In this genome-wide methylation study, we provide evidence that methylation of select genes occurs during chronic platinum exposure and can confer drug resistance. Over 50% of genes identified and studied were specifically methylated in at least one resistant cell line. Based on this data, and given the fact that there are hundreds of genes which fit our algorithm in each individual cell line pair, it is reasonable to assume that methylation-induced gene silencing is a frequent and relevant phenomenon during chronic drug exposure. It is also noteworthy that 6 of the 14 methylated genes identified by our selection algorithm are cisplatin inducible. This observation not only suggests their functional relevance to cisplatin cytotoxicity, but also provides a rationale for the methylation-induced silencing of these genes in cells undergoing selection pressure from cisplatin.
While these findings are in keeping with a temporal association between changes in cellular methylation patterns and the development of cisplatin resistance, we have furthered the causal relationship between the two as well by demonstrating that the silencing of two specific genes identified by our algorithm results in increased drug resistance in our cisplatin-sensitive cell lines. One of these genes, SAT, has previously been associated with cisplatin-induced cytotoxicity (17
). In our studies, SAT expression decreased in two resistant cell lines, both with differential promoter methylation. It has been shown that over-expression of SAT makes cells more sensitive to cisplatin (18
). Consistent with this finding, we have demonstrated that siRNA-induced gene silencing of SAT in KB-3-1 cells is associated with increased drug resistance. Taken together our data indicate that downregulation of SAT in resistant cell lines plays a role in drug resistance, and that it can be modulated through promoter methylation.
Our data suggest a different role for S100P in the development of cisplatin resistance. S100P, a 95-amino acid protein, can act as an autocrine growth and survival factor (22
). S100P displays minimal expression in normal tissue, but expression increases dramatically in some malignancies (23
). This increased expression is associated with demethylation of the S100P gene (28
). We and others have observed S100P expression to be drastically decreased (as compared to parental) in at least 5 platinum-resistant cell lines, including two of our cell lines, two bladder cancer cell lines (16
), and one colon cancer cell line (14
). Thus, we provide the first evidence of S100P re-methylation when cells that express S100P become cisplatin resistant. Although our findings are in vitro
, a clinical study of lung cancer patients has shown similar results. Bartling, et al.
, found S100P mRNA expression to be significantly elevated in lung adenocarcinomas as compared to normal lung tissue. However, this association was not observed in adenocarcinomas from patients who had received neoadjuvant chemotherapy prior to tumor resection (29
The relationship between slower cell cycle progression and the responsiveness of tumors to chemotherapeutic agents has been widely documented elsewhere. Stewart, et al.
, suggests that the main factor driving failure of chemotherapy to cure advanced NSCLC is deficiency or saturation of a factor required for cell killing, such as the presence of slow or non-cycling cells (30
). Cancer cells survive until therapy cessation by downregulating metabolism/cycling, thus becoming temporarily quiescent and resistant to chemotherapeutic agents (30
). In a study of 184 breast cancer patients treated with primary radiotherapy or neoadjuvant chemotherapy, overall response to neoadjuvant chemotherapy was significantly lower for tumors with low S-phase fraction (SFP) compared with tumors with high SPF (35
). Likewise, a study of 81 breast cancer patients found that the response to chemotherapy was significantly better in patients with tumors with high SPF (36
). We found in our study that S100P knockdown cells demonstrate slower cell cycle progression, less SPF, and increased resistance to cisplatin. Since the time course of downregulation/remethylation of S100P is within the time frame of the development of resistance, our data suggest that S100P, exerting its effects by regulating cellular proliferation, may be one of the factors that play a role in the failure of multiple cell cycle-sensitive chemotherapy drugs. By utilizing methylation and demethylation to modulate expression of S100P, tumor cells are able to provide themselves with both growth and survival advantages under different circumstances. This regulation through alternating methylation patterns is efficient, fast, and flexible.
It is clear, however, that cisplatin resistance has multiple mechanisms, and that changing S100P expression is not the only factor in the development of this resistance, even in the model described here. Gottesman has previously shown mislocalization of folate-binding protein (37
) and cytoskeletal defects including downregulation of the carboplatin-binding proteins, actin and filamin (39
) in cisplatin-resistant cell lines. We similarly found downregulation of FOLR1, as well as transcripts for the structural proteins keratin8, laminin, beta 3, and tubulin beta 2A. Interestingly, both laminin beta 3 and tubulin beta 2A displayed promoter methylation in the resistant cell lines. While these genes may play a role in cisplatin resistance, S100P-induced cisplatin-resistance is independent of these structural changes since it can be induced by silencing of the gene in parental cells.
In summary, this study provides evidence that epigenetic promoter methylation is a frequent event during chronic cisplatin exposure, and that secondary changes in gene regulation can play an important role in generating drug-resistant phenotypes. Differentially methylated genes, including those identified in this study, may provide informative drug resistance markers as well as therapeutic targets, potentially leading to improved therapies for cancer patients with better and more durable clinical responses.