Secreted Protein Acidic And Rich In Cysteine Protein Expression Research Articles

Secreted Protein Acidic and Rich in Cysteine (SPARC) Polymorphisms in Response to Neoadjuvant Chemotherapy in HER2-Negative Breast Cancer Patients.

Secreted protein acidic and rich in cysteine (SPARC) expression has been proposed as a prognostic and predictive biomarker for some cancer types, but knowledge about the predictive value of SPARC polymorphisms in the context of neoadjuvant therapy for breast cancer (BC) is lacking. In 132 HER2-negative BC patients treated with neoadjuvant chemotherapy, we determined polymorphisms in the SPARC gene and analyzed their association with outcome. We also determined SPARC protein expression in tumor tissue. SPARC rs19789707 was significantly associated with response to treatment according to the Miller and Payne system in the breast (multivariate: odds ratio (OR), 3.81; p = 0.028). This association was significant in the subgroup of patients with luminal tumors (univariate: p = 0.047). Regarding survival, two SPARC variants showed significant associations with event-free survival: the rs19789707 variant in the subgroup of luminal A tumors (univariate: p = 0.006), and the rs4958487 variant in the subgroup of luminal B tumors (univariate: p = 0.022). In addition, SPARC rs4958487, rs10065756, and rs12153644 were significantly correlated with SPARC protein expression. Our findings suggest that SPARC polymorphisms could be good predictors of treatment response and survival in BC patients treated with neoadjuvant chemotherapy, especially those with luminal tumors.

Association Between SPARC Polymorphisms and Ankylosing Spondylitis and Its mRNA and Protein Expression in a Chinese Han Population: A Case-Control Study.

We explore the association of polymorphisms in Secreted protein acidic and rich in cysteine (SPARC) with ankylosing spondylitis (AS) and detect SPARC mRNA and protein expression in a Chinese Han population. Nine single-nucleotide polymorphisms (SNPs) of SPARC were genotyped in 768 AS patients and 768 controls by TaqMan genotyping assay. mRNA expression of SPARC was detected by real-time polymerase chain reaction (RT-PCR), and serum level of SPARC protein was detected by ELISA. The frequency of A allele of rs171121187 was significantly higher in AS patients than in controls (Pc=0.003, odds ratio [OR]=1.45, 95% confidence interval [95% CI] = 1.18-1.77), the AA and AC genotypes increased the risk of AS when compared with CC genotype (Pc=0.003, OR=3.96, 95% CI=1.80-8.75, and Pc=0.003, OR=1.27, 95% CI=1.01-1.61, respectively). The frequency of G allele of rs4958487 was significantly lower in AS than in controls (Pc=0.001, OR=0.60, 95% CI=0.47-0.68), the GG and GA genotypes reduced the risk of AS when compared with AA genotype (Pc=0.005, OR=0.46, 95% CI 0.18-1.14, and Pc=0.005, OR=0.60, 95% CI=0.45-0.79, respectively). The haplotype AA of rs17112187/rs4958487 significantly increased the risk of AS (P=2.31E-5, OR=1.60, 95% CI=1.28-1.98), while haplotype CG decreased the risk of AS (P=5.42E-5, OR=0.55, 95% CI=0.41-0.74). Expression levels of SPARC mRNA were significantly lower in both Peripheral blood mononuclear cells (PBMC) and granulocytes in AS patients than in controls (P=0.008 and P=0.005, respectively). SPARC protein levels were also reduced in AS patients versus the controls (P=0.002). This study indicates that polymorphisms in SPARC are associated with AS susceptibility, and both mRNA and protein levels of SPARC are decreased in AS patients in a Chinese Han population.

The role of lymphoid tissue SPARC in the pathogenesis and response to treatment of multiple myeloma.

Despite the significant progress in the treatment of multiple myeloma (MM), the disease remains untreatable and its cure is still an unmet clinical need. Neoplastic transformation in MM is initiated in the germinal centers (GCs) of secondary lymphoid tissue (SLT) where B cells experience extensive somatic hypermutation induced by follicular dendritic cells (FDCs) and T-cell signals. We reason that secreted protein acidic and rich in cysteine (SPARC), a common stromal motif expressed by FDCs at the origin (SLTs) and the destination (BM) of MM, plays a role in the pathogenesis of MM, and, here, we sought to investigate this role. There were 107 BM biopsies from 57 MM patients (taken at different time points) together with 13 control specimens assessed for SPARC gene and protein expression and compared with tonsillar tissues. In addition, regulation of myeloma-promoting genes by SPARC-secreting FDCs was assessed in in vitro GC reactions (GCRs). SPARC gene expression was confirmed in both human primary (BM) and secondary (tonsils) lymphoid tissues, and the expression was significantly higher in the BM. Sparc was detectable in the BM and tonsillar lysates, co-localized with the FDC markers in both tissues, and stimulation of FDCs in vitro induced significantly higher levels of SPARC expression than unstimulated controls. In addition, SPARC inversely correlated with BM PC infiltration, ISS staging, and ECOG performance of the MM patients, and in vitro addition of FDCs to lymphocytes inhibited the expression of several oncogenes associated with malignant transformation of PCs. FDC-SPARC inhibits several myelomagenic gene expression and inversely correlates with PC infiltration and MM progression. Therapeutic induction of SPARC expression through combinations of the current MM drugs, repositioning of non-MM drugs, or novel drug discovery could pave the way to better control MM in clinically severe and drug-resistant patients.

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.

Role of secreted protein acidic and rich in cysteine (SPARC) in vascular endothelial cell dysfunction and inflammation

Secreted protein acidic and rich in cysteine (SPARC) is reported to induce collagen deposition through a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1) production and promote pro-inflammatory cytokine release in aging myocardium. The present study investigated the roles for SPARC in vascular endothelial cell dysfunction and inflammation using deoxycorticosterone acetate (DOCA)-salt hypertensive rats and cultured aortic endothelial cells. DOCA-salt treatment increased systolic blood pressure in 2 weeks and induced further hypertensin in 3 weeks. Acetylcholine-induced vasodilatory response impaired and macrophage infiltration increased in DOCA-salt rat aortas, both of which showed time-dependent changes. Immunoblot analysis revealed that SPARC and ADAMTS1 expression increased in DOCA-salt rats and peaked at 1 week. Angiotensin II treatment enhanced SPARC and ADAMTS1 protein expression in cultured aortic endothelial cells. Angiotensin II-induced overexpression of LOX-1 and MCP-1 mRNA was further augmented by SPARC or ADAMTS1 gene silencing. In conclusion, SPARC may be induced at the early stage of vascular injury to compensate inflammation. Further investigation into roles for SPARC in inflammation and relationship between SPARC and ADAMTS1 is needed.

Modulation of SPARC/Hevin Proteins in Alzheimer's Disease Brain Injury.

Alzheimer’s disease (AD) is an age-related progressive form of dementia that features neuronal loss, intracellular tau, and extracellular amyloid-β (Aβ) protein deposition. Neurodegeneration is accompanied by neuroinflammation mainly involving microglia, the resident innate immune cell population of the brain. During AD progression, microglia shift their phenotype, and it has been suggested that they express matricellular proteins such as secreted protein acidic and rich in cysteine (SPARC) and Hevin protein, which facilitate the migration of other immune cells, such as blood-derived dendritic cells. We have detected both SPARC and Hevin in postmortem AD brain tissues and confirmed significant alterations in transcript expression using real-time qPCR. We suggest that an infiltration of myeloid-derived immune cells occurs in the areas of diseased tissue. SPARC is highly expressed in AD brain and collocates to Aβ protein deposits, thus contributing actively to cerebral inflammation and subsequent tissue repair, and Hevin may be downregulated in the diseased state. However, further research is needed to reveal the exact roles of SPARC and Hevin proteins and associated signaling pathways in AD-related neuroinflammation. Nevertheless, normalizing SPARC/Hevin protein expression such as interdicting heightened SPARC protein expression may confer a novel therapeutic opportunity for modulating AD progression.

Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC

Ovarian cancer (OvCa) exhibits a specific predilection for metastasis to the omentum. Our earlier studies highlighted the tumour-suppressor effect of secreted protein acidic and rich in cysteine (SPARC) in OvCa through multi-faceted roles inhibiting cancer cell interactions within the peritoneal milieu. The goal of this study is to investigate the role of SPARC in OvCa interactions with omental adipocytes and its role in OvCa colonization in the omentum. We employed multi-pronged approach using primary omental adipocytes from Sparc knockout mice, genetically engineered human omental adipocytes in 3D co-cultures with OvCa cells, as well as treatment with recombinant SPARC protein. We show that SPARC suppresses multistep cascade in OvCa omental metastasis. SPARC inhibited in vivo and adipocyte-induced homing, proliferation, and invasion of OvCa cells. SPARC suppressed metabolic programming of both adipocytes and OvCa cells and exerted an inhibitory effect of adipocyte differentiation and their phenotypic switch to cancer-associated phenotype. Mechanistic studies revealed that this effect is mediated through inhibition of cEBPβ-NFkB-AP-1 transcription machinery. These findings define a novel and functionally important role of SPARC in OvCa and not only bridge the knowledge gap but highlight the need to consider SPARC protein expression in therapeutic development.

Expression and significance of secreted protein acidic and rich in cysteine in human osteosarcoma.

Osteosarcoma is the most common primary malignancy of bone, and is a high-grade malignant mesenchymal tumor with high recurrence and metastatic rates. Increased expression of secreted protein, acidic and rich in cysteine (SPARC) indicates poor prognosis in a number of malignances. However, the expression level of SPARC in human osteosarcoma and its associated mechanism remains unclear. To analyze the expression of SPARC in human osteosarcoma and its potential application in the diagnosis and treatment of osteosarcoma, the clinical records and samples of 20 cases of osteosarcoma were collected. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis applied to detect SPARC expression levels in osteosarcoma tissues, with normal bone tissue as control. Immunofluorescence detection was used to examine the distribution of SPARC. The association between SPARC level and clinical factors was analyzed. RT-qPCR (P=0.002) indicated that the SPARC level in osteosarcoma tissues was significantly increased compared with that in normal tissues. Immunofluorescence detection indicated that SPARC was widely distributed in tumor tissues. SPARC protein expression level was positively associated with lung metastasis (P=0.016). The results indicated that SPARC tends to be highly expressed in human osteosarcoma tissues. The expression level of SPARC is associated with lung metastasis, which may be an indicator of prognosis. Thus, SPARC may be a potential tumor marker and therapeutic target in osteosarcoma.

Abstract 2839: Systematic enrichment analysis of gene expression profiling studies identifies SPARC dependent consensus pathways implicated in bladder cancer

Abstract Bladder cancer (urothelial cancer of the bladder) is the most common malignancy affecting the urinary system with increasing incidence and mortality. Treatment of bladder cancer has not advanced in the past 30 years. Therefore, there is a crucial unmet need for novel therapies, especially for advanced disease. We have recently reported Secreted Protein Acidic and Rich in Cysteine (SPARC) as a tumor suppressor in urinary bladder cancer that inhibits the multistep cascades on tumor initiation, progression and metastasis. SPARC gene and protein expression was among candidates associated with the tumorigenicity of the isogenic T24/T24t cell lines. We reported that SPARC depletion in human bladder cancer cell lines including T24 cells increased their tumorigenicity and metastatic potential. In addition, SPARC protein and transcript expression are significantly downregulated in advanced urothelial cancer. To gain insight on the molecular mechanisms of the tumor suppressor effects of SPARC in urothelial cancer, we did whole transcriptome profiling of T24 cells depleted of SPARC to identify SPARC-signature associated with bladder cancer invasiveness, progression, and metastasis. Subsequently, we used gene signature enrichment analysis (GSEA) pathway enrichment tools to define functionally related genes that are consistently up- or down- regulated as a function of loss of SPARC. We found that loss of SPARC expression is associated with enrichment of multiple oncogenic signaling pathways that are enriched in muscle invasive urothelial cancer and have been associated with poor prognosis. These pathways include K-ras, Myc-oncogene, p53 pathways as well as DNA damage/repair. Consistent with our earlier reports, loss of SPARC is associated with genes and proteins involved in angiogenesis, inflammation and hypoxia. In addition, we have identified novel signatures involved in unfolded protein response, mTOR signaling, glycolysis and cholesterol metabolism. These comprehensive signatures not only reveal SPARC-dependent networks that cooperate to elicit its biological responses but enables coherent understanding of the etiology of complex bladder cancer disease. Importantly, these signatures identify novel therapeutic opportunities for bladder cancer. Citation Format: Bincy Anu John, Neveen Said. Systematic enrichment analysis of gene expression profiling studies identifies SPARC dependent consensus pathways implicated in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2839. doi:10.1158/1538-7445.AM2017-2839

Correlation between secreted protein acidic and rich in cysteine protein expression and the prognosis of postoperative patients exhibiting esophageal squamous cell carcinoma.

The aim of the present study was to investigate the association between the expression level of secreted protein acidic and rich in cysteine (SPARC) and the prognosis of postoperative patients with esophageal squamous cell carcinoma (ESCC). The expression level of SPARC was detected in the 89 ESCC tissue cases and 100 healthy esophageal mucosa cases, which served as the controls. Immunohistochemistry and reverse transcription‑polymerase chain reaction (RT‑PCR) were employed to evaluate the SPARC expression in cases with ESCC. RT‑PCR demonstrated that the positive rates of SPARC mRNA expression in ESCC were 71.91% (64/89). The positive rates of normal esophageal mucosa mRNA expression were 15.00% (15/100), which were significantly lower than that in the ESCC tissue samples. The difference was statistically significant (P<0.001). Immunohistochemical staining indicated that the positive expression rate of SPARC protein in the ESCC tissue samples was significantly higher than that in the esophageal mucosa tissue samples (65.17 vs. 8.00%; P<0.001). The expression of SPARC protein was negatively correlated with lymph node metastasis (P<0.05), which was not associated with the pathologic gross morphology, tumor differentiation degree or other clinical features. The survival of patients with ESCC was not associated with the expression level of SPARC protein (P>0.05), but was associated with the tumor location (P<0.05), differentiation (P<0.001) and staging (P<0.05). Thus, SPARC mRNA and protein were highly expressed in ESCC, and negatively correlated with lymph node metastasis, which was not associated with postoperative survival of ESCC patients. Thus, detection of SPARC mRNA and protein expression levels may facilitate early diagnosis and prognosis assessment of ESCC.

In phyllodes tumors of the breast expression of SPARC (osteonectin/BM40) mRNA by in situ hybridization correlates with protein expression by immunohistochemistry and is associated with tumor progression.

Secreted protein acidic and rich in cysteine (SPARC) plays an essential role in tumor invasion and metastasis. The present work was undertaken to detect expression of SPARC mRNA in phyllodes tumors (PTs) and its association with SPARC protein expression. This study also evaluated expression of SPARC mRNA and its correlation between grade and clinical behavior of PTs. In addition, we assessed in PTs the association of expression of SPARC with that of matrix metalloproteinase (MMP)-2 and of MMP-9. SPARC mRNA expression was determined by RNAscope in situ hybridization (ISH) in 50 benign, 22 borderline, and 10 malignant PTs using a tissue microarray. Furthermore, we applied immunohistochemistry (IHC) to examine expression of SPARC, MMP-2, and MMP-9. SPARC mRNA appeared to be concentrated mainly in the stromal compartment of PTs. IHC staining patterns of SPARC protein showed concordance with SPARC mRNA ISH results. Stromal SPARC expression increased continuously as PTs progress from benign through borderline to malignant PTs, both at mRNA (using ISH) (P=0.044) and protein level (using IHC) (P=0.000). The recurrence percentage was higher in the stromal SPARC mRNA or protein-positive group than in the SPARC-negative group but this difference was not statistically significant. Stromal SPARC mRNA and protein expression was associated with PT grade and correlated with MMP-2 expression. These results indicate that SPARC-mediated degradation of the extracellular matrix, and its possible association with MMPs, might contribute to progression of PTs.

Aberrant methylation of the SPARC gene promoter and its clinical implication in gastric cancer.

Secreted protein acidic and rich in cysteine (SPARC) gene has been shown to be epigenetically silenced in several cancers. We investigated the loss of expression and promoter methylation of this tumor suppressor gene in gastric cancers and correlated the data with clinicopathological features. We observed the loss of SPARC mRNA and SPARC protein expression in 7 of 10 (70%) gastric cancer cell lines. Upon treatment of expression-negative cell lines with a demethylating agent, expression of mRNA and protein was restored in all cells. Methylation rate of SPARC gene was 80% in ten gastric cancer cell lines and 74% (163 of 220) in primary tumors, while it was 5% in normal gastric mucosa (n = 40). In intestinal gastric cancer, SPARC methylation correlated with a negative prognosis (P < 0.001; relative risk 2.754, 95% confidence interval 1.780–4.261). Immunostaining revealed that SPARC protein was overexpressed in stromal fibroblasts adjacent to neoplastic epithelium but rarely expressed in the primary gastric cancer cells. These results implicate SPARC promoter methylation as an important factor in the tumorigenesis of gastric carcinomas and provide new insights into the potential use of SPARC as a novel biomarker and the potential clinical importance in human gastric cancers.

SPARC was overexpressed in human endometrial cancer stem-like cells and promoted migration activity

ObjectivesWe previously demonstrated that side-population (SP) cells found in human endometrial cancer tissue have features of cancer stem cells (CSCs). Endometrial cancer SP cells show enhanced migration, the potential to differentiate into the mesenchymal cell lineage, and they are associated with the epithelial–mesenchymal transition (EMT). In this study, we analyzed the expression and function of a specific protein, SPARC (secreted protein acidic and rich in cysteine) which we found to be up-regulated in endometrial cancer. MethodsWe performed microarray expression analysis to screen for up-regulated genes in CSCs using a set of RK12V-SP cells and -non-SP (NSP) cells. We used the MetaCore package to identify the Gene GO pathway MAPs associated with the up-regulated genes. Here, we investigated the expression and functions of SPARC, one of the genes up-regulated in endometrial CSCs.We established SPARC-overexpressing cells by transfecting endometrial cancer cells (Ishikawa cells [IK-SPARC cells]). We characterized these cells' growth rate, tumorigenicity, migration and invasion activity. The levels and locations of SPARC protein expression in Hec1SP cells-derived tumors and endometrial cancer tissues were examined by immunohistochemistry. ResultsSPARC was detected by microarray expression analysis during screens for up-regulated genes in SP and NSP CSC. The level of SPARC expression was enhanced in Hec1 SP cells compared with that in Hec1 non-SP cells. SPARC enhanced fibronectin expression and promoted migration activity in IK cells. SPARC expression suppressed tumor growth but promoted formation of tumor stroma.SPARC was expressed in endometrial cancer tissues, in particular, poorly differentiated endometrioid adenocarcinoma, clear and serous adenocarcinoma,but not in normal endometrial tissue. ConclusionThis is the first report of overexpression of SPARC in endometrial cancer stem-like cells. SPARC expression is associated with cell migration and stroma formation.

Abstract P3-06-05: Expression of SPARC in human breast cancer and its predictive value in the GeparTrio neoadjuvant trial.

Abstract Background: Secreted protein acidic and rich in cysteine (SPARC) is an albumin-binding protein and associated with poor prognosis in multiple cancers. The aim of this analysis was to determine the frequency of SPARC expression among different molecular breast cancer subtypes and to evaluate its predictive value for therapy response after neoadjuvant anthracycline/taxane based chemotherapy (CTX) in participants of the GeparTrio trial. Methods: We evaluated tumoral SPARC expression by immunohistochemistry on tissue microarrays (TMAs) constructed from formalin-fixed paraffin-embedded (FFPE) pre-treatment core biopsies from 667 patients (pts) of the GeparTrio trial. The details of the GeparTrio study design are described elsewhere (von Minckwitz, JNCI 2008). Cutoffs for SPARC expression were determined using the web-based software Cutoff Finder (http://molpath.charite.de/cutoff/). Results: SPARC protein expression was measurable by IHC on the TMAs and 176 (26.4 %) of 667 tumors were SPARC positive. SPARC expression was increased in pts with triple-negative breast cancer (TNBC) compared to hormone receptor or HER2 positive subtypes (p = 0.039). SPARC positivity was associated with an increased pCR rate in the overall population (p &amp;lt; 0.001). SPARC negative tumors had a pCR rate of 15%, which increased to 27% in SPARC positive tumors. Similarly, in TNBC the pCR rate increased from 26% in SPARC negative tumors to 47% in SPARC positive tumors (p = 0.032). In multivariate logistic regression analysis adjusted for standard clinicopathological factors, SPARC was independently predictive in the overall population (p = 0.010) as well as the subgroups of pts with TNBC (p = 0.036). Conclusions: SPARC is expressed in all biological breast cancer subtypes with TNBC revealing the highest expression rate. Our data suggest that SPARC expression may provide predictive information for response to neoadjuvant CTX. As SPARC is an albumin-binding protein and might mediate intratumoral accumulation of nab-Paclitaxel, prospective analysis of SPARC expression is planned in the GeparSepto trial. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-06-05.

The Role of SPARC Protein Expression in the Progress of Gastric Cancer

We aimed to investigate the expression of SPARC (secreted protein, acidic and rich in cysteine) in gastric cancer and its relationship with tumor angiogenesis and cancer cells proliferation. Protein expression of SPARC, VEGF, CD34 and Ki-67 in 80 cases of gastric cancer and 30 cases of normal gastric tissue was evaluated by immunohistochemistry. CD34 staining was used as an indicator of microvessel density (MVD). Ki-67 labeling Index (LI) indicated cancer cells proliferation. Statistical analysis was used to investigate its relationship with clinical characteristics, tumor angiogenesis and cancer cells proliferation. SPARC expression was mainly in the stromal cells surrounding the gastric cancer cells, and was statistically significant differences between gastric cancer and normal gastric tissue (P < 0.05). Both the expression of SPARC and VEGF were related to differentiation degree, clinical stage, Lauren classification and lymph node metastasis (P < 0.05). Expression of SPARC was significantly negatively correlated with the expression of VEGF and MVD in gastric cancer tissues. Expression of SPARC was also negatively correlated with Ki-67-LI. Our findings suggest that both the expression of SPARC and VEGF are closed to tumor angiogenesis in gastric cancer, SPARC inhibited tumor angiogenesis but VEGF promoted tumor angiogenesis. SPARC also inhibited cells proliferation of gastric cancer.

Prognostic value of NDRG1 and SPARC protein expression in breast cancer patients

An increasing number of studies have shown altered expression of secreted protein acidic and rich in cysteine (SPARC) and N-myc down-regulated gene (NDRG1) in several malignancies, including breast carcinoma; however, the role of these potential biomarkers in tumor development and progression is controversial. In this study, NDRG1 and SPARC protein expression was evaluated by immunohistochemistry on tissue microarrays containing breast tumor specimens from patients with 10 years of follow-up. NDRG1 and SPARC protein expression was determined in 596 patients along with other prognostic markers, such as ER, PR, and HER2. The status of NDRG1 and SPARC protein expression was correlated with prognostic variables and patient clinical outcome. Immunostaining revealed that 272 of the 596 cases (45.6%) were positive for NDRG1 and 431 (72.3%) were positive for SPARC. Statistically significant differences were found between the presence of SPARC and NDRG1 protein expression and standard clinicopathological variables. Kaplan-Meier analysis showed that NDRG1 positivity was directly associated with shorter disease-free survival (DFS, P < 0.001) and overall survival (OS, P < 0.001). In contrast, patients expressing low levels of SPARC protein had worse DFS (P = 0.001) and OS (P = 0.001) compared to those expressing high levels. Combined analysis of the two markers indicated that DFS (P < 0.001) and OS rates (P < 0.001) were lowest for patients with NDRG1-positive and SPARC-negative tumors. Furthermore, NDRG1 over-expression and SPARC down-regulation correlated with poor prognosis in patients with luminal A or triple-negative subtype breast cancer. On multivariate analysis using a Cox proportional hazards model, NDRG1 and SPARC protein expression were independent prognostic factors for both DFS and OS of breast cancer patients. These data indicate that NDRG1 over-expression and SPARC down-regulation could play important roles in breast cancer progression and serve as useful biomarkers to better define breast cancer prognosis.

Secreted protein, acidic and rich in cysteine (SPARC) expression in astrocytic tumour cells negatively correlates with proliferation, while vascular SPARC expression is associated with patient survival

Secreted protein, acidic and rich in cysteine (SPARC) is a regulator of cell-matrix interaction and has been associated with tumour stage and patient survival in various malignancies. As no large-scale study has yet been undertaken, we investigated human brain and astrocytomas for SPARC expression and associations with tumour grade, proliferation, vascular density and patient survival. A spectrum of 188 WHO grade I-IV astrocytic tumours and 24 autopsy cases were studied by immunohistochemistry for SPARC, MIB-1 proliferation index and CD31-positive vessels. SPARC protein expression was confirmed by quantitative real-time polymerase chain reaction and Western blot in 13 cases. In normal brain, SPARC is expressed in cortical marginal glia, cerebellar Bergmann glia and focally in white matter but is absent in neurones or vessels. High SPARC expression levels in the cytoplasm of astrocytic tumour cells decreased with the grade of malignancy but showed an increase with grade of malignancy in tumour vessels. SPARC negatively correlated with tumour proliferation but not with vascular density. While cytoplasmic SPARC staining was not associated with survival, vascular SPARC showed a significant association in the group of grade II-IV tumours (P = 0.02) and also in grade II astrocytomas alone (P = 0.01) with vascular SPARC associated with worse prognosis. SPARC is highly expressed in astrocytomas and decreases with tumour progression. We confirm an association of increased SPARC expression and decreased proliferation. While there is no association between the level of SPARC in the tumour cells and patient survival, increased tumour vascular SPARC expression is associated with decreased patient survival.

Glioma-astrocyte interaction modifies the astrocyte phenotype in a co-culture experimental model

As the majority of gliomas arise through malignant transformation of astrocytes, we aimed at investigating the interaction between malignant glioma cells and astrocytes in a co-culture experimental model. For this purpose we analyzed the expression of genes and proteins involved in tumor promotion and invasion, such as glial fibrillary acidic protein (GFAP), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of MMP-2 (TIMP-2), transforming growth factor-beta1 (TGF-beta1), secreted protein acidic and rich in cysteine (SPARC), and connexin 43 (CX43). Co-cultures of human neural stem cell-derived astrocytes and U87 MG astrocytoma cells were performed in a transwell system. Gene expression was evaluated by real-time RT-PCR, and protein analysis was performed by Western blotting, SDS-zymography, and immunofluorescence. GFAP tended to be up-regulated in astrocytes co-cultivated with U87, suggesting a reactive response induced by glioma cells. CX43 mRNA tended to be down- regulated in co-cultured astrocytes, as well as the non-phosphorylated isoform at the protein level. MMP-2 mRNA tended to be up-regulated, and MMP-2 protein levels were significantly increased in astrocytes co-cultivated with U87. TIMP-2 and SPARC mRNA decreased in astrocytes co-cultivated with U87, showing lower expression in glioma cells. By contrast, SPARC protein expression was strongly induced in supernatants of co-cultured astrocytes. TGF-beta1 was not modified. Our results suggest that U87 cells elicit phenotype modifications in the neighbouring resident astrocytes very likely mediated by soluble factors. Glioma/astrocyte interaction could possibly trigger an astrocyte phenotype modification consistent with a malignant transformation, and favouring a more permissive environment for glioma cells invasion.

Regulation of the Fibrosis and Angiogenesis Promoter SPARC/Osteonectin in Human Adipose Tissue by Weight Change, Leptin, Insulin, and Glucose

OBJECTIVEMatricellular Secreted Protein, Acidic and Rich in Cysteine (SPARC), originally discovered in bone as osteonectin, is a mediator of collagen deposition and promotes fibrosis. Adipose tissue collagen has recently been found to be linked with metabolic dysregulation. Therefore, we tested the hypothesis that SPARC in human adipose tissue is influenced by glucose metabolism and adipokines.RESEARCH DESIGN AND METHODSSerum and adipose tissue biopsies were obtained from morbidly obese nondiabetic subjects undergoing bariatric surgery and lean control subjects for analysis of metabolic markers, SPARC, and various cytokines (RT-PCR). Additionally, 24 obese subjects underwent a very-low-calorie diet of 1,883 kJ (450 kcal)/day for 16 weeks and serial subcutaneous-abdominal-adipose tissue (SCAT) biopsies (weight loss: 28 ± 3.7 kg). Another six lean subjects underwent fast-food–based hyperalimentation for 4 weeks (weight gain: 7.2 ± 1.6 kg). Finally, visceral adipose tissue explants were cultured with recombinant leptin, insulin, and glucose, and SPARC mRNA and protein expression determined by Western blot analyses.RESULTSSPARC expression in human adipose tissue correlated with fat mass and was higher in SCAT. Weight loss induced by very-low-calorie diet lowered SPARC expression by 33% and increased by 30% in adipose tissue of subjects gaining weight after a fast-food diet. SPARC expression was correlated with leptin independent of fat mass and correlated with homeostasis model assessment–insulin resistance. In vitro experiments showed that leptin and insulin potently increased SPARC production dose dependently in visceral adipose tissue explants, while glucose decreased SPARC protein.CONCLUSIONSOur data suggest that SPARC expression is predominant in subcutaneous fat and its expression and secretion in adipose tissue are influenced by fat mass, leptin, insulin, and glucose. The profibrotic effects of SPARC may contribute to metabolic dysregulation in obesity.

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.