ANTICANCER RESEARCH 33: 387-392 (2013)

ABCC4 Decreases Docetaxel and Not Cabazitaxel Efficacy in Prostate Cancer Cells In Vitro

DANIELA E. OPREA-LAGER1, IRENE V. BIJNSDORP2, REINDERT J.A. VAN MOORSELAAR2, ALFONS J.M. VAN DEN EERTWEGH3, OTTO S. HOEKSTRA1 and ALBERT A. GELDOF1,2

Departments of 1Radiology and Nuclear Medicine, 2Urology and 3Oncology, VU University Medical Center, Amsterdam, the Netherlands

Abstract. Background: This study aimed to investigate state is common within 18-36 months (2). In these patients, cabazitaxel efficacy in a model for docetaxel-resistant chemotherapy has proven to slow down androgen- prostate cancer cells and to evaluate the involvement of ATP- independent tumor growth. In particular, docetaxel, a cassette binding protein 4 (ABCC4) with regard to multidrug registered agent from the taxane class, was introduced as a resistance. Materials and Methods: Docetaxel and first-line chemotherapy (3). Its effect is largely based on cabazitaxel sensitivity was measured in PC3 and R3327- interaction with microtubules (4). Docetaxel plus MATLyLu (MLL) cell lines, using the sulforhodamine B estramustine or prednisone was found to significantly reduce (SRB) assay. ABCC4 expression was examined by western the prostate-specific antigen (PSA) value and improve blotting and its functional involvement in drug sensitivity by overall survival when compared to mitoxantrone-prednisone blocking with MK571 inhibitor. Results: The docetaxel- therapy. In combination with prednisone, docetaxel also resistant MLL cells (4.5-fold compared to cabazitaxel; appeared to be useful in achieving significant improvement p<0.001) were shown to express high levels of ABCC4, while of pain palliation and quality of life (3-5). These clinical non-resistant PC3 cells had no detectable ABCC4 results have resulted in the regimen of docetaxel and expression. Functional inhibition of ABCC4 in MLL cells prednisone once every three weeks becoming the current resulted in a two-fold decrease in effective concentration of recognized standard of treatment for castration-resistant PCa docetaxel and had no effect on toxicity of cabazitaxel. (CRPC) (5). Conclusion: Cabazitaxel showed an improved therapeutic Although these successes with docetaxel have improved efficacy over docetaxel in ABCC4-expressing prostate cancer clinical outcome of prostate malignancy, the progressive cells. ABCC4 appears to be an important determinant of development of chemotherapy resistance in PCa remains a docetaxel resistance, since its inhibition almost completely problem and new chemotherapeutic agents that circumvent reversed resistance. resistance are urgently needed. Cabazitaxel was found to be such an agent, being accepted as the second-line cytotoxic Prostate carcinoma (PCa) is one of the most common types modality in metastatic CRPC. Its role is based on the of cancer in men and it is treated depending on the clinical possibility of exerting antitumor activity on docetaxel-resistant stage (1). When patients develop metastatic disease, the neoplasms (6-7). Clinical studies, including the Phase III trial treatment of choice is hormonal suppression, with the on the treatment of hormone-refractory metastatic PCa purpose of disease regression due to obtaining castration previously treated with docetaxel-containing regimen levels of testosterone. After an initial response to anti- (TROPIC study), demonstrated a 30% reduced risk of death, hormonal therapy, progression towards a castration-resistant associated with a prolonged survival up to 15.1 months (8), when using therapy of cabazitaxel-prednisone. Currently, cabazitaxel-prednisone is the only chemotherapeutic option with demonstrated survival advantage for the treatment of Correspondence to: A.A. Geldof, Ph.D., Department of Radiology metastatic CRPC that had progressed under docetaxel-based and Nuclear Medicine, Department of Urology, VU University chemotherapy (8, 9). Medical Center, P.O. Box 7057, 1007 MB Amsterdam, the Since chemotherapy resistance may be mediated by tumor Netherlands. Tel: +31 204445104, Fax: +31 204446031, e-mail: [email protected] cell overexpression of multidrug-resistance proteins (MRPs), it would be beneficial for new drugs if they did not act as Key Words: Prostate cancer cell lines, cabazitaxel, ABCC4, substrates for drug efflux pumps. It has been reported that docetaxel resistance. PCa may express ATP-cassette binding subfamily C member

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Figure 1. Effect of cabazitaxel and docetaxel concentrations on cellular proliferation (SRB assay) after three days of drug incubation on PC3 (a) and R3327-MATLyLu (b) prostate cancer cells in vitro. Data are mean values ±SEM (n=3).

4, also known as ABCC4, as compared to benign prostate Sulforhodamine B (SRB) assay. Evaluation of drug cytotoxic tissue (10, 11). Therefore we investigated whether ABCC4 effects was performed using the Sulforhodamine B (SRB) assay, influences docetaxel and cabazitaxel resistance in two cell as described elswhere (13). In short: 1500 MLL cells or 3500 PC3 cells were seeded in each well of 96-well plates (Cellstar #655180; lines with different ABCC4 expression levels. Greiner BioOne, Frickenhausen, Germany). After 24 h drugs were added in appropriate concentrations and cells were allowed to Materials and Methods proliferate for an additional three days. After this incubation time wells were treated with trichloroacetic acid (1 h at 4˚C) and Cell lines. PC3 human prostate cancer cell line (derived from a stained using SRB solution (0.4% SRB in 1% acetic acid). The metastasis in a Caucasian patient with prostate cancer) was optical density was measured at 492 nm after reconstitution of the originally obtained from the American Type Culture Collection, dye in 150 μl 10 mM Tris buffer. Extinction values at termination Rockville, MD., USA (ATCC# CRL 1435). of the incubation period served to measure the resulting cell The R3327-MATLyLu (MLL) prostate tumor variant was density and were compared with values for day 0 plates (start of maintained in castrated male Copenhagen rats by trocar drug incubation) to compare the cellular proliferation in the transplantation, and has been used as a source for in vitro culture. absence and in the presence of increasing drug concentrations. The establishment of cell culture for this cell line has been Values were normalized to the cell density of control cultures described elswhere (12). In vitro, both cell lines were cultured in (100%). Values for the concentration inhibiting 50% of cells (IC50) RPMI-1640 culture medium (Gibco BRL, Life Technologies Europe were subsequently derived graphically and are given as the mean BV, Bleiswijk, the Netherlands), supplemented with 10% fetal calf ±SEM (n=3). serum (Cambrex Fetal Calf Serum EU Standard,#14-801F; Lonza To study the effect of ABCC4 on drug sensitivity, MK571 was Verviers, Belgium), 100 U/ml penicillin/streptomycin (Gibco BRL), used. MK571 was added 30 min before incubation with Cabazitaxel 1 mM sodium pyruvate and insulin/transferrin/ selenite medium and Docetaxel (with and without inhibitor) to the cultures in a series supplement (Sigma-Aldrich Chemicals, St. Louis MO, USA) at of SRB assays. 37˚C in a humidified atmosphere of 5% CO2/95% air. Semi-annual screening demonstrated the cultures to be mycoplasma-free. Western blot. Western blotting of untreated cells was performed as described previously (14). Cells grown in log-phase were scraped Drugs. Docetaxel was obtained from Sigma-Aldrich Chemicals in lysis buffer (Cell signalling, Denver, Co, USA) and centrifuged (Zwijndrecht, the Netherlands) and was dissolved in at 11,000 g at 4˚C for 10 min. The protein concentration in the dimethylsulfoxide to prepare stock concentrations of 10 nM. supernatant was determined using a Bio-Rad protein assay Cabazitaxel was obtained from Sanofi-Aventis (Aventis Pharma, according to the manufacturer’s instructions (Bio-Rad Antony, France) and was dissolved in phosphate-buffered salt Laboratories, Veenendaal, the Netherlands). From each condition, solution (10 nM). The MRP inhibitor MK571 (Enzo Life Sciences 30 μg of protein were separated on sodium dodecyl sulfate 10% BVBA, Antwerp, Belgium) was kindly provided (courtesy of Dr. G. polyacrylamide gel electrophoresis (SDS-PAGE) and Jansen, Department of Rheumatology, VU University Medical electroblotted onto a polyvinylidenedifluoride (PVDF) membrane. Center, Amsterdam, the Netherlands). All stock solutions were Blots were blocked for 1 h at room temperature in 5% milk in stored in aliquots at –20˚C. TBST (0.15 M NaCl, 0.05% Tween-20, 10 mM Tris-HCl, pH 8.0)

388 Oprea-Lager et al: ABCC4 Effect on Cabazitaxel vs. Docetaxel in Prostate Cancer Cells

Table I. Concentration inhibiting 50% of cells (IC50) for cabazitaxel and docetaxel in prostate cancer cell lines PC3 and R3327-MATLyLu (in nM). Data are mean values and SEM of 3 to 6 experiments when treated with (+) and without (–) MK571.

Docetaxel Cabazitaxel

–MK571 +MK571 –MK571 +MK571

PC3 cells 0.8±0.3 0.8±0.4 0.9±0.7 1.3±0.6 MLL cells 8.6±5.1 4.2±3.2 1.9±1.1 1.6±0.8

Figure 2. Western blot of ATP-cassette binding protein 4 (ABCC4) blotted on cell extracts of PC3 and R3327-MATLyLu prostate tumor cells (with β-actin as a control). and subsequently incubated at 4˚C overnight with the anti-ABCC4 antibody (courtesy of Georg Scheffer, Ph.D., Department of Pathology, VU University Medical Center, Amsterdam), diluted at 1: 500 in TBST with 5% milk. After washing, the secondary (p<0.05) increase in sensitivity to docetaxel incubation of antibody was added and signal was detected using Enhanced MLL cells (about two-fold, to a value of 4.2 nM). The Chemo-luminescence (ECL) or ECL-plus on Hyperfilms ABCC4 inhibitor did not affect docetaxel sensitivity of PC3 (Amersham International, Chalfont St. Giles, UK). cells and did not affect sensitivity to cabazitaxel in either cell line (Table I). Analysis and statistics. Potential differences between treatment effects of cabazitaxel and docetaxel and differences between effects with and without the ABCC4 inhibitor were evaluated using the Discussion two-tailed Student’s t-test. Treatment effects were considered to be significantly different when p<0.05. In the present study an ABCC4-negative cell line was used next to a ABCC4-positive PCa cell line. This permits direct Results comparison of the effects of cabazitaxel and docetaxel in the two models and relates the cytotoxic effects to ABCC4 Effects of cabazitaxel and docetaxel on prostate cancer cell status. Docetaxel was found to be far less effective in the proliferation. Effects of docetaxel and cabazitaxel on cancer ABCC4-positive cell line, while cabazitaxel was equally cell proliferation were investigated using colorimetric SRB effective in both models. This finding is in line with our assays after incubation of drugs with logarithmically hypothesis that ABCC4 plays a functional role in the growing cultures of PC3 and MLL PCa cell lines. PC3 cells docetaxel resistance of PCa cells. The subsequent exhibited comparable sensitivity towards cabazitaxel and abolishment of such a difference in treatment effect by a docetaxel, with IC50 values of 0.9 and 0.8 nM, respectively specific ABCC4 inhibitor underscores the functional (Figure 1). MLL cells were less sensitive to the drugs significance of this phenotype. The current experimental data compared to PC3 cells, especially to docetaxel, with an IC50 therefore do confirm the expression and functionality of value of 8.6 nM (Figure 1 and Table I). Remarkably, MLL ABCC4 in PCa cells. Moreover, the differential effects on cells were ±4.5-fold less susceptible to docetaxel compared cell proliferation and viability by two chemically related to cabazitaxel. taxanes point to differences in the roles of drug transporters in these cells in general and of ABCC4 in specific. However, Expression of the multidrug resistance pump protein ABCC4. in addition to ABCC4, there might be a functional role for To determine whether the expression level of ABCC4 is other drug transporters in docetaxel resistance, since MK571 related to docetaxel or cabazitaxel sensitivity, its protein did not completely restore efficacy of docetaxel in the expression was determined by western blotting. In MLL ABCC4-positive cell line. cells, a clear band identifying ABCC4 was observed, while In the current article, we describe the differential effects no ABCC4 was detectable in PC3 cells (Figure 2). of cabazitaxel compared with docetaxel in two PCa cell lines with different ABCC4 phenotypes. Cabazitaxel was Inhibition studies using MK571. To study whether ABCC4 developed as an approach for therapy of docetaxel-resistant contributed to the docetaxel resistance in MLL cells, PCa. In this resistance phenomenon, drug transporter pumps ABCC4 was inhibited by MK571 before treatment with the are purported to play an important role by eliminating drugs. The resulting IC50 values clearly show a significant intracellular cytotoxic drugs from the cancer cells (15-17).

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The ABC transporter superfamily of membrane proteins is References best known for its contribution to chemoresistance through efflux of cancer drugs from cancer cells (for overview see 1 Siegel R, Ward E, Brawley O and Jemal A: Cancer statistics, 15, 18). One of these MRPs, ABCC4, has been described to 2011: The impact of eliminating socioeconomic and racial be expressed in normal prostate and PCa (10-11, 19). disparities on premature cancer deaths. CA Cancer J Clin 61: 212-236, 2011. Elevated ABCC4 expression has been found in malignant, 2 Heidenreich A, Bellmunt J, Bolla M, Joniau S, Mason M, compared to benign prostate tissue (11, 15), and is highly Matveev V, Mottet N, Schmid HP, van der KT, Wiegel T and expressed in both the cytoplasm and cell membrane Zattoni F: EAU guidelines on prostate cancer. Part I: Screening, (www.proteinatlas.org). Additionally, Cai et al. (10) diagnosis, and treatment of clinically localised disease. Actas described ABCC4 as being regulated by the androgen Eur Urol 59(1): 61-71, 2011. receptor both in androgen-dependent and androgen- 3 Petrylak DP, Tangen CM, Hussain MH, Lara PN, Jr., Jones JA, independent PCa cells. The exact molecular role of ABCC4 Taplin ME, Burch PA, Berry D, Moinpour C, Kohli M, Benson in the origin of docetaxel-resistant phenotype in PCa MC, Small EJ, Raghavan D and Crawford ED: Docetaxel and estramustine compared with mitoxantrone and prednisone for remains to be elucidated. advanced refractory prostate cancer. N Engl J Med 351: 1513- The lack of ABCC4 expression in PC3 cells is in line with 1520, 2004. earlier findings by Cai et al. (10). The expression of ABCC4 4 Michielsen DP, Braeckman JG and Denis L: Cabazitaxel for the in MLL cells has not been described before, to the best of treatment of prostate cancer. Expert Opin Pharmacother 12: 977- our knowledge. The poor therapeutic effect of docetaxel on 982, 2011. these latter cells is in accordance with the observed poor 5 Tannock IF, De Wit R, Berry WR, Horti J, Pluzanska A, Chi KN, therapeutic effect on this cell line in vivo, when grown in Oudard S, Theodore C, James ND, Turesson I, Rosenthal MA and Eisenberger MA: Docetaxel plus prednisone or mitoxantrone syngeneic Copenhagen rats (studies from this laboratory, plus prednisone for advanced prostate cancer. N Engl J Med 351: Jacobs et al., unpublished data). 1502-1512, 2004. Since all patients with CRPC with a good clinical status 6 de Bono JS, Oudard S, Ozguroglu M, Hansen S, Machiels JP, may benefit from chemotherapy, independently of their age, Kocak I, Gravis G, Bodrogi I, Mackenzie MJ, Shen L, Roessner new treatment options that prolong survival and improve the M, Gupta S and Sartor AO: Prednisone plus cabazitaxel or quality of life are needed (20). Therefore it is essential to mitoxantrone for metastatic castration-resistant prostate cancer define new biomarkers that can predict docetaxel resistance progressing after docetaxel treatment: A randomised open-label trial. Lancet 376: 1147-1154, 2010. and, consequently, to early identify the category of patients 7 Paller CJ and Antonarakis ES: Cabazitaxel: A novel second-line in whom recurrence on docetaxel therapy may arise. This treatment for metastatic castration-resistant prostate cancer. Drug could result in improved patient selection strategies (20). For Des Devel Ther 5: 117-124, 2011. example, patients that have a high ABCC4 expression in PCa 8 Parente P, Parnis F and Gurney H: Emerging and second line tissue can be selected for first-line cabazitaxel therapy. therapies for the management of metastatic castration-resistant Moreover, ABCC4 as a potential predictive marker for prostate cancer: The Australian perspective. Asia-Pac J Clin docetaxel-based therapy responsiveness should be studied in Oncol 8: 31-42, 2012. 9 Higano CS and Crawford ED: New and emerging agents for the future trials. treatment of castration-resistant prostate cancer. Urol Oncol 29(6 Suppl): S1-S8, 2011. Conclusion 10 Cai C, Omwancha J, Hsieh CL and Shemshedini L: Androgen induces expression of the multidrug resistance protein gene MRP4 in prostate cancer cells. Prostate Cancer Prostatic Dis 10: In conclusion, our data indicate that ABCC4 may be an 39-45, 2007. important regulator for docetaxel sensitivity in PCa cells, 11 Ho LL, Kench JG, Handelsman DJ, Scheffer GL, Stricker PD, Grygiel JG, Sutherland RL, Henshall SM, Allen JD and Horvath while cabazitaxel has therapeutic efficacy independently of LG: Androgen regulation of multidrug resistance-associated protein ABCC4 expression. Since many prostate carcinomas have a 4 (MRP4/ABCC4) in prostate cancer. Prostate 68: 1421-1429, 2008. relatively high expression of ABCC4 protein (10), this 12 Geldof AA, Rao BR and De Voogt HJ: Direct effects of underlines the potential advantage of using cabazitaxel over chemotherapeutic agents on rat prostate tumor clonogenic cells. docetaxel. The utility of assessing ABCC4 expression in PCa Anticancer Res 6: 837-840, 1986. tissues, as a patient selection criterion for first-line 13 Bijnsdorp IV, Kruyt FA, Gokoel S, Fukushima M and Peters GJ: chemotherapy, should be explored. Synergistic interaction between trifluorothymidine and docetaxel is sequence dependent. Cancer Sci 99: 2302-2308, 2008. Disclosure 14 Bijnsdorp IV, Peters GJ, Temmink OH, Fukushima M and Kruyt FA: Differential activation of cell death and autophagy results in an increased cytotoxic potential for trifluorothymidine compared The Authors report they have no conflicts of interest in to 5-fluorouracil in colon cancer cells. Int J Cancer 126: 2457- regard to this work. 2468, 2010.

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15 Chen ZS and Tiwari AK: Multidrug-resistance proteins 18 Van de Ven R, Oerlemans R, van der Heijden JW, Scheffer GL, (MRPs/ABCCs) in cancer chemotherapy and genetic diseases. de Gruijl TD, Jansen G and Scheper RJ: ABC drug transporters FEBS J 278: 3226-3245, 2011. and immunity: Novel therapeutic targets in autoimmunity and 16 Diestra JE, Condom E, Del M, X, Scheffer GL, Perez J, Zurita cancer. J Leukoc Biol 86: 1075-1087, 2009. AJ, Munoz-Segui J, Vigues F, Scheper RJ, Capella G, Germa- 19 Rius M, Thon WF, Keppler D and Nies AT: Prostanoid transport Lluch JR and Izquierdo MA: Expression of multidrug resistance by multidrug-resistance protein 4 (MRP4/ABCC4) localized in proteins P-glycoprotein, multidrug-resistance protein 1, breast tissues of the human urogenital tract. J Urol 174: 2409-2414, cancer-resistance protein and lung-resistance-related protein in 2005. locally advanced bladder cancer treated with neoadjuvant 20 Sartor O: Progression of metastatic castrate-resistant prostate chemotherapy: Biological and clinical implications. J Urol 170: cancer: Impact of therapeutic intervention in the post-docetaxel 1383-1387, 2003. space. J Hematol Oncol 4: 18-25, 2011. 17 Sanchez C, Mercado A, Contreras HR, Mendoza P, Cabezas J, Acevedo C, Huidobro C and Castellon EA: Chemotherapy sensitivity recovery of prostate cancer cells by functional Received November 13, 2012 inhibition and knock-down of multidrug-resistance proteins. Revised November 30, 2012 Prostate 71: 1810-1817, 2011. Accepted December 3, 2012

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