High FREM2 Gene and Protein Expression Are Associated with Favorable Prognosis of IDH-WT Glioblastomas

Abstract

World Health Organization grade IV diffuse gliomas, known as glioblastomas, are the most common malignant brain tumors, and they show poor prognosis. Multimodal treatment of surgery followed by radiation and chemotherapy is not sufficient to increase patient survival, which is 12 to 18 months after diagnosis. Despite extensive research, patient life expectancy has not significantly improved over the last decade. Previously, we identified FREM2 and SPRY1 as genes with differential expression in glioblastoma cell lines compared to nonmalignant astrocytes. In addition, the FREM2 and SPRY1 proteins show specific localization on the surface of glioblastoma cells. In this study, we explored the roles of the FREM2 and SPRY1 genes and their proteins in glioblastoma pathology using human tissue samples. We used proteomic, transcriptomic, and bioinformatics approaches to detect changes at different molecular levels. We demonstrate increased FREM2 protein expression levels in glioblastomas compared to reference samples. At the transcriptomic level, both FREM2 and SPRY1 show increased expression in tissue samples of different glioma grades compared to nonmalignant brain tissue. To broaden our experimental findings, we analyzed The Cancer Genome Atlas glioblastoma patient datasets. We discovered higher FREM2 and SPRY1 gene expression levels in glioblastomas compared to lower grade gliomas and reference samples. In addition, we observed that low FREM2 expression was associated with progression of IDH-mutant low-grade glioma patients. Multivariate analysis showed positive association between FREM2 and favorable prognosis of IDH-wild type glioblastoma. We conclude that FREM2 has an important role in malignant progression of glioblastoma, and we suggest deeper analysis to determine its involvement in glioblastoma pathology.

Keywords: FREM2; SPRY1; TCGA; glioblastoma; malignancy.

Figure 1

Analysis of FREM2 (A,C) and SPRY1 (B,D) protein expression levels using immunoblotting. Glioblastoma samples (GBM: 1, 2, 3, 4, 15, 16, 17, 18, 23, 24, 25, 26), lower grade glioma samples (LGG: 5, 6, 7, 8, 19, 20, 21, 22, 27, 28, 29, 30) and reference brain samples (REF: 9, 10, hippocampus; 11, 12, periventricular zone; 13, 14, subventricular zone) are shown. (A,B) Representative immunoblots of the proteins of interest. (C,D) Quantification of the immunoblotting. Data are means ± standard deviation, and show significantly higher protein expression levels of FREM2 in GBM versus REF (****, p < 0.0001) and GBM versus LGG (***, p = 0.0009). Significant differences were not observed for SPRY1 expression levels among these analyzed samples.

Figure 2

Analysis of FREM2 (A) and SPRY1 (B) protein expression levels using ELISA. Quantification of the ELISA. GBM, glioblastoma; LGG, lower grade glioma; REF, reference brain samples. Data are means ± standard deviation, and show significantly higher ELISA signals for FREM2 in GBM versus REF (****, p < 0.0001) and GBM versus LGG (**, p = 0.0032). For SPRY1, there were significantly higher ELISA signals for GBM versus REF (*, p = 0.0120).

Figure 3

Analysis of FREM2 and SPRY1 protein expression levels using immunohistochemistry. (A) Representative immunohistochemistry samples. Magnification: glioblastoma, oligodendroglioma, reference (nonmalignant brain), 200×; (B) Quantification of the immunohistochemistry, with proportions of positive cells defined as: 0−25%, 1; 25−50%, 2; 50−75%, 3; and 75−100%, 4. Data are means ± standard deviation. Grade IV, primary and recurrent glioblastomas, secondary gliosarcomas and epithelioid gliomas; Grade II, diffuse astrocytomas and oligodendrogliomas. Significantly higher protein expression levels of FREM2 were shown for Grade IV versus Grade II (*, p = 0.0211). Differences were not observed for SPRY1 expression levels among these analyzed samples.

Figure 4

Analysis of FREM2 (A) and SPRY1 (B) mRNA levels using qPCR. GBM, glioblastoma; LGG, lower grade glioma; REF, reference brain samples. Data are means ± standard deviation, whereby both FREM2 and SPRY1 show significantly higher expression in GBM versus LGG (***, p = 0.0001; ****, p < 0.0001; respectively). FREM2 was expressed at significantly lower levels in LGG versus REF (*, p = 0.0137), while SPRY1 showed significantly increased expression in GBM versus REF (**, p = 0.0010).

Figure 5

Analysis of FREM2 and SPRY1 gene expression in different TCGA (The Cancer Genome Atlas) tumor samples using TCGA data, according to TCGA sites of cancers (A) and TCGA brain tumor samples (B). GBM, glioblastoma; LGG, lower grade gliomas; Norm, reference nonmalignant brain samples. FREM2 was expressed at significantly higher levels in GBM versus LGG (p < 2.2 × 10⁻¹⁶) and GBM versus normal (p = 0.003), but without significance in LGG versus normal (p = 0.689). Similarly, for SPRY1, in GBM versus LGG (p < 2.2 × 10⁻¹⁶) and GBM versus normal (p = 0.0004), and without significance in LGG versus normal (p < 0.931).

Figure 6

Analysis of FREM2 (A,B) and SPRY1 (C,D) for TGCA gene expression and temozolomide treatment responses in low grade glioma samples of different IDH mutation states, according to progressive disease and stabilization or response. (A) FREM2 expression in IDH mutation positive low-grade glioma samples. FREM2 gene expression levels were significantly lower in IDH-mutant gliomas that progressed after temozolomide treatment (p = 0.052); (B) FREM2 expression in IDH mutation negative (IDH-wild type [WT]) low grade glioma samples. No significant differences in gene expression were seen; (C) SPRY1 expression in IDH mutation positive low-grade glioma samples. SPRY1 gene expression levels were significantly lower in IDH-mutant positive gliomas that progressed after temozolomide treatment (p = 0.025); (D) SPRY1 expression in IDH mutation negative (IDH-WT) low grade glioma samples. No significant differences in gene expression were seen.

Figure 7

Multivariate survival analysis of overall survival probabilities with respect to FREM2 and SPRY1 gene expression, age, sex, and histological type (disease) in TCGA patients with different IDH mutation status. Exponential coefficients: according to Cox proportional-hazards models, these are scores that indicate possible impact of a given covariate (e.g., FREM2 expression, age, sex) on overall survival. An exponential coefficient >1.0 indicates that the given covariate probably decreases patient survival, whereas coefficients <1.0 indicates increased patient overall survival when this covariate in high. Confidence intervals: 95% for exponential coefficients. Generally, if a confidence interval does not contain the point 1.0, the value of the exponential coefficient is considered significant. P, Cox proportional-hazards p-value for given covariate. (A) Glioblastoma, IDH-WT, 105 patients; (B) Low grade glioma, IDH-WT, 35 patients; (C) Low grade glioma, IDH mutation positive, 63 patients.