R-RAS2 overexpression in tumors of the human central nervous system

Brain tissue samples were homogenized in 1 ml of lysis buffer (50 mM Tris [pH 8], 150 mM NaCl, 1% NP40 and a protease inhibitor cocktail [Sigma-Aldrich, USA]), incubated on ice for 45 minutes and centrifuged at 11,000 rpm. The protein concentration of the lysate was measured by spectrophotometry, and the proteins were then resolved on 15% SDS-PAGE gels (80 μg/well) and transferred to a nitrocellulose membrane. The membrane was probed overnight at 4°C with a specific rabbit antiserum against R-Ras2 (1:1000) purified with a GST-R-Ras2 fusion protein [12]. The GST-RRAS2 fusion protein was generated by the insertion of a cDNA encoding RRAS2 into the pGEX-4 T3 vector as recommended by the manufacturer (Pharmacia). Antibody binding was detected using a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (GE Healthcare UK), which was visualized by chemiluminescence (BioRad Laboratories, California, USA) after washing 3 times with 1% TBS Tween buffer and by exposure of the membrane to X-ray film.

R-RAS2 expression was assessed in the commercial Brain Tumor Screen (CC17-11-004: n = 200) and Brain Medulloblastoma (CC17-11-002: n = 60) tissue microarrays (Cybrdi). The arrays were equilibrated in 0.3 M NaCl, 0.1 M Tris–HCl [pH 7.4] containing 1% (v/v) newborn calf serum and then probed at 4°C overnight with the specific anti-α-R-RAS2 antibody (1:100). After washing thoroughly, antibody binding was detected with a biotinylated donkey anti-rabbit IgG (1 h) and then with a streptavidin-biotinylated horseradish peroxidase complex (1 h). The arrays were again washed and they were then incubated for a further 6 minutes in 0.1 M sodium phosphate in the presence of H₂O₂ (0.1 mg/ml) and diaminobenzidine (0.5 mg/ml), producing a colorimetric reaction that was stopped by adding 0.1 M sodium phosphate.

The expression of R-RAS2 in immunohistochemical (IHC) images was evaluated by calculating the integrated density of DAB in four equal and representative regions of interest in each sample using ImageJ 1.44c analysis software [23] The integrated density calculation represents the sum of the values of the pixels in the image or selection, which is equivalent to the product of the area and the mean grayscale value. For RGB images, the mean is calculated by converting each pixel to an 8-bit grayscale using the formula gray  =  (red  +  green  +  blue) ⁄ 3. The average density measurements of the samples were compared with those obtained from the controls and the results were expressed as a percentage of the corresponding control. Each sample was assessed by 5 independent evaluators.

All results are expressed as the mean ± standard deviation (SD). The experimental groups were compared using a two-sample Student’s t-test. To corroborate these statistical differences, we performed an additional ANOVA test, assembling the samples in groups according to tumor type. The Student’s t-test and ANOVA p values are indicated in the figure legends.

Samples exhibiting necrosis or more than 20% contamination with normal tissue were excluded from the analyses (C2, C9, C10, D7, D8, D9, D10, E11, E12, F4, F6 and F7 from Brain Cancer cDNA Array, sample number 178 from CC17-11-004 Array, and samples 13, 14 and 15 from CC17-01-002 Array). The tumors for which the degree of malignancy was not established were also excluded from the study (F8, F9, F10, F11 and F12 from Brain Cancer cDNA Array). Similarly, the samples whose values had a mean deviation ⁺/₋ 2SD were also eliminated from the study (C4, C11, D1, E1 and E12 from Brain Cancer cDNA Array; samples numbers 22, 37, 38, 75, 85, 150 and 152 from CC17-01-002 Array). The remaining samples were all included in the study.

We analyzed the expression of R-RAS2 in 199 human CNS tumor samples from 105 patients, with a wide range of tumor types (Table 1a). The expression of the R-RAS2 protein was assessed by immunohistochemistry using the polyclonal antiserum described previously, the specificity of which was confirmed by immunoblotting total brain homogenates of wild type and RRas2 ^−/− mice (Additional file 1: Figure S1c). R-RAS2 was overexpressed in all cancer types and grades when compared to the 23 normal brain tissue samples taken from patients (Figure 1, Additional file 1: Figure S1a and Additional file 2: Figure S3). Densitometric quantification revealed R-RAS2 protein expression to be between 3- and 7-fold higher in malignant tissue (n = 170) than in normal brain tissue (n = 23), although R-RAS2 overexpression appeared to be inversely correlated with the tumor grade for all the tumor types analyzed. Thus, R-RAS2 was overexpressed approximately 8-fold more in grade III glioblastomas (n = 13) when compared to normal tissue, while only a 4-fold increase in expression was observed in grade IV tumors (n = 15: Figure 1). Likewise, R-RAS2 expression increased 5-fold in grade I-II astrocytomas but only 3-fold in grade III astrocytomas. Finally, R-RAS2 expression increased approximately 7-fold in grade I-II oligodendrogliomas (n = 9) and 4-fold in grade III tumors (n = 4). Interestingly, R-RAS2 protein overexpression was also associated with non-malignant hyperplasia in the brain (Figure 1 and Additional file 2: Figure S3). Together, these results suggest that R-RAS2 is overexpressed in a wide variety of brain tumors, yet more intensely in pre-malignant conditions. Moreover, R-RAS2 overexpression is also more pronounced in lower grade tumors than in high-grade glioblastomas, astrocytomas and oligodendrogliomas (Figure 1, Additional file 1: Figure S1a and Additional file 2: Figure S3).

An analysis of RRAS2 expression in a second array of highly malignant CNS tumors, 57 samples from 19 patients (Table 1b), also revealed R-RAS2 overexpression in grade IV medulloblastomas (n = 18), cerebromas (n = 9), meningioma (n = 11), ependymomas (n = 3) and undifferentiated (n = 15) tumors (Figure 2 and Additional file 1: Figure S1b). Significant differences in R-RAS2 expression were observed depending of the type and origin of the tumor, with the strongest R-RAS2 expression detected in medulloblastomas and the weakest in undifferentiated ependymoma (Figure 2).

RRAS2 overexpression in human CNS tumors was confirmed by RT-qPCR in tissue samples from 32 patients (Table 1c). In different brain tumor types, RRAS2 mRNA expression was 2- to 22-fold higher than in normal tissue (Figure 3a). The highest levels of expression were found in grade II astrocytomas (22-fold increase, n = 2), whereas 4-fold increases were detected in grade III astrocytomas (n = 3), confirming that RRAS2 is more strongly overexpressed in lower-grade tumors. RRAS2 mRNA overexpression was also detected in pre-malignant hyperplasia (a 16-fold increase, n = 1: Figure 3a and Additional file 3: Figure S2). The only exception was a grade I astrocytoma sample in which low RRAS2 mRNA levels were detected (n = 1), although this may have been due to misclassification. even though the number of mRNA samples classified by tumor type was not sufficiently high to generate statistically significant differences, the mRNA expression data supported the protein expression results. However, when the tumors were grouped together in function of their degree of malignancy (grade I, n = 13; grade II, n = 8; and grade III, n = 8) statistically significant differences in expression were observed (Figure 3d).

We analyzed the expression of R-RAS2 in western blots of low grade (n = 5) and high grade (n = 5) malignant astrocytomas (Table 2). The results obtained indicate the existence of R-RAS2 overexpression associated with tumor progression as opposed to the control tissue (n = 3). Furthermore, stronger expression was observed in tumors with a lower degree of malignancy (Figure 4a and b). These data confirm the results obtained previously by immunohistochemistry and RT-qPCR, where R-RAS2 was seen to be expressed more strongly in the lower grade tumors as opposed to the higher grade tumors (p < 0.05; Figure 4b).

RRAS2 gene expression was compared by qPCR with that of the other 2 genes in this R-RAS subfamily (RRAS1 and RRAS3) in the same CNS tumor samples (n = 32). We observed a general increase in RRAS1 mRNA in CNS tumors and a decrease in RRAS3 expression in CNS tumors (Figure 3b, Additional file 3: Figure S2 and Table 3). When we assessed RRAS1, RRAS2 and RRAS3 expression according to tumor type and grade (Figure 3b-d), the expression of RRAS1 and RRAS2, but not that of RRAS3, was significantly stronger in grade I and II tumors than in grade III tumors (p < 0.01). However, while RRAS2 overexpression in grade I tumors was 13-fold higher than in normal tissue, RRAS1 levels only experienced a 5-fold increased (Figure 3b-e).

This study was performed in compliance with all applicable European and Spanish law.