Cancer Letters

Cancer Letters

Volume 247, Issue 1, 8 March 2007, Pages 137-142
Cancer Letters

Frequent epigenetic inactivation of p53 target genes in seminomatous and nonseminomatous germ cell tumors

https://doi.org/10.1016/j.canlet.2006.03.028Get rights and content

Abstract

Hypermethylation of tumor-suppressor genes has been implicated in the pathogenesis of human cancers. This study was designed to examine the methylation profiles of a selected group of p53 target genes (APAF-1, CASP-8, DAPK-1, IGFBP-3) and to correlate the findings with the histopathological characterization of testicular germ cell tumors (TGCT).

Promoter methylation status was analysed by highly sensitive real-time methylation-specific PCR in 46 primary TGCTs (26 seminomas and 20 nonseminomas) and 15 normal testicular tissue samples. APAF-1 methylation was detected in all of the seminomatous and nonseminomatous TGCTs as well as in 60% of normal testicular tissue. Methylation of DAPK-1 was frequent in seminomas (50%) and nonseminomas (20%), but not in normal testicular tissue (6%). The degree of DAPK-1 methylation correlated with the clinical stage of the disease (P=0.05) and was useful in differentiating seminomatous from nonseminomatous, and malignant from nonmalignant testicular tissue (P=0.04 and 0.02, respectively). The APAF-1 methylation index achieved a highly significant differentiation between seminomatous or nonseminomatous tissue and nonmalignant testicular tissue (P=0.0001). In testicular tumorigenesis, promoter methylation of specific p53 target genes occurs at early stage but to varying degrees. Methylation also occurs in normal testicular tissue, which is in contrast to findings in other urogenital malignancies. Further studies will be necessary to determine whether the methylation level may be used as marker for risk estimation, especially in clinical stage I disease.

Introduction

Testicular germ cell tumors (TGCT) represent the most common male malignancy in the third to fourth decade of life [1]. TGCTs are divided into two major histological groups and it is suggested that all tumors originate from carcinoma in situ and progress, after various cytogenetic and molecular alterations, first into seminoma and subsequently into nonseminoma. Fortunately, testicular cancer is a highly curable disease, even in advanced stages [2].

Several studies of TCGTs have demonstrated the presence of chromosomal alterations including allelic loss or the amplification of chromosome 12p [3], [4]. However, few genes have been identified to contribute to the tumorigenesis of testicular cancer. Recently, epigenetic silencing has emerged as one mechanism thought to impair the activity of specific genes in testicular cancer [5], [6]. Multipoint analysis of testicular neoplasms demonstrates that a minority of genes, typically those affected by promoter methylation such as RASSF1A, MGMT and PRSS21, is also altered in testicular cancer [7], [8], [9], [10]. To date, the findings of all previous studies are limited either by small sample size or the use of a nonquantitative method of methylation-specific PCR (MSP).

Since dysfunctional mutated p53 has been shown to play a role in the resistance of TGCT to chemotherapy, we focused on several downstream target genes of the p53 tumor suppressor gene to assess epigenetic silencing [11], [12]. Although, all the genes selected for investigation in this study are thought to be closely involved in the apoptotic cycle and harbor CpG islands in their promoter regions, and thus serving as potential targets for transcriptional silencing, none has ever been investigated for promoter methylation in testicular cancer.

One of the first p53 target genes found to be methylated and involved in the apoptotic pathway was the death-associated protein kinase (DAPK-1), identified in tumors of epithelial and nonepithelial origin [13], [14]. Another p53 target gene, shown to be methylated in both human leukemic cell lines and metastatic malignant melanoma, the apoptotic protein-activating factor (APAF-1) is thought to play an important role in the induction of apoptosis [15], [16]. One of the executioner caspases, Caspase 8 (CASP-8), plays an antagonistic role with anti-apoptotic survivin, although little is known about whether it is silenced via promoter hypermethylation [17]. Insulin-like growth-factor-binding protein-3 (IGFBP-3) was shown to have reduced mRNA expression in several tumors and also harbors CpG islands. However, no investigation of the IGFBP-3 promoter region has yet been reported [18].

In the current study, we investigated the frequency and percentage of promoter methylation for APAF-1, CASP-8, DAPK-1 and IGFBP-3 in 46 primary TGCTs (26 seminomas and 20 nonseminomas) and 15 normal testicular tissue samples using highly sensitive, quantitative real-time methylation specific PCR (Q-MSP).

Section snippets

Material and methods

Tissue specimens were collected at the Department of Urology, University Charité CBF, Berlin between 2001 and 2005. All patients signed a consent form approved by the Committee on Human Rights in Research of our institution. Normal testicular tissue biopsies (n=15) were obtained from seven patients undergoing subtotal orchiectomy for antihormonal therapy in prostate cancer and from eight patients who underwent a vasovasostomy and requested cryopreservation of a biopsy in case the intervention

Results

TCGT specimens included 26 seminomatous and 20 nonseminomatous testicular tumors. Testicular tissue obtained from 15 patients following testicular biopsy for nonmalignant disease were used as controls. Fifteen (57%) and Eleven (43%) of the seminoma patients were classified as clinical stage (CS) I and ≥II, respectively. In the nonseminomatous cohort, 9 (45%) and 11 (55%) had clinical stage I and ≥II disease, respectively. The median age in the seminoma cohort was 37.5 (±7.4) years as compared

Discussion

In this study, we report the first quantitative analysis of promoter hypermethylation in a subset of p53 target genes in testicular cancer. Promoter hypermethylation of APAF-1, DAPK-1 and IGFBP-3 occurs to varying degrees in testis cancer. For APAF-1, methylation was observed in all neoplastic testicular specimens and in almost two-thirds of the normal testicular tissue. Interestingly, the quantitative MSP analysis revealed that the degree of methylation within the normal tissue samples was

Acknowledgements

We thank Ms Petra von Kwiatkowski for her expert technical assistance and Dr Joanne Weirowski for her linguistic advice.

References (29)

  • S. Honorio et al.

    Frequent epigenetic inactivation of the RASSF1A tumour suppressor gene in testicular tumours and distinct methylation profiles of seminoma and nonseminoma testicular germ cell tumours

    Oncogene

    (2003)
  • B. Smith-Sorensen et al.

    Frequent promoter hypermethylation of the O6-methylguanine–DNA methyltransferase (MGMT) gene in testicular cancer

    Oncogene

    (2002)
  • S. Koul et al.

    Characteristic promoter hypermethylation signatures in male germ cell tumors

    Mol. Cancer

    (2002)
  • K.J. Manton et al.

    Hypermethylation of the 5′ CpG island of the gene encoding the serine protease Testisin promotes its loss in testicular tumorigenesis

    Br. J. Cancer

    (2005)
  • Cited by (0)

    View full text