Colao, A. (Annamaria)

Feelders, R.A. (Richard)

Herder, W.W. (Wouter) de

Hofland, L.J. (Leo)

Koetsveld, P.M. (Peter) van

Lamberts, S.W.J. (Steven)

Martino, M.C. (Maria Cristina) de

Pivonello, R. (Rosario)

Erasmus University Digital Repository

RESEARCH LETTEREffects of combination treatment with sirolimus and mitotaneon growth of human adrenocortical carcinoma cellsMaria Cristina De Martino1 • Peter M. van Koetsveld1 • Richard A. Feelders1 •Steven W. J. Lamberts1 • Wouter W. de Herder1 • Annamaria Colao2 •Rosario Pivonello2 • Leo J. Hofland1Received: 29 July 2015 / Accepted: 20 October 2015 / Published online: 8 December 2015 Springer Science+Business Media New York 2015IntroductionAdrenocortical carcinoma (ACC) is a rare but aggressivesolid cancer with a 5-year survival rate of \15 % at themetastatic stage [1]. Surgery remains the only curativetreatment in patients diagnosed at an early stage [1, 2].Mitotane, is currently the only drug approved in Europeand in the United States for the treatment of advanced ACC[2]. As monochemotherapy, a response rate of mitotanebetween 13–35 % has been reported [1, 2]. However,patients achieving a plasma mitotane level above 14 mg/Lshow a higher response rate and an improved survival [1,2]. In combination with chemotherapy, response rates ofmitotane range between 14–55 % [1–3]. No novel treat-ment option has emerged in the last four decades [1, 2],underlining the urgent need of new therapeutic options forpatients affected by this malignancy.The mammalian target of rapamycin (mTOR) pathway isconsidered a target for antineoplastic therapy, and in pre-clinical models of ACC, mTOR inhibitors such as sirolimus,temsirolimus, and everolimus inhibit cell proliferation in adose- and time-dependent manner [4–6]. However, differenthuman ACC cell lines and primary cultures show a variableresponse to mTOR inhibitors [7, 8]. In the absence of a clearpredictor of the effectiveness of mTOR inhibitors in thismalignancy, it is difficult to define selection criteria forpatients, who are candidates for these drugs [1, 7–9].Therefore, combination of mTOR inhibitors with mitotanecould be a more prudent clinical approach than the use ofthese inhibitors as monotherapy in unselected ACC patients.This study aimed at evaluating the effects of mitotane incombination with sirolimus in the two human ACC celllines H295 and the SW13.Materials and methodsTwo human ACC cell lines the NCI-H295R (H295) and theSW13, were obtained and cultured as previously described[7]. Mitotane was purchased from Sigma-Aldrich and dis-solved in ethanol as a concentrated (10-2 M) stock solution(stored at -20 C) and diluted in ethanol prior to use. ThemTOR inhibitor sirolimus was obtained from LC Labora-tories and used as previously described [7]. The doses ofsirolimus used were selected on the bases of the previouslyreported dose–response curves of sirolimus in the used celllines [7]. The measurement of total DNA content was usedas a measure of cell proliferation and performed as previ-ously described [7]. For the statistical analysis, statisticalsoftware of GraphPad Prism version 5 (GraphPhad Soft-ware, San Diego, CA) was used.ResultsIn both H295 and SW13, mitotane significantly inhibits cellproliferation in a dose-dependent manner (Fig. 1). Mito-tane was slightly, but significantly more potent in H295(IC50 4.5 9 10-6 M) than in SW13 (IC501.6 9 10-5 M)(p\ 0.01).& Leo J. Hoflandl.hofland@erasmusmc.nl1 Division of Endocrinology, Department of Internal Medicine,Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam,The Netherlands2 Dipartimento di Medicina Clinica e Chirurgia, Sezione diEndocrinologia, Uniniversita` Federico II di Napoli, Via S.Pansini 5, 80131 Naples, Italy123Endocrine (2016) 52:664–667DOI 10.1007/s12020-015-0818-0In both H295 and SW13, the selected concentrations ofsirolimus significantly inhibited cell proliferation (24–45 %and 57–70 %, respectively; p\ 0.001).When mitotane was used at low concentrations (between10-7 and 5 9 10-6M), sirolimus had a statistically sig-nificant additive effect, when compared with mitotanealone (Fig. 1).In H295, as compared with sirolimus alone, mitotane ata concentration higher than 10-7 M had significant addi-tive effects when combined with both the concentrations ofsirolimus tested (p\ 0.01 or p\ 0.001; Fig. 1a). In SW13,as compared with sirolimus alone, mitotane had significantadditive effects only when the highest concentration ofmitotane (10-5 M) was combined with the lowest con-centration of sirolimus (5 9 10-11 M) (p\ 0.05; Fig. 1b).DiscussionThe current study demonstrates that in human ACC celllines, sirolimus has additive antiproliferative effects whencombined with low mitotane doses.Mitotane is a referral drug in the treatment of ACCpatients, but unfortunately there are patients who do notControl M-7Mito 10M-6Mito 10M-6Mito 2.5*10M-6Mito 5*10M-6Mito 7.5*10M-5Mito 10 Control M-9S 5*10M-9M+S 5*10-7Mito 10M-9M+S 5*10-6Mito 10M-9M+S 5*10-6Mito 2.5*10M-9M+S 5*10-6Mito 5*10M-9M+S 5*10-6Mito 7.5*10M-9M+S 5*10-5Mito 10Control M-6S 10M-6M+S 10-7Mito 10M-6M+S 10-6Mito 10M-6M+S 10-6Mito 2.5*10M-6M+S 10-6Mito 5*10M-6M+S 10-6Mito 7.5*10M-6M+S 10-5Mito 10020406080100120****************xxx xxxxxxxxxxxxxxx xxxxxx xxxxxxxxxxxxxxx xxxxxxxxxxxxxxxx xxx% of controlControl M-7Mito 10M-6Mito 10M-6Mito 2.5*10M-6Mito 5*10M-6Mito 7.5*10M-5Mito 10 Control M-11S 5*10M-11+S 5*10-7Mito 10M-11+S 5*10-6Mito 10M-11+S 5*10-6Mito2.5*10M-11+S 5*10-6Mito 5*10M-11+S 5*10-6Mito 7.5*10M-11+S 5*10-5Mito 10Control M-10S 10M-10+S 10-7Mito 10M-10+S 10-6Mito 10M-10+S 10-6Mito 2.5*10M-10+S 10-6Mito 5*10M-10+S 10-6Mito 7.5*10-10M+S 10-5Mito 10020406080100120***********xxxxxxxxxxxxxxxxxxxxxxx xxx xxxxxxxxxxxx xxxxxx xxx xxxxxx xxx      SW13      H295ABFig. 1 Combined effects of a6-day treatment with increasingconcentrations of mitotane(Mito) and selectedconcentrations of sirolimus(S) in two human ACC celllines: H295 (panel a) and SW13(panel b). At some of thecombinations tested significantadditive inhibitory effects oncell growth are observed in bothcell lines. *p\ 0.05,**p\ 0.01 and ***p\ 0.001;xp\ 0.05, xxp\ 0.01 andxxxp\ 0.001 versus controlEndocrine (2016) 52:664–667 665123respond and/or tolerate the drug, raising the requirement ofnew treatment options [1, 2].The mitotane therapeutic window is very narrow.Therefore, monitoring mitotane plasma levels duringtreatment is very important. A response rate of up to 66 %has been reported in patients achieving mitotane plasmalevel above 14 mg/L, but a higher rate of adverse effects isreported when the plasma mitotane level exceeds 20 mg/L.A rapid achievement and long-term maintenance of thistherapeutic range (14–20 mg/L) has been suggested as apredictor of mitotane response in patients with ACC [1, 2,10, 11]. However, this clinical goal is sometimes difficultto reach because of the complex pharmacokinetic ofmitotane which causes a large variation in individual drugavailability. Additionally, the onset of adverse events canpreclude a fast drug escalation or the use of a high mitotanedose. Treatment strategies combining mitotane with otherdrugs could increase the response rate of patients, ascompared with monotherapy, for several reasons. Thecombination could have additive antiproliferative effectsthat potentially increase the in vivo antineoplastic effects.On the other hand, in absence of appropriate predictors of aclinical response that can help to select patients for themost appropriate treatment, the combination of two drugsacting with different mechanisms, could increase thechance of patients to get a clinical benefit from at least oneof the two treatments. Additionally, in particular in the caseof mitotane, the combined treatment with other activedrugs could reduce the risk of tumor progression during thetreatment initial mitotane dosage titration.Recently, mTOR inhibitors have been suggested as anew potential treatment for ACC. Preclinical data suggestsirolimus, temsirolimus, and everolimus can inhibit ACCcell proliferation [4–6]. However, preliminary experiencewith the use of everolimus as salvage treatment in fewACC patients did not show promising results [12], whereascombined treatment of an insulin like growth factor 1receptor (IGF1R) antibody with temsirolimus in a phase Istudy including ACC patients, showed more promisingresults [13]. Therefore, combination treatment with mTORinhibitors and other drugs might have higher effects thanmTOR inhibitors alone. To our knowledge, there are noin vitro studies evaluating the effects of mitotane in com-bination with mTOR inhibitors.In the current study, all the concentrations of mitotaneused (from 10-7 to 10-5 M) were lower than the concen-trations (4.3–6.3 9 10-5 M) corresponding to the mitotaneplasma level at the therapeutic range [14]. The addition ofsirolimus to these low concentrations of mitotane showedhigher antiproliferative effects than mitotane alone sug-gesting that combined treatment might have additiveeffects to the antineoplastic action of mitotane, permittingto reduce the dose required to obtain desired clinicaleffects. This additivity was higher when the concentrationof mitotane used were lower, suggesting that combinedtreatment might be particularly useful during the phases oftreatment in which mitotane plasma level are below thetherapeutic range, such as during the initial dose titrationand/or for those patients in which the therapeutic range ofmitotane is hardly maintained due to low tolerance or otherreasons.With the exception of 10-6 M sirolimus used in H295cells, all the tested concentrations where within the range ofblood drug levels reached in humans during sirolimustreatment (maximal concentration about 10-7 M) [15, 16].In both cell lines, the addition of mitotane to sirolimusshowed a significant additive inhibitory effect as comparedwith sirolimus alone, but only at some of the experimentalconditions tested. Particularly, in SW13 cells that are moresensitive to sirolimus, but less sensitive to mitotane thanH295, the addition of mitotane showed a significant additiveinhibitory effect as compared to sirolimus alone, only whenthe highest mitotane concentration was combined with avery low sirolimus concentration. This might be related tothe potent inhibitory effects of sirolimus alone in SW13cells, which induced already a near maximal inhibition ofcell proliferation. While H295 cells are well accepted as agood model of ACC, a debate is still open about theappropriateness of SW13 cells as a model for this type ofcancer [17]. Taking the other potential limitations of ACCcell lines as preclinical model of ACC, the results of thecurrent study might suggest that among ACC patients itcould be possible to find a subgroup of patients with a highsensitivity to sirolimus, in which the use of combinedtreatment could induce an important antineoplastic effecteven in the absence of mitotane effect, such as in mitotane-resistant patients.Among the potential limitations of the current study,there is the lack of possibility to explore pharmacokineticinteractions between drugs. In the in vivo setting, mitotaneis a well known inducer of microsomal liver enzymecytochrome P450 (CYP3A4/5) [18]. The induction of thisenzyme can reduce the circulating concentrations of drugsmetabolized by it, including mTOR inhibitors [19]. Thispharmacokinetic interaction should be kept in mind whentranslating the in vitro results to the potential in vivoapplications, and attention should be used in monitoring theblood concentrations of drugs to reach the desired thera-peutic levels.In conclusion, this study suggests a potential advantageof combining mitotane with sirolimus, but these data arestill preliminary. Clearly, additional studies, includinganimal models, are mandatory before moving from thebench to the bedside.666 Endocrine (2016) 52:664–667123Compliance with ethical standardsConflict of interest The authors declare that there is no conflict ofinterest that could be perceived as prejudicing the impartiality of theresearch reported.References1. E. Baudin, S. Leboulleux, A. AlGhuzlan, C. Chougnet, J. Young,D. Deandreis, F. Dumont, F. Dechamps, C. Caramella, P.Chanson, E. Lanoy, I. Borget, M. Schlumberger, Therapeuticmanagement of advanced adrenocortical carcinoma: what do weknow in 2011? Horm. Cancer 2(6), 363–371 (2011)2. A. Berruti, E. Baudin, H. Gelderblom, H.R. Haak, F. Porpiglia, G.Pentheroudakis, ESMO Guidelines: Working Group, Adrenalcancer: ESMO Clinical Practice Guidelines for diagnosis, treat-ment and follow-up. Ann. Oncol. 23((suppl 7)), Vii131 (2012)3. M. Fassnacht, M. Terzolo, B. Allolio, E. Baudin, H. Haak, A.Berruti, S. Welin, C. Schade-Brittinger, A. Lacroix, B. Jarzab, H.Sorbye, D.J. Torpy, V. Stepan, D.E. Schteingart, W. Arlt, M.Kroiss, S. Leboulleux, P. Sperone, A. Sundin, I. Hermsen, S.Hahner, H.S. Willenberg, A. Tabarin, M. Quinkler, C. de laFouchardiere, M. Schlumberger, F. Mantero, D. Weismann, F.Beuschlein, H. Gelderblom, H. Wilmink, M. Sender, M. Edgerly,W. Kenn, T. Fojo, H.H. Muller, B. Skogseid, Combinationchemotherapy in advanced adrenocortical carcinoma. N. Engl.J. Med. 366(23), 2189–2197 (2012)4. M.C. De Martino, P.M. van Koetsveld, R.A. Feelders, D. Sprij-Mooij, M.M. Waaijers, S.W. Lamberts, W.W. de Herder, A.A.Colao, R. Pivonello, L. Hofland, The role of mTOR inhibitors in theinhibition of growth and cortisol secretion in human adrenocorticalcarcinoma cells. Endocr Relat Cancer 19, 351 (2012)5. M.C. De Martino, P.M. van Koetsveld, R. Pivonello, L.J. Hof-land, Role of the mTOR pathway in normal and tumoral adrenalcells. Neuroendocrinology 92(Suppl 1), 28–34 (2010). doi:10.1159/0003142806. M. Doghman, A. ElWakil, B. Cardinaud, E. Thomas, J. Wang,W. Zhao, M.H. Peralta-DelValle, B.C. Figueiredo, G.P. Zambetti,E. Lalli, Regulation of insulin-like growth factor-mammaliantarget of rapamycin signaling by microRNA in childhoodadrenocortical tumors. Cancer Res. 70(11), 4666–4675 (2010)7. M.C. De Martino, P.M. van Koetsveld, R.A. Feelders, D. Sprij-Mooij, M. Waaijers, S.W. Lamberts, W.W. de Herder, A. Colao,R. Pivonello, L.J. Hofland, The role of mTOR inhibitors in theinhibition of growth and cortisol secretion in human adrenocor-tical carcinoma cells. Endocr. Relat. Cancer 19(3), 351–364(2012). doi:10.1530/ERC-11-02708. M.C. De Martino, A. AlGhuzlan, S. Aubert, G. Assie, J.Y.Scoazec, S. Leboulleux, C. DoCao, R. Libe, C. Nozieres, M.Lombes, F. Pattou, F. Borson-Chazot, S. Hescot, C. Mazoyer, J.Young, I. Borget, A. Colao, R. Pivonello, J.C. Soria, J. Bertherat,M. Schlumberger, L. Lacroix, E. Baudin, Molecular screening fora personalized treatment approach in advanced adrenocorticalcancer. J Clin Endocrinol Metab 98(10), 4080–4088 (2013).doi:10.1210/jc.2013-21659. M.C. De Martino, R.A. Feelders, W.W. de Herder, P.M. vanKoetsveld, F. Dogan, J.A. Janssen, A.M. Waaijers, C. Pivonello,S.W. Lamberts, A. Colao, R.R. de Krijger, R. Pivonello, L.J.Hofland, Characterization of the mTOR pathway in humannormal adrenal and adrenocortical tumors. Endocr. Relat. Cancer21(4), 601–613 (2014). doi:10.1530/ERC-13-011210. I.G. Hermsen, M. Fassnacht, M. Terzolo, S. Houterman, J. denHartigh, S. Leboulleux, F. Daffara, A. Berruti, R. Chadarevian,M. Schlumberger, B. Allolio, H.R. Haak, E. Baudin, Plasmaconcentrations of o, p’DDD, o, p’DDA, and o, p’DDE as pre-dictors of tumor response to mitotane in adrenocortical carci-noma: results of a retrospective ENS@T multicenter study.J. Clin. Endocrinol. Metab. 96(6), 1844–1851 (2011). doi:10.1210/jc.2010-267611. B. Wangberg, A. Khorram-Manesh, S. Jansson, B. Nilsson, O.Nilsson, C.E. Jakobsson, S. Lindstedt, A. Oden, H. Ahlman, Thelong-term survival in adrenocortical carcinoma with active sur-gical management and use of monitored mitotane. Endocr. Relat.Cancer 17(1), 265–272 (2010). doi:10.1677/ERC-09-019012. M. Fraenkel, M. Gueorguiev, D. Barak, A. Salmon, A.B.Grossman, D.J. Gross, Everolimus therapy for progressiveadrenocortical cancer. Endocrine 44(1), 187–192 (2013). doi:10.1007/s12020-013-9878-113. A. Naing, P. Lorusso, S. Fu, D. Hong, H.X. Chen, L.A. Doyle,A.T. Phan, M.A. Habra, R. Kurzrock, Insulin growth factorreceptor (IGF-1R) antibody cixutumumab combined with themTOR inhibitor temsirolimus in patients with metastaticadrenocortical carcinoma. Br. J. Cancer 108(4), 826–830 (2013).doi:10.1038/bjc.2013.4614. P.M. van Koetsveld, G. Vitale, R.A. Feelders, M. Waaijers, D.M.Sprij-Mooij, R.R. de Krijger, E.J. Speel, J. Hofland, S.W. Lam-berts, W.W. de Herder, L.J. Hofland, Interferon-beta is a potentinhibitor of cell growth and cortisol production in vitro andsensitizes human adrenocortical carcinoma cells to mitotane.Endocr. Relat. Cancer 20(3), 443–454 (2013). doi:10.1530/ERC-12-021715. A. O’Donnell, S. Faivre, H.A. Burris 3rd, D. Rea, V. Papadimi-trakopoulou, N. Shand, H.A. Lane, K. Hazell, U. Zoellner, J.M.Kovarik, C. Brock, S. Jones, E. Raymond, I. Judson, Phase Ipharmacokinetic and pharmacodynamic study of the oral mam-malian target of rapamycin inhibitor everolimus in patients withadvanced solid tumors. J. Clin. Oncol. 26(10), 1588–1595 (2008).doi:10.1200/JCO.2007.14.098816. M.B. Atkins, M. Hidalgo, W.M. Stadler, T.F. Logan, J.P.Dutcher, G.R. Hudes, Y. Park, S.H. Liou, B. Marshall, J.P. Boni,G. Dukart, M.L. Sherman, Randomized phase II study of multipledose levels of CCI-779, a novel mammalian target of rapamycinkinase inhibitor, in patients with advanced refractory renal cellcarcinoma. J. Clin. Oncol. 22(5), 909–918 (2004). doi:10.1200/JCO.2004.08.18517. T. Wang, W.E. Rainey, Human adrenocortical carcinoma celllines. Mol. Cell Endocrinol. 351(1), 58–65 (2012). doi:10.1016/j.mce.2011.08.04118. M. Kroiss, M. Quinkler, W.K. Lutz, B. Allolio, M. Fassnacht,Drug interactions with mitotane by induction of CYP3A4 meta-bolism in the clinical management of adrenocortical carcinoma.Clin. Endocrinol. (Oxf) 75(5), 585–591 (2011). doi:10.1111/j.1365-2265.2011.04214.x19. J. Boni, C. Leister, J. Burns, M. Cincotta, B. Hug, L. Moore,Pharmacokinetic profile of temsirolimus with concomitantadministration of cytochrome p450-inducing medications. J. Clin.Pharmacol. 47(11), 1430–1439 (2007). doi:10.1177/0091270007306957Endocrine (2016) 52:664–667 667123

Effects of combination treatment with sirolimus and mitotane on growth of human adrenocortical carcinoma cells

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Effects of combination treatment with sirolimus and mitotane on growth of human adrenocortical carcinoma cells

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