Loss of Sparc in p53-null Astrocytes Promotes Macrophage Activation and Phagocytosis Resulting in Decreased Tumor Size and Tumor Cell Survival.

Both the induction of SPARC expression and the loss of the p53 tumor suppressor gene are changes that occur early in glioma development. Both SPARC and p53 regulate glioma cell survival by inverse effects on apoptotic signaling. Therefore, during glioma formation, the upregulation of SPARC may cooperate with the loss of p53 to enhance cell survival. This study determined whether the loss of Sparc in astrocytes that are null for p53 would result in reduced cell survival and tumor formation and increased tumor immunogenicity in an in vivo xenograft brain tumor model. In vitro, the loss of Sparc in p53-null astrocytes resulted in an increase in cell proliferation, but a loss of tumorigenicity. At 7 days after intracranial implantation, Sparc-null tumors had decreased tumor cell survival, proliferation and reduced tumor size. The loss of Sparc promoted microglia/macrophage activation and phagocytosis of tumor cells. Our results indicate that the loss of p53 by deletion/mutation in the early stages of glioma formation may cooperate with the induction of SPARC to potentiate cancer cell survival and escape from immune surveillance.

PTEN suppresses SPARC-induced pMAPKAPK2 and inhibits SPARC-induced Ser78 HSP27 phosphorylation in glioma.

BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC) is overexpressed in astrocytomas (World Health Organization grades II-IV). We previously demonstrated that SPARC promotes glioma migration and invasion-in part, by activating the P38 mitogen-activated protein kinase (MAPK)-heat shock protein (HSP)27 signaling pathway. The commonly lost tumor suppressor phosphatase and tensin homolog (PTEN) suppresses SPARC-induced migration, which is accompanied by suppression of Shc-Ras-Raf-MEK-ERK1/2 and Akt signaling. As PTEN completely suppresses SPARC-induced migration, we proposed that PTEN must also interfere with SPARC-induced HSP27 signaling. Therefore, this study determined the effects of PTEN expression on SPARC-induced expression and phosphorylation of HSP27. METHODS: Control and SPARC-expressing clones transfected with control- or PTEN-expression plasmids were plated on fibronectin-coated tissue culture plates for 3, 6, 24, and 48 h and then lysed. Equal amounts of protein were subjected to Western blot and densitometric analyses. RESULTS: The results show that SPARC enhances phosphorylated (p)P38 MAPK, phosphorylated MAPK-activated protein kinase 2 (pMAPKAPK2), and serine (Ser)78 HSP27 phosphorylation relative to total HSP27. PTEN suppresses pAkt and pMAPKAPK2, suggesting that PTEN effects are downstream of pP38 MAPK. PTEN suppressed SPARC-induced sustained phosphorylation at Ser78 HSP27. As the level of total HSP27 differed based on the presence of SPARC or PTEN, the ratios of phosphorylation-specific to total HSP27 were examined. The data demonstrate that SPARC-induced phosphorylation at Ser78 remains elevated despite increasing levels of total HSP27. In contrast, PTEN inhibits SPARC-induced increases in Ser78 HSP27 phosphorylation relative to total HSP27. CONCLUSION: These data describe a novel mechanism whereby PTEN inhibits SPARC-induced migration through suppression and differential regulation of pAkt and the P38 MAPK-MAPKAPK2-HSP27 signaling pathway.

Inhibition of HSP27 alone or in combination with pAKT inhibition as therapeutic approaches to target SPARC-induced glioma cell survival.

BACKGROUND: The current treatment regimen for glioma patients is surgery, followed by radiation therapy plus temozolomide (TMZ), followed by 6 months of adjuvant TMZ. Despite this aggressive treatment regimen, the overall survival of all surgically treated GBM patients remains dismal, and additional or different therapies are required. Depending on the cancer type, SPARC has been proposed both as a therapeutic target and as a therapeutic agent. In glioma, SPARC promotes invasion via upregulation of the p38 MAPK/MAPKAPK2/HSP27 signaling pathway, and promotes tumor cell survival by upregulating pAKT. As HSP27 and AKT interact to regulate the activity of each other, we determined whether inhibition of HSP27 was better than targeting SPARC as a therapeutic approach to inhibit both SPARC-induced glioma cell invasion and survival. RESULTS: Our studies found the following. 1) SPARC increases the expression of tumor cell pro-survival and pro-death protein signaling in balance, and, as a net result, tumor cell survival remains unchanged. 2) Suppressing SPARC increases tumor cell survival, indicating it is not a good therapeutic target. 3) Suppressing HSP27 decreases tumor cell survival in all gliomas, but is more effective in SPARC-expressing tumor cells due to the removal of HSP27 inhibition of SPARC-induced pro-apoptotic signaling. 4) Suppressing total AKT1/2 paradoxically enhanced tumor cell survival, indicating that AKT1 or 2 are poor therapeutic targets. 5) However, inhibiting pAKT suppresses tumor cell survival. 6) Inhibiting both HSP27 and pAKT synergistically decreases tumor cell survival. 7) There appears to be a complex feedback system between SPARC, HSP27, and AKT. 8) This interaction is likely influenced by PTEN status. With respect to chemosensitization, we found the following. 1) SPARC enhances pro-apoptotic signaling in cells exposed to TMZ. 2) Despite this enhanced signaling, SPARC protects cells against TMZ. 3) This protection can be reduced by inhibiting pAKT. 4) Combined inhibition of HSP27 and pAKT is more effective than TMZ treatment alone. CONCLUSIONS: We conclude that inhibition of HSP27 alone, or in combination with pAKT inhibitor IV, may be an effective therapeutic approach to inhibit SPARC-induced glioma cell invasion and survival in SPARC-positive/PTEN-wildtype and SPARC-positive/PTEN-null tumors, respectively.

Deletion of the SPARC acidic domain or EGF-like module reduces SPARC-induced migration and signaling through p38 MAPK/HSP27 in glioma.

We previously demonstrated that secreted protein acidic and rich in cysteine (SPARC) increases heat shock protein 27 (HSP27) expression and phosphorylation and promotes glioma cell migration through the p38 mitogen-activated protein kinase (MAPK)/HSP27 signaling pathway. As different regions of the SPARC protein mediate different SPARC functions, elucidating which SPARC domains regulate HSP27 expression, signaling and migration might provide potential therapeutic strategies to target these functions. To investigate the roles of specific domains, we used an SPARC-green fluorescent protein (GFP) fusion protein and constructs of SPARC-GFP with deletions of either the acidic domain (ΔAcidic) or the epidermal growth factor (EGF)-like module (ΔEGF). GFP, SPARC-GFP and the two deletion mutants were expressed in U87MG glioma cells. Characterization of the derived stable clones by confocal imaging and western blotting suggests proper folding, processing and secretion of the deletion constructs. Uptake of the constructs by naive cells suggests enhanced internalization of ΔAcidic and reduced internalization of ΔEGF. Wound and transwell migration assays and western blot analysis confirm our previous results and indicate that ΔAcidic reduces SPARC-induced migration and p38 MAPK/HSP27 signaling and ΔEGF decreases SPARC-induced migration and dramatically decreases the expression and phosphorylation of HSP27 but is poorly internalized. Loss of the EGF-like module suppresses the enhanced HSP27 protein stability conferred by SPARC. In conclusion, deletions of the acidic domain and EGF-like module have differential effects on cell surface binding and HSP27 protein stability; however, both regions regulate SPARC-induced migration and signaling through HSP27. Our data link the domains of SPARC with different functions and suggest one or both of the constructs as potential therapeutic agents to inhibit SPARC-induced migration.

HSP27 mediates SPARC-induced changes in glioma morphology, migration, and invasion.

Secreted protein acidic and rich in cysteine (SPARC) regulates cell-extracellular matrix interactions that influence cell adhesion and migration. We have demonstrated that SPARC is highly expressed in human gliomas, and it promotes brain tumor invasion in vitro and in vivo. To further our understanding regarding SPARC function in glioma migration, we transfected SPARC-green fluorescent protein (GFP) and control GFP vectors into U87MG cells, and assessed the effects of SPARC on cell morphology, migration, and invasion after 24 h. The expression of SPARC was associated with elongated cell morphology, and increased migration and invasion. The effects of SPARC on downstream signaling were assessed from 0 to 6 h and 24 h. SPARC increased the levels of total and phosphorylated HSP27; the latter was preceded by activation of p38 MAPK and inhibited by the p38 MAPK inhibitor SB203580. Augmented expression of SPARC was correlated with increased levels of HSP27 mRNA. In a panel of glioma cell lines, increasing levels of SPARC correlated with increasing total and phosphorylated HSP27. SPARC and HSP27 were colocalized to invading cells in vivo. Inhibition of HSP27 mRNA reversed the SPARC-induced changes in cell morphology, migration, and invasion in vitro. These data indicate that HSP27, a protein that regulates actin polymerization, cell contraction, and migration, is a novel downstream effector of SPARC-regulated cell morphology and migration. As such, it is a potential therapeutic target to inhibit SPARC-induced glioma invasion.

Secreted protein acidic and rich in cysteine promotes glioma invasion and delays tumor growth in vivo.

Secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human astrocytomas, grades II-IV. We demonstrated previously that SPARC promotes invasion in vitro using the U87MG-derived clone U87T2 and U87T2-derived SPARC-transfected clones, A2b2, A2bi, and C2a4, in the spheroid confrontation assay. Additional in vitro studies demonstrated that SPARC delays growth, increases attachment, and modulates migration of tumor cells in extracellular matrix-specific and concentration-dependent manners. Therefore, we propose that SPARC functionally contributes to brain tumor invasion and delays tumor growth in vivo, and that the effects of SPARC are related to the level of SPARC secreted into the extracellular matrix. To test these hypotheses, we stereotactically injected these clones into nude rat brains (six animals were injected per clone). Animals were sacrificed on day 7 to assess growth and invasion for all clones at the same time in tumor development. To determine whether SPARC delayed but did not inhibit growth, rats were injected with U87T2 or clone A2b2, and the animals were sacrificed on days 9 (U87T2) and 20 (A2b2), when the animals demonstrated neurological deficit. Brains were removed, fixed, photographed, paraffin embedded, and sectioned. Sections were then serially stained with H&E for morphological assessment of invasion and to measure tumor volume, immunohistochemically stained to visualize SPARC, subjected to in situ hybridization with the human AluII DNA-binding probe to identify human cells, and immunohistochemically stained with MIB-1 to measure proliferation index. The results demonstrate that SPARC promotes invasion in vivo at day 7. Both the low (A2bi) and the high (A2b2) SPARC-secreting clones produced invasive tumors, invading with fingerlike projections and satellite masses into adjacent brain, as well as along the corpus collosum. The intermediate SPARC secreting clone (C2a4) primarily migrated as a bulk tumor along the corpus collosum. SPARC significantly decreased tumor growth at day 7, as measured both by adjusted MIB-1 proliferation indices (U87T2 = 95.3 +/- 1.4 versus A2bi = 73.4 +/- 4.0, A2b2 = 30.8 +/- 6.7 and C2a4 = 15.7 +/- 13.0) and tumor volumes (U87T2 = 13.4 +/- 0.6 mm(3) versus A2bi = 4.5 +/- 0.6 mm(3), A2b2 = 1.1 +/- 0.1 mm(3), and C2a4 = 0.4 +/- 0.1 mm(3)). Furthermore, SPARC delayed but did not inhibit tumor growth. The patterns of invasion and the extent of growth delay correlated with the level of SPARC expression. We propose that the ability of SPARC to promote invasion depends on the level of its secretion and the resultant modulation of the level of adherence and motility induced. This demonstration that SPARC functionally contributes to brain tumor invasion in vivo suggests that SPARC is a candidate therapeutic target for the design of therapies directed toward inhibition of the invasive phenotype.

cDNA array analysis of SPARC-modulated changes in glioma gene expression.

We have demonstrated that secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human gliomas and it promotes glioma invasion and delays tumor growth in vitro and in vivo. cDNA array analyses were performed to determine whether 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. Using a doxycycline (dox)-controlled gene expression system, two cDNA array analyses were performed using a parental U87T2 clone (-SPARC) transfected with the dox-controlled transactivator and a U87T2 parental-derived SPARC-transfected clone, A2b2 (+SPARC). Array analysis performed between the parental and the SPARC-transfected clone (-dox) identified 13 upregulated genes and 14 downregulated genes. With the exception of PAI-1 and MMP2, the identified genes are novel with respect to SPARC's mechanism of action. Array analysis performed using the SPARC-transfected clone ( +/- dox) identified 2 types of gene regulation; one reversible upon SPARC suppression, the other irreversible. Two of the SPARC-induced genes, BIGH3 (irreversible by dox) and PAI-1 (reversible by dox) were further studied in additional SPARC-transfected clones, human astrocytoma tissues, and human glioma cell lines by RT-PCR and Northern blot analyses. The results indicate that: (1) the array results were validated, (2) the dox regulation was validated, and (3) the differential expression identified by the array analyses was present between normal brain and in human astrocytoma tissues and cell lines. Therefore, we conclude that these cDNA array analyses provide candidate genes involved in SPARC-mediated effects on glioma cell cycle progression, signaling, and migration, and that SPARC may induce reversible and irreversible gene expression changes. Further investigation of these candidates may shed insights into SPARC's role in glioma cell proliferation and invasion, and potential use as a therapeutic target.