Expression Profile of ABC Transporter Genes in Breast Carcinoma

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Viktor Hlaváč (1), Radka Václavíková (1), Marie Ehrlichová (1), Ivona Hlavatá (1), Václav Pecha (2), Markéta Trnková (3), Ivan Gut (1), Pavel Souček (1)

Pracoviště:

  1. Toxicogenomics Unit, National Institute of Public Health, Prague
  2. Department of Oncosurgery, Medicon, Prague
  3. Biolab Ltd., Prague

Background: Worldwide, breast cancer comprises the fifth most common cause of cancer-related deaths in women. Chemotherapeutic treatment is limited by the interindividual variability in drug response and by the development of resistance of cancer cells. ATP-binding cassette (ABC) transporters belong to a family of transporter proteins that contribute to drug resistance via ATP-dependent drug efflux pumps, e.g. P-glycoprotein. Gene expression–based assays have the potential to improve prognostic accuracy, treatment choice, and disease outcomes in women diagnosed with breast cancer. A major goal of our study is to search for candidate molecular markers with predictive potential in terms of chemotherapy outcome. We followed the expression and variability of ABC transporter genes and intended to evaluate their associations with clinico-pathological data including therapy outcome of individual patients.
 
Material and methods: Expression profile of all known forty nine human ABC transporter genes was evaluated in postoperative tissue samples from 65 breast cancer patients treated by FAC, FEC or taxane-based neoadjuvant chemotherapy regimens. Non-neoplastic adjacent tissue was available for 41 patients. Gene expression was assessed using real-time PCR with relative quantification. High Resolution Melting Analysis (HRM) was developed for the study of single nucleotide polymorphisms (SNPs) in ABCB1. HRM analysis was confirmed by direct sequencing.
 
Results: ABC transporters were expressed in the majority of samples (tumors and paired adjacent non-neoplastic tissues) with striking inter-individual variability. Thirteen ABC transporters were significantly downregulated in tumor tissues. On the other side, nineteen ABC transporters were significantly upregulated in tumors (ABCA2, ABCA3, ABCA7, ABCA12, ABCB2, ABCB8, ABCB9, ABCB10, ABCC1, ABCC4, ABCC5, ABCC10, ABCC11, ABCC12, ABCD3, ABCF1, ABCF2 and ABCG1). Finally, SNPs in ABCB1 gene coding a prototypical anticancer drug efflux pump P-glycoprotein (1236C>T, Gly412Gly, rs1128503; 2677G>T/A, Ala893Ser/Thr, rs2032582 and 3435C>T, Ile1145Ile, rs1045642; all in strong linkage disequilibrium) were assessed by HRM with sensitivity 97.1% and accuracy 97.6%. On the basis of the examined SNPs, one strong haplotype block containing rs2032582 and rs1128503 SNPs was identified. In addition, significant associations of rs2032582 SNP with stage, tumor size, expression of HER2, and family history of cancer were found. Significant associations of ABCC8 transcript level with tumor grade, estrogen receptor (ER) expression (both p<0.001), Ki67 protein expression (p=0.032) and response to chemotherapy (p=0.025) were found. ABCA13 level significantly associated with ER expression and tumor grade. ER expression also associated with ABCC10, ABCC13 and ABCD4 transcript levels.
 
Conclusions: Our results revealed new candidate genes potentially causing the multidrug resistance of mammary tumors. Validation study on these candidates will be performed by absolute quantification in an independent patient cohort. The association of expression profiles with therapy outcome and disease-free survival will also be analyzed. In addition, new HRM method that seems to be rapid, accurate and cost-effective as well as time-effective was developed for screening of functional ABCB1 SNPs and can analogously be applied to other ABC gene(s).
This work was supported by grants of Grant Agency of the Ministry of Health of the Czech Republic, grants no.: NS9803-3 and NS9799-4.

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