Significance of Amphiregulin (AREG) for the Outcome of Low and High Grade Astrocytoma Patients

Abstract

Background: Amphiregulin (AREG) is one of the ligands of the epidermal growth factor receptor which levels was shown to have a tight coherence with various types of cancer. AREG was also designated to be a promising marker for several types of cancer however precious little data about AREG role in the most frequent and generally lethal human brain tumours - astrocytomas reported up to date. The aim of the study was to investigate how AREG changes at epigenetic and expression levels reflect on astrocytoma malignancy and patient outcome. Methods: In total 205 low and high grade astrocytoma samples (15 pilocytic astrocytomas, 56 diffuse astrocytomas, 32 anaplastic astrocytomas and 102 glioblastomas) were used for target mRNA, protein expression and DNA methylation analysis applying qRT-PCR, Western-Blot and MS-PCR assays, respectively. Results: Present research revealed that AREG expression level and methylation in cancer tissue is dependent on the grade of astrocytoma. GBM tissue disclosed elevated AREG mRNA expression but reduced AREG protein level as compared to grade II and grade III astrocytomas (p<0.001). Increased methylation frequency was also more abundant in GBM (74%) than grade I, II and III astrocytomas (25%, 34%, and 36%, respectively). The survival analysis revealed relevant differences in patient overall survival between AREG methylation, mRNA and protein expression groups. Kaplan-Meier analysis encompassing only malignant tumours showed similar results indicating that AREG is associated with astrocytoma patient survival independently from astrocytoma grade. Conclusions: Current findings demonstrate that AREG appearance is associated with patient survival as well as astrocytomas malignancy indicating its influence on tumour progression and suggest its applicability as a promising marker.

Keywords: amphiregulin; astrocytoma; expression; glioma; methylation.

Figure 1

Tukey Boxplot of AREG mRNA expression in WHO grade I-IV astrocytomas. (A) Boxplot with scatter dot of AREG mRNA expression normalized to TBP-ACTB. Glioblastoma tissue showed significantly upregulated AREG level compared to grade II astrocytomas. AI-AIII - WHO grade I-III astrocytoma, GBM - glioblastoma.

Figure 2

Tukey Boxplot of AREG protein level in in WHO grade I-IV astrocytomas. (A) AREG protein expression calculated including signals intensity of all detected isoforms. GBM tissue showed significantly downregulated AREG level compared to grade II and grade III astrocytomas. (B) AREG protein expression calculated including signal intensity of only 1st (heaviest) isoform (~50 kDa) showed similar associations as combined intensity calculations. (C) AREG protein level measured according to signal intensity of only 2nd (middle) isoform (~45 kDa) showed downregulated AREG in glioblastoma compared grade II astrocytoma. (D) AREG protein expression quantified including signal intensity of only 3rd (lightest) isoform (~42 kDa) showed significantly lower AREG levels in glioblastoma as compared to grade II astrocytoma tissue. Asterisk: *p<0.05; **p<0.01; ***p<0.001; AI-AIII - WHO grade I-III astrocytoma, GBM - glioblastoma.

Figure 3

Representative results of AREG protein Western blot analysis in WHO grade I-IV astrocytomas. Astrocytomas of WHO grade II, III, and IV (glioblastoma) are indicated as AII, AIII, and GBM, respectively. PL - protein ladder (Spectra™ Multicolor Broad Range Protein Ladder, Thermo Fisher Scientific Inc.). Three isoforms (~50; ~45; 42 kDa) of AREG protein were detected which are described in the literature .

Figure 4

AREG promoter methylation in WHO grades I-IV astrocytomas. (A) Methylation frequency (%) showed in darkened bars for each of astrocytoma grade. Data obtained using 1st set of primers (n=132). (B) Data obtained using 2nd set of primers (n=94). (C) Representative MS-PCR for AREG. Meth -„Standard Bisulfite Converted Universal Methylated Human DNA“ as positive methylation control, Unmeth - normal human peripheral lymphocyte DNA as negative methylation control, H2O - water control. AI, AII, AIII, GBM - astrocytoma grade I-IV samples. M - primer for methylated and U - primer for unmethylated DNA.

Figure 5

Kaplan-Meier curves representing patient survival and AREG mRNA and protein expression. (A) Kaplan-Meier curves of AREG mRNA data divided into two expression groups according to 50th percentile, Log-rank test, χ2=13.03, df=1, p<0.001. (B) Kaplan-Meier survival curves using mRNA data only from malignant (WHO grade III and grade IV) astrocytomas, Log-rank test, χ2=7.4, df=1, p=0.007. (C) Kaplan-Meier curves of AREG protein expression groups according to 50th percentile. All dataset used for the analysis. Log-rank test, χ2=10.52, df=1, p=0.001. (D) Kaplan-Meier curves of AREG protein expression groups. Only malignant astrocytomas (grade III and IV) used for the estimation. Log-rank test, χ2=4.26, df=1, p=0.039.

Figure 6

Kaplan-Meier curves representing AREG promoter methylation and patient survival. (A) Kaplan-Meier curves of AREG promoter methylation groups (using 1^st primer set), Log-rank test, N=132, χ2=18.61, df=1, p<0.001. (B) Kaplan-Meier curves of AREG promoter methylation groups in only malignant (WHO grade II-III) astrocytomas (using 1^st primer set), Log-rank test N=132, χ2=11.24, df=1, p=0.001. (C). Kaplan-Meier curves of AREG promoter methylation groups (using 2^nd primer set), Log-rank test N=94, χ2=5.88, df=1, p=0.015. (D) Kaplan-Meier curves of AREG promoter methylation groups in only malignant (grade II-III) astrocytomas (using 2^nd primer set), Log-rank test N=94, χ2=3.37, df=1, p=0.066.