Current diagnostics and treatment of fibrosarcoma -perspectives for future therapeutic targets and strategies.

Adult-type fibrosarcoma is a rare and highly aggressive subtype of soft tissue sarcomas. Due to the existence of other spindle-cell shaped sarcomas, its diagnosis is always one of exclusion. The likelihood of misdiagnoses between similar tumour entities is high, and often leads to inappropriate tumour treatment. We summarize here the main features of fibrosarcoma. When fibrosarcoma is appropriately diagnosed, the patient`s overall prognosis is generally quite poor. Fibrosarcoma is characterized by its low sensitivity towards radio- and chemotherapy as well as by its high rate of tumour recurrences. Thus it is important to identify new methods to improve treatment of this tumour entity. We discuss some promising new directions in fibrosarcoma research, specifically focusing on more effective targeting of the tumour microenvironment. Communication between tumour cells and their surrounding stromal tissue play a crucial role in cancer progression, invasion, metastasis and chemosensitivity. The therapeutic potential of targeting the tumour microenvironment is addressed.

Treatment of dermal fibroblasts with GPI-anchored human TIMP-1 protein moderates processes linked to scar formation.

Tissue inhibitors of metalloproteinases exhibit diverse physiological/biological functions including moderation of the proteolytic processing of growth factors and turnover of extracellular matrix. These various biological activities are linked in part to the stoichiometry of tissue inhibitor of metalloprotein/matrix metalloprotein (TIMP/MMP)/surface protein interactions. TIMP-1, a secreted protein, can be detected on the cell surface only through its interaction with surface-bound proteins. Proteins anchored by glycosylphosphatidylinositol (GPI), when purified and added to cells or tissues, are efficiently incorporated into their surface membranes. A GPI anchor was fused to TIMP-1 to focus defined concentrations of the inhibitory protein independently on the surface of primary dermal fibroblast cells. Exogenously added recombinant TIMP-1-GPI effectively inserted into the cell membrane of fibroblasts blocked the secretion of MMPs and markedly altered the stoichiometry of MMP association with the cell surface. TIMP-1-GPI treatment resulted in inhibition of fibroblast-reduced proliferation, and transiently reduced expression of fibrosis-associated genes. These effects were dose dependent. Treated cells also showed a more proapoptotic phenotype based on apoptotic assays and western blot analysis for apoptosis-associated protein expression. GPI-anchored TIMP-1 may represent a more effective version of the protein for use in therapeutic approaches to help control fibrosis and scar formation.

Cell surface engineering of renal cell carcinoma with glycosylphosphatidylinositol-anchored TIMP-1 blocks TGF- ß 1 activation and reduces regulatory ID gene expression.

Tissue inhibitor of metalloproteinase 1 (TIMP-1) controls matrix metalloproteinase activity through 1:1stoichiometric binding. Human TIMP-1 fused to a glycosylphosphatidylinositol(GPI) anchor (TIMP-1 - GPI) shifts the activity of TIMP-1 from the extracellular matrix to the cell surface. TIMP-1 - GPI treated renal cell carcinoma cells show increased apoptosis and reduced proliferation.Transcriptomic profiling and regulatory pathway mapping were used to identify the potential mechanisms driving these effects. Significant changes in the DNA binding inhibitors, TGF- ß 1/SMAD and BMP pathways resulted from TIMP-1 - GPI treatment. These events were linked to reduced TGF- ß 1 signaling mediated by inhibition of proteolytic processing of latent TGF- ß 1 by TIMP-1 - GPI.

Peritumoral administration of GPI-anchored TIMP-1 inhibits colon carcinoma growth in Rag-2 gamma chain-deficient mice.

Exogenous application of recombinant TIMP-1 protein modified by addition of a glycosylphosphatidylinositol (GPI) anchor allows efficient insertion of the fusion protein into cell membranes. This 'cell surface engineering' leads to changes in the proteolytic environment. TIMP-1-GPI shows enhanced as well as novel in vitro biological activities including suppression of proliferation, reduced migration, and inhibition of invasion of the colon carcinoma cell line SW480. Treatment of SW480 tumors implanted in Rag (-/-) common gamma chain (-/-) C57BL/6 mice with peritumorally applied TIMP-1-GPI, control rhTIMP-1 protein, or vehicle shows that TIMP-1-GPI leads to a significant reduction in tumor growth.

TIMP-1-GPI in combination with hyperthermic treatment of melanoma increases sensitivity to FAS-mediated apoptosis.

Resistance to apoptosis is a prominent feature of malignant melanoma. Hyperthermic therapy can be an effective adjuvant treatment for some tumors including melanoma. We developed a fusion protein based on the tissue inhibitor of matrix metalloproteinase-1 linked to a glycosylphosphatidylinositol anchor (TIMP-1-GPI). The TIMP-1-GPI-fusion protein shows unique properties. Exogenous administration of TIMP-1-GPI can result in transient morphological changes to treated cells including modulation of proliferation and decreased resistance to apoptosis. The effect of TIMP-1-GPI on the biology of melanoma in the context of a defined hyperthermic dose was evaluated in vitro. Clonogenic assays were used to measure cell survival. Gelatinase zymography determined secretion of MMP-2 and MMP-9. Monoclonal antibody against FAS/CD95 was applied to induce apoptosis. The expression of pro- and anti-apoptotic proteins and the secretion of immunoregulatory cytokines were then evaluated using Western blot and ELISA. TIMP-1-GPI combined with a sub-lethal hyperthermic treatment (41.8 degrees C for 2 h) suppressed tumor cell growth capacity as measured by clonogenic assay. The co-treatment also significantly suppressed tumor cell proliferation, enhanced FAS receptor surface expression increased tumor cell susceptibility to FAS-mediated killing. The increased sensitivity to FAS-induced apoptosis was linked to alterations in the apoptotic mediators Bcl-2, Bax, Bcl-XL and Apaf-1. The agent works in concert with sub-lethal hyperthermic treatment to render melanoma cells sensitive to FAS killing. The targeted delivery of TIMP-1-GPI to tumor environments in the context of regional hyperthermic therapy could be optimized through the use of thermosensitive liposomes.

Purification of Mg2+-dependent phosphatidate phosphohydrolase from rat liver: new steps and aspects.

A new procedure for the partial purification of Mg2+-dependent, N-ethylmaleimide-sensitive phosphatidate phosphohydrolase (Mg2+-PAP; EC 3.1.3.4) from rat liver cytosol is described, using protein precipitation with MgCl2, gel filtration on Sephacryl S-400, chromatography on DEAE-cellulose and affinity chromatography on calmodulin-agarose. From the parallel change in staining intensity and in the level of the specific activity of enzyme fractions, a relationship between a 90-kDa SDS gel band, identified as the beta-isoform of the 90-kDa heat shock protein, and Mg2+-PAP could be detected.