GSTP1 methylation and protein expression in prostate cancer: diagnostic implications

Gunelli Roberta1, Massimo Fiori1, Filippo Martignano2, Giorgia Gurioli2, Samanta Salvi2, Daniele Calistri2, Matteo Costantini3, Teo Zenico1, Ugo De Giorgi4, Flavia Foca5, Valentina Casadio2
  • 1 Ospadale Morgagni-Pierantoni, U.O. Urologia (Forlì)
  • 2 IRST Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori IRCCS, Laboratorio di Bioscienze (Meldola)
  • 3 Ospadale Morgagni-Pierantoni, U.O. Oncologia Medica (Forlì)
  • 4 IRST Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori IRCCS, Unità di Oncologia Medica (Meldola)
  • 5 IRST Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori IRCCS, Unità di Biostatistica e Sperimentazioni Cliniche (Meldola)

Objective

GSTP1 belongs to the GSTs family, a group of enzymes involved in detoxification of exogenous substances and it also plays an important role in cell cycle regulation [1].
GSTP1 plays an important role in the early process of cancerogenesis in prostate cancer (PCa) [2] and its methylation is present in about 70-80% of PCa, while benign prostatic hyperplasias are normally hypomethylated [3].
Methylation often produces loss of gene expression and some literature data have shown a correlation between GSTP1 promoter hypermethylation and reduction of expression in PCa [4] . However, these data still need to be confirmed.
Taking into account all the above considerations, GSTP1 expression could potentially represent a good histological marker in substitution of or in addition to AMACR, p63 or cytokeratin [5-6], already in use in clinical practice, so as to improve PCa detection when diagnosis is ambiguous due to the presence of cancer mimics such as PIN.
The aim of our study was to investigate GSTP1 gene methylation and its correlation with expression in cancer, preneoplastic lesions and healthy tissues obtained by prostatectomy, to clarify its involvement in tumour development.

Materials and Methods

Fifty-six prostatectomies were recruited. We isolated DNA from cancer and healthy tissues previously selected by a pathologist. DNA isolation was performed using QIAamp DNA FFPE Tissue Kit (Qiagen, Milan, Italy), according to the manufacturer’s instructions.We investigated GSTP1 methylation status with methylation specific PCR (MS-PCR). Briefly, we converted DNA with bisulphite and performed real-time PCR using SYBR-GREEN master mix (Biorad, Milan, Italy) and primers specific for bisulphite-converted DNA. We tested the DNA quality by Actin B amplification.
We also manually performed immunohistochemistry by immunostaining tissue samples with a rabbit monoclonal antibody anti GSTP1 (anti-GST3/GSTp, Abcam). Tissues were incubated with the antibody at a dilution of 1:500 for 60 minutes.

Results

Analysis of the PCa samples and the corresponding adjacent healthy prostatic tissue showed that GSTP1 is methylated in 51 tumor samples (91.1%) and in only 3 adjacent healthy tumor samples (5.4%).
All 56 prostatectomy samples analyzed showed GSTP1 expression in adjacent healthy tissue irrespective of methylation pattern. All 51 (100.0%) cases methylated for GSTP1 in PCa tissue showed no expression. We observed an inverse association between methylation and expression of GSTP1 in the overall case series with a p<0.001 (Fisher exact test).
GSTP1 is highly expressed in basal cell layer and lumen in benign glands while in prostatic intraepithelial neoplasia (PIN) it stains only basal cell layer and PCa glands are completely negative.
The results obtained from GSTP1 expression analysis on prostatectomies were confirmed on 16 biopsies taken before surgery (data not shown).

Discussions

GSTP1 promoter hypermethylation correlates with underexpression in malignant glands in almost every sample, whereas it is strongly expressed in healthy tissues.
It is interesting to observe that, while GSTP1 hypermethylation often results in gene silencing in PCa tissue, the unmethylated status for GSTP1 promoter with a loss of expression was observed in 3 cases of PCa. This suggests that GSTP1 suppression may be due also to other regulation mechanisms.
GSTP1 appears to be methylated and silenced also in patients with low grade and low PSA values, suggesting that GSTP1 alterations (methylation or expression) may be considered as useful early diagnostic markers, so as to avoid unnecessary re-biopsies, as recently demonstrated by Zelic R et al [7].
We also performed immunohistochemistry on 16 prostate needle biopsies (data not shown) containing both low grade PCa and PIN in order to understand whether GSTP1 staining could be helpful for the histological evaluation of core biopsies for diagnostic purpose. Thanks to its capacity of staining in basal cell layer, we hypothesized that GSTP1 could be used to discriminate benign prostatic hyperplasia and PIN (which maintains basal cell staining uniformity) from PCa (which lacks a basal layer). Unfortunately, GSTP1 did not prove satisfactorily reliable due to its not always homogeneous staining in morphologically normal glands that should be positive to GSTP1.

Conclusion

We demonstrated that gene methylation leads to underexpression of GSTP1. The progressive loss of GSTP1 expression from healthy glands to PIN and to PCa glands underlines its involvement in early carcinogenesis; its behavior in the various stages of tumor development is noteworthy and paves the way for further studies on larger case series.
We also confirmed that GSTP1 promoter methylation is an early epigenetic event related to PCa development. Methylation analysis could be helpful to reveal PCa even in patients with low grade tumors and low PSA values, moreover expression analysis further demonstrated that GSTP1 methylation leads to gene silencing in PCa tissues.

References

[1] E. Laborde, Glutathione transferases as mediators of signaling pathways involved in cell proliferation and cell death, Cell death and differentiation 17 (2010), 1373-1380.
[2] M. Schnekenburger, T. Karius and M. Diederich, Regulation of epigenetic traits of the glutathione S-transferase P1 gene: from detoxification toward cancer prevention and diagnosis, Frontiers in pharmacology 5 (2014), 170.
[3] D. Jiang, Y. Shen, D. Dai, et al. Meta-analyses of methylation markers for prostate cancer, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 35 (2014), 10449-10455.
[4] W. Zhang, H. Jiao, X. Zhang, et al, Correlation between the expression of DNMT1, and GSTP1 and APC, and the methylation status of GSTP1 and APC in association with their clinical significance in prostate cancer, Molecular medicine reports 12 (2015), 141-146.
[5] J.I. Epstein, L. Egevad, P.A. Humphrey, R. Montironi and Members of the ISUP Immunohistochemistry in Diagnostic Urologic Pathology Group, Best practices recommendations in the application of immunohistochemistry in the prostate: report from the International Society of Urologic Pathology consensus conference, The American Journal of Surgical Pathology 38 (2014), e6-e19.
[6] V. Singh, V. Manu, A. Malik, V. Dutta, N.S. Mani and S. Patrikar, Diagnostic utility of p63 and alpha-methyl acyl Co A racemase in resolving suspicious foci in prostatic needle biopsy and transurethral resection of prostate specimens, Journal of cancer research and therapeutics 10 (2014), 686-692.
[7] R. Zelic, V. Fiano, D. Zugna, C. et al, Global Hypomethylation (LINE-1) and Gene-Specific Hypermethylation (GSTP1) on Initial Negative Prostate Biopsy as Markers of Prostate Cancer on a Rebiopsy, Clinical cancer research : an official journal of the American Association for Cancer Research (2015).

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