Frans van Valen

Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer

Resistance of tumors to treatment with cytotoxic drugs, irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of different tumor cells including Ewing tumor, neuroblastoma, malignant brain tumors and melanoma that caspase-8 expression acts as a key determinant of sensitivity for apoptosis induced by death-inducing ligands or cytotoxic drugs. In tumor cell lines resistant to TRAIL, anti-CD95 or TNFα, caspase-8 protein and mRNA expression was decreased or absent without caspase-8 gene loss. Methylation-specific PCR revealed hypermethylation of caspase-8 regulatory sequences in cells with impaired caspase-8 expression. Treatment with the demethylation agent 5-Aza-2′-deoxycytidine (5-dAzaC) reversed hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and recruitment and activation of caspase-8 at the CD95 DISC upon receptor cross-linking thereby sensitizing for death receptor-, and importantly, also for drug-induced apoptosis. Inhibition of caspase-8 activity also inhibited apoptosis sensitization by 5-dAzaC. Similar to demethylation, introduction of caspase-8 by gene transfer sensitized for apoptosis induction. Hypermethylation of caspase-8 was linked to reduced caspase-8 expression in different tumor cell lines in vitro and, most importantly, also in primary tumor samples. Thus, these findings indicate that re-expression of caspase-8, e.g. by demethylation or caspase-8 gene transfer, might be an effective strategy to restore sensitivity for chemotherapy- or death receptor-induced apoptosis in various tumors in vivo.

Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas

Telomere maintenance is regarded as a key mechanism in overcoming cellular senescence in tumor cells and in most cases is achieved by the activation of telomerase. However there is at least one alternative mechanism of telomere lengthening (ALT) which is characterized by heterogeneous and elongated telomeres in the absence of telomerase activity (TA). We evaluated the prevalence of TA, gene expression of telomerase subunits and ALT in relation to telomere morphology and function in matrix producing bone tumors and in osteosarcoma cell lines and present evidence of a direct association of ALT with telomere dysfunction and chromosomal instability. Telomere fluorescence in situ hybridization (T-FISH) in ALT cells revealed elongated and shortened telomeres, partly in unusual configurations and loci, dicentric marker chromosomes and signal-free chromosome ends. Free ends give rise to end-to-end associations and may induce breakage-fusion-bridge cycles resulting in an increased number of complex chromosomal rearrangements, as detected by multiplex-FISH (M-FISH). We propose that ALT cannot be seen as an equivalent to telomerase activity in telomere maintenance. Its association with telomere dysfunction and chromosomal instability may have major implications for tumor progression.

Expression Profiling of t(12;22) Positive Clear Cell Sarcoma of Soft Tissue Cell Lines Reveals Characteristic Up-Regulation of Potential New Marker Genes Including ERBB3

Clear cell sarcoma of soft tissue (CCSST), also known as malignant melanoma of soft parts, represents a rare lesion of the musculoskeletal system usually affecting adolescents and young adults. CCSST is typified by a chromosomal t(12;22)(q13;q12) translocation resulting in a fusion between the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1), of which the activity in nontransformed cells is regulated by cyclic AMP. Our aim was to identify critical differentially expressed genes in CCSST tumor cells in comparison with other solid tumors affecting children and young adults to better understand signaling pathways regulating specific features of the development and progression of this tumor entity. We applied Affymetrix Human Genome U95Av2 oligonucleotide microarrays representing ∼12,000 genes to generate the expression profiles of the CCSST cell lines GG-62, DTC-1, KAO, MST2, MST3, and Su-CC-S1 in comparison with 8 neuroblastoma, 7 Ewing tumor, and 6 osteosarcoma cell lines. Subsequent hierarchical clustering of microarray data clearly separated all four of the tumor types from each other and identified differentially expressed transcripts, which are characteristically up-regulated in CCSST. Statistical analysis revealed a group of 331 probe sets, representing ∼300 significant (P < 0.001) differentially regulated genes, which clearly discriminated between the CCSST and other tumor samples. Besides genes that were already known to be highly expressed in CCSST, like S100A11 (S100 protein) or MITF (microphthalmia-associated transcription factor), this group shows an obvious portion of genes that are involved in cyclic AMP response or regulation, in pigmentation processes, or in neuronal structure and signaling. Comparison with other expression profile analyses on neuroectodermal childhood tumors confirms the high robustness of this strategy to characterize tumor entities based on their gene expression. We found the avian erythroblastic leukemia viral oncogene homologue 3 (ERBB3) to be one of the most dramatically up-regulated genes in CCSST. Quantitative real-time PCR and Northern blot analysis verified the mRNA abundance and confirmed the absence of the inhibitory transcript variant of this gene. The protein product of the member of the epidermal growth factor receptor family ERBB3 could be shown to be highly present in all of the CCSST cell lines investigated, as well as in 18 of 20 primary tumor biopsies. In conclusion, our data demonstrate new aspects of the phenotype and the biological behavior of CCSST and reveal ERBB3 to be a useful diagnostic marker.

Apoptotic responsiveness of the Ewing's sarcoma family of tumours to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL).

We investigated the cytotoxic responsiveness of 40 cell lines derived from representatives of the Ewings sarcoma family of tumours (ESFT), i.e., Ewings sarcoma (ES), peripheral primitive neuroectodermal tumour (pPNET) and Askin tumour (AT), to tumour necrosis factor–related apoptosis‐inducing ligand (TRAIL). Incubation with TRAIL at 100 ng/ml induced cell death at 24 hr in 19 of 26 ES, 11 of 12 pPNET and 2 of 2 AT cell lines. Half‐maximal cell death concentrations (IC50 values) varied from 0.1 to 20 ng/ml. TRAIL displayed potent cytotoxic activity against freshly derived ESFT cell isolates. Cytotoxicity was associated with phosphatidylserine expression and internucleosomal DNA fragmentation, features characteristic of apoptosis. The apoptotic programme in the sensitive ESFT VH‐64 cell line revealed TRAIL‐induced activation of FLICE/MACH1 (caspase‐8) and CPP32/Yama/apopain (caspase‐3) and processing of the prototype caspase substrate poly(ADP‐ribose) polymerase. In addition, TRAIL provoked a collapse of the mitochondrial transmembrane potential (ΔΨm), parallelled by a reduction in ATP levels and release of cytochrome c from mitochondria into the cytosol. Inhibition of caspase‐8 and caspase‐3 by zIETDfmk and zDEVDfmk, respectively, substantially prevented TRAIL‐induced apoptosis. However, zIETDfmk, but not zDEVDfmk, reduced TRAIL‐mediated ΔΨm dissipation, indicating that TRAIL causes mitochondrial dysfunction through caspase‐8 acting upstream of mitochondria. While macromolecule synthesis inhibitors (actinomycin D, cycloheximide) augmented susceptibility to TRAIL in TRAIL‐responsive cell lines, these agents did not render TRAIL‐resistant cell lines susceptible to TRAIL. However, the proteasome inhibitor MG132 sensitised to TRAIL in resistant cell lines. Collectively, these results show that TRAIL initiates effective death in the vast majority (80%) of cell lines derived from ESFT. Since TRAIL provoked cell death in ESFT ex vivo, this cytokine may be a promising drug for the treatment of ESFT in vivo. Int. J. Cancer 88:252–259, 2000.

Selective and nonselective toxicity of TRAIL/Apo2L combined with chemotherapy in human bone tumour cells vs. normal human cells

Although TRAIL/Apo2L preferably induces apoptosis in tumour cells without toxicity in normal cells, many tumour cell types display TRAIL/Apo2L resistance. Whether TRAIL/Apo2L in combination with chemotherapy may overcome TRAIL/Apo2L resistance while maintaining tumour selectivity remains to be determined. Here, we report that while ActD, DOX and CDDP sensitised both OS and Ewings tumour cell lines and normal cells (hOBs, synovial cells, fibroblasts) to TRAIL/Apo2L‐induced apoptosis, the combination of etoposide (VP16) and TRAIL/Apo2L was selectively active on tumour cells without affecting normal cells. Sensitisation of OS cells and hOBs to TRAIL/Apo2L did not correlate with a compatible change in the gene expression profile of the receptors for TRAIL/Apo2L determined by quantitative real‐time RT‐PCR. Also, sensitisation of the TRAIL/Apo2L death pathway did not rely entirely on the chemotherapy‐induced, caspase‐dependent cytotoxicity. Further, chemotherapy did not cause a compatible change in expression levels of proteins such as Bcl‐2, Bcl‐xL, Bax, cIAP2, XIAP and survivin. However, ActD, DOX and CDDP downregulated expression of cFLIP in OS cells as well as expression of p21 in normal hOBs. Interestingly, while VP16 also extinguished cFLIP in OS cells, which were sensitised for TRAIL/Apo2L by VP16, VP16 induced cFLIP and enhanced p21 levels in normal hOBs, which remained refractory to VP16 plus TRAIL/Apo2L. Together, our data reveal that TRAIL/Apo2L combined with certain chemotherapeutic drugs is toxic to bone tumour and normal human cells and suggest that cotreatment with TRAIL/Apo2L and VP16 provides an attractive approach for selective killing of tumour cells while leaving unaffected normal cells.

Ewing’s sarcoma and peripheral primitive neuroectodermal tumor cells produce large quantities of bioactive tumor necrosis factor-α (tnf-α) after radiation exposure

Abstract Purpose In the present study, we examined human Ewing’s sarcoma (ES) and peripheral primitive neuroectodermal tumor (pPNET) cell lines that are able to produce TNF-α as part of the response to irradiation. Radiation-induced tumor cell production of TNF-α may enhance irradiation efficacy and improve the effect of local tumor irradiation. On the other hand, radiation-induced tumor cell production of TNF-α may adversely affect the normal tissue. Methods and materials Twelve different ES/pPNET cell lines were investigated in vitro and (after establishment as tumor xenografts in athymic nude mice) in vivo for their TNF-α mRNA expression (real-time quantitative reverse transcriptase polymerase chain reaction) and TNF-α protein production ( in vitro : enhanced amplified sensitivity immunoassay; in vivo : immunohistochemistry) after exposure to different irradiation doses (2, 5, 10, 20, 30, or 40 Gy) and after different time intervals (1, 3, 6, 12, 24, 48, or 72 h after irradiation). The bioactivity of the TNF-α protein was evaluated in chromogenic cytotoxicity and neutralization assays. Results Nine out of 12 ES/pPNET cell lines express constitutively significant quantities of bioactive TNF-α in vitro . ES/pPNET cells originating from primary tumors secreted higher TNF-α levels than cells derived from metastatic lesions. In 5 of the 9 TNF-α–producing cell lines, TNF-α mRNA and protein levels were upregulated after irradiation exposure in a time- and dose-dependent manner. After establishment of the ES/pPNET cell lines in athymic nude mice, the radiation-induced TNF-α release could be demonstrated also in the xenograft tumors in vivo (analogous to the in vitro experiments). Using the same methods for quantitative analysis, it was determined that the TNF-α expression of the radiation-responsive tumor cells was up to 2000-fold higher compared to the maximal radiation-induced TNF-α release in normal lung tissue measured during the pneumonic phase. Conclusion Certain ES/pPNET cell lines produce extremely large quantities of bioactive TNF-α after radiation exposure in a time- and dose-dependent manner. Radiation-induced TNF-α production of tumor cells may be of paramount importance in respect to not only tumor behavior, but also to potential damage to normal tissue and the clinical status of the host.

Establishment of an In Vivo Model for Pediatric Ewing Tumors by Transplantation into NOD/ scid Mice

Ewing tumors are a clinically heterogeneous group of childhood sarcomas that represent a paradigm for understanding solid tumor biology, as they are the first group of sarcomas for which a chromosome translocation has been characterized at the molecular level. However, the biologic organization of the tumor, especially the processes that govern proliferation, differentiation, and metastasis of primitive tumor stem cells is poorly understood. Therefore, to develop a biologically relevant in vivo model, five different Ewing tumor cell lines and primary tumor cells from three patients were transplanted into immune-deficient mice via intravenous injection. NOD/ scid mice that carry a complex immune deficiency and thus nearly completely lack the ability to reject human cells were used as recipients. Overall, 26 of 52 mice (50%) transplanted with VH-64, WE-68, CADO-ES1, TC-71, and RM-82 cells and 4 of 10 mice (40%) transplanted with primary tumor cells engrafted. Moreover, primary cells that did not grow in vitro proliferated in mice. The pattern of metastasis was similar to that in patients with frequent metastases in lungs (62%), bone marrow (30%), and bone (23%). Using limiting dilution experiments, the frequency of the engraftment unit was estimated at 1 Ewing tumor-initiating cell in 3 × 105 VH-64 cells. These data demonstrate that we have been able to establish an in vivo model that recapitulates many aspects of growth and progression of human Ewing tumors. For the first time, this model provides the opportunity to identify and characterize primitive in vivo clonogenic solid tumor stem cells. This model will, therefore, be instrumental in studying many aspects of tumor cell biology, including organ-selective metastasis and tumor angiogenesis.

Characterization of the malignant melanoma of soft-parts cell line GG-62 by expression analysis using DNA microarrays

Abstract. GG-62 is a cell line previously thought to be derived from an atypical Ewing tumor (ET). Reverse-transcriptase polymerase chain reaction revealed an in-frame fusion between the Ewing sarcoma gene (EWS) codon 325 and the activating transcription factor 1 gene (ATF1) codon 65 which permits the production of chimeric EWS-ATF1 oncoproteins. We also identified the genomic breakpoint resulting from a reciprocal t(12;22)(q13;q12), which is the hallmark of malignant melanoma of soft parts (MMSP). We applied Affymetrix human cancer G110 arrays to compare the gene expression patterns of GG-62 and other cell lines derived from small blue round cell tumors of childhood. Hierarchical clustering of 463 differentially expressed genes distinguished GG-62 from the ETs, as well as the neuroblastomas, and revealed a cluster of 36 upregulated genes. Several of these genes are involved in signal transduction pathways that may be critical for maintaining cell transformation; some examples are avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3), neuregulin 1 (NRG1), fibroblast growth factor 9 (FGF9), and fibroblast growth factor receptor-1 (FGFR1). Furthermore, genes near the chromosome-12q13 breakpoint exhibited increased expression of GG-62 including ERBB3, NR4A1 (nuclear receptor subfamily 4, group A, member 1), cyclin-dependent kinase 2 (CDK2), and alpha 5 integrin (ITGA5). Altogether our findings demonstrate the MMSP derivation of GG-62 and may shed light on the mechanisms of tumorigenesis in this rare disease.

EWS-FLI-1-Targeted Cytotoxic T-cell Killing of Multiple Tumor Types Belonging to the Ewing Sarcoma Family of Tumors

Purpose: The Ewing sarcoma family of tumors (ESFT) comprises a group of aggressive, malignant bone, and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types. Design: Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of cytotoxic T-lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro. Results: EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing sarcoma, pPNET, Askins Tumor, and Biphenotypic sarcoma. Stimulation of human peripheral blood mononuclear cells with YLNPSVDSV peptide resulted in potent CTL-killing. Conclusions: These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development. Clin Cancer Res; 18(19); 5341–51. ©2012 AACR.

Adhesion of osteosarcoma cells to the 70-kDa core region of thrombospondin-1 is mediated by the α4β1 integrin

Abstract Thrombospondin-1 (TSP-1) is an extracellular glycoprotein that is involved in a variety of physiological processes such as tumor cell adhesion, invasion, and metastasis. It has been hypothesized that TSP-1 provides an adhesive matrix for osteosarcoma cells. Here we present data showing that TSP-1 can promote cell substrate adhesion to U2OS and SAOS cells through the α4β1 integrin. The dose-dependent adhesion to TSP-1 was inhibited by anti-integrin antibodies directed against the α4 or β1 subunit, but not by control antibodies against other integrins. To localize the potential α4β1-binding site within the TSP-1 molecule, the protein was subjected to limited proteolysis with chymotrypsin in the absence of calcium. The stable 70-kDa core fragment produced under these conditions promoted α4β1-dependent osteosarcoma cell adhesion in a manner similar to that of the intact protein. Moreover adhesion experiments with neutralizing antibodies revealed that the adhesion was totally dependent on the α4β1 interaction. Further blocking experiments with potential inhibitory peptides revealed that the α4β1-mediated adhesion was not influenced by peptides containing the RGD sequence. Attachment to the 70-kDa fragment was strongly inhibited by the CS-1 peptide, which represents the most active recognition domain for α4β1 integrin in fibronectin. The present data provide evidence that TSP-1 contains an α4β1 integrin-binding site within the 70-kDa core region.