Tumor Biol. DOI 10.1007/s13277-015-3635-4

RESEARCH ARTICLE

Glucose-regulated protein 94 is a novel glioma biomarker and promotes the aggressiveness of glioma via Wnt/β-catenin signaling pathway Tieyi Hu 1 & Niqi Xie 2 & Chuan Qin 3 & Jiasheng Wang 4 & Yi You 5

Received: 22 April 2015 / Accepted: 1 June 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015

Abstract Malignant glioma is the most common type of primary brain tumor and represents one of the most aggressive and lethal human cancer types. Glioma recurrence is a common event; however, the relevant molecular mechanisms in this setting are not well-understood. In this study, we investigated glucose-regulated protein 94 (GRP94) expressions in human glioma and aimed to determine the roles of GRP94 expression affects cell proliferation, invasion, and regulatory signaling in human glioma U87 cells. Our results showed that GRP94 was overexpressed at both mRNA and protein levels in high-grade glioblastoma as compared with normal brain tissues. High GRP94 levels also predict shorter overall survival of glioma patients. RNAi-mediated silencing of GRP94 suppressed cellular proliferation, colony formation ability in glioma cells. Depletion of GRP94 also inhibited cell migration and invasion ability in glioma cell. Furthermore, gene microarray analysis revealed that GRP94 depletion caused the dysregulation of critical pathway, Wnt/β-catenin signaling pathway. We next demonstrated GRP94 regulates Wnt/β-catenin

* Niqi Xie [email protected] 1

Department of Neurology, Dazu District People’s Hospital, Chongqing 402360, China

2

Department of Clinical Laboratory, Dazu District People’s Hospital, #1 affiliated #138 longgangxi Rd, longgang street Dazu District, Chongqing 402360, China

3

Department of Neurosurgery, Dazu District People’s Hospital, Chongqing 402360, China

4

Department of Intensive Care Unit, Dazu District People’s Hospital, Chongqing 402360, China

5

Department of Prevention and Health Care, Dazu District People’s Hospital, Chongqing 402360, China

signaling pathway to promote the proliferation of glioblastoma cells. Conclusion, our findings establish GRP94 as progression markers and druggable targets in glioblastoma, relating their oncogenic effects to activation of the Wnt/β-catenin signaling pathway. Keywords GRP94 . Proliferation . Migration . Invasion . Wnt/β-catenin pathway

Introduction Glioma is the most common form of brain tumor, accounting for more than 50 % of all brain tumors [1–3]. Despite significant improvements in neurosurgical approaches and chemotherapy/radiotherapy, grade 4 glioblastoma (GBM) remains a cancer type with dire prognosis, primarily because GBM cells are highly invasive and are capable of infiltrating into surrounding normal brain tissue, resulting in failure to completely remove the tumor by surgery [4–6]. To provide insight that will enable the development of new therapeutic strategies, it is crucial to elucidate the molecular mechanisms that promote the aggressiveness of human glioma. Glucose-regulated protein 94 (GRP94) is a type of stress protein, primarily localized in the endoplasmic reticulum [7–9]. Available data indicate that GRP94 high expression was detected in a variety of tumors and involved in tumorigenesis and development by regulating multiple signaling pathways. It is reported that silencing of GRP94 causes severe compromise in human multiple myeloma cell growth and induce apoptosis in pre-B leukemic cells through inhibiting Wnt pathway [10–13]. Several studies found that the underlying mechanisms for HCC development in cGrp94 (f/f) livers, they detected increase in TGF-β1, activation of SMAD2/3, ERK, and JNK, and cyclin D1 upregulation at the premalignant

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stage [14, 15]. However, studies for the expression level of GRP94 in human glioma and regulatory mechanisms are unknown. In this study, we determined the roles of GRP94 in human glioma tissue as well as human glioma cell lines. Our data indicates that GRP94 has an important role in regulating cell proliferation, survival, and invasiveness of glioma cells. GRP94 is a novel therapeutic target for human glioma. Mechanistically, we found that GRP94 critically controls the Wnt/ β-catenin pathway.

Materials and methods Human tissue samples Frozen glioma specimens’ classified as based on the World Health Organization (WHO) criteria were previously described [16]. These were obtained from The First Affiliated Hospital of Chongqing Medical University. The paraffinembedded human normal brain, grade 2 (oligodendroglioma, low grade astrocytoma, and mixed oligoastrocytoma), grade 3 (anaplastic astrocytoma and anaplastic oligodendroglioma), and grade 4 (GBM) glioma tissue microarrays (TMA) were also described [17, 18]. Each specimen was divided into two parts, one for mRNA expression analysis and the other for protein expression analysis. Cell culture Human glioma cell lines, U87 and U251, were obtained from ATCC and cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Hyclone) supplemented with 10 % fetal bovine serum, 100 units penicillin, and 100 units streptomycin at 37 °C in 5 % CO2 atmosphere in a humidified incubator.

Cell proliferation and soft agar colony formation assay Cell proliferation was measured using Cell Counting Kit8(CCK-8) and colony formation assay. U87 cells in each group were placed in 96-well plates, 2000 cells in 100 ul medium per well for incubation at 37 °C and 5 % CO2 supplied incubator for up to 5 days. A 10-ul CCK8 reagent was added to each well at the end of incubation, then 2 h later, the absorbance at 450 nm using a Microplate reader. All experiments were performed in triplicate. Colony formation ability was determined by anchorage independent soft agar assay on U87 cells. Briefly, 1.5 ml RPMI 1640 medium containing 0.6 % agarose were added to each well of six-well cell culture plates and allowed to solidify (base agar). U87 cells of 4×103 were then mixed with RPMI 1640 medium containing 0.35 % agarose and added to the top of base agar (top agar). The cells were then cultured for 14 days at 37 °C fewer than 5 % carbon dioxide. The plates were stained with 0.05 % crystal violet for 1 h, and the colonies were counted under a microscope. Wound-healing assays Cells of 5×105 were seeded on six-well plates, grown for overnight until confluence, and then the monolayer of the cells were scratched using a yellow pipette tip. Subsequently, phosphate buffered saline (PBS) was used to wash and remove floating and damaged cells, and the medium without fetal bovin serum (FBS) was then added to cells for culture. Cells migrated over the denuded area were observed and photographed with an inverted phase-contrast microscope equipped with a Quick Imaging system at the indicated times. The mean migration rate (um/h) was expressed by calculating the changes of wound widths measured directly on photos that were taken at the indicated time points. Cell invasion analysis

Stable knockdown cell line generation A vector-based RNAi approach was used to generate the stable GRP94 knockdown cell line. Briefly, according to the corresponding siRNA sequences used in transient experiment, the double-stranded short hairpin RNA (shRNA) template for both the negative control and GRP94 were designed and cloned into the Hind III/Bgl II sites of the pSUPER vector (Oligoengine Corporation), respectively. The recombinant RNAi constructs (pSUPER-ControlshRNA and pSUPER-GRP4-shRNA) were confirmed by direct sequencing and transfected into U87 cells by Lipofectamine 2000 (Invitrogen), respectively. The transfected cells were then subcultured and selected in the presence of G418 for generating the negative control and GRP94 stable knockdowncells, designated as pControl-sh and pGRP94sh, respectively.

U87 cells with GRP94 depletion or control were collected, washed, and resuspended in 1 % FBS Dulbecco’s modified eagle medium (DMEM), then seeded to the upper chamber of transwell chambers (Millipore, USA) with Matrigel (BD Biosciences, USA). Simultaneously, 600 ml DMEM medium supplemented with 20 % FBS in the lower chamber, and these chambers were cultured at 37 °C for over 24 h. Fixed in 4 % methanol and 10 min, 1 % crystal violet staining 15 min, PBS washed and counted by fluorescence microscope, all experiments were repeated 3 times. Gene microarray assay U87 cells with GRP94 depletion or control were collected; total RNA was isolated using TRIzol reagent and subjected to microarray analysis. The microarray experiments were

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performed at CapitalBio Corporation (Beijing, China) whose GeneChip microarray service was certificated by Affymetrix. To verify the microarray findings, several differentially expressed genes were further analyzed by quantitative realtime polymerase chain reaction (qRT-PCR) and Western blot.

Quantitative real-time PCR Total RNA was extracted from cultured cells using an EZNA Total RNA Kit (OMEGA Bio-tek, USA), and cDNA was generated using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Otsu, Japan). Quantitative real-time PCR was performed using the SYBR Premix ExTaqII (TLiRNaseH Plus) (TaKaRa, Otsu, Japan) with

Fig. 1 GRP94 is a progression marker in glioma. a Immunohistochemistry with GRP94 antibody of glioma TMA. b Kaplan-Meier plots of GRP94 expression in 20 cases of human glioma patients. Overall survival rate was performed by log-rank test. P<0.05 indicates significant differences between two groups. c Overexpression of GRP94 at mRNA level in glioma tissues. Expression of GRP94 in normal brain and glioma tissue was analyzed by qRT-PCR using the Origene TissueScan Glioma Panels with specific primers. The grades were classified as GI–G4. e, f qRT-PCR and Western blot analysis of cell extracts from NHA and 4 human glioblastoma cell lines showing GRP94 expression

a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Western blot analysis After measuring the protein concentration of the lysates using the BCA method (Beyotime, China), equal amounts of protein were separated by SDS-PAGE, blotted onto PVDF membranes (Millipore, Billerica, MA). The membranes were blocked with TBST containing 5 % skim milk for 1 h at room temperature, incubated with the corresponding primary antibodies overnight at 4 °C, and then with secondary antibodies conjugated to horseradish peroxide for 2 h at room temperature. The protein was visualized using enhanced chemiluminescence (GE Healthcare Biosciences).

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Statistical analysis The statistical analysis performed with the software of SPSS version 18.0 for Windows. All the data were expressed as mean ± SD. The statistical significance was evaluated by ANOVA or two-tailed t test, and the results were considered significant at a p value less than 0.05.

Results GRP94 is frequently upregulated in human glioma tissue To determine GRP94 expression levels to human glioma progression, we then focused on the expression profile analysis of GRP94 in glioma. Immunohistochemistry analysis of a glioma TMA containing normal brain and grades 2 to 4 gliomas confirmed the gradual increase of moesin staining in gliomas

Fig. 2 GRP94 depletion inhibits glioma tumorigenesis in vitro. a RT-PCR and Western blot analysis determined that the GRP94 expression was significantly decreased at both mRNA and protein levels. b Cell proliferation assay. GRP94 stable knockdown U87cells (shGRP94) and the negative control stable cells (shCtrl) were measured by CCK8 and counted at the indicated times. d Colony formation assay for GRP94 knockdown cells and control cells. e Numbers of colonies for GRP94 knockdown cells and control cells

of increasing severity (Fig. 1a). High GRP94 levels also predict shorter overall survival of glioma patients (Fig. 1b). RTPCR demonstrated that GRP94 was significantly upregulated at an mRNA level in grades 2 to 4 gliomas samples compared with normal brain tissues (Fig. 1c). Notably, overexpression of GRP94 mRNA was significantly associated with tumor grades and stages (Fig. 1d). We also examined the expression of GRP94 in five glioblastoma cell lines and a normal human astrocyte (NHA) cell line and showed increased GRP94 levels in all glioblastoma lines (Fig. 1e, f). These combined findings indicate GRP94 as glioma progression markers, with highly correlated expression levels. GRP94 depletion inhibits glioma tumorigenesis in vitro To further examine the potential role of GRP94 expression on human progression and aggressiveness, we generated U87 cells that constitutively expressed short hairpin RNA

Tumor Biol.

(shRNA) targeting either GFP (shCtrl) or GRP94-specific (shGRP94). RT-PCR and Western blot analysis determined that the GRP94 expression was significantly decreased at both mRNA and protein levels. Stable knockdown of GRP94 in these cells also resulted into a significant decrease in cell proliferation as measured by Cell Counting Kit-8. Then, the results of soft agar colony formation assay also demonstrated that the stable GRP94 knockdown cells showed a significantly reduced colony formation activity in both number and size, as compared with the control cells (Fig. 2).

GRP94 depletion inhibits migration and invasion in glioma cells We further examined whether inhibition of GRP94 expression affected cell migration and invasion ability in glioma cells. The cell migration and invasion ability was determined by wound healing assay and matrigel invasion assay. Wound healing assay revealed that depletion of GRP94 significantly decreased the rate of migration into a wound introduced in a

Fig. 3 GRP94 knockdown inhibits migration and invasion in glioma cells. Stable knockdown U87cells (shGRP94) and the negative control stable cells (shCtrl) cells were subjected to wound-healing assay (a, b), and transwell invasion assay (c, d), respectively

confluent monolayer of cells compared with controls. In the matrigel invasion assay (Fig. 3a, b), we also found that knockdown of GEP94 significantly decreased the number of cells that penetrated through the Matrigel-coated membrane (Fig. 3c, d). Therefore, these results strongly indicated that GRP94 might also regulate the cell migration and invasion ability of glioma cells.

Microarray analysis identifies the Wnt/β-catenin pathway as specific signaling downstream of the GRP94 To furthermore gain a better insight on potential molecular mechanism underlying the GRP94 depletion-induced suppression of tumor growth in glioblastoma, we test a transcription microarray assay to compare the differential gene expression profiles between GRP94 shRNA and negative control shRNA-treated U87 cells. This profiling revealed that: 520 genes were differentially expressed and classified into different groups. Among the differentially expressed genes, 123 genes were upregulated (ratio, >2.0), and 397 genes were downregulated (ratio, <0.5). Consistently, many of these

Tumor Biol. Fig. 4 GRP94 activates Wnt/βcatenin signaling pathway. a Important pathways affected by GRP94 knockdown. Microarraybased gene expression profiling. Stable knockdown U87cells (shGRP94) and the negative control stable cells (shCtrl) subsequently subjected to microarray analysis. b Decreased β-catenin nuclear expression in shGRP94 cells shown by nuclear immunofluorescence of U87 cells. c Stable knockdown U87cells (shGRP94) and the negative control stable cells (shCtrl) together with the pGL4-Snail/promoter and pRK-Luc vectors. After 72 h, luciferase activity was measured. Three independent experiments were carried out, and the relative luciferase activity is indicated (*P<0.05)

genes were related to cell proliferation. Using the Kyoto Encyclopedia of Genes and Genomes, BioCarta, and Gene Map annotator and Pathway Profiler databases, the significant signaling pathways were categorized into different groups including the Wnt/β-catenin signaling pathway, cell cycle regulation, and small glioma pathway etc. Important genes and pathways involved in this process are shown in Fig. 4a.

We next used the luciferase-reporter system with β-catenin responsive (TOP) or mutated (FOP) elements and observed that β-catenin transcriptional activity was significantly decreased in GRP94 depletion cells (Fig. 4b). The fractionation and immunofluorescence analysis of moesin-overexpressing cells showed that a fraction of β-catenin translocated from the membrane to the nuclear compartment in these cells (Fig. 4c). Overall, this comprehensive analysis showed that GRP94

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induces β-catenin nuclear translocation and increased transcriptional activity.

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Discussion Glioma is a common brain tumor. Despite enormous improvements in surgical techniques and development of therapeutic agents, the 5-year survival rate is lower than 30 % [19]. Cancer is essentially a genetic disease and during the process of carcinogenesis, genetic mutation influence key cellular pathways, eventually lead to unrestrained cell growth and malignant phenotype [17, 20, 21]. In this study, we founded expression of GRP94 was obviously higher than in normal tissue adjacent to carcinoma. Previous studies have shown that GRP94 overexpression may be a hallmark of aggressiveness and recurrence in breast cancers [12, 14]. In this study, we showed that GRP94 is upregulated in the majority of glioma cell lines and human glioma tissues. To the best of our knowledge, this is the first report of an association between GRP94 expression and glioma tissue in Chinese patients. GRP94 is known to be overexpressed in some tumors, such as hepatic carcinoma, lung cancer, cervical cancer etc. It plays an important role in the progression and metastasis. We identified that GRP94 depletion effectively suppress cell proliferation in U87 cells. Further functional analysis indicated that silencing of GRP94 via RNAi in glioma cells resulted in significant inhibition of cellular proliferation, cell migration, invasion, and colony formation in vitro. These findings strongly indicated that GRP94 played important role in tumorigenesis. Moreover, microarray analysis revealed that important pathways such as Wnt/β-catenin signaling pathway and DNA replication were deregulated after GRP94 knockdown. There are accumulating evidences that Wnt/β-catenin signaling plays a crucial role in the progression of human glioma, involved in regulating the cell proliferation and cell differentiation. GSK3b is a key molecule of Wnt/β-catenin signaling pathway which is involved in a widening differentiation, growth, motility, and apoptosis [22]. In conclusion, we described GRP94 as a marker for glioma progression that induces oncogenic growth through a mechanism involving the regulation Wnt/β-catenin pathway. We described for the first time a new therapy target consisting of the GRP94, whose disruption represses both the proliferation and β-catenin dependent signaling. This study adds GRP94 on the map of relevant molecular changes in glioblastoma and provides a druggable target for glioblastoma therapy.

Conflicts of interest None

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