Drug transporter regulation in tumors by DNA methylation

Epigenetic alterations, such as aberrant DNA methylation, are a hallmark of cancer. DNA hypermethylation of the promoter region affects, for example, the expression of tumor suppressor genes and is associated with their transcriptional silencing in tumors. A recent report has provided evidence for epigenetic silencing of the multispecific organic cation transporter SLC22A1 in hepatocellular carcinoma. Given the role of this transporter in the cellular uptake of several anticancer drugs, the study provided a novel mechanism to explain the substantial variability in treatment response, and it might provide a new strategy for optimization of pharmacotherapy of hepatocellular carcinoma. See research article http://www.genomemedicine.com/content/3/12/82

Development of hepatocellular carcinoma, in common with other types of tumor, is considered to be a multistep process. Genetic and epigenetic alterations accumulate in regulatory genes, leading to activation of oncogenes and inactivation or loss of tumor suppressor genes [1]. The concept that hepatocellular carcinoma is a disease of epigenetic as well as genetic alterations has been valid ated in the past two decades. The epigenetic pathway is, in contrast to the genetic events, a reversible alteration that is not caused by primary DNA sequence changes.
There are three main epigenetic mechanisms: (i) hyper methylation of CpG islands in promoter sequences lead ing to silencing of tumor suppressor genes; (ii) DNA hypo methylation, which causes genomic instability or induc tion of genes involved, for example, in cell growth and invasion [2]; and (iii) histone modification, which affects chromatin conformation. Because epigenetic mecha nisms may function as an interface between environmental factors and the genome [3], deregulation of the epi genome by environmental stressors (for example, hepatitis B and hepatitis C viruses, chronic alcohol intake, and afla toxins) is believed to disrupt cellular processes and contribute to the risk of hepatocellular carcinoma. The early appearance of epigenetic changes makes them attractive targets for biomarker discovery. Moreover, drugs to reverse the epigenetic abnormalities are under development and some have already been approved. Cancer epigenetics is continuously translating into clinical practice and will help to optimize cancer diagnostics and treatment. The recent observation by Schaeffeler et al. in Genome Medicine that uptake transporters for anticancer drugs are epigenetically regulated in hepatocellular carcinoma adds an important piece of information to the growing body of research on cancer epigenetics [4]. The potential implications of these findings for biomarker development and pharmacotherapy of hepatocellular carcinoma will be discussed.

Epigenetics of organic cation transporter genes in cancer
Whereas previous work has focused mainly on the role of CpG hypermethylation for regulating oncogenes, tumor suppressor genes or DNA repair genes in hepatocellular carcinoma, Schaeffeler et al. [4] investigated the role of DNA methylation in the expression of specific members of the solute carrier (SLC) gene superfamily, namely the organic cation transporter genes SLC22A1 to SLC22A3 (encoding proteins OCT1, OCT2 and OCT3, respect ively). In normal hepatocytes, SLC22A1 (solute carrier family 22 member 1), which encodes OCT1 (organic cation transporter 1), is highly expressed and is respon sible for the uptake of nutrients, metabolites and xeno biotics. The transporter is also crucial for the uptake of some drugs [5]. Whereas lipophilic antineoplastic agents can enter cells by passive diffusion, uptake of charged hydrophilic agents, such as the anticancer compounds imatinib, cisplatin, oxaliplatin, picoplatin, irinotecan and

Abstract
Epigenetic alterations, such as aberrant DNA methylation, are a hallmark of cancer. DNA hypermethylation of the promoter region affects, for example, the expression of tumor suppressor genes and is associated with their transcriptional silencing in tumors. A recent report has provided evidence for epigenetic silencing of the multispecific organic cation transporter SLC22A1 in hepatocellular carcinoma. Given the role of this transporter in the cellular uptake of several anticancer drugs, the study provided a novel mechanism to explain the substantial variability in treatment response, and it might provide a new strategy for optimization of pharmacotherapy of hepatocellular carcinoma.  cDNA microarray studies have demonstrated decreased expression of SLC22A1 in hepatocellular carcinoma com pared with normal liver tissue. However, the mechanisms of downregulation have remained unclear. Based on their findings, Scheffeler et al. [4] suggest SLC22A1 promoter hypermethylation as an important mechanism. Epigenetic silencing of OCT1 in hepatocellular carcinoma, as demonstrated by Schaeffeler et al. [4], most likely impairs uptake of some anticancer drugs into hepatocellular cancer cells and thus is likely to impair their efficacy. This might, in part, explain the low response rates and uncertain survival benefit of, for example, cisplatinbased chemotherapy for hepatocellular carcinoma. In contrast, the tyrosine kinase inhibitor sorafenib, which does not appear to require active transport to enter cells [6], induces better disease control, significantly extends survival, and has become a standard firstline option for systemic treatment of advanced hepatocellular carcinoma.

Biomarkers to guide individualization of chemotherapy
Given the important role of OCT1 in cancer cell uptake and thus treatment efficacy of several anticancer drugs, methylation of SLC22A1 might be used as a marker for predicting therapeutic response. The work by Schaeffeler et al. [4] has demonstrated substantial interindividual variability in the degree of SLC22A1 methylation and SLC22A1 production, as revealed by histochemical analysis. It is tempting to speculate that patients with a high SLC22A1 methylation phenotype and silenced transporter expression may exhibit chemoresistance, whereas patients with a low methylation phenotype and enhanced transporter expression may respond to chemo therapy. Most recently, Ma et al. [7] provided another example of the involvement of epigenetic mechanisms in determining chemoresistance and chemosensitivity in hepatocellular carcinoma. MIR193A promoter hypo methy la tion is associated with transcriptional induction of the microRNA 193a3p, which targets the serine/ argininerich splicing factor 2 (SRSF2). SRSF2 sensitizes hepatocellular cancer cells to the chemotherapeutic drug 5fluorouracil via upregulation of the proapoptotic splicing form of caspase 2. Accordingly, its repression by microRNA 193a3p reduces sensitivity of hepatocellular cancer cells to 5fluorouracil [7]. This experimental obser vation suggests that the DNA methylation state of MIR193A could function as a marker to predict the therapeutic response to 5fluorouracil.

Biomarker for cancer screening
As well as using DNA methylation as a biomarker for personalizing chemotherapy, evidence has emerged that detection of abnormal promoter CpG island hyper methylation is also a potential biomarker for risk of developing cancer. DNA methylation in adjacent histo logically normal liver tissue of hepatocellular carcinoma has been reported from several groups and is now accepted as an early event in cancer development [8]. Thus, aberrant DNA methylation might be useful for early cancer detection and for predicting prognosis. Comparisons of CpG island methylation in hepatocellular carcinoma with that in adjacent tumorfree tissue or normal control livers have produced a continuously expand ing list of potential marker genes. Interestingly, Schaeffeler et al. [4] demonstrated that SLC22A1 belongs to a group of genes with progressively increasing CpG methylation, from normal liver to hepatocellular carci noma, with intermediate methylation in precancerous tissues (that is, in adjacent tumorfree tissue). Similar methylation changes related to the progression of malig nant transformation have been observed previously for other genes, including RASSF1A, PRDM14 and TBX4, which encode Ras association domain family member 1, PR domain containing 14 and Tbox 4, respectively. [8]. Therefore, the CpG methylation phenotype of these genes may serve as a marker for early cancer detection or assessing cancer risk.
As biomarker strategies move towards actual clinical practice, these proofofprinciple findings should be validated in larger patient cohorts. For detection of other tumors, such as colorectal and lung cancers, assays that test methylation patterns of marker genes in DNA from the stool, blood or bronchoalveolar lavage are already commercially offered to clinicians [9].

Epigenetic therapy for cancer
The results reported by Shaeffeler et al. [4] also highlight the clinical implications of epigenetic modulation of trans porter expression. DNA methyltransferase inhibi tors, such as decitabine, have already been approved for treatment of lymphomas and are being tested as a therapeutic option against various solid tumors. If these agents are concomitantly administered with classical anticancer drugs, the resulting induction of transporter expression may influence the disposition and effect of these anticancer drugs. Specifically, repression of OCT1 might be reversed by treatment with the DNA methyl transferase inhibitor decitabine, enhancing uptake of cisplatin into hepatocellular tumor cells. However, the fascinating possibility of overcoming the problem of chemoresistance with an epigenetic therapy awaits proof of concept. Abbreviations OCT1, organic cation transporter 1; SRSF2, serine/arginine-rich splicing factor 2.