The Prostate 76:434–444 (2016) The Role of ATP-Binding Cassette Transporter Genes in the Progression of Prostate Cancer Omer F. Karatas,1 Esra Guzel,2,3 Mehmet B. Duz,2 Michael Ittmann,4,5 and Mustafa Ozen2,3,4* 1Department of Molecular Biology and Genetics, Erzurum Technical University, Erzurum, Turkey 2Department of Medical Genetics, Cerrahpasa Medical School, Istanbul University, Istanbul, Turkey 3Department of Molecular Biology and Genetics, Biruni University, Istanbul, Turkey 4Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas 5Michael E. DeBakey VAMC, Houston, Texas BACKGROUND. Prostate cancer (PCa) is the most commonly diagnosed neoplasm and the second leading cause of cancer-related death among men in developed countries. There is no clear evidence showing the success of current screening tests in reducing mortality of PCa. In this study, we aimed to profile expressions of nine ABC transporters, ABCA5, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC5, ABCC10, and ABCF2, in recurrent, non-recurrent PCa and normal prostate tissues. METHODS. A total of 77 (39 recurrent, 38 non-recurrent) radical prostatectomy and 20 normal prostate samples, obtained from Baylor College of Medicine Prostate Cancer program, were included into the study and divided into two independent groups as test and validation sample sets. Differential expression of selected ABC transporters was assessed using quantitative real-time PCR (qRT-PCR). Pearson’s correlation test, receiver operating characteristics (ROC) analysis and Kaplan–Meier test were used for statistical analysis. RESULTS. QRT-PCR results demonstrated the elevated expression of ABCA5, ABCB1, ABCB6, ABCC1, and ABCC2 as well as reduced expression of ABCC3 in PCa samples compared to normal prostate tissues. In addition, we found deregulation of ABCB1, ABCB6, ABCC3, and ABCC10 in recurrent PCa samples and validated differential expression of ABCB6, ABCC3, and ABCC10 in recurrent PCa compared to non-recurrent PCa. Pearson’s correlation, ROC and Kaplan–Meier analysis revealed the power of these three ABC transporters for estimating prognosis of PCa. CONCLUSIONS. We demonstrated differential expression of ABC transporters both in tumor versus normal and recurrent versus non-recurrent comparisons. Our data suggest ABCB6, ABCC3, and ABCC10 as valuable predictors of PCa progression. Prostate 76:434–444, 2016. # 2015 Wiley Periodicals, Inc. KEY WORDS: prostate cancer; recurrence; ABC transporters; progression; prognosis INTRODUCTION Prostate cancer (PCa) is the most commonly diag- nosed neoplasm and the second leading cause of Grant sponsor: The Scientific and Technological Research Council cancer-related death among men in the developed of Turkey (TUBITAK); Grant number: 108S051. countries [1]. Over 650,000 new cases are annually Conflicts of Interest: Authors declare no conflict of interest. diagnosed with PCa, which constitutes approximately ÃCorrespondence to: Mustafa Ozen, MD, PhD, Department of 10% of all new cancer cases in men worldwide [2]. In Medical Genetics/Molecular Biology and Genetics, Biruni Univer- Europe, incidence of PCa in elderly men has been sity, Topkapi, Istanbul, Turkey. E-mail: [email protected] Received 18 September 2015; Accepted 30 November 2015 rising rapidly [3]. DOI 10.1002/pros.23137 For early detection of PCa, prostate specific antigen Published online 28 December 2015 in Wiley Online Library (PSA) screening has been used widely as a diagnostic (wileyonlinelibrary.com). ß 2015 Wiley Periodicals, Inc. ABC Transporters in Prostate Cancer Progression 435 tool during the last two decades [4]. Biochemical demonstrated the differential expression of ABCA5, recurrence subsequent to radical prostatectomy ABCB1, ABCB6, ABCC1, ABCC2, and ABCC3 in tumor has been assessed using serum PSA level along samples when compared to the normal prostate sam- with T category, and Gleason score as prognostic ples. Furthermore, we found ABCB6, ABCC3, and variables [5]. However, there is no clear evidence ABCC10 as powerful biomarkers, which can serve for showing the success of PSA screening in reducing prediction of PCa progression. the morbidity and mortality of PCa [6]. Furthermore, current prognostic indicators fail to estimate the MATERIALS AND METHODS clinical outcome of the disease in most of the cases and a significant percentage (30%) of PCa patients Patients experience a recurrence following radical prostatec- A total of 77 (39 recurrent, 38 non-recurrent) radical tomy or other therapy [7]. prostatectomy materials, which contained at least 70% To enhance patient survival and success of the tumor tissue, and 20 normal prostate tissue samples, current treatment options, scientists focused on find- were obtained from Baylor College of Medicine ing of novel prognostic molecular targets and sug- Prostate Cancer program. Sample recruitment was in gested several putative biomarkers [8,9]. There is, accordance with internal institutional review board of however, still an urgent need to identify influential Baylor College of Medicine. Recurrence was described molecular predictors to more clearly understand the as two successive serum PSA levels greater than molecular basis of the disease, to predict the aggres- 0.2 ng/ml. Patients were followed until PSA recur- siveness of the tumor and to develop more effective, rence or at least 4 years for the non-recurrent cases. optimized and individualized therapy strategies. Thirty nine (20 recurrent, 19 non-recurrent) tumor ATP-binding cassette (ABC) efflux transporters and 20 normal tissue specimens were evaluated as the belong to a superfamily of integral membrane pro- “test set”, and the remaining 38 (19 recurrent and 19 teins, which is comprised of 49 genes [10] and divided non-recurrent) tumor tissue samples were assessed as into seven subtypes from ABC-A through -G based on “validation set”. A part of these samples has been used the amino acids sequence in the ATP-binding do- previously by our group to investigate the expression main [11]. ABC transporters are responsible for active profile of CSCs markers and specific microRNAs in transport of many substances including amino acids, recurrent and non-recurrent PCa tumors as well as in polysaccharides, peptides, lipids, drugs, and tox- adjacent normal prostate tissues [8,9]. ins [12]. Their differential expression has been demon- strated in several tumor types [13]. Resistance to chemotherapy can originate from RNA Isolation several mechanisms including resistance to drug infiltration into cancer cells, which is particularly Total RNA from 39 recurrent, 38 non-recurrent radical realized by ABC transporters [14]. However, indepen- prostatectomy materials and 20 normal prostate tissue dent of their role in the drug efflux and multidrug samples were isolated with TRIzol reagent (Invitrogen, ’ resistance, ABC transporters may contribute to the San Diego, CA) according to the manufacturer sinstruc- carcinogenesis process through actively transporting tions. Purities and quantities of RNA samples were endogenous substrates relevant to tumor biology [13]. assessed using a NanoDrop ND-2000c (Thermo Fisher Recently, several studies correlated the expression Scientific, Inc., Wilmington, DE) and their integrities levels of ABC transporters with tumor progression were tested with agarose gel electrophoresis and Spot and these transporters were suggested as powerful Check Nucleic Acid Quantitation Kit (Sigma). predictors of clinical outcome [15,16]. However, there is still lack of precise knowledge about the impact of cDNA synthesis and Quantitative Real-Time PCR certain ABC transporters on the progression and clinical outcome of PCa. Therefore, comprehensive Reverse transcription of the RNA samples were characterization of the expression profile of these carried out with the Transcriptor High Fidelity cDNA genes in association with the prognosis of the cancer synthesis kit (Roche, Switzerland) according to the cases is essential to decipher the underlying mecha- manufacturer’s instructions. The relative expression nisms of PCa recurrence. levels of ABCA5, ABCB1, ABCB6, ABCC1, ABCC2, Here,inthisstudy,weprofiledtheexpressionof ABCC3, ABCC5, ABCC10, and ABCF2 were evaluated nine important ABC transporter genes, ABCA5, with quantitative real time polymerase chain reaction ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC5, (qRT-PCR) using SYBR Green Master Mix of Roche ABCC10, and ABCF2, in recurrent and non-recurrent (Switzerland) in a LightCycler480-II real-time thermal PCa specimens as well as normal prostate tissues. We cycler (Roche, Switzerland). The primer sequences The Prostate 436 Karatas et al. used for RT-PCR are provided in Supplementary Recurrent PCa patients (18.32 Æ 2.06 ng/ml) had Table SI. The reactions were carried out as follows: higher preoperative PSA levels compared to non- initial denaturation (95°C for 5 min) followed by 40 recurrent PCa patients (11.28 Æ 1.67 ng/ml, P ¼ 0.048). cycles of 95°C for 10 sec, 60°C for 20 sec, and 72°C for Gleason Score’s of patients with recurrence were 25 sec. Fluorescence measurement was performed at between 2 þ 4 and 4 þ 5 with a median of 3 þ 4, the end of each cycle at 72°C. Expression data were however, non-recurrent PCa patients had Gleason normalized to housekeeping gene b-actin. All qRT-PCR Score’s varying from 2 þ 3to4þ 4 with a median of reactions were performed at least twice for each 3 þ 4. Months from operation to first recurrence in sample. The relative quantification analysis was done recurrent PCa patients were 23.45