Molecular studies of genetic changes in myxoid and round cell liposarcoma

Chromosomal translocations commonly result in the production of fusion genes and the fusion genes are often tumor-type specific. In myxoid and round cell liposarcomas (MLS/RCLS), almost 95% of the cases carry a t(12;16)(q13;p11). In the remaining 5% of the MLS/RCLS tumors, another translocation and fusion gene can be found, i.e. the t(12;22)(q13;q12). These translocations fuse CHOP on chromosome 12 to either TLS on chromosome 16 or EWSR1 on chromosome 22. The result is a TLS-CHOP or EWS-CHOP fusion protein that is believed to be a key player in the development of MLS/RCLS. Normally, CHOP plays a fundamental role in adipogenesis by forming heterodimers with members of the C/EBP family of transcription factors. TLS-CHOP is capable of dimerizing with the same proteins as CHOP, but the outcome is radically different. This thesis focuses on TLS-CHOP and the hypothesis that TLS-CHOP may function as an aberrant transcription factor. As such TLS-CHOP is believed to regulate the expression of a cascade of downstream genes that contribute to the phenotype and malignancy of MLS/RCLS. We have found that the TLS-CHOP fusion protein localizes to distinct nuclear structures which differs largely from the normal TLS and CHOP patterns. The nuclear structures do not overlap with PML nuclear bodies and furthermore, PML nuclear bodies are dislocated in TLS-CHOP-expressing cells. The abnormal localization of the PML tumor suppressor may be an important part of the transforming process in these tumors. Using a subtraction hybridization technique, we have found that several genes are aberrantly expressed in MLS/RCLS compared to normal adipose tissue and may have a function in the development of these tumors. This work resulted in the identification of a novel gene which seems to be associated with a low degree of differentiation. At present the normal function of this protein, LEPREL1, is not known. However, its different domains and intracellular distribution points in the direction that this protein functions as an enzyme in the ER and Golgi pathway. One of the genes identified as over-expressed in MLS/RCLS was the RET proto-oncogene. Further studies showed that the tumor cells produce the activating ligand persephin and that the receptor is phosphorylated. This indicates that it is activated and capable of signaling proliferation and anti-apoptosis into the cell nucleus. Thus, there seems to be an autocrine growth stimulatory loop running in MLS/RCLS tumors and disrupting the RET signaling pathway may be an attractive target for therapy in MLS/RCLS

Oncostatin M signaling in human glioma cell lines

We have recently found that oncostatin M (OSM) is overexpressed in most human brain tumors. The effects of OSM are unclear with conflicting reports of growth stimulatory or inhibitory effects in various cell types. The aim of this study was to investigate the effects of OSM in 5 glioma cell lines and 7 short-term cultures of human gliomas and in normal cultured human astrocytes. None of the cell lines and short-term cultured tumor cells expressed OSM in vitro. OSM signals through a gp130 containing receptor complex over the JAK/STAT pathway. Immunofluorescence and RT-PCR analysis showed that the tumor cells express gp130 and the other receptor components, LIFR beta and OSMR beta. OSM treatment induced phosphorylation of STAT3 and STAT1 indicating presence of a functional JAK/STAT pathway. No OSM effect on proliferation was observed. OSM gave no protective effects against tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced cytotoxicity

Enzymatic inactivation of endogenous IgG by IdeS enhances therapeutic antibody efficacy

Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells

LEPREL1, a novel ER and Golgi resident member of the Leprecan family.

We have identified a novel protein, Leprecan-like 1 (LEPREL1), with profound similarity to the Leprecan family of proteoglycans. The genomic organization of the Leprecan gene family was found to be highly conserved. Expression analysis shows that LEPREL1 is expressed in most tissues as a 3.4 kb transcript encoding an 80 kDa protein. A LEPREL1 specific antibody stains many cell types including adipocytes and neuroendocrine cells of the gastrointestinal epithelium. Muscle tissue contains a specific 6.5 kb transcript and a 200 kDa protein. The 3.4 kb LEPREL1 transcript encodes a 708 amino acid protein containing a signal sequence, four tetratricopeptide repeats (TPRs), a leucine zipper, a P-loop, a prolyl 4-hydroxylase α domain (P4Hα), and a C-terminal KDEL ER-retention motif. LEPREL1 is localized to the ER and Golgi network and over-expressing it affects normal protein disulfide isomerase staining patterns in the ER

Streptococcus pyogenes Infection and the Human Proteome with a Special Focus on the Immunoglobulin G-cleaving Enzyme IdeS

Infectious diseases are characterized by a complex interplay between host and pathogen, but how these interactions impact the host proteome is unclear. Here we applied a combined mass spectrometry-based proteomics strategy to investigate how the human proteome is transiently modified by the pathogen Streptococcus pyogenes, with a particular focus on bacterial cleavage of IgG in vivo. In invasive diseases, S. pyogenes evokes a massive host response in blood, whereas superficial diseases are characterized by a local leakage of several blood plasma proteins at the site of infection including IgG. S. pyogenes produces IdeS, a protease cleaving IgG in the lower hinge region and we find highly effective IdeS-cleavage of IgG in samples from local IgG poor microenvironments. The results show that IdeS contributes to the adaptation of S. pyogenes to its normal ecological niches. Additionally, the work identifies novel clinical opportunities for in vivo pathogen detection

The Myxoid/Round Cell Liposarcoma Fusion Oncogene FUS-DDIT3 and the Normal DDIT3 Induce a Liposarcoma Phenotype in Transfected Human Fibrosarcoma Cells

Myxoid/round cell liposarcoma (MLS/RCLS) is the most common subtype of liposarcoma. Most MLS/RCLS carry a t(12;16) translocation, resulting in a FUS-DDIT3 fusion gene. We investigated the role of the FUS-DDIT3 fusion in the development of MLS/RCLS in FUS-DDIT3- and DDIT3-transfected human HT1080 sarcoma cells. Cells expressing FUS-DDIT3 and DDIT3 grew as liposarcomas in severe combined immunodeficient mice and exhibited a capillary network morphology that was similar to networks of MLS/RCLS. Microarray-based comparison of HT1080, the transfected cells, and an MLS/RCLS-derived cell line showed that the FUS-DDIT3- and DDIT3-transfected variants shifted toward an MLS/RCLS-like expression pattern. DDIT3-transfected cells responded in vitro to adipogenic factors by accumulation of fat and transformation to a lipoblast-like morphology. In conclusion, because the fusion oncogene FUS-DDIT3 and the normal DDIT3 induce a liposarcoma phenotype when expressed in a primitive sarcoma cell line, MLS/RCLS may develop from cell types other than preadipocytes. This may explain the preferential occurrence of MLS/RCLS in nonadipose tissues. In addition, development of lipoblasts and the typical MLS/RCLS capillary network could be an effect of the DDIT3 transcription factor partner of the fusion oncogene