Association between DNA methylation of HSPB1 and death in low Gleason score prostate cancer

Abstract

Background: Heat shock protein 27 (Hsp-27) encoded by gene HSPB1 is a critical regulator of the behavioral phenotype of human prostate cancer (PCa) cells, enhanced expression being associated with highly aggressive disease and poor clinical outcome. In contrast, the protein is not expressed in PCas of low malignant potential. To gain insight into the mechanism regulating its expression, we tested the hypothesis that differential methylation of CpG islands within HSPB1 controls transcription and subsequent translation of the gene.

Methods: We studied prostate epithelial cell lines and tissue biopsies, including 59 BPH and 415 PCas, of which 367 were a cohort of men with up to 20 years of follow-up. Methylation across the gene (DNA methylation (DNAme)) was assayed by pyrosequencing. Hsp-27 expression was assessed by western blot and immunohistochemistry.

Results: In cancer tissues, methylation increased in a 3' direction (P < 0.0001) whereas in benign hyperplasia methylation was constantly below 5%, a cutoff giving a specificity of 100% and sensitivity of 50%. Although methylation of the promoter region was significantly discriminating between benign and malignant prostatic epithelia, it compared poorly with methylation of the first intron. The prognostic value of HSPB1 DNAme was confirmed by both univariate (hazard ratio 1.77 per 50% increment, P = 0.02) and multivariate models. Interaction between HSPB1 methylation and Gleason score revealed high DNAme to be a reliable prognostic marker of poor outcome in men with low Gleason score (P = 0.014).

Conclusions: Our data indicate CpG methylation of the first HSPB1 intron to be an important biomarker that identifies aggressive PCas otherwise regarded as low risk by current clinical criteria but that, biologically, require immediate active management.

Figures

Figure 1

Schematic overview of (a) the HSPB1 gene, (b) the CpG islands and the investigated sites and (c) the measurement of six methylation assays. (a) Transcription factor-binding sites (TFBS) and TATA box are located within the 200-bp region upstream of the transcriptional start site at position 156 (arrow). The HSPB1 gene, 1740-bp, and 500-bp upstream of the gene were searched with Methprimer, (b) identifying 5 CpG islands (gray) located in (a): the first in the promoter region (black solid line), a second covering exon 1 (striped box), a third and fourth within intron 1 (dotted line) and a fifth in exon 3. (b) CpG positions investigated by assay 1–6 are circled in the CpG map. (c) Median of methylation in fresh frozen 10 BPH (circle) and 27 prostate cancer (PCa) (square) show the increasing separation between BPH and PCa going in 3' direction, differences between BPH and PCa were all significant by the Wilcoxon test with the maximum difference (P<0.0001) provided by assay 5. The increase in DNAme across the six assays was highly significant (P<0.0001). The dashed line indicates 5% methylation.

Figure 2

Heat shock protein 27 (Hsp-27) expression and gene methylation in eight investigated cell lines. (a) Mean Hsp-27 generic expression (fold difference) relative to the immortalized prostate epithelial cell line PNT2 ± 1 s.d. was measured by western blot. (b) The methylation of six investigated regions in corresponding cell lines are shown in grayscale.

Figure 3

The diagnostic potential of HSPB1 DNA methylation (DNAme). (a) Methylation of HPSB1 was significantly (P<0.0001) higher in prostate cancer (PCa) tissue comparing with BPH in both fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples. The dotted line shows 5% methylation. (b) In order to visualize the diagnostic efficacy of HPSB1 methylation measured in the FFPE tissues in absence of an arbitrary cutoff value, the data were summarized using a receiver operating characteristic curve (ROC). Based on the sensitivity and specificity, highest possible specificity (100%) was obtained at cutoff 5%, with corresponding sensitivity 50%.

Figure 4

DNA methylation (DNAme) in patients who died (white box) of prostate cancer and patients alive or censored at the end of the study (shaded box) was stratified by Gleason score. In the low Gleason score group, higher median methylation (6%) was observed in patients who died than in patients alive or censored at the end of the study (median DNAme=0%). In the high Gleason score group, the reverse was observed with median methylation 8% vs 0%. Whiskers of the boxplot mark the 5th and 95th percentiles, the box 25th percentile, median and 75 percentile, whereas extreme values are shown by (•).