PGRN And CIP2A Are Overexpressed In Gliobiastoma And Involved In Tumor Cellular Invasion

IntroductionAstrocytomas are the most common primary malignant brain tumors, accounting for approximately75%of all types of glioma. They are classified into four malignancy grades (Grade Ⅰ-Ⅳ) according to the World Health Organization (WHO) criterion. Glioblastoma (Grade IV), the most prevalent and aggressive form of astrocytomas, has the poorest prognosis with median survival of less than1year despite multidisciplinary therapeutic approaches. Early diagnosis and personalized clinical intervention based on genetic profiles may be of paramount value in the treatment of astrocytomas. Thus, understanding the molecular basis of astrocytoma progression and identification of prognostic markers are needed to devise effective individual targeted therapy.Astrocytomas, particularly high grade types, are characterized by aggressive growth and intense vascularity, which are associated with their ability to produce growth factors such as endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and platelet derived growth factor (PDGF). Progranulin (PGRN), also known as PC-cell-derived growth factor, proepithelin or granulin-epithelin precursor, is an88KD secreted glycoprotein that belongs to a novel class of growth factors. Constitutive expression of PGRN at high levels occurs during remodeling processes such as embryonic development, tissue repair, inflammation and tumorigenesis. Previous studies have suggested that PGRN regulates a wide range of biological processes involved in tumor development and progression, such as enhanced cell proliferation, survival, migration, invasion and angiogenesis. These tumor-promoting functions of PGRN are known to be associated with activation of growth factor-related signaling system including PI3K/Akt, MAPK/ERK and FAK pathways. Moreover, PGRN has been found to be overexpressed in several human tumor types, including glioblastoma, breast cancer, ovarian cancer, hepatocellular carcinoma, prostate cancer as well as multiple myeloma.Despite that PGRN mRNA expression has been reported to be highly expressed in gioblastomas, the expression pattern and clinical significance of PGRN has not been described previously in various forms of human astrocytomas. In this study, we investigated the expression profile of PGRN protein and its possible prognostic relevance in a larger collective of patients with astrocytoma. We also analyzed whether knocked down PGRN affects the proliferation and invasion of GBM cells in vitro.MethodsA total of210samples were obtained from operated astrocytoma patients at the Department of Neurosurgery, Qilu Hospital of Shandong University from2005to2009. None of the patients had received radiotherapy or chemotherapy prior to surgery. The mRNA level of PGRN in35astrocytoma samples and glioblastoma cell lines(U87, U251, T98G) was examined by semi-quantitative RT-PCR. The protein level of PGRN was also measured by western blotting. Immunohistochemistry was further performed in a total of210astrocytoma samples. PGRN expression levels in specimens of different pathological grades were compared using ANOVA with Bonferroni correction. Chi-square test was used to analyze the relationship between PGRN expression and the clinical parameters. Survival curves were plotted using the Kaplan-Meier method and compared by the log-rank test. Survival data were evaluated using univariate and multivariate Cox regression analyses. The PGRN siRNA was prepared and transfected in a final concentration of30nM using Lipofectin2000. Cell proliferation assays were performed by using Cell Counting Kit-8according to the manufacturer's instructions. Matrigel invasion assay was evalu-ated using a24-well Transwell chamber with8.0μm pore polycarbonate filter inserts.Results1. The expression profiles and clinical significance of PGRN in human astrocytomas.We first examined the mRNA expression of PGRN by semi-quantitative RT-PCR in35clinical astrocytoma samples and GBM cell lines, and found its high level of expression in GBM cell lines and almost all of the samples, although no expression was detected in five normal brain tissues. Subsequently, we performed western blot analysis to quantify the relative level of PGRN protein expression. Samples of normal brains expressed very low levels of the PGRN protein, whereas significantly higher levels of this protein were observed in cell lines and tumor samples, particularly GBM tissues. Immunohistochemical staining was also performed to evaluate PGRN expression in normal brain and210astrocytoma tissues. The normal brain tissue was negative for PGRN except for weak cytoplasmic staining in a few neurons. In contrast, PGRN positive tumor cells were detected in96.2%of astrocytoma samples, and PGRN positive blood vessels were detected in82.4%of the tumors. PGRN immunostaining in tumor cells and blood vessels were scored separately. The tumor cellular PGRN scores, the blood vessels PGRN scores and total PGRN scores all increased with tumor grade. However, we did not find significant associations between PGRN expression and age, gender, KPS scores or tumor locations. In glioblastoma, univariate analysis revealed that high total PGRN expression, strong tumor cellar PGRN expression and strong vascular PGRN expression all associated with decreased overall survival of glioblastoma patients. In multivariate Cox regression model adjusting for age, gender and KPS score, only total PGRN expression and vascular PGRN expression were independently prognostic factors for overall survival of patients with glioblastoma.2. Depletion of PGRN expression inhibits GBM cells proliferation and invasionTo determine whether higher levels of PGRN contributed to the proliferation of GBM cells, we blocked PGRN expression in the U87and T98G GBM cell lines by siRNA. Cell proliferation rate was examined by CCK-8assay, and PGRN depletion resulted in a significant decrease in cell proliferation rate in T98G and U87cells. Additionally, GBM cells transfected with PGRN siRNA were more sensitive to oridonin than cells transfected with control siRNA. We further examined whether cell invasion capacity was altered in cells treated by PGRN siRNA. We found that PGRN knockdown suppressed their invasive ability. 3. Effect of PGRN depletion on MMP-9expression of GBN cellsSince the invasiveness of glioma largely depends on the proteolytic destruction of the extracellular matrix, we analyzed the expression of MMPs after PGRN siRNA treatment. We found that knockdown of PGRN by siRNA significantly decreased the mRNA levels of MMP-9, but not MMP-2and MMP-7in GBM cells. Western-blot analysis also showed the similar changes in protein level of MMP-9. We further performed gelatin zymogram to examine the effects of PGRN on MMP activation. It was found that PGRN siRNA significantly suppressed the activity of MMP-9, but not MMP-2in both GBM cell lines.ConclusionIn the current study, we demonstrate that elevated PGRN expression is a common event in various astrocytomas. PGRN is significantly up-regulated in glioblastoma as compared to normal brain and low-grade astrocytomas, and that its expression is inversely correlated with glioblastoma patient survival. Moreover, depletion of endogenous PGRN inhibited glioblastoma cells proliferation, and decreased their invasive capacity partly by suppressing MMP-9secretion. Thus, PGRN may be a novel biological marker and potential therapeutic target in astrocytomas, particularly glioblastoma. IntroductionGlioblastoma (GBM) is the most prevalent and malignant form of primary brain tumors. Although considerable improvements have been made in surgical and adjuvant therapies, the median survival time for patients with GBM remains only12-15months. Extensive infiltration of GBM cells into the surrounding normal brain tissues is a major reason for patient morbidity and mortality. These invading cells are extremely resistant to radiation and chemotherapy. Thus, investigation of tumor-specific molecules involved in GBM invasion is required for devising effective targeted therapy.Cancerous inhibitor of protein phosphatase2A (CIP2A) is a recently identified human oncoprotein expressed in various human malignancies including breast, tongue, lung, ovarian, and cervical cancers as well as renal cell carcinoma. CIP2A interacts with protein phosphatase2A (PP2A) and inhibits PP2A-mediated dephosphorylation of the oncogene c-Myc. It has been shown that high expression levels of CIP2A correlate with enhanced aggressiveness and/or poor patient survival in renal cell carcinoma, breast, gastric, tongue and serous ovarian cancers. However, its role in GBM has so far not been described. In the present study, we investigated whether expression of CIP2A in GBMs correlates with overall survival and clinicobiologic features representing tumor invasiveness in GBM patients.MethodsA total of79formalin-fixed, paraffin-embedded specimens of GBMs operated between2006and2010were retrieved form the archives of the Pathology Department of Qilu Hospital. None of the patients had received radiotherapy or chemotherapy prior to surgery. The diagnosis was confirmed by histopathological examination of the specimens.16GBMs specimens were snap-frozen in liquid nitrogen and stored at-80℃following surgery between2009and2010for analysis by western blot and semi-quantitative RT-PCR. The preoperative magnetic resonance image (MRI) studies were reviewed for evaluating the invasiveness of the tumours. The radiographic patterns of GBMs were classified into three categories according to the MRI features: solitary, diffuse, and multifocal types. Diffuse and multifocal imaging phenotypes are known to represent invasive features of the GBMs. The mRNA and protein level of CIP2A in16GBM samples were examined by semi-quantitative RT-PCR and western blotting. Immunostaining for CIP2A, MMP-9, EGFR, and Ki-67were further performed in a total of79GBM samples. Thex2-test was used to analyze the associations between CIP2A expression and clinicopathologic variables. Survival curves were plotted using the Kaplan-Meier method and compared by the log-rank test. Survival data were evaluated using univariate and multivariate Cox regression analyses. The CIP2A siRNA was prepared and transfected in a final concentration of30nM using Lipofectin2000. Matrigel invasion assay was evaluated using a24-well Transwell chamber with8.0μm pore polycarbonate filter inserts. The wound healing assay was used to assess the effeets of CIP2A siRNA on the migration capacity of GBMs cells.Results1. Expression of CIP2A in GBMRT-PCR and Western blot analysis were done in16clinical GBM specimens and7normal brain tissues. The presence of CIP2A transcripts was found in14of16(87.5%) of GBM samples, whereas only2of7(28.6%) normal brain controls exhibited detectable CIP2A mRNA expression. Similar to the mRNA results, CIP2A was overexpressed in11(68.8%) specimens of GBMs analyzed by Western blotting. However, in normal brain controls, CIP2A was undetectable in5of7(71.4%) samples. We further analyzed CIP2A protein level in79paraffin-embedded, archived GBM specimens using immunohistochemical staining. Cytoplasmic CIP2A immunoreactiviy in tumour cells was found in61of79(77.2%) of GBM samples. Of them, CIP2A staining was strongly positive in13specimens.2. Association of CIP2A expression with invasive phenotypes We found that CIP2A overexpression in GBMs were significantly with diffuse and multifocal features on MR images (P=0.000). On immunohistochemical analysis, CIP2A overexpression was also associated with MMP-9and EGFR (P=0.004and P=0.001, respectively), but not Ki-67protein expression (P=0.519). In addition, CIP2A knockdown significantly suppressed both motility and invasive abilities of GBM cells.3. Association of CIP2A expression with overall survivalThe Kaplan-Meier method was used to estimate overall survival. The median overall survival of patients with CIP2A overexpression was significantly shorter than that with no or low CIP2A expression (log-rank test, P=0.000). Multivariate analysis indicated that overexpression of CIP2A were independent predictors of poor overall survival (P=0.029).ConclusionOur results demonstrate that CIP2A overexpression positively correlates with the invasive phenotype of GBMs, and predicts poor clinical outcome. Thus, CIP2A may be a novel and promising therapeutic target for GBM therapy.