Lisa J. Guerrero

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.

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.

Anti-angiogenic SPARC peptides inhibit progression of neuroblastoma tumors

BackgroundNew, more effective strategies are needed to treat highly aggressive neuroblastoma. Our laboratory has previously shown that full-length Secreted Protein Acidic and Rich in Cysteine (SPARC) and a SPARC peptide corresponding to the follistatin domain of the protein (FS-E) potently block angiogenesis and inhibit the growth of neuroblastoma tumors in preclinical models. Peptide FS-E is structurally complex and difficult to produce, limiting its potential as a therapeutic in the clinic.ResultsIn this study, we synthesized two smaller and structurally more simple SPARC peptides, FSEN and FSEC, that respectively correspond to the N-and C-terminal loops of peptide FS-E. We show that both peptides FSEN and FSEC have anti-angiogenic activity in vitro and in vivo, although FSEC is more potent. Peptide FSEC also significantly inhibited the growth of neuroblastoma xenografts. Histologic examination demonstrated characteristic features of tumor angiogenesis with structurally abnormal, tortuous blood vessels in control neuroblastoma xenografts. In contrast, the blood vessels observed in tumors, treated with SPARC peptides, were thin walled and structurally more normal. Using a novel method to quantitatively assess blood vessel abnormality we demonstrated that both SPARC peptides induced changes in blood vessel architecture that are consistent with blood vessel normalization.ConclusionOur results demonstrate that SPARC peptide FSEC has potent anti-angiogenic and anti-tumorigenic effects in neuroblastoma. Its simple structure and ease of production indicate that it may have clinical utility in the treatment of high-risk neuroblastoma and other types of pediatric and adult cancers, which depend on angiogenesis.

Validation of a prognostic multi-gene signature in high-risk neuroblastoma using the high throughput digital NanoString nCounter™ system.

Microarray‐based molecular signatures have not been widely integrated into neuroblastoma diagnostic classification systems due to the complexities of the assay and requirement for high‐quality RNA. New digital technologies that accurately quantify gene expression using RNA isolated from formalin‐fixed paraffin embedded (FFPE) tissues are now available. In this study, we describe the first use of a high‐throughput digital system to assay the expression of genes in an “ultra‐high risk” microarray classifier in FFPE high‐risk neuroblastoma tumors. Customized probes corresponding to the 42 genes in a published multi‐gene neuroblastoma signature were hybridized to RNA isolated from 107 FFPE high‐risk neuroblastoma samples using the NanoString nCounter™ Analysis System. For classification of each patient, the Pearsons correlation coefficient was calculated between the standardized nCounter™ data and the molecular signature from the microarray data. We demonstrate that the nCounter™ 42‐gene panel sub‐stratified the high‐risk cohort into two subsets with statistically significantly different overall survival (p = 0.0027) and event‐free survival (p = 0.028). In contrast, none of the established prognostic risk markers (age, stage, tumor histology, MYCN status, and ploidy) were significantly associated with survival. We conclude that the nCounter™ System can reproducibly quantify expression levels of signature genes in FFPE tumor samples. Validation of this microarray signature in our high‐risk patient cohort using a completely different technology emphasizes the prognostic relevance of this classifier. Prospective studies testing the prognostic value of molecular signatures in high‐risk neuroblastoma patients using FFPE tumor samples and the nCounter™ System are warranted.

Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

BackgroundEpigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype.MethodsTwo NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity.ResultsPromoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation status and the histone code in the THBS-1 promoter modifies cell morphology, and inhibits their ability to form colonies in soft agar.ConclusionOur results suggest that epigenetic aberrations contribute to NB phenotype, and that tumorigenic properties can be inhibited by reversing the epigenetic changes with 5-Aza-dC.

Presence of cancer-associated fibroblasts inversely correlates with Schwannian stroma in neuroblastoma tumors.

Stromal cells have a central function in the regulation of tumor angiogenesis. Recent studies have shown that stromal myofibroblasts (cancer-associated fibroblasts) actively promote tumor growth and enhance tumor angiogenesis in many types of adult carcinomas. To evaluate the function cancer-associated fibroblasts have in neuroblastoma angiogenesis and investigate their relationship to stromal Schwann cells, we quantified cancer-associated fibroblasts in 60 primary neuroblastoma tumors and in a novel neuroblastoma xenograft model in which murine Schwann cells were induced to infiltrate into the tumor stroma. Tumor sections were examined for presence of microvascular proliferation, a hallmark of tumor angiogenesis. Cancer-associated fibroblasts were characterized by positive immunostaining for α-smooth muscle actin (α-SMA) and were distinguished from pericytes by staining negatively for high-molecular-weight caldesmon. α-SMA-positive cells were quantified and their number was defined as high when >1.0% of the area was positive. Associations between high cancer-associated fibroblast number, microvascular proliferation and established prognosticators were analyzed. High numbers of cancer-associated fibroblasts were associated with Schwannian stroma-poor histopathology and microvascular proliferation. Thirty-seven (80%) of the 46 Schwannian stroma-poor tumors had high numbers of cancer-associated fibroblasts in the tumor stroma compared to only 2 (14%) of the 14 Schwannian stroma-rich/dominant tumors (P<0.001). Thirty-three (89%) of 37 tumors with microvascular proliferation had high numbers of cancer-associated fibroblasts compared to 9 (40%) of 22 tumors without microvascular proliferation (P<0.001). In the xenografts with infiltrating Schwann cells (n=10), the number of cancer-associated fibroblasts per mm2 was approximately sevenfold less than in the control xenografts without stromal Schwann cells (n=9) (mean of 51±30 vs 368±105, respectively; P<0.001). Thus, cancer-associated fibroblasts were inversely associated with presence of Schwann cells, suggesting that Schwann cells may prevent the activation of fibroblasts. A deeper understanding of the function cancer-associated fibroblasts have in neuroblastoma angiogenesis may guide future development of stroma-directed therapeutic strategies.

Sorafenib inhibits neuroblastoma cell proliferation and signaling, blocks angiogenesis, and impairs tumor growth†

More effective therapy for children with high‐risk neuroblastoma is desperately needed. Preclinical studies have shown that neuroblastoma tumor growth can be inhibited by agents that block angiogenesis. We hypothesized that drugs which target both neuroblastoma cells and tumor angiogenesis would have potent anti‐tumor activity. In this study we tested the effects of sorafenib, a multi‐kinase inhibitor, on neuroblastoma cell proliferation and signaling, and in mice with subcutaneous human neuroblastoma xenografts or orthotopic adrenal tumors.

The quinoxaline anti-tumor agent (R+)XK469 inhibits neuroblastoma tumor growth.

The quinoxaline anti‐tumor agent (R+)XK469 mediates its effects by topoisomerase IIB inhibition. This report describes a 14‐year old with relapsed neuroblastoma who experienced disease stabilization for 14 months while receiving (R+)XK469 monotherapy. Due to this favorable response, laboratory studies were undertaken to determine efficacy in the preclinical setting. (R+)XK469 inhibited proliferation, caused G2 cell cycle arrest of neuroblastoma cells in vitro, and inhibited growth of neuroblastoma xenograft tumors. These preclinical results, coupled with the favorable clinical response, demonstrate that (R+)XK469 and similar anti‐tumor agents may be effective in the treatment of high‐risk neuroblastoma and warrant further testing. Pediatr Blood Cancer. 2010;56:164–167.

Secreted protein acidic and rich in cysteine (SPARC) induces lipotoxicity in neuroblastoma by regulating transport of albumin complexed with fatty acids

SPARC is a matrix protein that mediates interactions between cells and the microenvironment. In cancer, SPARC may either promote or inhibit tumor growth depending upon the tumor type. In neuroblastoma, SPARC is expressed in the stromal Schwannian cells and functions as a tumor suppressor. Here, we developed a novel in vivo model of stroma-rich neuroblastoma using non-tumorigenic SHEP cells with modulated levels of SPARC, mixed with tumorigenic KCNR cells. Tumors with stroma-derived SPARC displayed suppressed growth, inhibited angiogenesis and increased lipid accumulation. Based on the described chaperone function of SPARC, we hypothesized that SPARC binds albumin complexed with fatty acids and transports them to tumors. We show that SPARC binds albumin with Kd=18.9±2.3 uM, and enhances endothelial cell internalization and transendothelial transport of albumin in vitro. We also demonstrate that lipids induce toxicity in neuroblastoma cells and show that lipotoxicity is increased when cells are cultured in hypoxic conditions. Studies investigating the therapeutic potential of SPARC are warranted.