Original article
Common germline MDR1/ABCB1 functional polymorphisms and haplotypes modify susceptibility to colorectal cancers with high microsatellite instability

https://doi.org/10.1016/j.cancergencyto.2008.01.023Get rights and content

Abstract

Altered expression of P-glycoprotein (P-gp) encoded by the multidrug resistance (MDR1/ABCB1) gene, as well as somatic mutations and hypermethylation in the MDR1/ABCB1 gene, were identified in a proportion of previously untreated colorectal cancers (CRC) exhibiting high microsatellite instability (MSI-H), which suggested that MDR1/ABCB1 acts as a candidate gene contributing to the initiation and progression of MSI-H tumors. Here we report germline functional single nucleotide polymorphisms (SNPs) and haplotypes in the MDR1/ABCB1 gene, which could contribute to genetic risk or increase susceptibility to MSI-H cancers. We have confirmed disease association by comparing the MDR1/ABCB1 genotype, allele, and haplotype frequencies between healthy controls and patients with MSI-H tumors. In particular, carriers of the T/T genotype in exon 12 (1236 C→T) SNP and the T/T genotype in exon 21 (2677G→T) SNP were most significantly associated with a higher risk for developing MSI-H CRC compared to controls (P=0.01, OR=3.182 and P=0.005, OR=3.594, respectively). The most significant MSI-H–associated risk haplotypes include the most frequent haplotype H1 (T-C-T-T) defined by SNPs in exon 12, intron 13 (rs2235035), exon 21, and exon 26 (3435 C→T; P=0.004, OR= 0.476). Our results suggest that ABCB1/MDR1 is a novel low-penetrance gene for susceptibility to MSI-H tumors. The present study provides additional evidence for the role that the MDR1/ABCB1 gene plays in the initiation and progression of MSI-H CRC development. ©2008 Elsevier Inc. All rights reserved.

Introduction

A proportion of sporadic colorectal cancers (CRC) and 90% of cancers from patients with hereditary non-polyposis colorectal cancer (HNPCC) exhibit high microsatellite instability (MSI) [1]. MSI tumors differ in clinicopathologic and molecular genetic features and are associated with a better prognosis than CRC without MSI. Patients with MSI tumors have a higher mean age and are mostly women. The tumors, mostly located in the proximal colon, are predominantly mucinous, poorly differentiated, diploid, diagnosed at a higher Dukes’ stage, and have increased lymphoid cell infiltrates [2]. Interestingly, MSI CRC are also more often resistant to several chemotherapeutics; the selection of cells for resistance to cisplatin can result in the loss of DNA mismatch repair, and loss of DNA mismatch repair in turn contributes to cisplatin resistance [3]. Tumors with high microsatellite instability (MSI-H) are the result of deficient mismatch repair (MMR), and mutations in MMR genes are associated with HNPCC patients. More than 50% of HNPCC patients will have germline mutations in MMR genes, mainly in hMLH1 and hMSH2, and only in some cases in other MMR genes, including hMSH6 (GTBP), PMS1, and PMS2[1]. The genetic predisposition for the approximately other 50% HNPCC families and other hereditary colorectal cancers that do not completely fulfill the clinical criteria for HNPCC still need to be identified. If randomly selected CRC patients were to be analyzed for MSI and subsequent mutational analysis performed in major MMR genes, germline mutations could be found in 30% of MSI-H–positive CRC patients [4]. Sporadic MSI-H cancers arise mainly as a result of hypermethylation in the hMLH1 gene promoter [5], but germline genetic risk factors that would affect the susceptibility for sporadic MSI-H cancer development are poorly understood.

P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporterm, was initially identified as a protein responsible for multidrug resistance in cancer cells [6]. While the role of MDR1 in multidrug resistance after chemotherapy is well understood, its physiological function and role in the pathogenesis of cancer needs further clarification. The highest levels of P-gp are found in epithelial cells of the kidney, liver, pancreas, and colon, as well as in cells forming the blood/brain barrier and in lymphocytes. P-gp plays a role in the secretion of toxic compounds [7], apoptosis [8], and immune response [9]. High expression of P-gp at the atypical surface of differentiated tubular structures was identified in previously nontreated CRC [10], and its high expression at the leading edge of colorectal carcinoma has been associated with tumor progression [11]. A transcription factor complex TCF4/ß-catenin–responsive element was identified recently in the MDR1 promoter region, pointing to a direct link between the MDR1 gene and the WNT signalling pathway, the most important pathway that is altered in colorectal cancers [12]. We have identified hypermethylation and somatic mutations in the MDR1 gene in untreated MSI-H CRC [13].

Germline frequent single-nucleotide polymorphisms (SNPs) and haplotypes within the MDR1 gene were recently suggested to alter MDR1 functional expression and predispose to altered pharmacokinetics [14] and complex diseases, including renal cell carcinoma [15] and inflammatory bowel diseases [16], [17]. SNP in exon 26 was initially used for MDR1 association studies, but more significant results were obtained when haplotype analysis was performed [17], [18]. Haplotypes significantly associated with disease were defined by SNPs in exons 12 (1236 C→A), 21(A893S), and 26 (3435 C→T). We recently identified a SNP in intron 13 (rs2235035) in linkage disequilibrium (LD) with the disease haplotype, which is most significantly associated with refractory Crohn's disease and ulcerative colitis. The aim of the present study was to determine whether common germline polymorphisms in the MDR1/ABCB1 gene associated with functional haplotypes could affect the risk for MSI-H CRC.

Section snippets

Patients

In a period between 1996 and 2000, 400 newly diagnosed CRC patients from clinics all over Slovenia participated in this study. None of these patients received chemotherapy treatment before operation. Primary colorectal adenocarcinomas and corresponding normal colorectal mucosa taken from a site distant several centimeters from the tumor were used in the study. Tumors were evaluated histopathologically according to Jass's classification [19]. The lymphoid infiltration of the tumors was assessed

Results

We genotyped 355 healthy blood donors and 400 CRC patients, including 38 CRC patients with MSI-H, for SNPs previously found to be most significantly associated with functional haplotypes in the MDR1/ABCB1 gene. We found SNP in exon 12, intron 13, and exon 21 of the MDR1/ABCB1 gene associated with higher risk for developing MSI-H cancer when comparing the genotype and allele frequencies between MSI-H cancers and controls (Table 1). In particular, carriers of the T/T genotype in exon 12 (1236C→T)

Discussion

We identified the multidrug resistance 1 gene (MDR1/ABCB1) coding for P-glycoprotein (P-gp) as a novel low-penetrance susceptibility gene increasing risk for CRC with MSI-H. Here, we report common germline functional polymorphisms and haplotypes in the MDR1/ABCB1 gene associated with MSI-H colorectal cancers. To the best of our knowledge, this is the first report of common frequent germline polymorphisms affecting the genetic risk for sporadic MSI-H tumors. Mostly unique or rare high-penetrance

Acknowledgments

The Agency for development and research of the Republic of Slovenia supported this study (projects Z3-6028-0381-04 and J3-7141). The Medical Ethical Commission of the Republic of Slovenia agreed to the scope of the study in October 1995. We are grateful to patients who participated in the study. We thank Rastko Golouh and Stanislav Repše for the patients’ tissue samples.

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