysis. The upregulated miRNAs included two cancer-associated species (miR-21 and miR-26a), and the downregulated miRNAs included two species that were highly abundant in adipose tissue (miR-143 and miR-145). Restoring miR-143 expression in dedifferentiated liposarcoma cells inhibited proliferation, induced apoptosis, and decreased expression of BCL2, topoisomerase 2A, protein regulator of cytokinesis 1 (PRC1), and polo-like kinase 1 (PLK1). The downregulation of PRC1 and its docking partner PLK1 suggests that miR-143 inhibits cytokinesis in these cells. In support of this idea, treatment with a PLK1 inhibitor potently induced G(2)-M growth arrest and apoptosis in liposarcoma cells. Taken together, our findings suggest that miR-143 re-expression vectors or selective agents directed at miR-143 or its targets may have therapeutic value in dedifferentiated liposarcoma.

Expression profiling of liposarcoma yields a multigene predictor of patient outcome and identifies genes that contribute to liposarcomagenesis.

Liposarcomas are the most common type of soft tissue sarcoma but their genetics are poorly defined. To identify genes that contribute to liposarcomagenesis and serve as prognostic candidates, we undertook expression profiling of 140 primary liposarcoma samples, which were randomly split into training set (n = 95) and test set (n = 45). A multigene predictor for distant recurrence-free survival (DRFS) was developed by the supervised principal component method. Expression levels of the 588 genes in the predictor were used to calculate a risk score for each patient. In validation of the predictor in the test set, patients with low risk score had a 3-year DRFS of 83% versus 45% for high risk score patients (P = 0.001). The HR for high versus low score, adjusted for histologic subtype, was 4.42 (95% CI, 1.26-15.55; P = 0.021). The concordance probability for risk score was 0.732. In contrast, the concordance probability for histologic subtype, which had been considered the best predictor of outcome in liposarcoma, was 0.669. Genes related to adipogenesis, DNA replication, mitosis, and spindle assembly checkpoint control were all highly represented in the multigene predictor. Three genes from the predictor, TOP2A, PTK7, and CHEK1, were found to be overexpressed in liposarcoma samples of all five subtypes and in liposarcoma cell lines. RNAi-mediated knockdown of these genes in liposarcoma cell lines reduced proliferation and invasiveness and increased apoptosis. Taken together, our findings identify genes that seem to be involved in liposarcomagenesis and have promise as therapeutic targets, and support the use of this multigene predictor to improve risk stratification for individual patients with liposarcoma.

mRNA and protein levels of FUS, EWSR1, and TAF15 are upregulated in liposarcoma.

Translocations or mutations of FUS, EWSR1, and TAF15 (FET) result in distinct genetic diseases. N-terminal translocations of any FET protein to a series of transcription factors yields chimeric proteins that contribute to sarcomagenesis, whereas mutations in the conserved COOH-terminal domain of wild-type FUS were recently shown to cause familial amyotrophic lateral sclerosis. We thus investigated whether the loss of one FUS allele by translocation in liposarcoma may be followed by mutations in either the remaining FUS allele or the paralogous EWSR1. Furthermore, we investigated the strength of the FET promoters and their contributions to sarcomagenesis given the proteins' frequent involvement in oncogenic translocations. We sequenced the respective genomic regions of both FUS and EWSR1 in 96 liposarcoma samples. Additionally, we determined FET transcript and protein levels in several liposarcoma cell lines. We did not observe sequence variations in either FUS or EWSR1. However, protein copy numbers reached an impressive 0.9 and 5.5 Mio of FUS and EWSR1 per tumor cell, respectively. Compared with adipose-derived stem cells, FUS and EWSR1 protein expression levels were elevated on average 28.6-fold and 7.3-fold, respectively. TAF15 mRNA levels were elevated on average 3.9-fold, although with a larger variation between samples. Interestingly, elevated TAF15 mRNA levels did not translate to strongly elevated protein levels, consistent with its infrequent occurrence as translocation partner in tumors. These results suggest that the powerful promoters of FET genes are predominantly responsible for the oncogenic effect of transcription factor translocations in sarcomas.

Frequent alterations and epigenetic silencing of differentiation pathway genes in structurally rearranged liposarcomas.

UNLABELLED: We explored diverse alterations contributing to liposarcomagenesis by sequencing the genome, exome, transcriptome, and cytosine methylome of a primary and recurrent dedifferentiated liposarcoma (DLPS) from distinct chemotherapy/radiotherapy-naïve patients. The liposarcoma genomes had complex structural rearrangements, but in different patterns, and with varied effects on the structure and expression of affected genes. While the point mutation rate was modest, integrative analyses and additional screening identified somatic mutations in HDAC1 in 8.3% of DLPS. Liposarcoma methylomes revealed alterations in differentiation pathway genes, including CEBPA methylation in 24% of DLPS. Treatment with demethylating agents, which restored CEBPA expression in DLPS cells, was anti-proliferative and pro-apoptotic in vitro and reduced tumor growth in vivo. Both genetic and epigenetic abnormalities established a role for small RNAs in liposarcomagenesis, typified by methylation-induced silencing of microRNA-193b in DLPS but not its well-differentiated counterpart. These findings reveal an unanticipated role for epigenetic abnormalities in DLPS tumors and suggest demethylating agents as potential therapeutics. SIGNIFICANCE: Multimodality sequence analysis of DLPS revealed recurrent mutations and epigenetic abnormalities critical to liposarcomagenesis and to the suppression of adipocyte differentiation. Pharmacologic inhibition of DNA methylation promoted apoptosis and differentiated DLPS cells in vitro and inhibited tumor growth in vivo, providing a rationale for investigating methylation inhibitors in this disease.

ZIC1 overexpression is oncogenic in liposarcoma.

Liposarcomas are aggressive mesenchymal cancers with poor outcomes that exhibit remarkable histologic diversity (there are five recognized subtypes). Currently, the mainstay of therapy for liposarcoma is surgical excision because liposarcomas are often resistant to traditional chemotherapy. In light of the high mortality associated with liposarcoma and the lack of effective systemic therapy, we sought novel genomic alterations driving liposarcomagenesis that might serve as therapeutic targets. ZIC1, a critical transcription factor for neuronal development, is overexpressed in all five subtypes of liposarcoma compared with normal fat, and in liposarcoma cell lines compared with adipose-derived stem cells. Here, we show that ZIC1 contributes to the pathogenesis of liposarcoma. ZIC1 knockdown inhibits proliferation, reduces invasion, and induces apoptosis in dedifferentiated and myxoid/round cell liposarcoma cell lines, but not in either adipose-derived stem cells or in a lung cancer cell line with low ZIC1 expression. ZIC1 knockdown is associated with increased nuclear expression of p27 proteins and the downregulation of prosurvival target genes BCL2L13, JunD, Fam57A, and EIF3M. Our results show that ZIC1 expression is essential for liposarcomagenesis and that targeting ZIC1 or its downstream targets might lead to novel therapy for liposarcoma.

Subtype-specific genomic alterations define new targets for soft-tissue sarcoma therapy.

Soft-tissue sarcomas, which result in approximately 10,700 diagnoses and 3,800 deaths per year in the United States, show remarkable histologic diversity, with more than 50 recognized subtypes. However, knowledge of their genomic alterations is limited. We describe an integrative analysis of DNA sequence, copy number and mRNA expression in 207 samples encompassing seven major subtypes. Frequently mutated genes included TP53 (17% of pleomorphic liposarcomas), NF1 (10.5% of myxofibrosarcomas and 8% of pleomorphic liposarcomas) and PIK3CA (18% of myxoid/round-cell liposarcomas, or MRCs). PIK3CA mutations in MRCs were associated with Akt activation and poor clinical outcomes. In myxofibrosarcomas and pleomorphic liposarcomas, we found both point mutations and genomic deletions affecting the tumor suppressor NF1. Finally, we found that short hairpin RNA (shRNA)-based knockdown of several genes amplified in dedifferentiated liposarcoma, including CDK4 and YEATS4, decreased cell proliferation. Our study yields a detailed map of molecular alterations across diverse sarcoma subtypes and suggests potential subtype-specific targets for therapy.

Functional copy-number alterations in cancer.

Understanding the molecular basis of cancer requires characterization of its genetic defects. DNA microarray technologies can provide detailed raw data about chromosomal aberrations in tumor samples. Computational analysis is needed (1) to deduce from raw array data actual amplification or deletion events for chromosomal fragments and (2) to distinguish causal chromosomal alterations from functionally neutral ones. We present a comprehensive computational approach, RAE, designed to robustly map chromosomal alterations in tumor samples and assess their functional importance in cancer. To demonstrate the methodology, we experimentally profile copy number changes in a clinically aggressive subtype of soft-tissue sarcoma, pleomorphic liposarcoma, and computationally derive a portrait of candidate oncogenic alterations and their target genes. Many affected genes are known to be involved in sarcomagenesis; others are novel, including mediators of adipocyte differentiation, and may include valuable therapeutic targets. Taken together, we present a statistically robust methodology applicable to high-resolution genomic data to assess the extent and function of copy-number alterations in cancer.

Resolution of creatine and phosphocreatine 1H signals in isolated human skeletal muscle using HR-MAS 1H NMR.

Proton NMR spectra of freshly isolated human skeletal muscle samples contain creatine and phosphocreatine resonances with distinct chemical shifts that are easily visualized with magic angle spinning (MAS, spinning the sample rapidly at 54.7 degrees with respect to the magnetic field) methods. The identification of the phosphocreatine resonance was based on two findings: that (i) the possible small dipolar coupling does not contribute to line splitting under rapid MAS, and (ii) the 1H signal decreases concurrently with the phosphocreatine resonance observed in 31P NMR experiments. In the MAS 1H spectra, the phosphocreatine resonance remains a singlet with a linewidth of less than 3 Hz. The creatine resonances are split into two peaks with linewidths at half height of approximately 2 and 6 Hz, respectively. The resonance with the broader linewidth represents creatine that is significantly motion-restricted and suggests that a creatine pool in muscle tissue is highly compartmentalized.

Gene expression profiling of liposarcoma identifies distinct biological types/subtypes and potential therapeutic targets in well-differentiated and dedifferentiated liposarcoma.

Classification of liposarcoma into three biological types encompassing five subtypes, (a) well-differentiated/dedifferentiated, (b) myxoid/round cell, and (c) pleomorphic, based on morphologic features and cytogenetic aberrations, is widely accepted. However, diagnostic discordance remains even among expert sarcoma pathologists. We sought to develop a more systematic approach to liposarcoma classification based on gene expression analysis and to identify subtype-specific differentially expressed genes that may be involved in liposarcoma genesis/progression and serve as potential therapeutic targets. A classifier based on gene expression profiling was able to distinguish between liposarcoma subtypes, lipoma, and normal fat samples. A 142-gene predictor of tissue class was derived to automatically determine the class of an independent validation set of lipomatous samples and shows the feasibility of liposarcoma classification based entirely on gene expression monitoring. Differentially expressed genes for each liposarcoma subtype compared with normal fat were used to identify histology-specific candidate genes with an in-depth analysis of signaling pathways important to liposarcoma pathogenesis and progression in the well-differentiated/dedifferentiated subset. The activation of cell cycle and checkpoint pathways in well-differentiated/dedifferentiated liposarcoma provides several possible novel therapeutic strategies with MDM2 serving as a particularly promising target. We show that Nutlin-3a, an antagonist of MDM2, preferentially induces apoptosis and growth arrest in dedifferentiated liposarcoma cells compared with normal adipocytes. These results support the development of a clinical trial with MDM2 antagonists for liposarcoma subtypes which overexpress MDM2 and show the promise of using this expression dataset for new drug discovery in liposarcoma.

Flavopiridol targets c-KIT transcription and induces apoptosis in gastrointestinal stromal tumor cells.

Gastrointestinal stromal tumors (GIST) are characterized by activating mutations in the c-KIT gene which confers ligand-independent activation of the KIT receptor. Imatinib mesylate has been shown to effectively block constitutively active KIT and delay tumor growth. However, resistance to imatinib mesylate is emerging as a major clinical problem and novel therapies are needed. We report that treatment of GIST cells with the transcriptional inhibitor flavopiridol, initially down-regulates the antiapoptotic proteins bcl-2, mcl-1, and X-linked inhibitor of apoptosis protein which occurs as early as 4 hours after exposure. This is followed at 24 hours by the transcriptional suppression of KIT resulting in poly(ADP-ribose) polymerase cleavage and apoptosis. To separate the apoptotic effect of KIT suppression relative to the down-regulation of antiapoptotic proteins, we used small interfering RNA-directed knockdown of KIT. Results show that focused suppression of KIT alone is sufficient to induce apoptosis in GIST cells, but not to the same extent as flavopiridol. In contrast, imatinib mesylate, which inhibits KIT kinase activity but does not suppress total KIT expression, fails to cause apoptosis. We also show that flavopiridol suppresses KIT mRNA expression through positive transcriptional elongation factor inhibition and decreases KIT promoter activity. This causes a global decrease in the level of functionally mature KIT at the cell surface, resulting in a decrease in autophosphorylation at tyrosine residues 703 and 721, which characterizes activated KIT. Our results indicate that targeting KIT expression and these antiapoptotic proteins with flavopiridol represents a novel means to disrupt GIST cell dependence on KIT signaling and collectively renders these cells sensitive to apoptosis.

High-resolution MAS NMR spectroscopy detection of the spin magnetization exchange by cross-relaxation and chemical exchange in intact cell lines and human tissue specimens.

High-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy detects resolved signals from membrane phospholipids and proteins in intact cell and tissue samples. MAS has the additional advantage of quenching spin-diffusion through a mutual "flip-flop" of neighbor spins by time-independent dipolar coupling as long as the dipolar coupling is "inhomogeneous." Under MAS, significant magnetization transfer (MT) was observed between water and each proton site in membrane phospholipid and between water and the NMR-observable protein proton signals. The MT rates between water and membrane phospholipids are lower than those between water and protein proton signals. The interaction of water to other small molecules is selective with the observation of MT from water to creatine, lactate, taurine, and glycine, but not to triglyceride, phosphocholine, choline, or myo-inositol. HR-MAS NMR allows the detection of a complete MT network between water and each proton group of creatine. Two creatine pools (one motion-restricted and one motion-free) were identified in skeletal muscle.

Water suppression without signal loss in HR-MAS 1H NMR of cells and tissues.

In cell and tissue samples, water is normally three orders of magnitude more abundant than other metabolites. Thus, water suppression is required in the acquisition of NMR spectra to overcome the dynamic range problem and to recover metabolites that overlap with the broad baseline of the strong water resonance. However, the heterogeneous cellular environment often complicates water suppression and the strong coupling of water to membrane lipids interferes with the NMR detection of membrane associated lipid components. The widely used water suppression techniques including presaturation and double pulsed field gradient selective echo result in more than a 70% reduction in membrane associated lipid components in proton spectra of cells and tissues compared to proton spectra acquired in the absence of water suppression. A water suppression technique based on the combination of selective excitation pulses and pulsed field gradients is proposed to use in the acquisition of high resolution MAS NMR spectra of tissue specimens and cell samples. This pulse sequence methodology enables efficient water suppression for intact cells and tissue samples and eliminates signal loss from cellular metabolites.