The Role And Mechanism Of Gab2in Invasion Of Glioma

Glioma, the most common malignant primary tumor in the central nervous system, accounts for about40%of all intracranial tumors. Glioma is characterized by high invasiveness and poor prognosis. As high invasiveness leads to high recurrence rates and high mortality, a better understanding of the molecular mechanism mediating the invasion of glioma is imperative to the development of effective therapy that prevents the invasion of glioma cells and to improve the prognosis of glioma patients.Gab2is a member of the DOS/Gab family of scaffolding adapters that include mammalian Gab1and Gab3. Gab2contains an N-terminal pleckstrin homology domain and C-terminal portion with multiple tyrosine phosphorylation sites and proline rich motifs. Gab proteins are recruited to activated receptors mostly indirectly by Grb2. On stimulation, Gab2undergoes tyrosine phosphorylation, creating a number of docking sites to mediate interactions with SH2domain-containing proteins such as the p85subunit of PI3K. The interaction of Gab2with the p85subunit of PI3K is crucial in mediating the PI3K-Akt-mTOR signaling.Recently, a series of studies have shown that Gab2is involved in tumor cells migration and invasion. Firstly, high expression of Gab2was detected in human breast cancer cell lines and primary tumors. In the Her2/Neu-induced Gab2knockout mouse model, Gab2-deficient cells exhibit decreased migration and mice show reduced lung metastasis, suggesting its role in promoting mammary tumor metastasis. Secondly, some researchers found over-expression of Gab2in ovarian tumors and that Gab2can regulate the migratory behaviors by activating the PI3K pathway in ovarian cancer cells. Moreover, Gab2is also over-expressed in metastatic melanoma, gastric cancer, lung cancer and acute myeloid leukemia. However, till now, it is not known whether the Gab2protein is also over-expressed in gliomas and whether Gab2has any role in the migration and invasion of glioma cells.In this study, for the first time, we measured the expression of Gab2in glioma to explore its role in the progression of glioma. Our data suggest that Gab2plays a role in glioma invasion and could be a molecular marker of prognostic value for glioma.Methods1. Patients and tissue specimensThis study was conducted on a total of163paraffin-embedded glioma specimens, which were histopathologically diagnosed at the Affiliated Hospital of Weifang Medical University and the Weifang People’s Hospital from1997to2007, including23cases of grade Ⅰ (pilocytic astrocytoma)(14.1%),48cases of grade Ⅱ (29.5%),52cases of grade Ⅲ (31.9%) and40cases of grade Ⅳ gliomas (24.5%) by immunohistochemistry. For the use of these clinical materials for research purposes, prior patient’s consents and approval from the Institutional Research Ethics Committee were obtained.2. ImmunohistochemistryImmunohistochemistry was performed as described bySanta Cruz. Briefly,4um slides were deparaffinized in xylene and transferred through two changes of100%ethanol. For antigen retrieval, the slides were boiled in a pressure cooker at maximum heat for2minutes containing0.01mol/L sodium citrate. Endogenous peroxidase activity was blocked in0.3%H2O2. Then slides were incubated with the primary antibody overnight at4℃. After washing with PBS, the bound primary antibody was detected by Biotinylated Goat Anti-Rabbit IgG (H+L) and DAB. The specimens were counterstained with hematoxylin, mounted, and examined by light microscopy.3. Immunohistochemical EvaluationThe degree of immunostaining of the sections was viewed and scored separately by two independent investigators, who were blinded to the histopathologic features and patient data of the samples. The results were evaluated by an immunohisto-chemical score (IHS). Theoretically, the scores could range from0to12. An HIS score of9-12was considered strong immunoreactivity,5-8was considered moderate,1-4was considered weak, and0was scored as negative.0-4were regarded as low expression,5-12were regarded as high expression.4. Western blotting assayFor Western blot, cells or tissues were directly lysed in1×SDS sample buffer. Protein samples were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, immunoblotted using the appropriate primary and the HRP conjugated secondary antibodies, and were visualized by using enhanced chemiluminescence reagents ECL. The intensities of bands in Western blots were quantified by densitometry analysis Western blot data shown in the paper are representatives from three independent experiments. using AlphaImager HP (Alpha Innotech, USA) and NIH Image J software (Rockville, MD, USA).5. Cell culture and and plasmid transfectionHuman glioblastoma U87, LN229and U251cells were cultured in RPMI1640supplement with10%fetal bovine serum. The transfection was performed with Lipofectamine2000according to the manufacturer’s instructions. For transient transfections, two Stealth siRNAs against human Gab2 (#1:5’-GTGAGAACGATGAGAAATA-3’;#2:5’-GATGCAGGCCTGACCTTTA-3’) and a scrambled siRNA were synthesized by Invitrogen. RT-PCR and Western-blot was used to examine the transfection efficiency. Stable transfectants were selected by using700μg/mL neomycin.6. PR-PCRThe total RNA from cells was extracted by Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA was reverse-transcribed to cDNA using the One Step RNA PCR Kit (AMV). This cDNA chain was used as a template on the gradient PCR amplification of objective product. Took amplification products5ul on2%agarose gel for electrophoresis, gel image was analyzed on the photo analysis system, various grey value was analyzed. β-actin was used as loading control, each cell line was repeated3times.7. Wound healing/scratch assayAll cells were seeded in six-well plates and grown until100%confluence. After making a straight scratch by using a10μl pipette tip, cells were incubated in a minimum medium (containing0.1%BSA) in a37%humidified incubator at various time points and the wound distances were measured under a light microscope.8. Chemotaxis assayThe chemo-attractant (IGF-1) was loaded into the lower chemotaxis chamber and5×105cells/mL cells suspended in the binding medium were added to the upper chambers. The polycarbonate filter was pretreated with10ug/mL fibronectin overnight and inserted between the upper and lower chambers. Then, the chamber was incubated at37℃in5%CO2for3h. The filter membrane was then rinsed, fixed, and stained. The numbers of migrating cells were counted at200×in three separate fields by light microscope.9. Cellular F-actin measurement The control cells and the Gab2deficient cells were followed by the stimulation of10ng/mL IGF-1at37℃at different time points. The cells were then fixed, permeablized, and incubated with Oregon Alexa-fluro568phalloidin in F-actin buffer at room temperature for2h. The labeled phalloidin that were bound to F-actin was extracted by using methanol at4℃for90min. The fluorescence was captured at Ex/Em578/600run in each sample (Hitachi, F-7000) and normalized against the total protein content as analyzed by a BCA kit (Pierce, Rockford, IL). The relative F-actin content over different time periods was calculated. All samples were tested in triplicate, and the data is expressed as the mean±S.D.10. Matrigel invasion assayA Boyden chamber invasion assay was performed. The cells were suspended in serum-free medium at a final concentration of4×105/mL and incubated at37℃for30min. Then, the cells, in the presence or absence of10ng/mL IGF-1, were promptly added to a35mm dish containing dried fibronectin-coated glass coverslips. After an incubation of24h, the cells were washed gently twice with cold PBS and fixed. The attached cells were counted under a light microscope at200×. All assays were repeated at least three times independently. The differences in the invasion rates between control and Gab2deficient cells were analyzed.11. Enzyme-linked immunosorbent assay (ELISA)ELISA was done to determine the MMP-2and MMP-9expression levels in the culture medium of Scr/U87and siGab2/U87cells. All cells were seeded in six-well plates and grown until60-70%confluence, then the growth medium was removed and replaced with fresh RPMI-1640containing1%fetal bovine serum.10ng/mL IGF-1was added to growth medium to stimulate the expression of MMP-2and MMP-9. The cells were incubated for a further24h until about80%confluence was attained. The medium was then harvested and filtered for the measurement of MMP-2 and MMP-9using the human MMP-2and MMP-9ELISA Kit (R&D Systems, USA) according to manufacturer instructions. The remaining cells were directly lysed in1×SDS sample buffer and protein samples were for MMP-2and MMP-9Western blot assay.12. Intracranial brain tumor xenografts and H&E stainingScr/U87(5×105) or siGab2/U87(5×105) cells were stereotactically implanted into four-week-old male SCID mice brains individually and the morphologies of implanted glioma tumors were examined. The survival time of both groups were observed and5-μm sections of intracranial brain tumor xenografts were cut and subjected to H&E staining. The number of satellite tumors were counted to measure invasiveness in vivo. The images were captured using the light microscope system.13. Statistical analysisAll statistical analyses were carried out using the SPSS17.0statistical software package. Survival curves were compared by the log-rank test. Chi-square test was used for analysis of the relationship between Gab2expression level and clinical pathological features; T test for mean differences between the two groups compared with the independent sample; One-Way ANOVA for quantitative data of three groups or above; LSD test for multiple comparisons if data satisfy the homogeneity of variance assumption, if not satisfied, use Welch Correction; A repeated measures analysis for the repeated measurement data; The two-factor factorial analysis was used for the main effects and interaction effects. a=0.05was considered as test level.Results1. Upregulation of Gab2in primary glioma and glioma cell linesWestern blot analyses on paired glioma tumor and adjacent nontumor tissues showed that the protein levels of Gab2were0.83±0.062and0.37±0.067respectively, expression level of Gab2in glioma samples was obviously higher when compared with each corresponding ANT, t=20.432, P=0.000。And the expression of Gab2remarkably increased as glioma progressed, the high Gab2expression rates of low grade glioma and high grade glioma were36.8%and62.1%respectively, there was significent difference in low and high groups, P=0.001. In addition, analyses on glioma cell lines revealed that the expression of Gab2was also markedly high in U87, LN229and U251cell lines.2. Increased expression of Gab2correlates with progression of gliomasQuantitative analysis indicated that the average mean score of Gab2staining increased as glioma progressed from histologic grade Ⅰ to grade Ⅳ. The expression of Gab2closely correlated with WHO grading (χ2=10.189, P=0.001) and survival status of glioma patients (χ2=8.710, P=0.003). Gab2expression has no significant correlation with gender and age. Log-rank survival analysis showed significant correlation between high Gab2expression and shorter overall survival time in WHO grading subgroups of gliomas.3. Increased expression of Gab2is relevant to increased activation of pAktGab2expression coincides with pAkt expression in glioma samples. ANOVA analysis showed a strong correlation between Gab2and pAkt expression in the tested tissue samples (χ2=24.280, P=0.000). There was significant difference of pAkt expression between low grade glioma (Ⅰ+Ⅱ) and high grade glioma (Ⅲ+Ⅳ), χ2=10.189, P=0.001. These results suggest that upregulation of Gab2is clinically relevant to increased expression of pAkt in human gliomas.4. Identification of stable transfection for human glioma U87, LN229and of U251cellsRT-PCR and western blot showed that the expression of Gab2mRNA and protein in siGab2/U87, siGab2/LN229and siGab2/U251cells are significantly reduced when compared with the control cells Scr/U87, Scr/LN229and Scr/U251respectively, so as to proved that the expression of Gab2is stably inhibited in siGab2/U87, siGab2/LN229and siGab2/U251cells.5. Reducing the expression of Gab2inhibit the migration ability of glioma cellsThe wound healing experiment results showed that the migration distance of control Scr/U87cells and Gab2reduced siGab2/U87cells were278.222±29.269um and530.556±27.758um respectively, there are significant differences, t=18.766, P<0.001; Similarly, the migration distance of Scr/LN229and siGab2/LN229cells were309.556±26.378um and560.667±41.286um respectively, t=15.376, P<0.001; The migration distance of Scr/U251and siGab2/U251cells were409.889±25.487um and593.333±33.982um respectively, t=12.956, P<0.001. These results indicated that Gab2reduced cells took a longer time to fill the gap than control cells, which further supports the role of Gab2in directional migration.6. Reduction of Gab2by siRNA inhibits chemotaxis of glioma cellsUsing chemotaxis chamber, glioma cells were induced by different concentrations of IGF-1for3h and the optimal IGF-1concentration of10ng/mL was determined. When induced by10ng/mL IGF-1, Scr/U87, Scr/LN229and Scr/U251cells have strong chemotactic movement ability and more visible cells migrate through the membrane filters. But siGab2/U87, siGab2/LN229and siGab2/U251cells showed decreased chemotaxis, there are significant differences, t=8.147, P=0.001; t=4.448, P=0.011;t=3.517, P=0.025respectively. This result indicated that reduced expression of Gab2decreaseed chemotaxis of glioma cells.7. Knockdown of Gab2impaired glioma cells invasionIGF-1at lOng/mL was used as a chemoattractant to stimulate the cells to penetrate through the matrigel and to migrate through the filters. The number of cells that migrated through the matrix and the membrane filter for Gab2reduced cells siGab2/U87, siGab2/LN229and siGab2/U251were20.750±4.113,27.500±6.455and25.000±7.165respectively; But for control cell groups Scr/U87, Scr/LN229and Scr/U251, the number of cells that migrated through the matrix and the membrane filter were40.750±6.850,45.500±7.853and43.250±4.992respectively.Compared with control cells, siGab2/U87cells showed dramatically reduced invasive ability, there were significant differences, t=5.007, P=0.002. In addition, we found that the invasiveness of U251and LN229glioma cells was also greatly impaired after reduction of Gab2, t=3.541, P=0.012; t=4.180, P=0.006respectively. This result indicated that reduced expression of Gab2decreaseed invasion ability of glioma cells.8. Reduction of Gab2impaired the IGF-1-induced F-actin polymerization in U87cellsQuantitive F-actin polymerization assay showed that IGF-1elicited a transient actin polymerization at15s and60s in Scr/U87cells while in siGab2/U87cells F-actin polymerization was significantly inhibited. F-actin polymerization in Scr/U87cells was higher than that in siGab2/U87cells, there was significant difference between Scr/U87and siGab2/U87cells, F=29.819, P<.001. The results indicate that Gab2plays an important role in cytoskeleton reorganization.9. Gab2regulates the phosphorylation of cofilinWestern blot was used to test IGF-1(10ng/mL) induced phosphorylation of cofilin and LIMK1/2in Scr/U87and siGab2/U87cells. Ins iGab2/U87cells, there was a marked decrease activation of both cofilin and LIMK1/2, whereas both total cofilin and LIMK1levels remained unchanged. IGF-1-induced relative phosphorylation levels of cofilin and LIMK1/2in Scr/U87and siGab2/U87cells showed significant difference,F=11.781, P=0.005; F=24.206, P<0.001respectively. Taken together, our results indicate that Gab2regulates IGF-1-induced glioma cell chemotaxis, probably by mediating cell actin polymerization.10. Reduction of Gab2reduce the expression of MMP-2and MMP-9of glioma cellsELISA assay was used to determine the dissociated contents of MMP-2and MMP-9in cell culture medium. Results showed that IGF-1(10ng/mL) induced secretion of MMP-2and MMP-9obviously increased in Scr/U87cells, MMP-2and MMP-9contents were3.36±0.38and4.09±0.19respectively. Conversly, there was no obvious MMP-2and MMP-9increase in siGab/U87cells induced by IGF-1(10ng/mL), MMP-2and MMP-9contents were1.14±0.09and1.05±0.11respectively.There were statistical significance when compared with Scr/U87cells with IGF-1stimulation, F=72.853, P<0.001; F=321.272, P<0.001. Consistent with the ELISA data, Western blot results also showed that IGF-1(lOng/mL) induced secretion of MMP-2and MMP-9of siGab2/U87cells was inhibited. IGF-1(10ng/mL) induced relative MMP-2and MMP-9expression in Scr/U87cells were1.62±0.25and0.95±0.13respectively while in siGab2/U87cells were0.69±0.12和0.27±0.09respectively. There were statistical significance between Scr/U87and siGab2/U87cells, F=39.891, P<0.001; F=30.648, P=0.001.11. Reduction of Gab2decreased phosphorylation of AktCompared with Scr/U87cells, IGF-1-induced phosphorylation of pAkt was significantly decreased in siGab2/U87cells, there was statistical significance between Scr/U87and siGab2/U87cells, F=72.100, P<0.001. The result was consistent with the immunohistochemical staining of Gab2and pAkt in glioma tissues.12. Reduction of Gab2decreased phosphorylation of mTORCompared with Scr/U87cells, IGF-1-induced phosphorylation of pmTOR was significantly decreased in siGab2/U87cells, there was statistical significance between Scr/U87and siGab2/U87cells, F=139.792, P<0.001. The result was consistent with the phosphorylation of Akt and the immunohistochemical staining of Gab2and pAkt in glioma tissues. These data indicate that Gab2-mediated signaling probably results in Akt activation and promotes invasion in glioma cells via Akt-mTOR pathway.13. Survival time of tumor-burdened nude miceThe median survival time of Scr/U87and siGab2/U87nude mice is22.5days and26.5days respectively, siGab2/U87group with low Gab2expression had a longer survival time than Scr/U87group, there was statistical significance between Scr/U87and siGab2/U87groups,χ2=11.47, P=0.001.14. Reduction of Gab2decreased glioma cells invasion in vivoSCID mice had a considerable high number of satellite tumors (mean=27.00±4.04) infiltrating into surrounding normal tissue in the brains implanted with Scr/U87. In contrast, in the brains implanted with siGab2/U87cells, most tumor cells were limited in the injection site and less invasive satellite intracranial tumors(mean=12.13±2.75) in the normal brain tissue was detected. The siGab2/U87group had a much less number of satellite tumors and there was statistical significance between Scr/U87and siGab2/U87groups,t=8.617, P<0.001. This result indicated that reduction of Gab2inhibited cell invasion in vivo.Conclusion1. Gab2is over expressed in both primary glioma tissues and glioma cell lines, and there is a significant correlation between the expressions of Gab2and the WHO grade and prognosisof glioma patients.2. The expressions of pAkt has a significant correlation between the expressions of Gab2and the WHO grade of glioma patients.3. Reduction of Gab2inhibits migration, chemotaxis and invasive ability of glioma cells. 4. By regulating F-actin polymerization, Gab2plays important roles in cytoskeleton reorganization.5. By regulating cofilin and LIMK1/2phosphorylation, Gab2affects F-actin polymerization and chemotaxis of glioma cells.6. Reduction of Gab2led to a drastic decreased expression of MMP-2and MMP-9and then finally a decrease in the invasive ability.7. Gab2mediated phosphorylation of both Akt and mTOR suggests that one possible molecular mechanism of Gab2participating in the invasion of glioma is to regulate the PI3K/Akt/mTOR signaling pathway.8. Downregulation of Gab2through siRNA inhibited invasive ability of glioma cells into the brain of SCID mice, slowed the malignancy progression and prolonged the survival time.