Background
Adult male germ cell tumors (GCTs) are considered to be a model system for a curable malignancy because of their exquisite sensitivity to cisplatin (CDDP)-based combination (cisplatin, etoposide, with or without bleomycin) chemotherapy. Histologically, GCTs present as a germ cell (GC)-like undifferentiated seminoma (SGCT) or a differentiated nonseminoma (NSGCT). NSGCTs display complex differentiation patterns that include embryonal, extra-embryonal, and somatic tissue types [
1]. Furthermore, embryonal lineage teratomas differentiate into various somatic cell types that may undergo malignant transformation to epithelial, mesenchymal, neurogenic, or hematologic tumors [
2]. Seminomas are exquisitely sensitive to radiation therapy while NSGCTs are highly sensitive to treatment with CDDP-based chemotherapy. Despite this sensitivity to chemotherapy, 20–30% of metastatic tumors are refractory to initial treatment, requiring salvage therapy and accounting for high mortalitiy. Such patients are treated with high dose and experimental chemotherapy protocols [
3]. The underlying molecular basis of this exquisite drug responsiveness of GCT remains to be fully understood [
4].
Little is known about the genetic basis of chemotherapy response in GCT. Studies have previously identified that TP53 mutations and gene amplification may play a role in GCT resistance [
5,
6]. It has also been recently shown that microsatellite instability is associated with the treatment resistance in GCT [
7]. An epigenetic alteration by promoter hypermethylation that plays a role in inactivating tumor suppressor genes in a wide-variety of cancers also has been shown to occur in GCT [
8‐
10]. We previously showed the absence of promoter hypermethylation in SGCT and acquisition of unique patterns of promoter hypermethylation in NSGCT [
8]. However, the role of such epigenetic changes in GCT resistance and sensitivity remains unknown.
In the present study, we evaluated the status of hypermethylation in 22 gene promoters in 39 resistant and 31 sensitive NSGCTs. We found that RASSF1A and HIC1 promoter hypermethylation was associated with highly resistant tumors. Evidence was also obtained suggesting that promoter hypermethylation is induced against the initial CDDP treatment and that this hypermethylation plays a crucial role in further treatment response. We show that changes in the patterns of gene expression occur during the in vitro acquisition of a highly refractory tumor to CDDP, which irreversibly affects the response to demethylating and histone deacetylase inhibiting agents.
Discussion
The molecular mechanisms that determine the curability of GCT to CDDP-based combination chemotherapy are unclear [
17‐
20]. Understanding the genetic basis of this exquisite sensitivity could lead to the development of a more effective treatment for resistant tumors. A number of genetic mechanisms for CDDP resistance, such as enhanced adduct repair, drug inactivation, or tolerance to DNA damage, have been proposed [
21].
We and others previously reported that epigenetic alterations in the promoters of specific genes occur in NSGCT [
8‐
10,
22]. We also showed that promoter hypermethylation was associated with gene repression in NSGCT and this down regulated expression is reactivated upon demethylation suggesting a potential role for epigenetic changes in GCT biology [
8]. These results prompted us to examine the possible involvement of epigenetic changes in chemo-sensitivity and resistance in GCT. To achieve this, we investigated epigenetic changes in resistant and sensitive NSGCTs and found a high incidence of promoter hypermethylation of
RASSF1A and
HIC1 in resistant tumors, while promoter hypermethylation of
MGMT and
RARB genes was associated with sensitive tumors.
RASSF1A has shown to be epigenetically inactivated in a wide variety of tumor types suggesting a major role for this gene in cancer [
23]. In the present study, we demonstrated that a higher frequency of resistant tumors carry promoter methylation compared to sensitive GCT, suggesting that
RASSF1A hypermethylation is associated with the resistance phenotype.
RASSF1A represents a long isoform of human
RASSF1 gene, which encodes a diacylglycerol (DAG)-binding domain at the NH
2 terminus, a RAS-association domain at COOH terminus, which interacts with the XPA protein.
RASSF1A gene functions as a negative regulator of cell growth [
23].
Hic1encoding a zinc finger transcription factor acts as a tumor suppressor gene [
24].
HIC1 is silenced by promoter hypermethylation in several types of human cancer [
25]. We found a higher frequency of resistant tumors harboring
HIC1 promoter hypermethylation.
On the other hand, we showed promoter hypermethylation of
MGMT and
RARB genes associated with CDDP sensitivity.
MGMT gene encodes O(6)-methylguanine-DNA methyltransferase and plays an important role in removing DNA adducts formed by alkylating agents [
11]. Epigenetic silencing of
MGMT has been shown to confer enhanced sensitivity on cancer cell to alkylating agents, while the lack of methylation and high-levels of protein expression contribute to drug-resistance phenotype [
12,
26,
27]. We showed here that either complete or partial methylation of
MGMT occurs in a majority of GCT. These data suggest that the complete promoter methylation of
MGMT plays a role in favorable response to CDDP treatment. However, the demonstration here of partial methylation in most GCTs provides a possible mechanism for down-regulated expression of
MGMT, which is commonly seen in this tumor. These results, thus, support the view that the epigenetic alteration in
MGMT may be a factor in the exquisite sensitivity of GCT to CDDP. Such a model provides opportunities to alter MGMT pathway and chemosensitize relapsed tumor to CDDP.
Retinoids control gene transcription by activating retinoic acid receptors (RAR∝, β and γ) and retinoid X receptors (RXR∝, β and γ). Expression of these receptors regulates organogenesis, organ homeostasis, cell growth, and differentiation and death [
28]. It is well established that changes in expression of RARs play a major role in cancer development and response of tumor cells to treatment of all-trans retinoic acid (ATRA). A number of premalignant lesions and cancers have been shown to exhibit a loss of expression of
RARB due to promoter hypermethylation [
28]. In the present study, we found
RARB promoter hypermethylation only in sensitive NSGCTs. The data, therefore, suggest that
RARB down-regulation may favor response to CDDP treatment. The mechanisms regulated by
RARB responsive genes in GCT require further studies to understand the role of this gene in chemotherapy response.
Previous studies indicated that tumor cells exposed to anticancer agents induce DNA hypermethylation resulting in the silencing of genes that play role in drug metabolism and resistance [
29]. Human tumor cells exposed to high concentrations of CDDP
in vitro induces alterations in 5-methyl Cytosine (5-mCyt) [
30]. Similarly,
in vivo exposure of bone marrow cells to cytosine arabinoside (araC) alone or a combination of hydroxyurea, VP-16 and araC also result in a several-fold increase of 5-mCyt content in leukemic blasts ([
30]. Thus, the exposure of tumor cells to cytotoxic chemotherapy agents
in vitro and
in vivo causes an induction of DNA hypermethylation. In the present study, we examined whether CDDP treatment
in vivo causes such a hypermethylation in GCT by studying specific gene promoters. Our results suggest that initial CDDP treatment in tumors induces promoter hypermethylation of certain gene promoters such as
MGMT,
RARB, and
BRCA1 (Fig.
2). This induction of methylation in these genes hypersensitize the tumor to further treatment, while tumors that had promoter hypermethylation of
RASSF1A,
HIC1, and
APC are selected upon further treatment to develop drug resistance (Figs.
1 and
2). Such a model of CDDP-induced non-random hypermethylation can predict response to further treatment and allows specific gene targeted therapeutic approaches for resistant GCTs. Hypermethylation of
RASSF1A,
HIC1, and
APC genes provides a plausible mechanism for the propensity of these tumors to CDDP resistance, and demethylation could result in restoration of hypersensitivity. Well documented evidence in certain tumor types suggest that drug resistance disrupts general mechanisms of chemosensitivity by targeting mutations and gene amplifications [
31]. Here, we demonstrate that epigenetic alterations in specific genes also play a role in chemosensitivity to CDDP in GCT. The present data, thus, suggest that specific gene promoter hypermethylation induced by drugs may serve as prognostic indicator of treatment response in NSGCT. In view of the biological relevance of DNA methylation, CDDP-induced hypermethylation shown here in GCTs will have clinical significance in drug-response phenotypes.
CDDP-induced promoter hypermethylation in tumor cells might set in motion a cascade of ectopic gene expression events that might release tumor from normal homeostatic controls. These changes include deamination of 5-methyl cytosine in CpG causing genetic instability (i.e., mutations), transducing epigenetic changes into genetic alterations, or inactivation of methylated genes. To test the latter possibility, we tested gene expression in four different clones from two highly resistant cell lines. We could not reactivate the gene expression by exposure to the demethylating agent 5-Aza-C or histone deacetylase inhibitor TSA, implying that a common epigenetic and/or genetic mechanisms that regulate transcriptional activation of hypermethylated genes was affected in highly resistant cells rather than simple methylation changes in specific gene promoters.
The cytotoxic effectiveness of CDDP against tumor cell is believed to be mediated through the formation of DNA adducts, which inhibit DNA replication and transcription [
32,
33]. Cisplatin primarily forms intra-strand GpG cross-links, which are removed by neucleotide excision repair (NER) [
34]. Highly regulated steps involving a number of proteins coordinate the NER in human cells. One hypothesis to explain the hypersensitivity of GCT to CDDP is that there is a deficiency in one or more components of this repair machinery [
35]. Recently, it has been shown that elevated testis-specific high-mobility group (ts-HMG) DNA-binding proteins may enhance sensitivity to CDDP [
34]. Our results suggest a potential molecular mechanism of CDDP-induced transcriptional inactivation of genes prone to hypermethylation. The CDDP exposure may cause genetic damage that might sequester essential proteins from their designated function such as elements of DNA repair pathways. The results presented here support the notion that epigenetic mechanisms play a role in CDDP-response in a gene specific manner. As cellular response to CDDP treatment in GCT is believed to be a complex process, future studies to address this issue need to examine both epigenetic and genetic alterations.
Authors' contributions
SK carried out the methylation, cloning, sequencing and gene expression analysis. JMM participated in selection of tumor specimens, isolation of DNA and RNA. GN participated in the analysis of gene expression. JH coordinated the selection of tumors, tissue culture, isolation of genomic DNA and RNA. JB performed statistical analysis. DLB participated in obtaining the follow up on patients. AMA and MM participated in the preparation of tissue array and gene expression analysis. VER participated in histologic diagnosis. GJB was responsible for referring the patients and clinical information. RSKC and VVVSM have conceived and coordinated the study. All authors read and approved the final manuscript.