Thrombospondin-1 Gene Research Articles

Regulation of thrombospondin-1 expression in alternatively activated macrophages and adipocytes: role of cellular cross talk and omega-3 fatty acids

Thrombospondin-1 (TSP-1) expression in human adipose positively correlates with body mass index and may contribute to adipose dysfunction by activating transforming growth factor-β and/or inhibiting angiogenesis. Our objective was to determine how TSP-1 is regulated in adipocytes and polarized macrophages using a coculture system and to determine whether fatty acids, including the ω-3 fatty acid docosahexaenoic acid (DHA), regulate TSP-1 expression. Coculture of M1, M2a or M2c macrophages with adipocytes induced TSP-1 gene expression in adipocytes (from 2.4- to 4.2-fold, P<.05), and adipocyte coculture induced TSP-1 gene expression in M1 and M2c macrophages (M1: 8.6-fold, M2c: 26-fold; P<.05). TSP-1 protein levels in the shared media of adipocytes and M2c cells were also strongly induced by coculture (>10-fold, P<.05). DHA treatment during the coculture of adipocytes and M2c macrophages potently inhibited the M2c macrophage TSP-1 mRNA level (97% inhibition, P<.05). Adipocyte coculture induced interleukin (IL)-10 expression in M2c macrophages (10.1-fold, P<.05), and this increase in IL-10 mRNA expression was almost completely blocked with DHA treatment (96% inhibition, P<.05); thus, IL-10 expression closely paralleled TSP-1 expression. Since IL-10 has been shown to regulate TSP-1 in other cell types, we reduced IL-10 expression with siRNA in the M2c cells and found that this caused TSP-1 to be reduced in response to adipocyte coculture by 60% (P<.05), suggesting that IL-10 regulates TSP-1 expression in M2c macrophages. These results suggest that supplementation with dietary ω-3 fatty acids could potentially be beneficial to adipose tissue in obesity by reducing TSP-1 and fibrosis.

SiRNA Transfection With Cholesterol Modification From Electrospun PET Facilitates Gene Silencing in Human Aortic Smooth Muscle Cells

Anastomotic intimal hyperplasia (AIH) remains the leading cause for prosthetic arterial graft failure. RNAi is a promising tool to silence genes such as Thrombospondin-2 (TSP-2), previously shown by our group to be upregulated in AIH. The present study is aimed at delivering modified TSP-2 siRNA (3’cholesterol tagged TSP-2 siRNA) from electrospun polyethylene terephthalate (ePET) and therefore knocking down TSP-2 gene and ultimately inhibiting AIH.

TP53 Status is Associated with Thrombospondin1 Expression In vitro

Objectives: To elucidate the association between thrombospondin1 (THBS1) expression and TP53 status and THBS1 promoter methylation in epithelial ovarian cancer (EOC).Methods: Epithelial ovarian cancer cell lines with known TP53 status were analyzed for THBS1 gene expression using Affymetrix U133 microarrays and promoter methylation by pyrosequencing. THBS1 mRNA expression was obtained pre- and post-exposure to radiation and hypoxia treatment in A2780 parent wild-type (wt) and mutant (m)TP53 cells. THBS1 expression was compared to tumor growth properties.Results: THBS1 gene expression was higher in cells containing a wtTP53 gene or null TP53 mutation (p = 0.005) and low or absent p53 protein expression (p = 0.008) compared to those harboring a missense TP53 gene mutation and exhibiting high p53 protein expression. Following exposure to radiation, there was a 3.4-fold increase in THBS1 mRNA levels in the mTP53 versus wtTP53 A2780 cells. After exposure to hypoxia, THBS1 mRNA levels increased approximately fourfold in both wtTP53 and mTP53 A2780 cells. Promoter methylation levels were low (median = 8.6%; range = 3.5–88.8%). There was a non-significant inverse correlation between THBS1 methylation and transcript levels. There was no association between THBS1 expression and population doubling time, invasive capacity, or anchorage-independent growth.Conclusion: THBS1 expression may be regulated via the TP53 pathway, and induced by hypoxic tumor microenvironment conditions. Overall low levels of THBS1 promoter methylation imply that methylation is not the primary driver of THBS1 expression in EOC.

Thrombospondin-4 Contributes to Spinal Sensitization and Neuropathic Pain States

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Thrombospondin-1 plays a profibrotic and pro-inflammatory role during ureteric obstruction

Thrombospondin-1 (TSP-1) is an endogenous activator of transforming growth factor-β (TGF-β), and an anti-angiogenic factor, which may prevent kidney repair. Here we investigated whether TSP-1 is involved in the development of chronic kidney disease using rats with unilateral ureteral obstruction, a well-known model to study renal fibrosis. Obstruction of 10 days duration induced inflammation, tubular cell atrophy, dilation, apoptosis, and proliferation, leading to interstitial fibrosis. TSP-1 expression was increased in parallel to that of collagen III and TGF-β. Relief of the obstruction at day 10 produced a gradual improvement in renal structure and function, the reappearance of peritubular capillaries, and restoration of renal VEGF content over a 7- to 15-day post-relief period. TSP-1 expression decreased in parallel with that of TGF-β1 and collagen III. Mice in which the TSP-1 gene was knocked out displayed less inflammation and had better preservation of renal tissue and the peritubular capillary network compared to wild-type mice. Additional studies showed that the inflammatory effect of TSP-1 was mediated, at least in part, by monocyte chemoattractant protein-1 and activation of the Th17 pathway. Thus, TSP-1 is an important profibrotic and inflammatory mediator of renal disease. Blockade of its action may be a treatment against the development of chronic kidney disease.

Two Molecular Weight Species of Thrombospondin-2 Are Present in Bone and Differentially Modulated in Fractured and Nonfractured Tibiae in a Murine Model of Bone Healing

We report two immuoreactive species of thrombospondin-2 (TSP2), sized approximately 200 and 125kDa, in the long bones of growing, but not skeletally mature, mice. In vitro osteoblasts secrete a 200-kDa species into the culture medium as early as day 3, and it appears in the cell-matrix layer by day 7. A 125-kDa species appears in the cell-matrix layer in parallel with mineralization; it is not detected in cell-conditioned medium. Unilateral tibial fracture induced a time-dependent upregulation of the 200-kDa species at the site of trauma. By contrast, relative levels of the 125-kDa species at the fracture site were lower than in bones from naive control animals. In the contralateral untouched control tibia, the 200-kDa species was rapidly and substantially reduced compared to bone harvested from naive control mice. Levels of the 125-kDa species in the untouched tibia declined gradually with time postfracture. TSP2 gene expression in uninjured control bone decreased modestly by 21days postfracture. On the day of fracture, the osteoblast differentiation potential of MSCs harvested from uninjured bones decreased compared to those harvested from naive control animals. The presence of two isoforms suggests that TSP2 may undergo posttranscriptional or posttranslational processing in skeletal tissue. Our data also suggest that, in the context of trauma, the two TSP2 isforms are differentially modulated at injured and noninjured skeletal sites in an animal undergoing fracture healing.

Association of thrombospondin 1 gene with schizophrenia in Korean population

Thrombospondin 1 (THBS1), a multi-domain glycoprotein, is secreted from astrocytes and promotes synaptogenesis. Increasing evidence has suggested that not only various markers for synaptic pathology, but also astrocytes are affected in schizophrenia. In this study, we investigated whether coding region single nucleotide polymorphisms (cSNPs) of the THBS1 gene were associated with schizophrenia and with the clinical symptoms of schizophrenia patients. We genotyped two cSNPs [rs2228261 (Asn470Asn) and rs2292305 (Thr523Ala)] using direct sequencing in 220 schizophrenia patients and 376 control subjects. In this study, rs2228261 revealed significant association with schizophrenia in both codominant (TT vs. CC, P = 0.009, OR = 2.10, 95% CI = 1.23-3.59) and recessive models (TT vs. CC/CT, P = 0.0012, OR = 2.28, 95% CI = 1.38-3.77). Also, rs2292305 was associated with schizophrenia in the recessive model (GG vs. AA/AG, P = 0.0052, OR = 2.05, 95% CI = 1.24-3.38). Additionally, in the analysis of the haplotype, the CA and TG haplotypes consisting of rs2228261 and rs2292305 were associated with schizophrenia in the dominant (P = 0.019, OR = 1.79, 95% CI = 1.10-2.90) and recessive models, respectively (P = 0.0086, OR = 0.51, 95% CI = 0.31-0.84). In further analysis according to the clinical symptoms, rs2292305 showed a weak association with the poor concentration symptoms of schizophrenia patients in the dominant model (AG/GG vs. AA, P = 0.024, OR = 2.04, 95% CI = 1.09-3.83). The results suggest that the THBS1 gene may contribute to the susceptibility of schizophrenia.

Loss-of-function thrombospondin-1 mutations in familial pulmonary hypertension

Most patients with familial pulmonary arterial hypertension (FPAH) carry mutations in the bone morphogenic protein receptor 2 gene (BMPR2). Yet carriers have only a 20% risk of disease, suggesting that other factors influence penetrance. Thrombospondin-1 (TSP1) regulates activation of TGF-β and inhibits endothelial and smooth muscle cell proliferation, pathways coincidentally altered in pulmonary arterial hypertension (PAH). To determine whether a subset of FPAH patients also have mutations in the TSP1 gene (THBS1) we resequenced the type I repeats of THBS1 encoding the TGF-β regulation and cell growth inhibition domains in 60 FPAH probands, 70 nonfamilial PAH subjects, and in large control groups. We identified THBS1 mutations in three families: a novel missense mutation in two (Asp362Asn), and an intronic mutation in a third (IVS8+255 G/A). Neither mutation was detected in population controls. Mutant 362Asn TSP1 had less than half of the ability of wild-type TSP1 to activate TGF-β. Mutant 362Asn TSP1 also lost the ability to inhibit growth of pulmonary arterial smooth muscle cells and was over threefold less effective at inhibiting endothelial cell growth. The IVS8+255 G/A mutation decreased and/or eliminated local binding of the transcription factors SP1 and MAZ but did not affect RNA splicing. These novel mutations implicate THBS1 as a modifier gene in FPAH. These THBS1 mutations have implications in the genetic evaluation of FPAH patients. However, since FPAH is rare, these data are most relevant as evidence for the importance of TSP1 in pulmonary vascular homeostasis. Further examination of THBS1 in the pathogenesis of PAH is warranted.

Alteration of HGF and TSP‐1 expression in ovarian carcinoma associated with clinical features

The aim of this study was to investigate the expression levels of hepatocyte growth factor (HGF) and thrombospondin-1 (TSP-1) with the clinical pathological factors in ovarian cancer, and the correlation between HGF and TSP-1 expression at the protein level. Immunohistochemistry was applied to detect the location and expression of HGF and TSP-1 protein in ovarian cancer and benign ovarian tumor tissue. Real-time quantitative polymerase chain reaction was applied to detect HGF and TSP-1 gene mRNA expression in ovarian cancer and benign ovarian tumor tissue. The level and positive expression rate of HGF mRNA in ovarian cancer tissue was significantly higher than in ovarian adenoma tissues. The positive expression of HGF protein in ovarian cancer was related with International Federation of Gynecology and Obstetrics (FIGO) stage and lymph node metastasis. The level and positive expression rate of TSP-1 mRNA in ovarian cancer tissue was lower than in ovarian adenoma. The absence expression of TSP-1 protein in ovarian cancer was significantly related with FIGO stage and histological grade. The intensity of these positive expressions in ovarian cancer tissues were significant negatively associated with each other. Abnormal expression of HGF and TSP-1 may be related to malignant progression of ovarian cancer and associated in the pathogenesis of ovarian cancer.

TSP-1 promotes glomerular mesangial cell proliferation and extracellular matrix secretion in Thy-1 nephritis rats

The proliferation of glomerular mesangial cells (GMC) and secretion of the extracellular matrix (ECM) in rat with Thy-1 nephritis (Thy-1N) resembling human mesangioproliferative glomerulonephritis have been explored for many years; however, the molecular mechanisms of GMC proliferation and ECM production remain unclear. Our previous studies have demonstrated that the thrombospondin-1 (TSP-1) gene was involved in mediating rat GMC proliferation and ECM synthesis induced by sublytic C5b-9 in vitro. In the present study, the roles of the TSP-1 gene in GMC proliferation, ECM production, and urinary protein secretion in Thy-1N rats were determined by using TSP-1 small hairpin RNA, and the results revealed that silencing of the TSP-1 gene in rat renal tissues could diminish GMC proliferation ( P < 0.01) and ECM secretion ( P < 0.01) as well as urinary protein secretion ( P < 0.05) in Thy-1N rats. Together, the current findings suggested that TSP-1 gene expression was required for GMC proliferation and ECM production in Thy-1N rats.

A functional variant in the thrombospondin-1 gene and the risk of small for gestational age infants.

Thrombospondin-1 (TSP-1) is a prothrombotic and anti-angiogenic glycoprotein expressed in the placenta. A functional single nucleotide polymorphism in the TSP-1 gene (TSP-1 A2210G) is a risk factor for familial premature myocardial infarction. Small for gestational age (SGA) infants are at increased risk of coronary artery disease in adult life and common genetic factors may underlie both conditions. We investigated the association of TSP-1 A2210G in SGA infants and their parents. The 3234 nulliparous pregnant women, their partners and babies were recruited in Adelaide and Auckland to a prospective multicenter cohort study. Amongst 2123 Caucasian women, 216 (10.2%) delivered an SGA infant, defined as birth weight < 10th customized centile adjusted for maternal height, weight, parity and ethnicity, as well as gestational age at delivery and infant sex. Uncomplicated pregnancies served as controls (n = 1185). DNA extracted from peripheral/cord blood or buccal swabs was genotyped using Sequenom MassARRAY. Multivariable logistic regression was used to compare the odds of SGA between the genotype groups adjusting for potential confounders. Paternal (adjOR, 1.4; 95% CI 1.0-2.0) and neonatal (adjOR, 1.8; 95% CI, 1.1-2.7) TSP-1 A2210G associates with SGA. The maternal polymorphism approaches significance for an association with SGA (adjOR, 1.3; 95% CI, 0.9-1.9). Maternal TSP-1 A2210G associates with a reduced maternal birth weight adjusted for gestational age at delivery (P = 0.03). The TSP-1 A2210G polymorphism, which is a risk factor for myocardial infarction, is associated with SGA pregnancies, suggesting that this polymorphism may associate with the risk of vascular disorders across the life course.

Polymorphism ofTHBS1rs1478604 A&gt;G in 5-Untranslated Region Is Associated with Lymph Node Metastasis of Gastric Cancer in a Southeast Chinese Population

Thrombospondin-1 plays an important role in cancer development and progression. This study investigated if a correlation exists between single-nucleotide polymorphisms (SNPs) in the Thrombospondin-1 gene (THBS1) and gastric cancer. We conducted a case-control study on a randomly recruited population of 283 patients and 283 healthy individuals from the city of Fuzhou in Southeast China. Individuals were genotyped for four SNPs (rs1478604 A>G, rs2228261 C>T, rs2292305 T>C, and rs3743125 C>T) in THBS1 using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. THBS1 genotypic distributions between the case and control groups were tested for correlations with cancer development. Comparisons between the case and control groups showed no significant differences in the genotypic distributions of rs1478604 A>G, rs2228261 C>T, and rs3743125 C>T. However, we found a statistically significant association between homozygous CC of THBS1 rs2292305 T>C and development of highly differentiated carcinoma (HDC). The rs1478604 A>G variant was found to be associated with invasion and lymph node metastasis in gastric cancer. After logistic regression and stratification analysis, rs1478604 A>G was more strongly associated with lymph node metastasis in HDC gastric cancer. The power to detect an effect for rs1478604 A>G in HDC was 90%. These findings indicate that the THBS1 rs1478604 A>G variant is linked with differential risks for gastric cancer nodal metastasis. These results support further investigation of THBS1 as a potential therapeutic target in gastric cancer.

Leiomyoma growth mediated by disruptions in injury response and angiogenesis

Thrombospondin 1 (TSP1) is an extracellular matrix component that interacts with proteoglycans, integrins, fibrinogen, and fibronectin. It is involved in cell proliferation, apoptosis, angiogenesis inhibition, and activates latent transforming growth factor beta 1. TSP1 is secreted in response to injury, and high expression is restricted to sites of tissue remodeling or injury. Given dependence of leiomyoma formation on both TGF-beta mediated aberrant injury response and angiogenesis, we investigated the role of TSP1 expression in human leiomyoma surgical specimens and in immortalized leiomyoma and patient-matched myometrial cultures. Laboratory study. Surgical specimens and immortalized leiomyoma and patient-matched myometrial cell lines were analyzed using qRT-PCR and western blot analysis. In untreated leiomyoma surgical specimens, there was a 30.2% decrease in TSP-1 mRNA expression when compared to patient matched myometrium. Conversely, TSP-1 protein was nearly undetectable in myometrial tissue, while TSP1 protein expression in leiomyoma surgical specimens remained elevated. We demonstrated a similar pattern in immortalized cell lines. In order to determine if we could alter TSP1 expression in leiomyoma cells to patterns comparable to myometrial cells, we treated leiomyoma cells with the retinoic acid metabolic blocking agent liarozole at concentrations ranging from 10−9M to 10−6M. Compared to untreated leiomyoma cells, leiomyoma cells treated with liarozole demonstrated an additional 35% decrease in TSP1 mRNA expression. Leiomyoma surgical specimens had decreased TSP1 gene expression but much greater TSP1 protein expression compared to patient matched myometrium, a pattern mirrored by in vitro cultures. Treatment with liarozole decreased mRNA expression further. Inhibition of TSP1 expression could disrupt both the fibrotic process and angiogenesis, resulting in potential improvement of leiomyoma bulk symptoms.

Thrombospondin-2 Gene Silencing in Human Aortic Smooth Muscle Cells Improves Cell Attachment

Despite decades of research, anastomotic intimal hyperplasia remains a major cause of delayed prosthetic arterial graft failure. Previously, we reported profound upregulation of thrombospondin-2 (TSP-2) mRNA in neointimal smooth muscle cells after prosthetic arterial bypass graft placement. TSP-2 is an antiangiogenic matricellular protein with specific functions yet unknown. In this study, we hypothesized that inhibition of TSP-2 in human aortic smooth muscle cells (HAoSMCs) would reduce cell proliferation and migration in vitro, providing a therapeutic target to mitigate intimal hyperplasia. HAoSMCs were transfected with TSP-2 small interfering ribonucleic acid (siRNA) using a commercial transfection reagent. Gene silencing was evaluated using semiquantitative real-time polymerase chain reaction. ELISA was used to measure TSP-2 protein levels in cell culture supernatants. Cell migration and proliferation were assessed using scratch wound assays and alamar blue assays, respectively. Attachment assays were performed to assess the effect of TSP-2 silencing on HAoSMC adhesion to fibronectin. TSP-2 siRNA achieved consistent target gene silencing at 48 hours post-transfection in HAoSMCs. This single transfection allowed suppression of TSP-2 protein expression for more than 30 days. TSP-2 gene silencing did not affect HAoSMC migration or proliferation. MMP-2 levels were also unaffected by changes in TSP-2 protein levels. However, HAoSMC attachment to fibronectin improved significantly in cells treated with TSP-2 siRNA. siRNA-mediated TSP-2 silencing of human aortic HAoSMCs improved cell attachment but had no effect on cell migration or proliferation. The effect on cell attachment was unrelated to changes in MMP activity.

Association of thrombospondin-1 gene N700S polymorphism with coronary artery disease

To investigate the association of thrombospondin-1 (TSP- 1) gene A8831G (N700S) polymorphism with coronary artery disease (CAD). This study was conducted with a case-control design including 178 patients with CAD (55 AMI) and 158 healthy subjects. The TSP-1 N700S polymorphism was determined by polymerase chain reaction and restriction fragment length polymorphism analysis. No significant difference of the AG genotype in CAD group and control group (1.7% compared with 0.6%, P=0.375) was detected. None of the homozygotes was detected for the G allele. The prevalence of the G allele was not significantly different between CAD group and controls (0.8% compared with 0.3%, P=0.376). No significant difference of the AG genotype in AMI group and control group (3.6% compared with 0.6%, P=0.104). The prevalence of G allele was not significantly different between AMI patients and controls (1.8% compared with 0.3%, P=0.364). There are TSP-1 N700S polymorphisms in Chinese Zhejiang Han people, but the TSP-1 N700S variant shows a much lower prevalence compared with Western populations and may be not a potential risk for CAD and AMI.

Expression of Thrombospondin-1 Gene mRNA and Protein in the Placenta in Gestosis

The expression of TSP-1 gene mRNA and TSP-1 protein in the placental tissue was studied during normal pregnancy and in gestosis. The formation of placental tissue in normal gestation was associated with expression of TSP-1 gene mRNA and of TSP-1 protein. Gestosis was associated with inflammatory reaction in the placenta characterized by increased counts of lymphocytes and macrophages in the villous stroma and involution degenerative changes in tissue. Disorders in placental villi maturation and branching in gestosis were paralleled by hyperexpression of TSP-1 gene mRNA by placental cells and hyperexpression of TSP-1 protein predominating in the stromal elements of terminal villi and near villous vessels.

Abstract 3275: miR-194 counterbalances transcriptional activation of the anti-angiogenic factor thrombospondin-1 by p53

Abstract Thrombospondin-1 (TSP-1) is a key endogenous inhibitor of angiogenesis, negatively regulated by oncogenes (e.g., c-Myc) and transcriptionally activated by the key tumor suppressor p53 (1). Yet in some cancers, such as colon adenocarcinomas, tumor progression accompanied by the loss of p53 does not result in TSP-1 down-regulation (2, 3). This paradox could involve promoter-independent mechanisms, for example microRNA-mediated regulation. In order to test this hypothesis, we conducted a global screen for microRNAs that regulate TSP-1 levels. Gain-of-function experiments and microarray data showed that in addition to the previously identified mir-17-92 cluster members (4, 5), several other miRs, including let-7, mir-199a-3p, mir-218 and miR-194, downregulate TSP-1. mir-194 was of particular interest, since its expression is known to be largely colon-specific (6), maintained by p53 and thus frequently reduced in advanced cancers (7). This reduced miR-194 expression could account for unexpectedly high TSP-1 levels in p53-null tumors. Indeed, mir-194 inhibition with antisense 2’-O-methyl ribonucleotides in p53-sufficient colon cancer cell lines strongly restored TSP-1 levels. Conversely, miR-194 mimics reduced TSP-1 expression at both RNA and protein levels. Furthermore, dual luciferase sensor assay demonstrated that this regulation was mediated by the single mir-194 site in the 3’UTR of the thbs1 gene. We then set up a retroviral transduction-based system to test the involvement of this miR-194 binding site in TSP-1 regulation by p53. Viruses expressing the thrombospondin-1 gene with 3’UTR in either wild type or miR-194-mutated configurations were introduced into HCT116-p53 sufficient cells (miR-194 levels are high) and their p53-null derivatives (miR-194 levels are low). In parental HCT116 cells, the mutation in the mir-194 site sharply increased TSP-1 levels, resulting in reduced angiogenesis in Matrigel plugs and tumor xenografts. However, the effects of this mutation disappeared in the absence of p53, resulting in lower TSP-1 expression levels. These results are consistent with the idea that miR-194 counterbalances transcriptional activation of TSP-1 by p53 and underscore the complex relationship between promoter-dependent and microRNA-mediated effects of p53. Grant Support: R01 CA122334 (ATT) and T32 CA009140 (PS) 1. K. M. Dameron, O. V. Volpert, M. A. Tainsky, N. Bouck, Science 265, 1582 (1994). 2. S. Kaiser et al., Genome Biol 8, R131 (2007). 3. D. A. Notterman, U. Alon, A. J. Sierk, A. J. Levine, Cancer Res 61, 3124 (2001). 4. M. Dews et al., Nat Genet 38, 1060 (2006). 5. M. Dews et al., Cancer Res 70, 8233 (2010). 6. K. Hino et al., RNA. 14, 1433 (2008). 7. C. J. Braun et al., Cancer Res 68, 10094 (2008). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3275. doi:10.1158/1538-7445.AM2011-3275

Role of thrombospondin 1 (THBS1) expression in peritoneal metastasis of gastric cancer treated with taxanes.

57 Background: To clarify the utility of taxanes and to pick up the prognostic factors in the treatment with 5FU- based chemotherapy for peritoneal metastasis of gastric cancer. Methods: Responder analysis: 18 patients with peritoneal metastasis of gastric cancer were enrolled in phase I study. The regimen consists of S-1 (80-120 mg) for 14 days and intraperitoneal infusion of PTX (dose escalation: 40 - 100 mg/m2) at day 1 and 14, which was repeated 2 cycles. To pick up the predictive factors, the 137 genes, which were selected in the viewpoint of sensitivity of 5FU, CPT11 and taxanes, were analyzed using focused DNA microarray for the 12 patients. Role of THBS1 expression: THBS1 expressions were evaluated in immunohistochemical (IHC) staining of surgical specimens of 59 patients with peritoneal metastasis of gastric cancer who were administered 5FU-based chemotherapy. THBS1 positive was defined &gt; 30% of the population stained moderate to strong. Results: Responder analysis: Expressions of THBS1 gene of the 6 patients who had clinical benefits (2: partial response, 2: positive adenocarcinoma cells in peritoneal cytology became negative, 2: remarkable decrease of ascites) were more than 2 folds higher than those of no responders. THBS1 expression was confirmed in 5 (83.3%) using IHC staining, who had significantly higher survival rate compared with that of the negative. Role of THBS1 expression: 17 patients (28.8%) was THBS1 positive and had significantly better prognosis compared with negative patients (1year survival: 64.7% vs 34.7%). 38 patients treated with regimes including taxanes revealed a tendency of improved overall survival rates (p=0.05). Overall survival of 15 patients with THBS1 positive administered taxanes was significantly higher than that of the negative patients. (1 year survival: 66.7% vs 42.4%) Conclusions: PTX could improve the survival of peritoneal metastasis of gastric cancer. THBS1 is a prognostic factor especially in the patients treated with taxanes, which leads to tailor-made therapy. [Table: see text]

Thrombospondin-4 is Necessary for the Increased Calcium Cycling Associated with the Slow Force Response

Thrombospondin-4 (TSP4) is a matricellular protein found in the heart and upregulated in heart failure; however, its role in cardiac regulation is unknown. Our previous work showed that mice lacking the TSP4 gene (tsp4-/-) respond normally to acute (seconds) pressure overload, but rapidly deteriorate minutes after. Thus, we hypothesize that TSP4 is involved in the slow force response (SFR), the second phase of stretch-mediated adaptation to loading, discovered a century ago, yet not fully understood. One proposed mechanism for the SFR is an increase in ERK phosphorylation that leads to increased Na+/H+ exchanger-1 activity. The latter increases intracellular Na+, causing the Na+/Ca2+ exchanger (reverse mode) to rise intracellular Ca2+, therefore increasing force. To investigate this, cardiac papillary muscles were isolated from tsp4-/- and wild-type (WT) littermates. Muscles were stretched from 92% of the length that generated maximum force (Lmax ) to 98% Lmax. This length was maintained for 15 min while force and Ca2+ transients (fura-2AM) were simultaneously recorded. The immediate rise in force without change in Ca2+ (Frank-Starling) was similar in both groups. However, whereas a positive SFR occurred in WT (33 ± 7%, n=5), tsp4-/- muscle displayed a negative SFR (−14 ± 2%, n=5). The difference in force was accompanied by a rise in Ca2+ in WT but not in tsp4-/- (7 ± 2% vs. 1± 2%, P<0.05). Next, hearts were excised from mice 15 minutes following transaortic constriction (pressure overload) and phosphorylation of ERK1/2 and Akt was found to be decreased in the tsp4-/- mice (P<0.05). We conclude that TSP4 is necessary for cardiac adaptation to stretch. Its absence blunts the SFR (force and Ca2+ rise). Moreover, TSP4 seems to be involved in this mechanism upstream of ERK1/2 and Akt.

Regulation of thrombospondin-1 expression through AU-rich elements in the 3'UTR of the mRNA.

Thrombospondin-1 (TSP-1) is a matricellular protein that participates in numerous normal and pathological tissue processes and is rapidly modulated by different stimuli. The presence of 8 highly-conserved AU rich elements (AREs) within the 3′-untranslated region (3′UTR) of the TSP-1 mRNA suggests that post-transcriptional regulation is likely to represent one mechanism by which TSP-1 gene expression is regulated. We investigated the roles of these AREs, and proteins which bind to them, in the control of TSP-1 mRNA stability. The endogenous TSP-1 mRNA half-life is approximately 2.0 hours in HEK293 cells. Luciferase reporter mRNAs containing the TSP-1 3′UTR show a similar rate of decay, suggesting that the 3′UTR influences the decay rate. Site-directed mutagenesis of individual and adjacent AREs prolonged reporter mRNA halflife to between 2.2 and 4.4 hours. Mutation of all AREs increased mRNA half life to 8.8 hours, suggesting that all AREs have some effect, but that specific AREs may have key roles in stability regulation. A labeled RNA oligonucleotide derived from the most influential ARE was utilized to purify TSP-1 AREbinding proteins. The AU-binding protein AUF1 was shown to associate with this motif. These studies reveal that AREs in the 3′UTR control TSP-1 mRNA stability and that the RNA binding protein AUF1 participates in this control. These studies suggest that ARE-dependent control of TSP-1 mRNA stability may represent an important component in the control of TSP-1 gene expression.