Secreted protein acidic and rich in cysteine (SPARC), a calcium-binding matricellular glycoprotein, is implicated in the progression of many cancers. In this study, we investigated the expression and function of SPARC in ovarian cancer.
cDNA microarray analysis was performed to compare gene expression profiles of the highly invasive and the low invasive subclones derived from the SKOV3 human ovarian cancer cell line. Immunohistochemistry (IHC) staining was performed to investigate SPARC expression in a total of 140 ovarian tissue specimens. In functional assays, effects of SPARC knockdown on the biological behavior of ovarian cancer cells were investigated. The mechanisms of SPARC in ovarian cancer proliferation, apoptosis and invasion were also researched.
SPARC was overexpressed in the highly invasive subclone compared with the low invasive subclone. High SPARC expression was associated with high stage, low differentiation, lymph node metastasis and poor prognosis of ovarian cancer. Knockdown of SPARC expression significantly suppressed ovarian cancer cell proliferation, induced cell apoptosis and inhibited cell invasion and metastasis.
SPARC is overexpressed in highly invasive subclone and ovarian cancer tissues and plays an important role in ovarian cancer growth, apoptosis and metastasis.
Secreted protein acidic and rich in cysteine (SPARC) is a glycoprotein that functions to inhibit angiogenesis, proliferation, and invasion in different types of cancer. The ability of SPARC to modulate neovascularisation is believed to be mediated in part by its ability to modulate the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). In this study, we aimed to determine the effect of SPARC expression in gastric cancer cells on proliferation and angiogenesis in vitro and in vivo.
We evaluated expression of SPARC in seven human gastric cancer cell lines. Then we established a stably transfected SPARC overexpressed cell line (BGC-SP) and a stably transfected SPARC knock-down cell line (HGC-sh). The effect of SPARC overexpression and SPARC silencing was studied by examining capillary formation of HUVECs in vitro and a dorsal skin-fold chamber model in vivo. Quantitative real-time PCR and western blotting were performed to detect if the expressions of VEGF and MMP-7 were modulated by SPARC expression. To further determine the effect of SPARC expression on angiogenesis in vivo, xenograft models were established and microvessel density (MVD) of different clones were detected by immunohistochemistry.
Endogenous SPARC overexpression inhibited the expression of VEGF and MMP-7, as well as the angiogenesis induced by BGC-SP cells. Correspondingly, SPARC silencing increased the expression of VEGF and MMP-7, as well as the angiogenesis induced by HGC-sh cells. Elevated angiogenesis induced by SPARC silencing in HGC-sh cells was decreased when VEGF was neutralised by antibodies, and MMP-7 was knocked down in vitro.
SPARC suppresses angiogenesis of gastric cancer by down-regulating the expression of VEGF and MMP-7.
SPARC is a matricellular glycoprotein with growth-inhibitory and antiangiogenic activity in some cell types. The study of this protein in hematopoietic malignancies led to conflicting reports about its role as a tumor suppressor or promoter, depending on its different functions in the tumor microenvironment. In this study we investigated the variations in SPARC production by peripheral blood cells from chronic myeloid leukemia (CML) patients at diagnosis and after treatment and we identified the subpopulation of cells that are the prevalent source of SPARC.
We evaluated SPARC expression using real-time PCR and western blotting. SPARC serum levels were detected by ELISA assay. Finally we analyzed the interaction between exogenous SPARC and imatinib (IM), in vitro, using ATP-lite and cell cycle analysis.
Our study shows that the CML cells of patients at diagnosis have a low mRNA and protein expression of SPARC. Low serum levels of this protein are also recorded in CML patients at diagnosis. However, after IM treatment we observed an increase of SPARC mRNA, protein, and serum level in the peripheral blood of these patients that had already started at 3 months and was maintained for at least the 18 months of observation. This SPARC increase was predominantly due to monocyte production. In addition, exogenous SPARC protein reduced the growth of K562 cell line and synergized in vitro with IM by inhibiting cell cycle progression from G1 to S phase.
Our results suggest that low endogenous SPARC expression is a constant feature of BCR/ABL positive cells and that IM treatment induces SPARC overproduction by normal cells. This exogenous SPARC may inhibit CML cell proliferation and may synergize with IM activity against CML.
CML; Imatinib; SPARC; Granulocytes; Monocytes
Secreted protein acidic and rich in cysteine (SPARC), a matricellular glycoprotein, modulates cellular interaction with the extracellular matrix and is capable of altering the growth of various cancers. We therefore sought to determine the effect of SPARC expression on medulloblastoma tumour growth and angiogenesis.
To this extent, we selected three SPARC full-length cDNA overexpressed clones (Daoy-SP). Consequences of SPARC overexpression were studied in terms of cell growth, angiogenesis using co-culture assay in vitro, dorsal skin-fold chamber assay in vivo, PCR Array for human angiogenic genes, as well as western blotting for angiogenic molecules and tumour growth, in an orthotopic tumour model.
The SPARC protein and mRNA levels were increased by approximately three-fold in Daoy-SP cells compared with parental (Daoy-P) and vector (Daoy-EV) controls. Daoy-SP clones reduced tumour cell-induced angiogenesis in vitro and in vivo, and formed small tumours with fewer blood vessels when compared with controls. Matrix metalloprotease-9 (MMP-9) and vascular endothelial growth factor (VEGF) expression were decreased in Daoy-SP clones. Further, inhibition of MMP-9 expression caused SPARC-mediated inhibition of angiogenesis and tumour growth as MMP-9 rescued SPARC-mediated anti-angiogenic effect in vitro and tumour growth inhibition in vivo.
Overexpression of SPARC decreases angiogenesis, which leads to decreased tumour growth. Further, the role of MMP-9 could be attributed to the anti-angiogenic effect of SPARC.
angiogenesis; SPARC; VEGF; MMP-9; CD-31
Successful pregnancy depends on the precise regulation of extravilloustrophoblast (EVT) invasion into the uterine decidua. SPARC (secreted protein acidic and rich in cysteine) is a matricellular glycoprotein that plays critical roles in the pathologies associated with obesity and diabetes, as well as tumorigenesis. The objective of this study was to investigate the role of SPARC in the process of trophoblast invasion which shares many similarities with tumor cell invasion. By Western blot, higher expression of SPARC was observed in mouse brain, ovary and uterus compared to other mouse tissues. Immunohistochemistry analysis revealed a spatio-temporal expression of SPARC in mouse uterus in the periimplantation period. At the implantation site of d8 pregnancy, SPARC mainly accumulated in the secondary decidua zone (SDZ), trophoblast cells and blastocyst. The expression of SPARC was also detected in human placental villi and trophoblast cell lines. In a Matrigel invasion assay, we found SPARC-specific RNA interference significantly reduced the invasion of human extravilloustrophoblast HTR8/SVneo cells. Microarray analysis revealed that SPARC depletion upregulated the expression of interleukin 11 (IL11), KISS1, insulin-like growth factor binding protein 4 (IGFBP4), collagen type I alpha 1 (COLIA1), matrix metallopeptidase 9 (MMP9), and downregulated the expression of the alpha polypeptide of chorionic gonadotropin (CGA), MMP1, gap junction protein alpha 1 (GJA1), et al. The gene array result was further validated by qRT-PCR and Western blot. The present data indicate that SPARC may play an important role in the regulation of normal placentation by promoting the invasion of trophoblast cells into the uterine decidua.
Secreted protein acidic and rich in cysteine (SPARC) plays a key role in the development of many tissues and organ types. Aberrant SPARC expression was found in a wide variety of human cancers, contributes to tumor development. Because SPARC was found to be overexpressed in human gastric cancer tissue, we therefore to explore the expression of SPARC in gastric cancer lines and the carcinogenic mechanisms.
SPARC expression was evaluated in a panel of human gastric cancer cell lines. MGC803 and HGC 27 gastric cancer cell lines expressing high level of SPARC were transiently transfected with SPARC-specific small interfering RNAs and subsequently evaluated for effects on invasion and proliferation.
Small interfering RNA-mediated knockdown of SPARC in MGC803 and HGC 27 gastric cancer cells dramatically decreased their invasion. Knockdown of SPARC was also observed to significantly increase the apoptosis of MGC803 and HGC 27 gastric cancer cells compared with control transfected group.
Our data showed that downregulating of SPARC inhibits invasion and growth of human gastric cancer cells. Thus, targeting of SPARC could be an effective therapeutic approach against gastric cancer.
Pancreatic adenocarcinoma, a desmoplastic disease, is the fourth leading cause of cancer-related death in the Western world due, in large part, to locally invasive primary tumor growth and ensuing metastasis. SPARC is a matricellular protein that governs extracellular matrix (ECM) deposition and maturation during tissue remodeling, particularly, during wound healing and tumorigenesis. In the present study, we sought to determine the mechanism by which lack of host SPARC alters the tumor microenvironment and enhances invasion and metastasis of an orthotopic model of pancreatic cancer. We identified that levels of active TGFβ1 were increased significantly in tumors grown in SPARC-null mice. TGFβ1 contributes to many aspects of tumor development including metastasis, endothelial cell permeability, inflammation and fibrosis, all of which are altered in the absence of stromal-derived SPARC. Given these results, we performed a survival study to assess the contribution of increased TGFβ1 activity to tumor progression in SPARC-null mice using losartan, an angiotensin II type 1 receptor antagonist that diminishes TGFβ1 expression and activation in vivo. Tumors grown in SPARC-null mice progressed more quickly than those grown in wild-type littermates leading to a significant reduction in median survival. However, median survival of SPARC-null animals treated with losartan was extended to that of losartan-treated wild-type controls. In addition, losartan abrogated TGFβ induced gene expression, reduced local invasion and metastasis, decreased vascular permeability and altered the immune profile of tumors grown in SPARC-null mice. These data support the concept that aberrant TGFβ1-activation in the absence of host SPARC contributes significantly to tumor progression and suggests that SPARC, by controlling ECM deposition and maturation, can regulate TGFβ availability and activation.
SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.
In in vitro studies, skin fibroblasts obtained from a Tgfbr1 knock-in mouse (TBR1CA; Cre-ER) were transfected with SPARC siRNA. Gene and protein expressions of the Col1a2 and the Ctgf were examined by real-time RT-PCR and Western blotting, respectively. In in vivo studies, C57BL/6 mice were induced for skin and lung fibrosis by bleomycin and followed by SPARC siRNA treatment through subcutaneous injection and intratracheal instillation, respectively. The pathological changes of skin and lungs were assessed by hematoxylin and eosin and Masson's trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays.
SPARC siRNA significantly reduced gene and protein expression of collagen type 1 in fibroblasts obtained from the TBR1CA; Cre-ER mouse that was induced for constitutively active TGF-β receptor I. Skin and lung fibrosis induced by bleomycin was markedly reduced by treatment with SPARC siRNA. The anti-fibrotic effect of SPARC siRNA in vivo was accompanied by an inhibition of Ctgf expression in these same tissues.
Specific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases.
SPARC (secreted protein, acidic and rich in cysteine) is closely related with the progress, invasion and metastasis of malignant tumor and angiogenesis.
Using human colon adenocarcinoma tissues (hereinafter referred to as colon cancer) and their corresponding non-diseased colon from 114 patients' biopsies, the expression of SPARC and vascular endothelial growth factor (VEGF) were investigated by immunohistochemistry staining to assessment the relationship between SPARC and VEGF, as well as their prognostic significance in patients. Evaluation of VEGF expression level with the same tissues was used to establish the antigenic profiles, and the marker of CD34 staining was used as an indicator of microvessel density (MVD).
SPARC expression was mainly in the stromal cells surrounding the colon cancer, and was significant difference in those tissues with the lymph node metastasis and differentiation degree of tumor. Expression of SPARC was significantly correlated with the expression of VEGF and MVD in colon cancer tissues. Patients with low or absence expressing SPARC had significantly worse overall survival and disease-free survival in a Single Factor Analysis; Cox Regression Analysis, SPARC emerged as an overall survival and disease-free survival independent prognostic factor for colon cancer.
The low expression or absence of stromal SPARC was an independent prognostic factor for poor prognosis of colon cancer. SPARC maybe involved in the regulation of anti-angiogenesis by which it may serve as a novel target for colon cancer treatment as well as a novel distinctive marker.
Malignant glioma is a highly invasive brain tumor resistant to conventional therapies. Secreted protein acidic and rich in cysteine (SPARC) has been shown to facilitate glioma invasion. However, the effects of SPARC on cell growth have yet to be adequately elucidated. In this study, we constructed a plasmid expressing shRNA against SPARC, evaluated the effect of SPARCshRNA on SPARC expression and then assessed its effect on cell growth in U-87MG cells. Using plasmid-delivered shRNA, we effectively suppressed SPARC expression in U-87MG cells. Cell growth curves and colony formation assay suggested that the introduction of SPARCshRNA resulted in an increase of cell growth and colony formation. We also showed that knockdown of SPARC expression was capable of promoting the cell cycle progression from the G1 to S phase. However, no difference was found in the level of apoptosis. A molecular analysis of signal mediators indicated that the inhibition of p-c-Raf (Ser259) and accumulation of p-GSK-3β (Ser9) and p-AKT (Ser473) may be connected with the growth promotion by SPARC shRNA. Our study may provide an insight into the biological function of SPARC in glioma.
The aim of the present study was to analyse the expression of Secreted protein acidic and rich in cysteine (SPARC) in nasopharyngeal carcinoma (NPC) specimens, and to evaluate its correlation with clinicopathologic features, including survival of patients with NPC
NPC tissue microarrays (TMAs) were constructed from Sun Yat-sen University Cancer Center (SYSUCC), another three centers on mainland China, Singapore and Hong Kong. Using quantitative RT-PCR and Western-blotting techniques, we detected mRNA and protein expression of SPARC in NPC cell lines and immortalized nasopharyngeal epithelial cells (NPECs) induced by Bmi-1 (NPEC2 Bmi-1). The difference of SPARC expression in the cell lines was tested using a t-test method. The relationship between the SPARC expression and clinicopathological data was assessed by chi-square. Survival analysis was estimated using the Kaplan-Meier approach with log-rank test. Univariate and multivariate analyses of clinical variables were performed using Cox proportional hazards regression models.
The expression levels of SPARC mRNA and protein were markedly higher in NPC cell lines than in NPEC2 Bmi-1. Especially, the expression levels of SPARC mRNA and protein were much lower in the 6-10B than in the 5-8 F (P = 0.002, P = 0.001). SPARC immunostaining revealed cytoplasmic localization in NPC cells and no staining in the stroma and epithelium.
In addition, high level of SPARC positively correlated with the status of distant metastasis (P = 0.001) and WHO histological classification (P = 0.023). NPC patients with high SPARC expression also had a significantly poorer prognosis than patients with low SPARC expression (log-rank test, P < 0.001), especially patients with advanced stage disease (log-rank, P < 0.001). Multivariate analysis suggested that the level of SPARC expression was an independent prognostic indicator for the overall survival of patients with NPC (P < 0.001).
SPARC expression is common in NPC patients. Our data shows that elevated SPARC expression is a potential unfavorable prognostic factor for patients with NPC.
SPARC; Nasopharyngeal carcinoma; Metastasis
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.
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.
osteonectin; SPARC; fibroblast growth factor receptor; pancreatic cancer; pancreatic stellate cells
Secreted protein acidic and rich in cysteine (SPARC), also known as osteonectin or basement-membrane-40 (BM-40), is a member of a family of matricellular proteins, whose functions are to modulate cell-matrix interactions, growth and angiogenesis in colorectal cancer. In this study, the expression of SPARC was evaluated and its correlations with clinicopathological parameters were investigated.
The researchers analyzed the expression patterns of SPARC by using immunohistochemistry in 332 cases of colorectal cancer of tissue microarray. The clinicopathological characteristics were defined by using the TNM criteria of the Union for International Cancer Control. Clinicopathological factors such as age, sex, histologic type of the tumor, pathologic tumor stage, TNM stage, and lymphovascular invasion were evaluated according to the SPARC expression.
The hazard ratios expressing SPARC in tumor cells, in the stroma, and in both tumor cells and the stroma were 2.10 (P = 0.036), 3.27 (P = 0.003) and 2.12 (P = 0.038), respectively. Patient survival was decreased in patient expressing SPARC in the stroma, and this result showed statistical significance (P = 0.016).
These findings suggest that SPARC expression in a tumor and in the stroma correlates with disease progression and may be used as a prognostic marker for colorectal cancer.
SPARC; Osteonectin; Colorectal neoplasms; Prognosis
To investigate the expression of the matricellular protein SPARC (secreted acidic cysteine-rich glycoprotein) in scarred human Tenon’s capsule and in cultured human Tenon’s fibroblasts (HTF), and to analyze the influence of SPARC on cell proliferation and collagen matrix contraction in vitro.
Human Tenon's capsule scars obtained from surgical revisions after filtration surgery were analyzed for SPARC expression by immunohistochemistry. In cultured HTF cells, SPARC expression was assessed by northern and western blot analyses after incubation with transforming growth factor (TGF)-β1 and TGF-β2. Cell proliferation was determined by bromodeoxyuridine (BrdU)–labeling and HTF cells-mediated collagen matrix contraction by morphometric measurements of three-dimensional collagen lattices after treatment with SPARC and/or TGF-β1.
In scarred human Tenon’s capsule specimens, an increased expression of SPARC was mainly localized to the extracellular matrix and to blood vessel walls as compared to healthy control Tenon’s capsule. In cultured HTF cells, treatment with TGF-β1 more than TGF-β2 induced the expression of SPARC both on the mRNA and protein level. Incubation of HTF cells with SPARC resulted in an increase in collagen matrix contraction and cell proliferation. Moreover, a combined incubation of SPARC and TGF-β1 stimulated HTF cell proliferation significantly over the levels that were observed after single treatment.
Our data provide evidence that SPARC contributes to excessive wound healing and scar formation in human Tenon’s capsules after filtration surgery and may thus represent a novel target for anti-fibrotic strategies.
SPARC (secreted protein acidic and rich in cysteine) is an extracellular Ca2+-binding matricellular glycoprotein associated with the regulation of cell adhesion and growth. We investigated loss of expression of SPARC gene and promoter methylation in lung cancers and correlated the data with clinicopathological features. We observed loss of SPARC expression in 12 of 20 (60%) lung cancer cell lines. Treatment of expression-negative cell lines with a demethylating agent restored expression in all cases. Methylation frequencies of SPARC gene were 55% in 20 lung cancer cell lines. Primary tumours had methylation at a rate of 69% (119 of 173), while nonmalignant lung tissues (n=60) had very low rates (3%). In lung adenocarcinomas, SPARC methylation correlated with a negative prognosis (P=0.0021; relative risk 4.65, 95% confidence interval 1.75–12.35, multivariate Cox's proportional-hazard model). Immunostaining revealed protein expression in bronchial epithelium (weak intensity) and in juxtatumoral stromal tissues (strong intensity) accompanied by frequent loss in cancer cells that correlated with the presence of methylation (P<0.001). Our findings are of biological interest and potentially of clinical importance in human lung cancers.
methylation; SPARC; lung cancer; immunostaining
SPARC belongs to a class of extracellular matrix-associated proteins that have counteradhesive properties. The ability of SPARC to modulate cell-cell and cell-matrix interactions provides a strong rationale for studies designed to determine its expression in cancer. The objective of this study was to determine if SPARC expression was altered in cadmium (Cd+2) and arsenite (As+3) induced bladder cancer and if these alterations were present in archival specimens of human bladder cancer. The expression of SPARC was determined in human parental UROtsa cells, their Cd+2 and As+3 transformed counterparts and derived tumors, and in archival specimens of human bladder cancer using a combination of real time reverse transcriptase polymerase chain reaction, western blotting, immunofluoresence localization and immunohistochemical staining. It was demonstrated that SPARC expression was down-regulated in Cd+2 and As+3 transformed UROtsa cells. In addition, the malignant epithelial component of tumors derived from these cell lines were also down-regulated for SPARC expression, but the stromal cells recruited to these tumors was highly reactive for SPARC. This finding was shown to translate to specimens of human bladder cancer where tumor cells were SPARC negative, but stromal cells were positive. Acute exposure of UROtsa cells to both cadmium and arsenite reduced the expression of SPARC through a mechanism that did not involve changes in DNA methylation or histone acetylation. These studies suggest that environmental exposure to As+3 or Cd+2 can alter cell-cell and cell-matrix interactions in normal urothelial cells through a reduction in the expression of SPARC. The SPARC associated loss of cell-cell and cell-matrix contacts may participate in the multi-step process of bladder carcinogenesis.
Arsenite; cadmium; SPARC; and bladder cancer
The molecular mechanism leading to the cancer metastasis to bone is poorly understood but yet determines prognosis and therapy. Here, we define a new molecular pathway that may account for the extraordinarily high osteotropism of prostate cancer. By using SPARC (secreted protein, acidic and rich in cysteine)-deficient mice and recombinant SPARC, we demonstrated that SPARC selectively supports the migration of highly metastatic relative to less metastatic prostate cancer cell lines to bone. Increased migration to SPARC can be traced to the activation of integrins αvβ and αvβ5 on tumor cells. Such activation is induced by an autocrine vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-2 loop on the tumor cells, which also supports the growth and proliferation of prostate cancer cells. A consequence of SPARC recognition by αvβ5 is enhanced VEGF production. Thus, prostate cancer cells expressing VEGF/VEGFR-2 will activate αvβ5 and αvβ5 on their surface and use these integrins to migrate toward SPARC in bone. Within the bone environment, SPARC engagement of these integrins will stimulate growth of the tumor and further production of VEGF to support neoangiogenesis, thereby favoring the development of the metastatic tumor. Supporting this model, activated integrins were found to colocalize with VEGFR-2 in tissue samples of metastatic prostate tumors from patients.
The matricellular glycoprotein Secreted Protein Acidic and Rich in Cysteine (SPARC) plays an important role in the regulation of cell adhesion and proliferation as well as in tumorigenesis and metastasis. Earlier, we reported that, in addition to its potent anti-angiogenic functions, SPARC also induces apoptosis in medulloblastoma cells, mediated by autophagy. We therefore sought to investigate the underlying molecular mechanism through which SPARC inhibits migration and invasion of Daoy medulloblastoma cells, both in vitro and in vivo. For this study, we used SPARC-overexpressing stable Daoy medulloblastoma cells. SPARC overexpression in Daoy medulloblastoma cells inhibited migration and invasion in vitro. Additionally, SPARC overexpression significantly suppressed the activity of Rho, Rac and Cdc42, which all regulate the actin cytoskeleton. This suppression was accompanied by an increase in the phosphorylation of Src at TYR-416, which led to a loss of actin stress fibers and focal contacts and a decrease in the phosphorylation level of cofilin. The reduced phosphorylation level of cofilin, which is indicative of receding Rho function, in turn led to inhibition of active Rho A. To confirm the role of SPARC in inhibition of migration and invasion of Daoy medulloblastoma cells, we transfected parental and SPARC-overexpressing Daoy cells with a plasmid vector carrying siRNA against SPARC. Transfection with SPARC siRNA reversed Src-mediated disruption of the cytoskeleton organization as well as dephosphorylation of cofilin and activation of Rho A. Taken together, these results establish SPARC as an effector of Src-induced cytoskeleton disruption in Daoy medulloblastoma cells, which subsequently led to decreased migration and invasion.
SPARC; Src; Migration; Invasion; Rho; Rac; Cdc42
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 is a key determinant of invasion and metastasis in some tumors, such as gliomas, melanomas and prostate tumors. SPARC can change the composition and structure of the matrix and promote angiogenesis; these effects are closely related to clinical stage and the prognosis of tumors such as meningiomas. However, little is known about the expression of SPARC in intracranial aneurysms. The goal of this study was to establish the role of SPARC in human intracranial aneurysms.
Thirty-one intracranial aneurysms were immunohistochemically stained for SPARC, MMP-2 and MMP-9. As controls, normal Circle of Willis arteries were similarly immunostained. All specimens were retrieved during autopsies and were embedded in paraffin. To evaluate the expression levels of SPARC, MMP-2 and MMP-9, western blotting was also performed in three available intracranial aneurysm specimens. The limited availability of fresh intracranial aneurysm tissue was the result of the majority of patients choosing endovascular embolization.
The results showed that SPARC, MMP-2 and MMP-9 were strongly expressed in intracranial aneurysm tissues; however, these proteins were expressed minimally or not at all in normal Circle of Willis arteries. The western blot results showed that the expression levels of SPARC, MMP-2 and MMP-9 were significantly up-regulated in intracranial aneurysms relative to the expression levels in the normal Circle of Willis arteries. Data analysis showed that SPARC was significantly correlated with MMP-2 and MMP-9, also with age and risk factors but not with the Hunt-Hess grade or with sex.
The results indicate that SPARC is widely expressed in human intracranial aneurysms, and its expression correlates with MMP-2 and MMP-9 expression, age and risk factors but not with the Hunt-Hess grade. The results of this study suggest that SPARC has a pathogenic role in the alteration of the extracellular matrix of intracranial arteries during aneurysm formation.
Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that mediates cell-matrix interactions. It has been shown, depending on the type of cancer, to possess either pro- or anti-tumorigenic properties. The transcriptional regulation of the SPARC gene expression has not been fully elucidated and the effects of anti-cancer drugs on this process have not been explored.
In the present study, we demonstrated that chromatin remodeling factor Brg-1 is recruited to the proximal SPARC promoter region (-130/-56) through an interaction with transcription factor Sp1. We identified Brg-1 as a critical regulator for the constitutive expression levels of SPARC mRNA and protein in mammary carcinoma cell lines and for SPARC secretion into culture media. Furthermore, we found that Brg-1 cooperates with Sp1 to enhance SPARC promoter activity. Interestingly, fenretinide [N-4(hydroxyphenyl) retinamide, 4-HPR], a synthetic retinoid with anti-cancer properties, was found to up-regulate the transcription, expression and secretion of SPARC via induction of the Brg-1 in a dose-dependent manner. Finally, our results demonstrated that fenretinide-induced expression of SPARC contributes significantly to a decreased invasion of mammary carcinoma cells.
Overall, our results reveal a novel cooperative role of Brg-1 and Sp1 in mediating the constitutive and fenretinide-induced expression of SPARC, and provide new insights for the understanding of the anti-cancer effects of fenretinide.
Secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human gliomas where it promotes invasion and delays tumor growth, both in vitro and in vivo. SPARC, which interacts at the cell surface, has an impact on intracellular signaling and downstream gene expression changes, which might account for some of its effects on invasion and growth. Additional in vitro studies demonstrated that SPARC delays growth, increases attachment, and modulates migration of tumor cells in an extracellular matrix-specific and concentration-dependent manner. Because the signaling aspect of this migration is neither well understood nor characterized, we overexpressed SPARC in both the minimally-invasive U87 cell line and in the most aggressive invasive cell line, SNB19. We first performed RT-PCR analysis and observed an upregulation of uPA and its receptor, uPAR. We also observed increased expression levels of matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9). Western blot analysis confirmed these results, and the enzymatic activity of the metalloproteinases and uPA was further supported by zymography.
Downstream of the uPA-uPAR interaction, upregulation of PI3-K occurred in cells overexpressing SPARC. Using GST-TRBD, we showed the upregulation of active GTP-bound RhoA, but neither Rac1 nor Cdc42 were activated. The inhibition of uPA and uPAR downregulated PI3-K activity and cell migration, as shown by matrigel invasion assay. A dorsal skin-fold chamber model revealed the high angiogenic activity of SPARC, though the proliferation of SPARC overexpressing cells was unaffected. Our results show that the small GTPase RhoA was a critical mediator of invasion or migration in the uPA-uPAR/PI3-K signaling pathway.
Glioblastoma; SPARC; uPA-uPAR signaling; small GTPase RhoA; Migration; SPARC Secreted protein acidic and rich in cysteine, MMP-2 and MMP-9 matrix metalloproteinases-2 and -9, RNAi, RNA interference; siRNA, short interfering RNA; uPA, urokinase-type plasminogen activator (receptor); uPAR, urokinase-type plasminogen activator receptor; CMV, cytomegalovirus; PBS, phosphate-buffered saline; FITC, fluorescein 5-isothiocyanate; EV, empty vector; H&E, hematoxylin & eosin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; pU2, plasmid siRNA vector for uPA and uPAR; pGFP, plasmid siRNA for GFP; puPAR,plasmid siRNA vector for uPAR; puPA,plasmid siRNA vector for uPA; PI3-K/Akt, Phosphoinositide 3-kinase/Serine and threonine kinase; FAK, focal adhesion kinase; RT-PCR, reverse transcriptase polymerase chain reaction; ECM, extra-cellular matrix; VEGF, vascular endothelial growth factor; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; GPCR, G-protein coupled receptors; BFGF, Basic fibroblast growth factor; TNF-α, tumor necrosis factor; ANG-1 Angiopoietin-1; HBEGF, heparin-binding EGF-like growth factor; IGF-1 insulin-like growth factor 1
Pancreatic adenocarcinoma is characterized by desmoplasia, local invasion, and metastasis. These features are regulated in part by MMP9 and SPARC. To explore the interaction of SPARC and MMP9 in cancer, we first established orthotopic pancreatic tumors in SPARC-null and wild-type mice with the murine pancreatic adenocarcinoma cell line, PAN02. MMP9 expression was higher in tumors from wild-type compared to SPARC-null mice. Coincident with lower MMP9 expression, tumors grown in SPARC-null mice were significantly larger, had decreased ECM deposition and reduced microvessel density compared to wild-type controls. In addition, metastasis was enhanced in the absence of host SPARC. Therefore, we next analyzed the orthotopic tumor growth of PAN02 cells transduced with MMP9 or a control empty vector. Forced expression of MMP9 by the PAN02 cells resulted in larger tumors in both wild-type and SPARC-null animals compared to empty vector controls and further diminished ECM deposition. Importantly, forced expression of MMP9 within the tumor reversed the decrease in angiogenesis and abrogated the metastatic potential displayed by control tumors grown in SPARC-null mice. Finally, contrary to the in vivo results, MMP9 increased cell migration in vitro, which was blocked by the addition of SPARC. These results suggest that SPARC and MMP9 interact to regulate many stages of tumor progression including ECM deposition, angiogenesis and metastasis.
SPARC; MMP9; tumor microenvironment; ECM; pancreatic; metastasis
Idiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the recurrent damage of alveolar epithelial cells as well as inappropriate expansion and activation of fibroblasts resulting in pronounced extracellular matrix (ECM) deposition. Although recent studies have indicated the involvement of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein regulating ECM deposition, in the pathogenesis of fibrosis, factors regulating SPARC expression or roles of SPARC in fibrosis have not been fully elucidated.
Among the profibrotic factors examined in cultured fibroblasts, we showed that SPARC expression was upregulated mainly by transforming growth factor (TGF)-β. We also showed that expression of SPARC in the lung was upregulated in the murine bleomycin-induced pulmonary fibrosis model, which was inhibited by TGF-β receptor I inhibitor. Knockdown of SPARC in fibroblasts using siRNA or treatment with the antioxidant N-acetylcysteine attenuated epithelial cell injury induced by TGF-β-activated fibroblasts in a coculture system. We also demonstrated that SPARC was required for hydrogen peroxide (H2O2) production in fibroblasts treated with TGF-β. Furthermore, TGF-β activated integrin-linked kinase (ILK), which was inhibited by SPARC siRNA. Knockdown of ILK attenuated extracellular H2O2 generation in TGF-β-stimulated fibroblasts. Our results indicated that SPARC is upregulated by TGF-β and is required for TGF-β-induced H2O2 production via activation of ILK, and this H2O2 production from fibroblasts is capable of causing epithelial cell injury.
The results presented in this study suggest that SPARC plays a role in epithelial damage in the IPF lung via enhanced H2O2 production from fibroblasts activated by TGF-β. Therefore, SPARC inhibition may prevent epithelial injury in IPF lung and represent a potential therapeutic approach for IPF.
SPARC; TGF-β; Hydrogen peroxide; Fibroblast; Pulmonary fibrosis
The matricellular protein SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin) mediates cell–matrix interactions during wound healing and regulates the production and/or assembly of the extracellular matrix (ECM). This study investigated whether SPARC functions in infarct healing and ECM maturation after myocardial infarction (MI). In comparison with wild-type (WT) mice, animals with a targeted inactivation of SPARC exhibited a fourfold increase in mortality that resulted from an increased incidence of cardiac rupture and failure after MI. SPARC-null infarcts had a disorganized granulation tissue and immature collagenous ECM. In contrast, adenoviral overexpression of SPARC in WT mice improved the collagen maturation and prevented cardiac dilatation and dysfunction after MI. In cardiac fibroblasts in vitro, reduction of SPARC by short hairpin RNA attenuated transforming growth factor β (TGF)–mediated increase of Smad2 phosphorylation, whereas addition of recombinant SPARC increased Smad2 phosphorylation concordant with increased Smad2 phosphorylation in SPARC-treated mice. Importantly, infusion of TGF-β rescued cardiac rupture in SPARC-null mice but did not significantly alter infarct healing in WT mice. These findings indicate that local production of SPARC is essential for maintenance of the integrity of cardiac ECM after MI. The protective effects of SPARC emphasize the potential therapeutic applications of this protein to prevent cardiac dilatation and dysfunction after MI.