RNAi mediated MMP-1 silencing inhibits human chondrosarcoma invasion.

Previous work indicates that matrix metalloproteinase-1 (MMP-1) gene expression is a prognostic factor for local recurrence and metastasis in human chondrosarcoma. The current study evaluates the effects of MMP-1 knock-down via RNA interference on chondrosarcoma metastasis in vitro. Of nine siRNA oligos tested, one showed a 90% inhibitory effect on MMP-1 gene expression. Stable attenuation of MMP-1 in chondrosarcoma cells was achieved by using a plasmid vector-based siRNA system. Pronounced MMP-1 activity suppression was accompanied by marked inhibition of tumor cell invasion. Cells in which MMP-1 has been stably silenced are useful tools to elucidate the mechanism of MMP-1 mediated tumor metastasis.

siRNA mediated inhibition of MMP-1 reduces invasive potential of a human chondrosarcoma cell line.

Matrix metalloproteinases (MMPs) play a crucial role in tumor cell invasion and metastasis. Expression of MMP-1 has been reported as a prognostic predictor of recurrence in human chondrosarcoma, and studies using human chondrosarcoma cell lines indicate that MMP-1 expression levels correlate with in vitro invasiveness. These observations suggest that MMP-1 activity has a central role in cell egress from the primary tumor at an early step in the metastatic cascade. In this study, siRNA was used to investigate whether knock down of the MMP-1 gene could be used to inhibit invasiveness in a human chondrosarcoma cell line. The inhibitory effect of siRNA on endogenous MMP-1 gene expression and protein synthesis was demonstrated via RT-PCR, Northern blotting, Western blotting, collagenase activity assay, and an in vitro cell migration assay. The siRNA inhibited MMP-1 expression specifically, since it did not affect the expression of endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) nor other collagenases. Most importantly, the siRNA mediated reduction in MMP-1 expression correlated with a decreased ability of chondrosarcoma cells to invade a Type I collagen matrix. The reduction of invasive behavior demonstrated by human chondrosarcoma cells transfected with MMP-1 siRNA and the specificity of this inhibition supports the hypothesis that this metalloproteinase molecule is involved in initiation of chondrosarcoma metastasis.

Absence of a correlation between the presence of a single nucleotide polymorphism in the matrix metalloproteinase 1 promoter and outcome in patients of chondrosarcoma.

PURPOSE: Increased levels of matrix metalloproteinase 1 (MMP-1) expression have been associated with poor outcome in chondrosarcoma. The existence of a single nucleotide polymorphism creating an Ets-binding site in the MMP-1 promoter may be one mechanism for elevated MMP-1 transcription. The aim of our study was to identify the prevalence of this single nucleotide polymorphism (SNP) in chondrosarcoma patients, to determine its correlation with disease outcome, and to discern whether it could serve as a prognostic marker in patients with chondrosarcoma. EXPERIMENTAL DESIGN: Sixty-seven chondrosarcoma specimens were selected sequentially from an established tumor bank with a median duration of 47 months follow-up (range, 24 to 179 months). DNA was extracted, amplified with PCR, and sequenced to determine presence (GG) or absence of the Ets-binding site created by the SNP. RESULTS: Eighteen (27%) samples were homozygous for the absence of the Ets site, 34 (51%) were heterozygous for the SNP, and 15 (22%) were homozygous for the SNP. The 5-year overall survival rate for patients was 78, 80, and 84%, respectively (P = 0.5527). The disease-free survival rate was 16, 63, and 76%, respectively (P = 0.0801). The 5-year disease-free survival rate for patients with the homozygous G/G genotype was 16%, compared with 71% for patients who were either homozygous or heterozygous for the GG allele (P = 0.0444). CONCLUSIONS: Despite a statistical correlation between MMP-1 gene expression and outcome in chondrosarcoma, this study demonstrates an absence of a correlation between the presence of the SNP and prognosis in patients with chondrosarcoma.

Signaling "cross-talk" between TGF-beta1 and ECM signals in chondrocytic cells.

The objective of this investigation was to clarify how the integrin pathway modulates downstream effectors of the TGF-beta1 pathway in chondrocytic cell signaling. The levels of Smad2 and Smad3 phosphorylation upon TGF-beta1 or alpha2beta1 integrin (Type II collagen) stimulation were analyzed by Western blotting techniques. Cellular response was determined by quantitation of procollagen gene expression. Stimulation of cells with TGF-beta1 and Type II collagen led to rapid phosphorylation of Smad2 and 3 with phosphorylation peaking between 15 min and 1 h. Combined stimulation led to a synergistic increase in the phosphorylation of Smad2 and Smad3. Type II collagen gene expression paralleled Smad phosphorylation. Type II collagen modulates the TGF signaling cascade involving Smad2 and Smad3 leading to an increase in Type II collagen transcription. Therefore, we conclude that TGF-beta1 and integrin stimuli interact prior to Smad2 and 3 phosphorylation in the cytoplasm of chondrocytic cells and regulates the expression of ECM components in chondrocytes.

Inhibition of MMP-1 expression by antisense RNA decreases invasiveness of human chondrosarcoma.

We previously reported that an elevated level of matrix metalloproteinase-1 (MMP-1) gene expression in patients with chondrosarcoma has a strong statistical correlation with recurrence and in vitro invasion. In the present study, we used an antisense RNA strategy for MMP-1 inhibition to determine if this would affect the invasive characteristics of the cells. We transfected a human chondrosarcoma cell line with a retroviral plasmid expressing a 770 bp genomic fragment of the human MMP-1 gene in the sense or antisense orientation. The results show that cells transfected with the MMP-1 antisense fragment had a significant decrease in both MMP-1 protein and enzyme activity (p<0.05) as compared to cells transfected with an empty plasmid or the parental cells. Cells transfected with the MMP-1 antisense fragment demonstrated a significant decrease in their ability to invade the collagen I barrier (p<0.05). The gene expression for MMP-8 and MMP-13 were unaffected in cells transfected with the MMP-1 antisense fragment, MMP-1 sense fragment, or empty plasmid. These results support the hypothesis that MMP-1 facilitates tumor cell egress from chondrosarcoma tissue and demonstrate the potential of MMP-1 as a promising target for a novel biologic therapy in chondrosarcoma.

Stimulation of adipogenesis, peroxisome proliferator-activated receptor-gamma (PPARgamma), and thyrotropin receptor by PPARgamma agonist in human orbital preadipocyte fibroblasts.

The symptoms and signs of Graves' ophthalmopathy (GO) result from both an accumulation of hydrated hyaluronan in the orbital muscles and connective tissues and an expansion of the orbital adipose tissues. Recent studies have suggested a link between the stimulation of adipogenesis within the orbit in GO and the expression in these tissues of TSH receptor (TSHR), the putative orbital autoantigen. To further investigate this association, we treated orbital fibroblasts from patients with GO with rosiglitazone, a thiazolidinedione agonist of the PPARgamma receptor that stimulates adipocyte differentiation. We found this compound to be a potent stimulator of functional TSHR expression as well as TSHR and PPARgamma mRNA levels in differentiated cultures. In addition, rosiglitazone treatment stimulated recruitment and differentiation of a subset of cells within these cultures into mature lipid-laden adipocytes. These results suggest that TSHR expression in GO orbital preadipocyte fibroblasts is linked to adipogenesis, and that ligation of the PPARgamma receptor results in differentiation of these cells. It is possible that endogenous PPARgamma ligands play a role in stimulating orbital adipogenesis in GO, and that future treatments may be aimed at antagonism of various components of the PPARgamma signaling system.