Expression Of Asporin Research Articles

Abstract P4-06-18: Asporin Is Associated with Invasive Growth and Bone Metastasis of Breast Cancer

Abstract Background: In an earlier publication we identified asporin as a novel cancer related protein (BMC Cancer 2007, 7:55), also reported among bone metastasis specific genes (Cancer Letters 2009, 276:212-220). Asporin is a member of the class I small leucine-rich repeat proteoglycan family and besides affinity to collagen, it binds calcium and initiates collagen mineralization. Asporin is abundantly expressed in tissues with high remodeling rate (e.g. peridontal ligament) whereas extremely high turnover of matrix proteins is also associated with invasive cancer. Material and methods: Formalin-fixed paraffin-embedded tissues [invasive ductal (101) and lobular (39) carcinomas classified according to hormone receptors and Her-2; 5 bone metastases from autopsies] were immunohistochemically stained with in-house polyclonal peptide antibody against asporin. The antibody was validated on uterus tissue as a positive control together with a commercial antibody (Abcam). Data were evaluated by non-parametric Mann-Whitney U test and Spearman correlation coefficient. Expression of asporin in both tissues and cell lines was also queried in databases Gene Expression Omnibus, ArrayExpress and Oncomine. Expression of asporin was further tested by RT-PCR in selected breast cancer cell lines. Migration and invasion of MDA-MB-231 cells were tested using real-time cell analysis Xcelligence system (Roche). Transwells were coated by solidifying collagen for 6 hours (with or without recombinant asporin). Starved cells were seeded on the collagen in transwells while FBS was used as a chemoattractant in bottom wells. Results: Levels of asporin were significantly higher in invasive lobularcarcinomas than ductal ones (P<0.001). Asporin was also highly expressed in all bone metastases. Asporin expression positively correlated with tumor grade within the triple negative subgroup (Rs 0.4, P<0.05) but not in luminal and Her-2 subtypes. Expression of asporin in invasive breast carcinomas was also confirmed in other public datasets (e.g. Karnoub breast and Hess breast in Oncomine database). Surprisingly, we found no breast cancer cell line overexpressing asporin, which underlines the importance of the tumor microenvironment in vivo. MDA-MB-231 cells migrated faster through collagen I with recombinant asporin. Conclusion: Asporin is expressed by both invasive breast carcinomas and bone metastases. Mechanistically, asporin may affect extracellular matrix structure and promote invasive growth of breast cancer cells. Further investigation of the role of asporin in metastasizing to bone is additionally justified given its affinity for calcium. Acknowledgements This work was supported by grants NS 9956-4 from the Czech Ministry of Health and MSM 6198959216 from the Czech Ministry of Education. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-18.

Comparative proteomic analysis between normal skin and keloid scar

Keloids are pathological scars and, despite numerous available treatment modalities, continue to plague physicians and patients. Identification of molecular mediators that contribute to this fibrotic phenotype. Two-dimensional gel electrophoresis, MALDI-TOF, Mascot online database searching algorithm and Melanie 5 gel analysis software were employed for comparative proteomic analysis between normal skin (NS) and keloid scar (KS) tissue extracts. Seventy-nine protein spots corresponding to 23 and 32 differentially expressed proteins were identified in NS and KS, respectively. Isoforms of heat shock proteins, gelsolin, carbonic anhydrase and notably keratin 10 were strongly expressed in NS along with manganese superoxide dismutase, immune components, antitrypsin, prostatic binding protein and crystalline. Various classes of proteins were found either to be present or to be upregulated in keloid tissue: (i) inflammatory/differentiated keratinocyte markers: S100 proteins, peroxiredoxin I; (ii) wound healing proteins: gelsolin-like capping protein; (iii) fibrogenetic proteins: mast cell β-tryptase, macrophage migration inhibitory factor (MIF); (iv) antifibrotic proteins: asporin; (v) tumour suppressor proteins: stratifin, galectin-1, maspin; and (vi) antiangiogenic proteins: pigment epithelium-derived factor. Significant increases in expression of asporin, stratifin, galectin-1 and MIF were observed by Western blot analysis in KS. This work has identified differentially expressed proteins specific to KS tissue extracts which can potentially be used as specific targets for therapeutic intervention.

Asporin and transforming growth factor-β gene expression in osteoblasts from subchondral bone and osteophytes in osteoarthritis

BackgroundTo clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-β1 (TGF-β1), TGF-β2, TGF-β3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism. MethodsOsteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-βi, TGF-β2, TGF-β3, and Runx2 was analyzed using real-time RT-PCR. ResultsExpression of ASPN, TGF-β1, and TGF-β3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-β1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage. ConclusionsThe increased ratio of ASPN mRNA to TGF-β1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-β1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

Asporin, a susceptibility gene in osteoarthritis, is expressed at higher levels in the more degenerate human intervertebral disc

IntroductionAsporin, also known as periodontal ligament-associated protein 1 (PLAP1), is a member of the family of small leucine-rich proteoglycan (SLRP) family. It is present within the cartilage extracellular matrix (ECM), and is reported to have a genetic association with osteoarthritis. Its D14 allele has recently been found to be associated with lumbar disc degeneration in Asian subjects. There have been no studies, however, of this gene's normal immunohistochemical localization within the human intervertebral disc, or of expression levels in Caucasian individuals with disc degeneration.MethodsStudies were approved by our human subjects Institutional Review Board. Methods included immunohistochemical localization of asporin in the disc of humans and the sand rat (a small rodent with spontaneous age-related disc degeneration), and Affymetrix microarray analysis of asporin gene expression in vivo and in vitro.ResultsImmunohistochemical studies of human discs revealed that some, but not all, cells of the outer annulus expressed asporin. Fewer cells in the inner annulus contained asporin, and it was rarely present in cells in the nucleus pulposus. Similar patterns were found for the presence of asporin in lumbar discs of sand rats. Substantial relative gene expression levels were seen for asporin in both disc tissue and in annulus cells grown in three-dimensional culture. More degenerate human discs (Thompson grade 4) showed higher expression levels of asporin than did less degenerate (grade 1, 2 and 3) discs, P = 0.004.ConclusionsIn the discs of Caucasian subjects studied here, and in the sand rat, greater immunolocalization levels were found in the outer compared to inner annulus. Localization was rare in the nucleus. Gene expression studies showed greatest expression of asporin in the more degenerate human discs in vivo.

Expression, Regulation and Function of Asporin, A Susceptibility Gene in Common Bone and Joint Diseases

Asporin is an extracellular matrix protein that belongs to the small leucine-rich repeat proteoglycan (SLRP) family of proteins. It is unique among SLRPs in that it lacks a glycosaminoglycan attachment site and contains an asparatic acid (D) repeat at its amino terminus. Its biological role has been unclear, but recent genetic studies have demonstrated association between asporin and various bone and joint diseases, including osteoarthritis, rheumatoid arthritis and lumbar disc disease. Each of these common diseases presents a substantial medical, social and economical burden to societies worldwide. This paper reviews recent progress in the study of asporin, focusing on its expression, regulation and function as well as its role in the molecular pathogenesis of common bone and joint diseases. Asporin is found primarily in regions surrounding skeletal tissue and is up-regulated in disease states. It binds to various growth factors, including TGF-beta and BMP-2, and negatively regulates their activity. By inhibiting binding of TGF-beta1 to its type II receptor, asporin forms a functional feedback loop with TGF-beta1 and regulates its chondrogenic potential. As an extracellular, tissue-specific protein, asporin represents a promising target for phamacogenomic approaches to common bone and joint diseases.

Expression Pattern and Gene Characterization ofAsporin: A NEWLY DISCOVERED MEMBER OF THE LEUCINE-RICH REPEAT PROTEIN FAMILY

We have discovered a new member of the class I small leucine-rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9q21.3 where asporin is part of a SLRP gene cluster that includes extracellular matrix protein 2, osteoadherin, and osteoglycin. Further analysis shows that, with the exception of biglycan, all known SLRP genes reside in three gene clusters.