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 .
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 . 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 . 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 NH2 terminus, a RAS-association domain at COOH terminus, which interacts with the XPA protein. RASSF1A gene functions as a negative regulator of cell growth .
Hic1encoding a zinc finger transcription factor acts as a tumor suppressor gene . HIC1 is silenced by promoter hypermethylation in several types of human cancer . 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 . 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 . 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 . 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 . Human tumor cells exposed to high concentrations of CDDP in vitro induces alterations in 5-methyl Cytosine (5-mCyt) . 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 (. 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 . 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) . 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 . Recently, it has been shown that elevated testis-specific high-mobility group (ts-HMG) DNA-binding proteins may enhance sensitivity to CDDP . 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.