Exogenous Secreted Protein Acidic And Rich In Cysteine Research Articles

SPARC expression in tumor microenvironment induces partial epithelial-to-mesenchymal transition of esophageal adenocarcinoma cells via cooperating with TGF-β signaling.

Secreted protein, acidic and rich in cysteine (SPARC) has been characterized as an oncoprotein in esophageal squamous cell carcinoma (ESCC), but its involvement in the pathological development of esophageal adenocarcinoma (ESAD) remains poorly understood. In this study, we aimed to explore the sources of SPARC in the tumor microenvironment (TME) and its functional role in ESAD. Bioinformatic analysis was conducted using data from The Cancer Genome Atlas (TCGA)-esophageal cancer (ESCA) and Genotype-Tissue Expression (GTEx). ESAD tumor cell line OE33 and OE19 cells were used as in vitro cell models. Results showed that SPARC upregulation was associated with unfavorable disease-specific survival (DSS) in ESAD. ESAD tumor cells (OE33 and OE19) had no detectable SPARC protein expression. In contrast, IHC staining in ESAD tumor tissues suggested that peritumoral stromal cells (tumor-associated fibroblasts and macrophages) were the dominant SPARC source in TME. Exogenous SPARC induced partial epithelial-to-mesenchymal transitionof ESAD cells, reflected by reduced CDH1 and elevated ZEB1/VIM expression at both mRNA and protein levels. Besides, exogenous SPARC enhanced tumor cell invasion. When TGFBR2 expression was inhibited, the activation of TGF-β signaling induced by exogenous SPARC was impaired. However, the activating effects were rescued by overexpressing mutant TGFBR2 resistant to the shRNA sequence. Copresence of exogenous SPARC and TGF-β1 induced higher expression of mesenchymal markers and enhanced the invading capability of ESAD cells than TGF-β1 alone. In conclusion, this study suggests a potential cross-talk between ESAD tumor stromal cells and cancer cells via a SPARC-TGF-β1 paracrine network.

SPARC Overexpression Promotes Liver Cancer Cell Proliferation and Tumor Growth.

Background: Secreted protein acidic and rich in cysteine (SPARC) plays an important role in cancer development. The roles of SPARC in the liver hepatocellular carcinoma (LIHC) are unclear. Methods: GEPIA2 and UALCAN were used to analyze the SPARC mRNA expression levels in LIHC based on the TCGA database. The GEO database was used to verify the analysis results. Immunohistochemical (IHC) analysis was used to investigate the SPARC protein levels in LIHC tissues. The Kaplan–Meier (KM) plotter was used to analyze the correlation between SPARC and prognosis. The serum SPARC levels were measured by ELISA. CCK8 and murine xenograft models were used to investigate the effect of SPARC on the liver cancer growth in vitro and in vivo. SPARC-correlated genes were screened by LinkedOmics. Results: Based on the TCGA and GEO databases, the analysis showed that the SPARC mRNA expression levels were increased in tumor tissues and peripheral blood mononuclear cell (PBMC) from LIHC compared to normal controls. The IHC analysis showed an increased level of SPARC in LIHC tissues compared to adjacent non-tumor tissues. However, we found that the serum SPARC levels were lower in LIHC than those in healthy controls. The KM plotter showed that there was no significant correlation between the SPARC mRNA levels and overall survival. However, in sorafenib-treated LIHC patients, the high SPARC expression predicts favorable prognosis. Furthermore, the endogenous SPARC overexpression promotes liver cancer cell proliferation in vitro and tumor growth in vivo, while there was no significant effect of exogenous SPARC treatment on liver cancer cell proliferation. Function enrichment analysis of SPARC-correlated genes indicated a critical role of interaction with an extracellular matrix in SPARC-promoting cancer cell proliferation. Conclusion: SPARC mRNAs were increased in LIHC tumor tissues, and SPARC overexpression may promote the liver cancer growth. Further studies are needed to clarify the potential prognostic value of SPARC, both in tissues and in circulation.

The Effect of SPARC on the Proliferation and Migration of Limbal Epithelial Stem Cells During the Corneal Epithelial Wound Healing.

Secreted protein acidic and rich in cysteine (SPARC) shows a specific colocalization with limbal epithelial stem cells (LESCs) in vivo; however, the inherent relationship between SPARC and LESCs is still unclear. This study investigated the effects of SPARC on the maintenance of LESC stemness and corneal wound healing. To test the influence of different concentration of exogenous SPARC on the proliferation of LESCs, cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine staining were performed and the results indicated that 1 μg/mL SPARC was the optimum concentration for enhanced LESC proliferation. Compared with a control group, SPARC-treated group showed a higher expression of LESC-positive markers p63α, ABCG-2, and Bmi-1, and a lower level of differentiation marker cytokeratin-3 (CK3), thereby suggesting that SPARC could maintain LESC characteristic phenotype and suppress spontaneous epithelial differentiation in vitro. In vivo, exogenous SPARC accelerated the wound-healing process by both the enhancement of LESC proliferation and promoting the migration of the proliferating cells. However, the intact epithelium impaired this function of SPARC by contact inhibition.

Efficacy and Safety of Human Serum Albumin-Cisplatin Complex in U87MG Xenograft Mouse Models.

Cisplatin (cis-diamminedichloroplatinum (II), CDDP) is a chemotherapeutic drug widely used against many solid tumors. A pharmacokinetics study found that CDDP can bind to human serum albumin (HSA), which is the most abundant plasma protein in serum. HSA has the advantage of being a nanocarrier and can accumulate in tumors by passive targeting and active targeting mediated by the secreted protein acidic and rich in cysteine (SPARC). In this study, we investigated the possibility of using a CDDP–HSA complex (HSA–CDDP) as a SPARC-mediated therapeutic agent. To investigate the HSA-dependent therapeutic effect of HSA–CDDP, we used two types of U87MG glioma cells that express SPARC differently. HSA–CDDP was highly taken up in SPARC expressing cells and this uptake was enhanced with exogenous SPARC treatment in cells with low expression of SPARC. The cytotoxicity of HSA–CDDP was also higher in SPARC-expressing cells. In the tumor model, HSA–CDDP showed a similar tumor growth and survival rate to CDDP only in SPARC-expressing tumor models. The biosafety test indicated that HSA–CDDP was less nephrotoxic than CDDP, based on blood markers and histopathology examination. Our findings show that HSA–CDDP has the potential to be a novel therapeutic agent for SPARC-expressing tumors, enhancing the tumor targeting effect by HSA and reducing the nephrotoxicity of CDDP.

Extracellular SPARC cooperates with TGF-β signalling to induce pro-fibrotic activation of systemic sclerosis patient dermal fibroblasts.

ObjectivesSSc is an autoimmune disease characterized by inflammation, vascular injury and excessive fibrosis in multiple organs. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that regulates processes involved in SSc pathology, such as inflammation and fibrosis. In vivo and in vitro studies have implicated SPARC in SSc, but it is unclear if the pro-fibrotic effects of SPARC on fibroblasts are a result of intracellular signalling or fibroblast interactions with extracellular SPARC hampering further development of SPARC as a potential therapeutic target. This study aimed to analyse the potential role of exogenous SPARC as a regulator of fibrosis in SSc.MethodsDermal fibroblasts from both healthy controls and SSc patients were stimulated with SPARC alone or in combination with TGF-β1, in the absence or presence of a TGF receptor 1 inhibitor. mRNA and protein expression of extracellular matrix components and other fibrosis-related mediators were measured by quantitative PCR and western blot.ResultsExogenous SPARC induced mRNA and protein expression of collagen I, collagen IV, fibronectin 1, TGF-β and SPARC by dermal fibroblasts from SSc patients, but not from healthy controls. Importantly, exogenous SPARC induced the activation of the tyrosine kinase SMAD2 and pro-fibrotic gene expression induced by SPARC in SSc fibroblasts was abrogated by inhibition of TGF-β signalling.ConclusionThese results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-β dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.

SPARC acts as a mediator of TGF-β1 in promoting epithelial-to-mesenchymal transition in A549 and H1299 lung cancer cells.

Migration and metastasis of tumor cells greatly contributes to the failure of cancer treatment. Recently, the extracellular protein secreted protein acidic and rich in cysteine (SPARC) has been reported closely related to tumorigenesis. Some articles have suggested that SPARC promoted metastasis in several highly metastatic tumors. However, there are also some studies shown that SPARC acted as an antitumor factor. SPARC-induced epithelial-to-mesenchymal transition (EMT) in melanoma cells and promoted EMT in hepatocellular carcinoma. Therefore, the role of SPARC in tumorigenesis and its relationship with EMT is still unclear. In this study, we investigated the expression change of SPARC in A549 and H1299 lung cancer cells undergoing EMT process. Our study indicated that SPARC was upregulated in A549 and H1299 cells EMT process. We further investigated the function of SPARC on proliferation, migration, and EMT process of A549 and H1299 cells. Overexpression of SPARC promoted the migration and EMT of A549 and H1299 cells. Knockdown SPARC inhibited the EMT of A549 cells. Overexpression of SPARC induced the increased expression of p-Akt and P-ERK. Furthermore, exogenous SPARC peptide promoted transforming growth factor (TGF)-β1-induced EMT of A549 and H1299 cells. SPARC knockdown partially eliminated TGF-β1 function in inducing EMT of A549 cells. SPARC follistatin-like functional domain reduced the expression of E-cadherin, but had no effect on the expression of p-Akt and p-ERK. In conclusion, we elucidated that SPARC contributes to tumorigenesis by promoting migration and EMT of A549 and H1299 lung cancer cells. These results will provide some new suggestion for lung cancer treatment. © 2018 BioFactors, 44(5):453-464, 2018.

Implication of SPARC in the modulation of the extracellular matrix and mitochondrial function in muscle cells.

Secreted protein, acidic and rich in cysteine (SPARC) is differentially associated with cell proliferation and extracellular matrix (ECM) assembly. We show here the effect of exogenous SPARC inhibition/induction on ECM and mitochondrial proteins expression and on the differentiation of C2C12 cells. The cells were cultured in growth medium (GM) supplemented with different experimental conditions. The differentiation of myoblasts was studied for 5 days, the expressions of ECM and mitochondrial proteins were measured and the formation of the myotubes was quantified after exogenous induction/inhibition of SPARC. The results indicate that the addition of recombinant SPARC protein (rSPARC) in cell culture medium increased the differentiation of C2C12 myoblasts and myogenin expression during the myotube formation. However, the treatment with antibody specific for SPARC (anti-SPARC) prevented the differentiation and decreased myogenin expression. The induction of SPARC in the proliferating and differentiating C2C12 cells increased collagen 1a1 protein expression, whereas the inhibition decreased it. The effects on fibronectin protein expression were opposite. Furthermore, the addition of rSPARC in C2C12 myoblast increased the expression of mitochondrial proteins, ubiquinol-cytochrome c reductase core protein II (UQCRC2) and succinate dehydrogenase iron-sulfur subunit (SDHB), whereas the anti-SPARC decreased them. During the differentiation, only the anti-SPARC had the effects on mitochondrial proteins, NADH dehydrogenase ubiquinone 1 beta subcomplex subunit 8 (NADHB8), SDHB and cytochrome c oxidase 1 (MTCO1). Thus, SPARC plays a crucial role in the proliferation and differentiation of C2C12 and may be involved in the link between the ECM remodeling and mitochondrial function.

Secreted Protein Acidic and Rich in Cysteine (SPARC) Enhances Cell Proliferation, Migration, and Epithelial Mesenchymal Transition, and SPARC Expression is Associated with Tumor Grade in Head and Neck Cancer.

Secreted protein acidic and rich in cysteine (SPARC) is a secreted protein which is involved in various biological processes. SPARC expression is associated with tumor metastasis and poor prognosis in several types of cancer. However, the SPARC-induced signaling pathway was not fully understood in head and neck cancer. In this study, our results showed that SPARC treatment promoted cell proliferation and migration in head and neck cancer cell lines FaDu and Detroit 562. In addition, SPARC induced expression of epithelial mesenchymal transition (EMT) regulators, including Slug, Snail, and Twist in Detroit 562. The results of phospho-kinase array analysis showed that SPARC treatment increased phosphorylation of some molecules including protein kinase B (PKB/AKT), ribosomal S6 kinase (RSK), and extracellular signal–regulated kinases (ERK). The expression of SPARC-induced EMT regulator Slug was suppressed by AKT inhibitor, but not ERK and RSK inhibitors. The SPARC expression in grade IV tumor samples is higher when compared to that in grade I–III tumor samples. Our results suggest that SPARC treatment enhances the EMT signaling pathway via activation of AKT, and exogenous SPARC and tumor expressing SPARC might be associated with tumor progression in head and neck cancers.

Novel Role for Matricellular Proteins in the Regulation of Islet β Cell Survival

Understanding the mechanisms regulating islet growth and survival is critical for developing novel approaches to increasing or sustaining β cell mass in both type 1 and type 2 diabetes patients. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that is important for the regulation of cell growth and adhesion. Increased SPARC can be detected in the serum of type 2 diabetes patients. The aim of this study was to investigate the role of SPARC in the regulation of β cell growth and survival. We show using immunohistochemistry that SPARC is expressed by stromal cells within islets and can be detected in primary mouse islets by Western blot. SPARC is secreted at high levels by pancreatic stellate cells and is regulated by metabolic parameters in these cells, but SPARC expression was not detectable in β cells. In islets, SPARC expression is highest in young mice, and is also elevated in the islets of non-obese diabetic (NOD) mice compared with controls. Purified SPARC inhibits growth factor-induced signaling in both INS-1 β cells and primary mouse islets, and inhibits IGF-1-induced proliferation of INS-1 β cells. Similarly, exogenous SPARC prevents IGF-1-induced survival of primary mouse islet cells. This study identifies the stromal-derived matricellular protein SPARC as a novel regulator of islet survival and β cell growth.

Exogenous SPARC Suppresses Proliferation and Migration of Prostate Cancer by Interacting With Integrin β1

The matricellular protein secreted protein acidic and rich in cysteine (SPARC) plays an important role on tumor metastasis and progression in several cancers. However, the roles of SPARC in prostate cancer (PCa) remain unclear. To identify SPARC protein in prostate tissue, immunohistochemical analysis of SPARC was conducted using human prostate tissue microarray. To detect SPARC expression in prostate cancer (LNCaP, DU145, and PC-3) and stromal cells, RT-PCR, western blot analysis, and ELISA was conducted. To reveal the function of exogenous SPARC in PCa cells, AKT phosphorylation was confirmed by western blot analysis after coculture with stromal cells. Proliferation and migration of PCa cells were examined by addition of SPARC. The interaction between SPARC and integrin β1 was confirmed by western blot analysis after immunoprecipitation. SPARC protein was expressed well in normal tissue compared with PCa tissue. ELISA showed high secreted SPARC protein in normal prostate-derived stromal cell (PrSC) compared with PCa-derived stromal cell (PCaSC) and PCa. PCa cells cocultured with PrSC showed reduced AKT phosphorylation more than with PCaSC. PCa cells cocultured with PrSC whose SPARC was knocked-down restored AKT phosphorylation. Moreover, PCa cells treated with SPARC led to reduced AKT phosphorylation. Immunoprecipitation with SPARC revealed interaction of SPARC and integrin β1 in PCa cells. Inhibited proliferation and migration of PCa cells by SPARC was restored by integrin β1 neutralizing antibody. Reduced SPARC secretion from stromal cells might affect PCa progression mediating through limiting AKT phosphorylation after interaction with integrin β1.

SPARC promotes the development of erythroid progenitors

Secreted protein acidic and rich in cysteine (SPARC) is a well-recognized regulator that affects cell proliferation, differentiation, and survival. Here, we investigated the impact of SPARC on erythropoiesis. Erythropoiesis in bone marrow (BM) from SPARC(-/-) mice was analyzed by flow cytometry and colony-forming assays. The mechanisms that affected erythropoiesis were investigated by BM transplantation experiments. CD34(+) cells from umbilical cord blood were isolated by MiniMACS, and the effect of SPARC on human erythropoiesis was assessed by colony-formation assay and erythroid differentiation culture. The hemoglobin level in peripheral blood (PB) was lower in SPARC(-/-) mice. Neither red blood cell count in PB nor the percentage of Ter119(+) erythrocytes in BM showed significant difference between SPARC(-/-) and WT mice. However, the ability of SPARC(-/-) bone marrow cells (BMCs) to form erythroid burst-forming units (BFU-E), as well as spleen colony-forming units (CFU-S8), was significantly decreased. The addition of exogenous SPARC could promote the formation of BFU-E from SPARC(-/-) BMCs in vitro. And the impaired ability of SPARC(-/-) BMCs to form BFU-E could be restored by transplanted into WT BM microenvironment, whereas the BFU-E formation of WT BMCs was impaired after transplanted into SPARC(-/-) recipients. Furthermore, exogenous SPARC promoted umbilical cord blood CD34(+) cells to form erythroid colonies and the hemoglobinization of erythroid, but did not affect the expression levels of glycophorin A and CD71. In conclusion, our results indicate that SPARC promotes the development of erythroid progenitors, but does not affect terminal erythroid differentiation.

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.

Secreted protein acidic and rich in cysteine-induced cellular senescence in colorectal cancers in response to irinotecan is mediated by P53

Cellular senescence is another mechanism that can be exploited to achieve better chemosensitivity and greater tumor regression. Unlike apoptosis, cellular senescence can be induced at much lower concentrations of chemotherapy that are better tolerated by patients. We previously revealed that secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, may function as a modulator of chemotherapy sensitivity by enhancing apoptosis. Here, we examine the effects of SPARC on cellular senescence in the presence of chemotherapy. Cellular senescence is induced only in sensitive colorectal cancer (CRC) cells with low concentrations of irinotecan (CPT-11). However, CPT-11-resistant cells exposed to endogenous or exogenous SPARC can also be triggered into cellular senescence. This induction is associated with higher levels of p16(INK4A) and phosphorylated p53. Knock down of p16(INK4A) reduces drug-induced senescence in all cells, but knock down and overexpression of p53 modulates senescence only in cells exposed to SPARC. Furthermore, treatment of mice with SPARC and CPT-11 leads to significantly increased cellular senescence and tumor regression. The chemosensitizing effects of SPARC in CRCs are, therefore, probably mediated in part by activating cellular senescence.

Inhibition of endogenous SPARC enhances pancreatic cancer cell growth: modulation by FGFR1-III isoform expression

Background:Secreted protein acidic and rich in cysteine (SPARC) is a multi-faceted protein-modulating cell–cell and cell–matrix interactions. In cancer, SPARC can be not only associated with a highly aggressive phenotype, but also acts as a tumour suppressor. The aim of this study was to characterise the function of SPARC and its modulation by fibroblast growth factor receptor (FGFR) 1 isoforms in pancreatic ductal adenocarcinoma (PDAC).Methods and results:Exogenous SPARC inhibited growth, movement, and migration. ShRNA inhibition of endogenous SPARC in ASPC-1 and PANC-1 cells resulted in increased anchorage-dependent and -independent growth, transwell migration, and xenograft growth as well as increased mitogenic efficacy of fibroblast growth factor (FGF) 1 and FGF2. Endogenous SPARC expression in PANC-1 cells was increased in FGFR1-IIIb over-expressing cells, but decreased in FGFR1-IIIc over-expressing cells. The up-regulation of endogenous SPARC was abrogated by the p38-mitogen-activated protein kinase inhibitor SB203580. SPARC was detectable in conditioned medium of pancreatic stellate cells (PSCs), but not PDAC cells. Conditioned medium of PDAC cells reduced endogenous SPARC expression of PSCs.Conclusion:Endogenous SPARC inhibits the malignant phenotype of PDAC cells and may, therefore, act as a tumour suppressor in PDAC. Endogenous SPARC expression can be modulated by FGFR1-III isoform expression. In addition, PDAC cells may inhibit endogenous SPARC expression in surrounding PSCs by paracrine actions.

Aberrant Promoter Methylation of Sparc in Ovarian Cancer

Epigenetic silencing of tumor suppressor genes is a new focus of investigation in the generation and proliferation of carcinomas. Secreted protein acidic and rich in cysteine (SPARC) is reportedly detrimental to the growth of ovarian cancer cells and has been shown to be epigenetically silenced in several cancers. We hypothesized that SPARC is downregulated in ovarian cancer through aberrant promoter hypermethylation. To that end, we analyzed SPARC expression in ovarian cancer cell lines and investigated the methylation status of the Sparc promoter using methylation-specific polymerase chain reaction. Our results show that SPARC mRNA expression is decreased in three (33%) and absent in four (44%) of the nine ovarian cancer cell lines studied, which correlated with hypermethylation of the Sparc promoter. Treatment with the demethylating agent 5-aza-2′-deoxycytidine rescued SPARC mRNA and protein expression. Addition of exogenous SPARC, as well as ectopic expression by an adenoviral vector, resulted in decreased proliferation of ovarian cancer cell lines. Investigation of primary tumors revealed that the Sparc promoter is methylated in 68% of primary ovarian tumors and that the levels of SPARC protein decrease as the disease progresses from low to high grade. Lastly, de novo methylation of Sparc promoter was shown to be mediated by DNA methyltransferase 3a. These results implicate Sparc promoter methylation as an important factor in the genesis and survival of ovarian carcinomas and provide new insights into the potential use of SPARC as a novel biomarker and/or treatment modality for this disease.

The identification of new tumour suppressor micrornas epigenetically silenced in drug resistant cancer cells

Epigenetic silencing of tumor suppressor genes is a new focus of investigation in the generation and proliferation of carcinomas. Secreted protein acidic and rich in cysteine (SPARC) is reportedly detrimental to the growth of ovarian cancer cells and has been shown to be epigenetically silenced in several cancers. We hypothesized that SPARC is downregulated in ovarian cancer through aberrant promoter hypermethylation. To that end, we analyzed SPARC expression in ovarian cancer cell lines and investigated the methylation status of the Sparc promoter using methylation-specific polymerase chain reaction. Our results show that SPARC mRNA expression is decreased in three (33%) and absent in four (44%) of the nine ovarian cancer cell lines studied, which correlated with hypermethylation of the Sparc promoter. Treatment with the demethylating agent 5-aza-2′-deoxycytidine rescued SPARC mRNA and protein expression. Addition of exogenous SPARC, as well as ectopic expression by an adenoviral vector, resulted in decreased proliferation of ovarian cancer cell lines. Investigation of primary tumors revealed that the Sparc promoter is methylated in 68% of primary ovarian tumors and that the levels of SPARC protein decrease as the disease progresses from low to high grade. Lastly, de novo methylation of Sparc promoter was shown to be mediated by DNA methyltransferase 3a. These results implicate Sparc promoter methylation as an important factor in the genesis and survival of ovarian carcinomas and provide new insights into the potential use of SPARC as a novel biomarker and/or treatment modality for this disease.

SPARC Endogenous Level, rather than Fibroblast-Produced SPARC or Stroma Reorganization Induced by SPARC, Is Responsible for Melanoma Cell Growth

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein whose overexpression in malignant or tumor-stromal cells is often associated with increased aggressiveness and bad prognosis in a wide range of human cancer types, particularly melanoma. We established the impact that changes in the level of SPARC produced by malignant cells and neighboring stromal cells have on melanoma growth. Melanoma cell growth in monolayer was only slightly affected by changes in SPARC levels. However, melanoma growth in spheroids was strongly inhibited upon SPARC hyperexpression and conversely enhanced when SPARC expression was downregulated. Interestingly, SPARC overexpression in neighboring fibroblasts had no effect on spheroid growth irrespective of SPARC levels expressed by the melanoma cells, themselves. Downregulation of SPARC expression in melanoma cells induced their rejection in vivo through a mechanism mediated exclusively by host polymorphonuclear cells. On the other hand, SPARC hyperexpression enhanced vascular density, collagen deposition, and fibroblast recruitment in the surrounding stroma without affecting melanoma growth. In agreement with the in vitro data, overexpression of SPARC in co-injected fibroblasts did not affect melanoma growth in vivo. All the data indicate that melanoma growth is not subject to regulation by exogenous SPARC, nor by stromal organization, but only by SPARC levels produced by the malignant cells themselves.

SPARC, an upstream regulator of connective tissue growth factor in response to transforming growth factor β stimulation

To differentiate the effects of inhibition of specific small interfering RNA (siRNA) of SPARC (secreted protein, acidic and rich in cysteine) and siRNA of connective tissue growth factor (CTGF) in cultured human fibroblasts, and to identify potential interrelationships between SPARC and CTGF. Fibroblasts from skin biopsy specimens of 2 normal individuals were transfected with siRNA of SPARC and siRNA of CTGF. The fibroblasts were stimulated with or without transforming growth factor beta1 (TGFbeta1) and examined by real-time quantitative reverse transcription-polymerase chain reaction to determine the transcription levels of several extracellular matrix genes. After exogenous TGFbeta1 stimulation, both SPARC siRNA and CTGF siRNA showed a protective role against overexpression of collagen genes. Following TGFbeta1 stimulation, SPARC siRNA-transfected fibroblasts showed a greater reduction in expression of the collagen genes compared with CTGF siRNA-transfected fibroblasts, as well as a significantly decreased expression of CTGF (P < 0.05). Using linear structure equations to quantitatively model a genetic network based on expression levels of each gene, a positive regulatory role of SPARC on CTGF, COL1A2, COL3A1, COL11A1, and TIMP3 was observed. However, the regulatory role of CTGF on SPARC appeared to be negative and very small, while the positive regulatory effects of CTGF on COL1A2, COL3A1, COL11A1, and TIMP3 were less than those of SPARC. The results of this quantitative comparison support the hypothesis that in these cultured fibroblasts, the regulatory effects of SPARC on some major extracellular matrix structural components are greater than those of CTGF. In addition, SPARC appears to regulate CTGF in a predominantly positive manner, while CTGF may act as a negative feedback control on SPARC following TGFbeta stimulation.

Low or absent SPARC expression in acute myeloid leukemia with MLL rearrangements is associated with sensitivity to growth inhibition by exogenous SPARC protein

Secreted protein, acidic and rich in cysteine (SPARC), is a matricellular glycoprotein with growth-inhibitory and antiangiogenic functions. Although SPARC has been implicated as a tumor suppressor in humans, its function in normal or malignant hematopoiesis has not previously been studied. We found that the leukemic cells of AML patients with MLL gene rearrangements express low to undetectable amounts of SPARC whereas normal hematopoietic progenitors and most AML patients express this gene. SPARC RNA and protein levels were also low or undetectable in AML cell lines with MLL translocations. Consistent with its tumor suppressive effects in various solid tumor models, exogenous SPARC protein selectively reduced the growth of cell lines with MLL rearrangements by inhibiting cell cycle progression from G1 to S phase. The lack of SPARC expression in MLL-rearranged cell lines was associated with dense promoter methylation. However, we found no evidence of methylation-based silencing of SPARC in primary patient samples. Our results suggest that low or absent SPARC expression is a consistent feature of AML cells with MLL rearrangements and that SPARC may function as a tumor suppressor in this subset of patients. A potential role of exogenous SPARC in the therapy of MLL-rearranged AML warrants further investigation.

Inhibition of PDGF-stimulated and matrix-mediated proliferation of human vascular smooth muscle cells by SPARC is independent of changes in cell shape or cyclin-dependent kinase inhibitors.

Interactions among growth factors, cells, and extracellular matrix regulate proliferation during normal development and in pathologies such as atherosclerosis. SPARC (secreted protein, acidic, and rich in cysteine) is a matrix-associated glycoprotein that modulates the adhesion and proliferation of vascular cells. In this study, we demonstrate that SPARC inhibits human arterial smooth muscle cell proliferation stimulated by platelet-derived growth factor or by adhesion to monomeric type I collagen. Binding studies with SPARC and SPARC peptides indicate specific and saturable interaction with smooth muscle cells that involves the C-terminal Ca2+-binding region of the protein. We also report that SPARC arrests monomeric collagen-supported smooth muscle cell proliferation in the late G1-phase of the cell cycle in the absence of an effect on cell shape or on levels of cyclin-dependent kinase inhibitors. Cyclin-dependent kinase-2 activity, p107 and cyclin A levels, and retinoblastoma protein phosphorylation are markedly reduced in response to the addition of exogenous SPARC and/or peptides derived from specific domains of SPARC. Thus, SPARC, previously characterized as an inhibitor of platelet-derived growth factor binding to its receptor, also antagonizes smooth muscle cell proliferation mediated by monomeric collagen at the level of cyclin-dependent kinase-2 activity.