Shuqing Liu

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.

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.

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.

The regulation of angiogenesis in neuroblastoma

Angiogenesis is required for the growth and metastasis of malignant tumors, and high vascular density has been correlated with aggressive tumor growth in many types of cancer. This process is regulated by the local balance of stimulatory and inhibitory molecules produced by tumor cells, stromal cells, and the organ-specific environment. In neuroblastoma, a pediatric malignancy that is characterized by a broad spectrum of clinical behavior, angiogenesis also appears to play an important role in determining tumor phenotype. The nature of the angiogenic balance in neuroblastoma is complex, and a spectrum of angiogenesis stimulators and inhibitors has been detected in neuroblastoma tumors. This review summarizes our current understanding of the regulation of angiogenesis in neuroblastoma.

Cross-Talk between Schwann Cells and Neuroblasts Influences the Biology of Neuroblastoma Xenografts

Neuroblastoma (NB) tumors with abundant schwannian stroma have a differentiated phenotype, low vascularity, and are associated with a favorable prognosis. These observations suggest that cross-talk between Schwann cells and neuroblasts may influence tumor biology. To test this hypothesis, we developed a novel NB xenograft model with infiltrating mouse Schwann cells. Human SMS-KCNR NB cells were injected intrafascicularly (sciatic nerve-engrafted NB, n = 19) or outside the sciatic nerve (control, n = 12). Xenografts were harvested 4 to 12 weeks after tumor cell inoculation for histological studies. Schwann cells were immunostained with S-100 and species-specific p75(NGFR), major histocompatibility complex, and human leukocyte antigen antibodies. The number of proliferating cells, infiltrating Schwann cells, apoptotic cells, differentiated neuroblasts, and blood vessels in the sciatic nerve-engrafted NB tumors were compared to controls. Significantly more Schwann cells were detected in the sciatic nerve-engrafted NB xenografts than controls (P < 0.001). The infiltrating Schwann cells were S-100-positive and reacted with anti-mouse major histocompatibility complex class Ib and p75(NGFR) but not anti-human p75(NGFR) and human leukocyte antigen class I antibodies. The sciatic nerve-engrafted tumors also had lower numbers of proliferating neuroblasts, higher numbers of differentiated neuroblasts and apoptotic cells, and decreased vascular density compared to controls. Our results indicate that infiltrating Schwann cells of mouse origin are capable of promoting human neuroblast differentiation, inducing apoptosis, and inhibiting proliferation and angiogenesis in vivo.

Methylation-Associated Silencing of the Heat Shock Protein 47 Gene in Human Neuroblastoma

Hypermethylation of gene promoter CpG islands is a frequent mechanism for gene inactivation in a variety of human cancers, including neuroblastoma (NB). We demonstrated recently that treatment with the demethylating agent 5′-aza-2′-deoxycytidine (5-Aza-dC) significantly inhibited NB growth in vivo. In an effort to identify the genes and biological pathways that are responsible for the impaired NB tumor growth observed after treatment with 5-Aza-dC, we performed genome-wide gene expression analysis of control and treated NBL-W-S NB cells. We found ≥3-fold changes in expression of 44 genes that play roles in angiogenesis, apoptosis, cell adhesion, transcriptional regulation, and signal transduction. The gene encoding heat shock protein 47 (Hsp47), a collagen-specific molecular chaperon, was up-regulated >80-fold after 5-Aza-dC treatment. Expression studies confirmed that Hsp47 is silenced in a subset of NB cell lines and tumors. We also show that silencing of Hsp47 in NB cells is associated with aberrant methylation of promoter CpG islands and that Hsp47 expression can be restored after treatment with 5-Aza-dC. A strong correlation between Hsp47 and collagen type I and IV expression was seen in NB cells. Interestingly, tumorigenicity was inversely correlated with the level of collagen expression in NB cell lines, and higher levels of collagen were detected in mature NB tumors that are associated with favorable outcome compared with undifferentiated, advanced-stage NBs. Our studies support a role for Hsp47 in the regulation of collagen type I and IV production in NB cells and suggest that the level of collagen expression may influence NB tumor phenotype.