A Complex Mechanism For HDGF-mediated Cell Growth, Migration,Invasion, And TMZ Chemosensitivity In Glioma

HDGF is overexpressed in gliomas as compared to normal brain. We therefore analyzed the molecular mechanisms of HDGF action in gliomas. HDGF was downregulated in normal brain tissue as compared to glioma specimens at both the mRNA and the protein levels. In glioma samples, increased HDGF expression was associated with disease progression. Knocking down HDGF expression not only significantly decreased cellular proliferation, migration, invasion, and tumorgenesis, but also markedly enhanced TMZ-induced cytotoxicity and apoptosis in glioma cells. Mechanistic analyses revealed that CCND1, c-myc, and TGF-β were downregulated after stable HDGF knockdown in the U251and U87glioma cells. HDGF knockdown restored E-cadherin expression and suppressed mesenchymal cell markers such as vimentin,β-catenin, and N-cadherin. The expression of cleaved caspase-3increased, while Bcl-2decreased in each cell line following treatment with shHDGF and TMZ, as compared to TMZ alone. Furthermore, RNAi-based knockdown study revealed that HDGF is involved in the activation of both the PI3K/Akt and the TGF-β signaling pathways. Together, our data suggested that HDGF regulates glioma cell growth, apoptosis and epithelial-mesenchymal transition (EMT) through the Akt and the TGF-β signaling pathways. These results provide evidence that targeting HDGF or its downstream targets may lead to novel therapies for gliomas.IntroductionGliomas can be classified according to the cell type that they mostly resemble with, into several categories, such as astrocytomas, oligodendrogliomas, or oligoastrocytomas. Over half of the gliomas are glioblastoma multiforme (GBM), the most malignant form, which is characterized by rapid progression, high resistance to radiotherapy and chemotherapy, and an extremely poor prognosis. Although surgical resection, adjuvant radiotherapy, and chemotherapy improve survival, death occurs inevitably from either recurrent or progressive disease. Understanding the molecular alterations in the signaling pathways and the subsequent pathological changes that characterize GBM have greatly improved in recent years and started to match the knowledge from other cancer types.HDGF is an acidic heparin-binding protein that was originally purified from media conditioned with the HuH7human hepatoma cell line. It is highly expressed in fetal tissues and may be involved in the development of organs, including cardiovascular, liver, and kidney development. HDGF is translocated to the nucleus via nuclear localization signals, a process that is essential for inducing cell growth. Concerning neoplasms, a growing body of evidence suggests that HDGF is closely linked to the aggressive biological potential of cancer cells. These findings suggest that HDGF expression in cancer cells could be used as a prognostic factor.Previous studies indicated that HDGF participates to many cellular processes, including astrocyte proliferation, renal development, vascular lesion formation, and cardiovascular differentiation. Several findings suggest that HDGF overexpression is associated with aggressive tumor behavior, such as proliferation, angiogenesis, and metastasis. Therefore, HDGF may prove useful as a prognostic factor for patients with hepatocellular carcinoma, gastric, pancreatic, and breast cancers.Recently, a significant correlation has been reported between HDGF expression and the prognosis of patients with glioma. Increasing evidence suggests that HDGF might be implicated in the malignant phenotype of glioma cells. However, the molecular mechanisms involved in HDGF-associated tumorigenesis and in the aggressive behavior of glioma cells remain unclear.Therefore, to investigate the oncogenic potential of HDGF in stable cell lines, we silenced its expression using lentiviral shRNA. Subsequently, we found that HDGF downregulation significantly suppressed glioma cell proliferation, migration and invasion, enhanced TMZ-induced cytotoxicity and apoptosis in vitro, as well as inhibited tumorigenesis in vivo. Our study revealed that HDGF is involved in the activation of the PI3K/Akt and the TGF-β signaling pathways, suggesting potential molecular mechanisms whereby HDGF regulates tumor growth and emigration.Materials and methodsCell culture and sample collectionCell culture was carried out as described. The human U251and U87glioma cell lines were purchased from the Chinese Academy of Sciences (Shanghai, China) and grown in Dulbecco’s modified Eagle’s medium (DMEM)(Hyclone, Logan, UT) supplemented with10%fetal calf serum (ExCell, Shanghai, China). All cell lines were cultured at37℃in a humidified atmosphere with5%CO2. A total of117paraffin-embedded glioma and18normal brain (NB) tissue samples were obtained from the Nanfang Hospital of Southern Medical University, Guangzhou, China. These cases were from79males and38females with ages between10and71years (median,43.3years). Prior consent from patients and approval from the Ethics Committees of Nanfang Hospital was obtained for using these clinical materials for research purposes. All specimens had confirmed pathological diagnosis and were classified according to the World Health Organization (WHO) criteria.Establishment of a Glioma Cell Line with Stable Expression of HDGF Short Hairpin RNAThe preparation of lentiviruses expressing human HDGF short hairpin RNA was performed using the pLVTHM-GFP lentiviral RNAi expression system. U87and U251cells were infected with lentiviral particles containing specific or negative control vectors, and polyclonal cells with GFP signals were selected for further experiments using FACS flow cytometry. Total RNA from these clones was isolated, and HDGF mRNA levels were measured using real-time PCR analysis.EdU incorporation assayU251and U87cells were cultured in96-well plates at5000cells per well, transfected with siHDGF or empty vector (si-Ctr) for48h, and then exposed to50μmol/L5-ethynyl-20-deoxyuridine (EdU, Ribobio, China) for an additional2h at37℃. The cells were fixed with4%formaldehyde for15min and treated with0.5%Triton X-100for20min at room temperature. After washing three times with phosphate buffered saline, the cells from each well were exposed to100μL1X Apollo reaction cocktail for30min. Subsequently, the DNA from cells in each well was stained with100μL of Hoechst33342(5μg/mL) for30min and visualized under a fluorescent microscope.TMZ chemosensitivity test and IC50definitionTransfected U251and U87cells were seeded at a density of6000cells/well, associated with aggressive tumor behavior, such as proliferation, angiogenesis, and metastasis. Therefore, HDGF may prove useful as a prognostic factor for patients with hepatocellular carcinoma, gastric, pancreatic, and breast cancers.Recently, a significant correlation has been reported between HDGF expression and the prognosis of patients with glioma. Increasing evidence suggests that HDGF might be implicated in the malignant phenotype of glioma cells. However, the molecular mechanisms involved in HDGF-associated tumorigenesis and in the aggressive behavior of glioma cells remain unclear.Therefore, to investigate the oncogenic potential of HDGF in stable cell lines, we silenced its expression using lentiviral shRNA. Subsequently, we found that HDGF downregulation significantly suppressed glioma cell proliferation, migration and invasion, enhanced TMZ-induced cytotoxicity and apoptosis in vitro, as well as inhibited tumorigenesis in vivo. Our study revealed that HDGF is involved in the activation of the PI3K/Akt and the TGF-β signaling pathways, suggesting potential molecular mechanisms whereby HDGF regulates tumor growth and emigration.Materials and methodsCell culture and sample collectionCell culture was carried out as described. The human U251and U87glioma cell lines were purchased from the Chinese Academy of Sciences (Shanghai, China) and grown in Dulbecco’s modified Eagle’s medium (DMEM)(Hyclone, Logan, UT) supplemented with10%fetal calf serum (ExCell, Shanghai, China). All cell lines were cultured at37℃in a humidified atmosphere with5%CO2. A total of117paraffin-embedded glioma and18normal brain (NB) tissue samples were obtained from the Nanfang Hospital of Southern Medical University, Guangzhou, China. These cases were from79males and38females with ages between10and71years (median,43.3years). Prior consent from patients and approval from the Ethics Committees of Nanfang Hospital was obtained for using these clinical materials for research purposes. All specimens had confirmed pathological diagnosis and were classified according to the World Health Organization (WHO) criteria.Establishment of a Glioma Cell Line with Stable Expression of HDGF Short Hairpin RNAThe preparation of lentiviruses expressing human HDGF short hairpin RNA was performed using the pLVTHM-GFP lentiviral RNAi expression system. U87and U251cells were infected with lentiviral particles containing specific or negative control vectors, and polyclonal cells with GFP signals were selected for further experiments using FACS flow cytometry. Total RNA from these clones was isolated, and HDGF mRNA levels were measured using real-time PCR analysis.EdU incorporation assayU251and U87cells were cultured in96-well plates at5000cells per well, transfected with siHDGF or empty vector (si-Ctr) for48h, and then exposed to50μmol/L5-ethynyl-20-deoxyuridine (EdU, Ribobio, China) for an additional2h at37℃. The cells were fixed with4%formaldehyde for15min and treated with0.5%Triton X-100for20min at room temperature. After washing three times with phosphate buffered saline, the cells from each well were exposed to100μL1X Apollo reaction cocktail for30min. Subsequently, the DNA from cells in each well was stained with100μL of Hoechst33342(5μg/mL) for30min and visualized under a fluorescent microscope.TMZ chemosensitivity test and IC50definitionTransfected U251and U87cells were seeded at a density of6000cells/well, and after24h the cells were treated with25μM,50μM,100μM,200μM, and400μM TMZ (Merck product) for48h. MTT assays were then performed to determine the fraction of cells surviving after TMZ exposure. The resistance of the induced cells was measured by IC50. The TMZ concentration causing a50%inhibition in glioma cell activity was defined as IC50.Statistical analysisAll quantified data represented an average of at least three samples. SPSS13.0and Graph Pad Prism4.0software were used for statistical analysis. Data are presented as mean±SD. One-way ANOVA or two-tailed Student’s t-test were used for comparisons between groups. The Chi-square test or Fischer’s test were used to identify differences between categorical variables. Survival analysis was performed using the Kaplan-Meier method. The multivariate Cox proportional hazards method was used to analyze the relationship between variables and the patients’ survival. Differences were considered statistically significant for P<0.05.ResultsHDGF expression in glioma and NB cellsIn order to assess the role of HDGF in glioma, we performed real-time PCR to measure the expression of HDGF mRNA transcripts in45freshly collected glioma tissues and15freshly collected NB tissues. Compared with the NB tissues, glioma tissues exhibited higher HDGF mRNA expression levels (P<0.0001). Furthermore, HDGF protein expression was investigated by Western blotting and found to be upregulated in3cases of glioma, as compared to3NB tissues (P<0.0001). We also measured the expression levels and the subcellular localization of the HDGF protein in117archived paraffin-embedded glioma samples and18NB tissues using immunohistochemical staining. HDGF was highly expressed in70.9%(83/117) of the glioma samples but in only in27.8%(5/18) of the NB samples, a difference that was statistically significant (P<0.001).The relationship between clinicopathological characteristics and HDGF expression levels in individuals with glioma is summarized in Table S2. We found no significant associations between HDGF expression and patients’age, sex, or tumor histological type in the117glioma cases. However, we observed that, in glioma patients, HDGF expression levels were positively correlated with the status of the pathological classification (WHO Ⅰ-Ⅱ vs. WHO Ⅲ-Ⅳ)(P=0.000).HDGF downregulation suppresses cell proliferation, colony formation, and in vivo tumorigenicityWe used a lentiviral shRNA vector to specifically and stably knock down HDGF expression in the U87and U251cell lines that were established from high-grade tumors. HDGF transcription levels were assessed by RT-PCR, and the most efficient knockdowns were compared to the empty vector controls [pLVTHM-GFP-Control (PLV-Ctr)](P<0.01). Consistent results for protein levels were observed by Western blotting. We also detected the interference effect of small interfering RNA (siRNA) used to knock down endogenous HDGF expression, as compared to the negative control (si-Ctr) groups.Subsequently, we examined the effect of decreased HDGF expression on glioma cell growth in vitro. Using the MTT assay, we found that growth of the shHDGF U251and U87cells was significantly slower than that of PLV-Ctr cells (P<0.05). Interestingly, similar results were observed for the siRNA-mediated suppression of HDGF in glioma cells. The EdU incorporation assay revealed that the percentage of cells in S phase decreased after HDGF expression was downregulated. The colony formation assay further showed that suppressing HDGF significantly decreased the degree of cell proliferation compared to PLV-Ctr cells. To confirm the growth-enhancing effects of HDGF, we performed an in vivo tumorigenesis study by inoculating shHDGF U251cells into nude mice. Mice from the shHDGF-U251and PLV-Ctr groups were sacrificed18days after inoculation, and their average tumor weights were0.258g and0.726g, respectively (P<0.001). These results suggested a significant in vivo inhibitory effect of decreased HDGF on tumorigenesis.HDGF knockdown suppresses glioma, cell migration, and invasion in vitroTo determine the function of HDGF in glioma cell migration and invasion, the transwell chamber, scratch assay, and Boyden chamber assays were performed using the U87and U251cells. After incubation for24h, the percentage of cells that migrated in both the shHDGF-U251and the shHDGF-U87glioma cell groups was significantly lower than in the PLV-Ctr cells (with P<0.01for both). In addition, shHDGF transfection into U251and U87cells impaired the migration capacity (P<0.01) when compared to the pLV-Ctr cells, as evidenced by the scratch migration assay. As shown in Figure3C, decreased HDGF expression led to a significant decrease in the invasion activity of U251and U87cells in the transwell assay. Similar to the results obtained after stably suppressing HDGF expression, HDGF downregulation using siRNA-HDGF also inhibited cell migration and invasion in U251and U87cells.HDGF controls the expression of cell cycle-and EMT-associated genes via the The PI3K/Akt and TGF-β signaling pathways in gliomaTo further study the mechanism by which HDGF regulates cell proliferation, migration, and invasion, we examined protein levels of cell cycle-and EMT-associated genes in U251and U87glioma cells with stably suppressed HDGF. Knocking down endogenous HDGF not only inhibited the activation of oncogenic cell cycle regulators, including TGF-β, CCND1, and c-myc, but also depressed the expression of the p16and p21tumor suppressors. However, p16and p21protein levels were decreased in siTGF-β-treated U251and U87cells compared to the negative control groups. Furthermore, we found that suppressing HDGF expression decreased the expression of β-catenin, vimentin, and N-cadherin, while increasing E-cadherin expression.PI3K/Akt has been reported as an upstream signaling pathway that modulates cell cycle and EMT signals. We found that reduced HDGF significantly decreased the expression of phosphorylated PI3K and Akt. These results suggested that HDGF is an upstream factor modulating the PI3K/Akt and TGF-β pathways in glioma.HDGF downregulation enhances TMZ-induced cytotoxicity and apoptosis in U251and U87cellsTo determine whether HDGF inhibition sensitizes U251and U87cells to TMZ-induced cell death, dose-response curves were examined for TMZ and in combination with shHDGF treatment, and compared with the PLV-Ctr groups (Figure5A). The results revealed that HDGF knockdown decreases proliferation and increases U251and U87cell sensitivity to TMZ. Figure5B shows that the TMZ concentration causing IC50in U251cells decreased from487to202μM after shHDGF treatment (P<0.01). Similarly, treatment with shHDGF reduced the IC50to TMZ in U87cells from808to406μM (P<0.01).DiscussionHDGF is a mitogenic growth factor in glioma progression and can be a useful prognostic marker for GBM. Increasing evidence suggests that HDGF might be implicated in the malignant phenotype of glioma cells. However, the molecular mechanism involved in HDGF-associated glioma cell proliferation, invasiveness, and metastasis remains elusive.In the present study, we first confirmed that HDGF mRNA levels were higher in45glioma samples than in15NB tissue samples. Consistent with the mRNA levels, HDGF protein expression was significantly elevated in3glioma samples compared to3NB tissue samples. In agreement with a previous study, we found that HDGF was mainly localized to the nucleus of glioma cells, while being weakly expressed in the cytoplasm and the nucleus of NB cells. We also observed that HDGF expression was positively correlated with the tumor grade, as shown by a significant difference between high-and low-grade gliomas. Consistent with a previous study, our results showed that HDGF knockdown significantly inhibited the malignant phenotype not only in U87cells, but also in U251cells, including the in vitro cell proliferation, migration, and invasion, as well as in vivo tumorigenesis.In summary, we provide compelling evidence that HDGF downregulation significantly suppresses glioma cell proliferation, migration, invasion in vitro and tumorigenesis in vivo is involved in the activation of both the PI3K/Akt and the TGF-P signaling pathways. Meanwhile, the PI3K/Akt pathway also activates caspase-3, which contributes to TMZ-induced cytotoxicity and apoptosis after HDGF downregulation. The results of the present study indicate that HDGF can serve as a potential target for anticancer drug design.