Predicting Chemosensitivity to Gemcitabine and Cisplatin Based on Gene Polymorphisms and mRNA Expression in Non-small-cell Lung Cancer Cells

Xiangning Meng; Geng Wang; Rongwei Guan; Xueyuan Jia; Wei Gao; Jie Wu; Jingcui Yu; Peng Liu; Yang Yu; Wenjing Sun; Haiying Dong; Songbin Fu

Disclosures

Pharmacogenomics. 2015;16(1):23-44. 

In This Article

Abstract and Introduction

Abstract

Aim We used a panel of 17 non-small-cell lung cancer cell lines to investigate whether the presence of polymorphisms in the RRM1, ERCC1, ABCB1 and MTHFR genes and alterations in their mRNA expression can affect the in vitro chemosensitivity to cisplatin and gemcitabine.

Materials & methods Polymorphisms in these genes were evaluated by direct sequencing. mRNA expression levels were assessed by realtime PCR. In vitro chemosensitivity to cisplatin and gemcitabine was expressed as IC50 values, using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.

Results There was a significant, positive correlation between RRM1 mRNA expression and IC50 values for gemcitabine (r = 0.6533, p = 0.0045), and there was a significant, negative correlation between ABCB1 mRNA expression and IC50 values for cisplatin (r = -0.5459, p = 0.0287). When examining the association between the polymorphisms and IC50, we found that only the MTHFR 1298A>C polymorphism showed a tendency to be more chemosensitive to gemcitabine (p = 0.0440).

Conclusion These in vitro results suggest that mRNA expression levels of the RRM1 and ABCB1 genes may be useful indicators of chemosensitivity to gemcitabine and cisplatin, respectively. The MTHFR 1298A>C polymorphism was associated with gemcitabine chemosensitivity, which require further functional analysis with co-expressed genes and should be explored in prospective clinical studies to determine its potential clinical application as a predictive biomarker.

Introduction

Cytotoxic chemotherapy is the standard of care for patients with advanced non-small-cell lung cancer (NSCLC).[1–3] As with many cancer therapeutic agents, resistance is a significant problem with the use of cisplatin and gemcitabine to treat NSCLC. Therefore, the clinical integration of molecular biomarkers that predict responses to chemotherapeutic or molecularly targeted agents, leading ultimately to individualized chemotherapy, may be important for improving clinical outcomes in advanced NSCLC.

Cisplatin is a platinum agent that is known to act through the formation of DNA adducts that inhibit DNA synthesis and transcription. Although drug resistance is caused by multiple genetic factors, DNA repair genes play a key role in cisplatin resistance. DNA repair mechanisms are important determinants of the sensitivity to platinum-based chemotherapy, especially those involving the nucleotide excision repair (NER) pathway.[4,5] Gemcitabine, a potent and specific pyrimidine nucleoside antimetabolite agent, is active against NSCLC, especially when administered with platinum derivatives.[3,6] Evidence has shown that sequence variation in genes involved in gemcitabine and cisplatin transport, metabolism and bioactivation pathways contributes to the variation in gemcitabine and cisplatin response.[7–13]

RRM1, a regulatory subunit of ribonucleotide reductase (RR), is required for deoxynucleotide production, which is the rate-limiting step in DNA synthesis and repair. Gemcitabine is a potent and widely used RR inhibitor. Gemcitabine (2′,2′-difluorodeoxycytidine) is a deoxycytidine analogue that is activated by deoxycytidine kinase to its monophosphate form. Subsequent phosphorylation by uridylate-cytidylate monophosphate kinase generates difluorodeoxycytidine diphosphate, which binds to the substrate binding site and inactivates the RRM1 subunit, reducing the pool of deoxyribonucleotide diphosphate available for DNA synthesis.[14–16] Retrospective studies of stage IV NSCLC patients treated with gemcitabine-based chemotherapy have shown that patients with low RRM1 mRNA levels live longer than patients with higher expression levels.[17–20] Preclinical studies have shown that the expression of RRM1 is involved in sensitivity to gemcitabine in NSCLC.[21–23] Zheng et al.[24] studied RRM1 and ERCC1 expression in vitro and in vivo and found that tumoral RRM1 expression is a major predictor of disease response to gemcitabine/platinum chemotherapy. Kwon et al.[7] have found that the RRM1 2464G>A polymorphism is associated with gemcitabine sensitivity using a panel of 62 human cancer cell lines. Thus, RRM1 expression and polymorphisms may be used to develop optimal, customized chemotherapy regimens in clinical practice.

ERCC1 is a DNA damage repair gene that encodes the 5′ endonuclease of the NER complex. An increase in ERCC1 expression is likely to cause the cisplatin resistance phenotype, which causes the cytotoxicity of cancer cells by forming adducts that result in DNA cross-links. The NER complex recognizes and removes these adducts, which may trigger resistance to platinum agents. High tumor tissue levels of ERCC1 mRNA have been associated with clinical resistance to cisplatin-based chemotherapy in human ovarian, gastric, cervical, colon and NSCLC carcinomas.[25] The ERCC1 8092C>A polymorphism has a statistically significant association with a decreased platinum-based chemotherapy response.[26] Patients who had the ERCC1 8092AA genotype showed a better response to gemcitabine plus cisplatin than patients who had the CC or CA genotype.[27] Thus, the polymorphic status of ERCC1 might be a promising ancillary marker for predicting the treatment response of advanced-stage NSCLC patients.

Pgp is a transmembrane transporter that belongs to the superfamily of ATP-binding cassette (ABC) transporters and is encoded by the human ABCref-1 (also known as MDR1) gene. Pgp is responsible for cytotoxic product elimination at the cell membrane by the hydrolysis of ATP.[28] Pgp plays a major role in drug resistance by impairing the intracellular retention of multiple anticancer drugs.[29,30] Stordal et al.[31] found that Pgp was overexpressed in a cisplatin-resistant ovarian cancer cell line. Bergman et al.[32] observed that Pgp overexpression may have caused cellular stress, resulting in increased gemcitabine metabolism and sensitivity. Additionally, the reversal of collateral gemcitabine sensitivity by verapamil also suggests a direct relationship between the presence of membrane efflux pumps and gemcitabine sensitivity.

MTHFR, a key enzyme in folate metabolism, catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate (5,10-methyleneTHF) to 5-methyltetrahydrofolate (5-methyl-THF), which is required for methylation reactions, including the conversion of homocysteine to methionine. Methionine, in turn, is the precursor of S-adenosylmethionine, the universal methyl donor for methylation reactions, including DNA methylation.[33] Genomic DNA methylation is directly correlated with folate status and inversely correlated with plasma homocysteine levels. Changes in the status of DNA methylation, known as epigenetic alterations, are one of the most common molecular signatures in cancer. The reduction of MTHFR enzyme activity can ultimately lead to impaired methylation of homocysteine to methionine and subsequent hyperhomocysteinemia. The aberrant methylation of cell-cycle checkpoint and DNA repair genes has been shown to be indicative of sensitivity to cytotoxic drugs.[34] We therefore reasoned that the MTHFR gene associated with hypomethylation could be surrogate markers for predicting chemosensitivity to cisplatin and gemcitabine.

In this study, we first examined the relationship between mRNA expression (RRM1, ERCC1, ABCref-1 and MTHFR) and chemosensitivity to cisplatin and gemcitabine in 17 NSCLC cell lines. In addition, polymorphisms of these genes and their associations with drug chemosensitivity and mRNA expression were analyzed to determine whether polymorphisms influence the in vitro chemosensitivity to cisplatin and gemcitabine due to differential gene expression.

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