Testin (TES) as a candidate tumour suppressor and prognostic marker in human astrocytoma

Abstract

Astrocytomas are one of the most common brain tumours; however, the current methods used to characterize these tumours are inadequate. The establishment of molecular markers may identify variables required to improve tumour characterization and subtyping, and may aid to specify targets for improved treatment with essential prognostic value for patient survival. One such candidate is testin (TES), which was reported to have prognostic value for glioblastoma patients. However, the role of TES protein in gliomagenesis is currently unknown. In the present study, the methylation status of the TES promoter was investigated in post-operative astrocytoma tumours of different malignancy grade, and its association with the survival of astrocytoma patients was evaluated. In addition, the expression of TES protein was investigated in the same set of astrocytoma tumours tissue, and the association of protein expression with glioma patients survival was evaluated. The methylation status of TES was assessed by methylation-specific polymerase chain reaction in 138 different grade astrocytoma samples. Western blot analysis was used to characterize the expression pattern of TES in 86 different grade astrocytoma specimens: 13 of pathological grade I, 31 of pathological grade II, 17 of pathological grade III and 25 of pathological grade IV (glioblastoma). Statistical analyses were conducted to investigate the association between tumour molecular pattern, patient clinical variables and overall survival. The methylation analysis of the TES promoter exhibited a distinct profile between astrocytomas of different malignancy grade (P<0.001). Furthermore, gene promoter methylation was significantly associated with patients' age, survival and pathological grade (P<0.001). The protein expression level of TES was significantly lower in glioblastoma (grade IV astrocytoma) than in lower grade (II-III) astrocytoma tissue (P=0.028 and P=0.04, respectively). Additionally, short overall survival of patients was markedly associated with low TES protein expression (P=0.007). However, no association between TES methylation and TES protein expression was noticed. The present study demonstrated that decreased expression of TES may be important in tumour progression and prognosis in human astrocytomas. TES may be a useful marker for predicting the clinical outcome of astrocytoma patients.

Keywords: DNA methylation; astrocytoma; expression; glioblastoma; glioma; survival; testin; testin LIM domain protein; testis-derived transcript.

Figure 1.

TES promoter methylation analysis in astrocytomas. (A) Frequency of methylated TES gene promoter in different malignancy grade astrocytomas. (B) Kaplan-Meier estimation of overall survival of astrocytoma patients (all grades) according to the methylation status of the TES gene promoter (log-rank test, χ2=17.864, degrees of freedom =1, P<0.001). TES, testin; WHO, World Health Organization.

Figure 2.

Analysis of TES protein expression in astrocytomas. (A) Relative TES protein expression in different malignancy grade astrocytomas. Box plots of relative expression measurements of TES obtained by western blot analysis of astrocytoma samples. Pathological grades I, II, III and IV are indicated as AI, AII, AIII and GBM, respectively. The line inside each box represents the median; the lower and upper edges of the boxes represent the 25th (1st quartile) and 75th (3rd quartile) percentiles, respectively; and the upper and lower edges of the boxes represent the Tukey's whiskers. The plus (+) symbols represent the outliers (values greater than 1.5 interquartile ranges below the 1st or above the 3rd quartile). (B) Representative western blot results of TES protein expression in astrocytoma. The highest molecular weight band represents the TES protein (~48 kDa), while the other bands observed below may be possible degradation products of TES. In addition, the ~42 kDa band that is steadily visible in all samples could be a plausible isoform of TES. GADPH in this example was re-probed on the same membrane, while β-actin was detected on other membrane with the same set and arrangement of samples. (C) Kaplan-Meier survival curves of astrocytoma patients stratified by TES protein expression groups (high, medium and low) (log-rank test, χ2=6.285, degrees of freedom =2, P=0.043). TES, testin; GBM, glioblastoma; WHO, World Health Organization; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3.

Distributions of relative TES protein expression in different astrocytoma grade and methylation groups. The middle line in each group represents the median, while and the upper and lower lines represent the upper and lower limits of the interquartile range, respectively. No significant association between TES promoter methylation status and TES protein expression was observed. TES, testin; M, methylated; U, unmethylated.