Alexandre Chlenski

Modulation of matrix remodeling by SPARC in neoplastic progression

SPARC is a matricellular glycoprotein that mediates interactions between cells and their microenvironment. It is produced at sites of tissue remodeling, where it regulates matrix deposition and turnover, cell adhesion, and signaling by extracellular factors, exerting profound effects on tissue architecture and cell physiology. During extensive matrix remodeling in neoplastic progression, SPARC is expressed in cancer-associated stroma and in malignant cells of some types, affecting tumor development, invasion, metastases, angiogenesis and inflammation. SPARC-induced changes in the tumor microenvironment can suppress or promote progression of different cancers depending on the tissue and cell type. Understanding the mechanism of matrix remodeling and its regulation by SPARC is essential for the development of new treatment strategies for highly aggressive cancers.

SPARC expression is associated with impaired tumor growth, inhibited angiogenesis and changes in the extracellular matrix

Secreted protein, acidic and rich in cysteine (SPARC), is a multifunctional matricellular glycoprotein. In vitro, SPARC has antiangiogenic properties, including the ability to inhibit the proliferation and migration of endothelial cells stimulated by bFGF and VEGF. Previously, we demonstrated that platelet‐derived SPARC also inhibits angiogenesis and impairs the growth of neuroblastoma tumors in vivo. In the present study, we produced rhSPARC in the transformed human embryonic kidney cell line 293 and show that the recombinant molecule retains its ability to inhibit angiogenesis. Although 293 cell proliferation was not affected by exogenous expression of SPARC in vitro, growth of tumors formed by SPARC‐transfected 293 cells was significantly impaired compared to tumors comprised of wild‐type cells or 293 cells transfected with a control vector. Consistent with its function as an angiogenesis inhibitor, significantly fewer blood vessels were seen in SPARC‐transfected 293 tumors compared to controls, and these tumors contained increased numbers of apoptotic cells. Light microscopy revealed small nests of tumor cells surrounded by abundant stromal tissue in xenografts with SPARC expression, whereas control tumors were comprised largely of neoplastic cells with scant stroma. Mature, covalently cross‐linked collagen was detected in SPARC‐transfected 293 xenografts but not in control tumors. Our studies suggest that SPARC may regulate tumor growth by inhibiting angiogenesis, inducing tumor cell apoptosis and mediating changes in the deposition and organization of the tumor microenvironment.

Methylation of CASP8, DCR2, and HIN-1 in Neuroblastoma Is Associated with Poor Outcome

Purpose: Epigenetic aberrations have been shown to play an important role in the pathogenesis of most cancers. To investigate the clinical significance of epigenetic changes in neuroblastoma, we evaluated the relationship between clinicopathologic variables and the pattern of gene methylation in neuroblastoma cell lines and tumors. Experimental Design: Methylation-specific PCR was used to evaluate the gene methylation status of 19 genes in 14 neuroblastoma cell lines and 8 genes in 70 primary neuroblastoma tumors. Associations between gene methylation, established prognostic factors, and outcome were evaluated. Log-rank tests were used to identify the number of methylated genes that was most predictive of overall survival. Results: Epigenetic changes were detected in the neuroblastoma cell lines and primary tumors, although the pattern of methylation varied. Eight of the 19 genes analyzed were methylated in >70% of the cell lines. Epigenetic changes of four genes were detected in only small numbers of cell lines. None of the cell lines had methylation of the other seven genes analyzed. In primary neuroblastoma tumors, high-risk disease and poor outcome were associated with methylation of DCR2, CASP8, and HIN-1 individually. Although methylation of the other five individual genes was not predictive of poor outcome, a trend toward decreased survival was seen in patients with a methylation phenotype, defined as ≥4 methylated genes (P = 0.055). Conclusion: Our study indicates that clinically aggressive neuroblastoma tumors have aberrant methylation of multiple genes and provides a rationale for exploring treatment strategies that include demethylating agents.

Association of epigenetic inactivation of RASSF1A with poor outcome in human neuroblastoma

Purpose: To investigate the prevalence and potential clinical significance of epigenetic aberrations in neuroblastoma (NB). Experimental Design: The methylation status of 11 genes that are frequently epigenetically inactivated in adult cancers was assayed in 13 NB cell lines. The prevalence of RASSF1A and TSP-1 methylation was also analyzed in 56 NBs and 5 ganglioneuromas by methylation-specific PCR. Associations between the methylation status of RASSF1A and TSP-1 and patient age, tumor stage, tumor MYCN status, and patient survival were evaluated. Results: Epigenetic changes were detected in all 13 NB cell lines, although the pattern of gene methylation varied. The putative tumor suppressor gene RASSF1A was methylated in all 13 cell lines, and TSP-1 and CASP8 were methylated in 11 of 13 cell lines. Epigenetic changes of DAPK and FAS were detected in only small numbers of cell lines, whereas none of the cell lines had methylation of p16, p21, p73, RAR-β2, SPARC, or TIMP-3. RASSF1A was also methylated in 70% of the primary NB tumors tested, and TSP-1 methylation was detected in 55% of the tumors. RASSF1A methylation was significantly associated with age >1 year (P < 0.01), high-risk disease (P < 0.016), and poor survival (P < 0.001). In contrast, no association between TSP-1 methylation and prognostic factors or survival was observed. Conclusions: Our results suggest that epigenetic inactivation of RASSF1A may contribute to the clinically aggressive phenotype of high-risk NB.

Role of Peroxisome Proliferator-Activated Receptor γ and Its Ligands in Non-Neoplastic and Neoplastic Human Urothelial Cells

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors and is expressed in several types of tissue. Although PPARgamma reportedly is expressed in normal urothelium, its function is unknown. We examined the expression of PPARgamma in normal urothelium and bladder cancer in an attempt to assess its functional role. Immunohistochemical staining revealed normal urothelium to express PPARgamma uniformly. All low-grade carcinomas were positive either diffusely or focally, whereas staining was primarily focal or absent in high-grade carcinomas. A nonneoplastic urothelial cell line (1T-1), a low-grade (RT4) carcinoma cell line, and two high-grade (T24 and 253J) carcinoma cell lines in culture expressed PPARgamma mRNA and protein. Luciferase assay indicated that PPARgamma was functional. PPARgamma ligands (15-deoxy-Delta(12,14)-prostaglandin J(2), troglitazone and pioglitazone) suppressed the growth of nonneoplastic and neoplastic urothelial cells in a dose-dependent manner. However, neoplastic cells were more resistant than were nonneoplastic cells. Failure to express PPARgamma or ineffective transcriptional activity may be some of the mechanisms responsible for resistance to the inhibitory action of PPARgamma ligands.

Organization and expression of the human zo-2 gene (tjp-2) in normal and neoplastic tissues.

One of the tight junction components, zonula occludens protein 2 (ZO-2), is expressed as two isoforms, ZO-2A and ZO-2C, in normal epithelia. In pancreatic adenocarcinoma of the ductal type ZO-2A is absent, but none of the common mechanisms of gene inactivation is responsible for lack of ZO-2A expression. In the current study, we report the complete organization of the human zo-2 gene (tjp-2), its alternative splicing, and its expression in normal and neoplastic tissues of several organ sites. In addition to pancreatic adenocarcinoma, ZO-2 was found to be de-regulated in breast adenocarcinoma, but not in colon or prostate adenocarcinoma. The latter are considered to be of acinar rather than ductal type. Thus, our data indicate the importance of zo-2 (tjp-2) gene regulation in ductal cancer development and should help to understand the defects of intercellular interactions, critical for suppressing the malignant phenotype.

Tight junction protein zo‐2 is differentially expressed in normal pancreatic ducts compared to human pancreatic adenocarcinoma

Differential display of hamster mRNA identified a fragment present in normal pancreatic duct cells that is not expressed in pancreatic duct carcinoma cells. Sequence analysis showed an 88% and 82% identity, respectively, to the cDNA of the canine and human tight junction zo‐2 gene. Semi‐quantitative RT‐PCR analysis of human ZO‐2 revealed a striking difference in the expression of various regions of the ZO‐2 transcript in normal and neoplastic cells and the presence of an abnormality at the 5′‐end of mRNA. RACE analysis identified 2 human ZO‐2 mRNAs that encode proteins of different lengths, designated as ZO‐2A and ZO‐2C. The difference between the 2 forms of ZO‐2 is the absence of 23 amino acid residues at the N terminus of ZO‐2C compared with ZO‐2A. Although ZO‐2C was expressed in normal pancreatic cells and a majority of neoplastic tissues analyzed, ZO‐2A was undetectable except in one case in all of the pancreatic adenocarcinomas analyzed. This suggests the presence of a yet to be identified motif important for cell‐growth regulation within the 23–amino acid residue N‐terminal peptide of ZO‐2A, MPVRGDRGFPPRRELSGWLRAPG. Int. J. Cancer 82:137–144, 1999.

Neuroblastoma Angiogenesis Is Inhibited with a Folded Synthetic Molecule Corresponding to the Epidermal Growth Factor-Like Module of the Follistatin Domain of SPARC

Secreted protein acidic and rich in cysteine (SPARC) is a multifunctional matricellular glycoprotein. In vitro, SPARC inhibits the proliferation and migration of endothelial cells stimulated by growth factors and induces endothelial cell apoptosis. We previously showed that SPARC also inhibits angiogenesis in vivo and impairs the growth of the pediatric tumor neuroblastoma (NB). SPARC comprises three domains that are independently folded by a complex pattern of disulfide bonds and have a high degree of structural conservation. In this study, separate modules of the SPARC domains were synthesized as cysteine-linked peptides and tested for their ability to inhibit angiogenesis. Peptide FS-E, representing the epidermal growth factor (EGF)-like module of the follistatin (FS) domain, did not cause endothelial cell apoptosis but strongly inhibited basic fibroblast growth factor (bFGF)–induced endothelial cell migration with an ED50 = 10 pmol/L. In vivo, peptide FS-E blocked bFGF-stimulated angiogenesis and neovascularization induced by NB cells. The EGF-like conformation was essential for peptide FS-E function because reduction of its two disulfide bonds completely abrogated peptide activity. Peptides FS-K and EC-N, corresponding to part of the Kazal module of the FS domain and the conserved α-helix in the extracellular calcium-binding domain, respectively, had minimal to no inhibitory activity. Our data show that the EGF-like module of the SPARC FS domain is angiosuppressive, and its structural conformation is critical for antiangiogenic activity.

SPARC enhances tumor stroma formation and prevents fibroblast activation

Tumor growth is influenced by interactions between malignant cells and the tumor stroma. Although the normal host microenvironment is nonpermissive for neoplastic progression, tumor-reactive stroma, characterized by the presence of activated fibroblasts, promotes neoplastic growth and metastasis. Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that is capable of inhibiting the growth of several different types of cancer. Recently, we reported that SPARC also impairs the growth of xenografts comprised of 293 cells. In this study, we show that in addition to enhancing stroma formation, SPARC prevents fibroblast activation in 293 xenografts, suggesting that the anti-cancer effects of SPARC may be due, at least in part, to the formation of tumor stroma that is not supportive of tumor growth. In vitro, 3T3 fibroblasts cocultured with SPARC-transfected 293 cells remain negative for α-smooth muscle actin, whereas wild-type 293 cells induce fibroblast activation. Moreover, activation of 3T3 cells and primary fibroblasts by transforming growth factor β is blocked by SPARC treatment. We also demonstrate that SPARC significantly increases basic fibroblast growth factor-induced fibroblast migration in vitro, indicating that it may recruit host fibroblasts to the tumor stroma. Taken together, our results suggest that in addition to blocking angiogenesis, SPARC may inhibit tumor growth by promoting the assembly of stroma that is non-permissive for tumor progression.

Secreted Protein Acidic and Rich in Cysteine Is a Matrix Scavenger Chaperone

Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca2+ concentrations are low, high extracellular concentrations of Ca2+ activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca2+ concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimentional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the pathogenesis of matrix-associated disorders and lead to the novel treatment strategies.