Abstract
Purpose
Mitotane is the only chemotherapeutic agent available for the treatment of adrenocortical carcinoma (ACC), however, the anti-neoplastic efficacy is limited due to several side-effects in vivo. There is, therefore, a need of exploring for new anti-tumoral agents which can be used either alone or in combination with mitotane. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) acts as an anti-proliferative agent in human cancer by inhibiting the Wnt/beta-catenin pathway through the vitamin D receptor (VDR). The aim of this study was to study the effects of mitotane and 1α,25(OH)2D3, individually or in combination, in an in vitro model with H295R ACC cells, and to elucidate the molecular events behind their effects involving the Wnt/beta-catenin signaling.
Methods and results
Multiple concentrations of mitotane and 1α,25(OH)2D3, individually or in combination, were tested on H295R cells for 24–96 h, and the effects analysed by MTT. A reduction in cell growth was observed in a dose/time-dependent manner for both mitotane and 1α,25(OH)2D3. In addition, a combination of clinically sub-therapeutic concentrations of mitotane with 1α,25(OH)2D3, had an additive anti-proliferative effect (Combination Index = 1.02). In a wound healing assay, individual treatments of both mitotane and 1α,25(OH)2D3 reduced the migration ability of H295R cells, with the effect further enhanced on combining both the agents. Western blotting and qRT-PCR analysis showed a modulation of the Wnt/beta-catenin and VDR signaling pathways.
Conclusion
Our results show an additive effect of mitotane and 1α,25(OH)2D3 on the inhibition of H295R ACC cell growth and viability, and suggest that molecular mechanisms of their effects involve a functional link between VDR and Wnt/beta-catenin pathways.
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References
Else T, Kim AC, Sabolch A, Raymond VM, Kandathil A, Caoili EM, Jolly S, Miller BS, Giordano TJ, Hammer GD (2014) Adrenocortical carcinoma. Endocr Rev 35(2):282–326
Stigliano A, Chiodini I, Giordano R, Faggiano A, Canu L, Della Casa S, Loli P, Luconi M, Mantero F, Terzolo M (2016) Management of adrenocortical carcinoma: a consensus statement of the Italian Society of Endocrinology (SIE). J Endocrinol Investig 39(1):103–121
Paragliola RM, Torino F, Papi G, Locantore P, Pontecorvi A, Corsello SM (2018) Role of mitotane in adrenocortical carcinoma—review and state of the art. Eur Endocrinol 14(2):62–66
Fassnacht M, Dekkers OM, Else T, Baudin E, Berruti A, de Krijger R, Haak HR, Mihai R, Assie G, Terzolo M (2018) European Society of Endocrinology Clinical Practice Guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol 179(4):G1–G46
Waszut U, Szyszka P, Dworakowska D (2017) Understanding mitotane mode of action. J Physiol Pharmacol 68(1):13–26
Terzolo M, Baudin AE, Ardito A, Kroiss M, Leboulleux S, Daffara F, Perotti P, Feelders RA, deVries JH, Zaggia B, De Francia S, Volante M, Haak HR, Allolio B, Al Ghuzlan A, Fassnacht M, Berruti A (2013) Mitotane levels predict the outcome of patients with adrenocortical carcinoma treated adjuvantly following radical resection. Eur J Endocrinol 169(3):263–270
Rosen CJ, Adams JS, Bikle DD, Black DM, Demay MB, Manson JE, Murad MH, Kovacs CS (2012) The nonskeletal effects of vitamin D: an Endocrine Society scientific statement. Endocr Rev 33(3):456–492
Ma Y, Trump DL, Johnson CS (2010) Vitamin D in combination cancer treatment. J Cancer 1:101–107
Vuolo L, Di Somma C, Faggiano A, Colao A (2012) Vitamin D and cancer. Front Endocrinol (Lausanne) 3:58
Feldman D, Krishnan AV, Swami S, Giovannucci E, Feldman BJ (2014) The role of vitamin D in reducing cancer risk and progression. Nat Rev Cancer 14(5):342–357
Yang L, Ma J, Zhang X, Fan Y, Wang L (2012) Protective role of the vitamin D receptor. Cell Immunol 279(2):160–166
Leyssens C, Verlinden L, Verstuyf A (2013) Antineoplastic effects of 1,25(OH)2D3 and its analogs in breast, prostate and colorectal cancer. Endocr Relat Cancer 20(2):R31–R47
Umar M, Sastry KS, Chouchane AI (2018) Role of vitamin D beyond the skeletal function: a review of the molecular and clinical studies. Int J Mol Sci 19(6):1618
Johnson AL, Zinser GM, Waltz SE (2015) Vitamin D3-dependent VDR signaling delays ron-mediated breast tumorigenesis through suppression of β-catenin activity. Oncotarget 6(18):16304–16320
Ferrer-Mayorga G, Larriba MJ, Crespo P, Muñoz A (2019) Mechanisms of action of vitamin D in colon cancer. J Steroid Biochem Mol Biol 185:1–6
Muscogiuri G, Altieri B, Penna-Martinez M, Badenhoop K (2015) Focus on vitamin D and the adrenal gland. Horm Metab Res 47(4):239–246
Tirabassi G, Salvio G, Altieri B, Ronchi CL, Della Casa S, Pontecorvi A, Balercia G (2017) Adrenal disorders: is there any role for vitamin D? Rev Endocr Metab Disord 18(3):355–362
Pilon C, Urbanet R, Williams TA, Maekawa T, Vettore S, Sirianni R, Pezzi V, Mulatero P, Fassina A, Sasano H, Fallo F (2014) 1α,25-Dihydroxyvitamin D3 inhibits the human H295R cell proliferation by cell cycle arrest: a model for a protective role of vitamin D receptor against adrenocortical cancer. J Steroid Biochem Mol Biol 140:26–33
Pilon C, Rebellato A, Urbanet R, Guzzardo V, Cappellesso R, Sasano H, Fassina A, Fallo F (2015) Methylation status of vitamin D receptor gene promoter in benign and malignant adrenal tumors. Int J Endocrinol 2015:375349
Chou TC (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70(2):440–446
Fallo F, Pilon C, Barzon L, Pistorello M, Pagotto U, Altavilla G, Boscaro M, Sonino N (1996) Effects of taxol on the human NCI-H295 adrenocortical carcinoma cell line. Endocr Res 22(4):709–715
Drelon C, Berthon A, Mathieu M, Ragazzon B, Kuick R, Tabbal H, Septier A, Rodriguez S, Batisse-Lignier M, Sahut-Barnola I, Dumontet T, Pointud JC, Lefrançois-Martinez AM, Baron S, Giordano TJ, Bertherat J, Martinez A, Val P (2016) EZH2 is overexpressed in adrenocortical carcinoma and is associated with disease progression. Hum Mol Genet 25(13):2789–2800
Aronova A, Min IM, Crowley MJP, Panjwani SJ, Finnerty BM, Scognamiglio T, Liu YF, Whitsett TG, Garg S, Demeure MJ, Elemento O, Zarnegar R, Fahey TJ III (2018) STMN1 is overexpressed in adrenocortical carcinoma and promotes a more aggressive phenotype in vitro. Ann Surg Oncol 25(3):792–800
Rubin B, Regazzo D, Redaelli M, Mucignat C, Citton M, Iacobone M, Scaroni C, Betterle C, Mantero F, Fassina A, Pezzani R, Boscaro M (2010) Investigation of N-cadherin/β-catenin expression in adrenocortical tumors. Tumour Biol 37(10):13545–13555
Wang T, Rainey WE (2012) Human adrenocortical carcinoma cell lines. Mol Cell Endocrinol 351(1):58–65
Minisola S, Ferrone F, Danese V, Cecchetti V, Pepe J, Cipriani C, Colangelo L (2019) Controversies surrounding vitamin D: focus on supplementation and cancer. Int J Environ Res Public Health 16(2):189
Ebeling PR, Adler RA, Jones G, Liberman UA, Mazziotti G, Minisola S, Munns CF, Napoli N, Pittas AG, Giustina A, Bilezikian JP, Rizzoli R (2018) Management of endocrine disease: therapeutics of vitamin D. Eur J Endocrinol 179(5):R239–R259
Scragg RKR (2019) Overview of results from the vitamin D assessment (ViDA) study. J Endocrinol Investig. https://doi.org/10.1007/s40618-019-01056-z(Epub ahead of print)
Poli G, Guasti D, Rapizzi E, Fucci R, Canu L, Bandini A, Cini N, Bani D, Mannelli M, Luconi M (2013) Morphofunctional effects of mitotane on mitochondria in human adrenocortical cancer cells. Endocr Relat Cancer 20(4):537–550
Germano A, Rapa I, Volante M, Lo Buono N, Carturan S, Berruti A, Terzolo M, Papotti M (2014) Cytotoxic activity of gemcitabine, alone or in combination with mitotane, in adrenocortical carcinoma cell lines. Mol Cell Endocrinol 382(1):1–7
De Martino MC, van Koetsveld PM, Feelders RA, Lamberts SW, de Herder WW, Colao A, Pivonello R, Hofland LJ (2016) Effects of combination treatment with sirolimus and mitotane on growth of human adrenocortical carcinoma cells. Endocrine 52(3):664–667
Boulate G, Amazit L, Naman A, Seck A, Paci A, Lombes A, Pussard E, Baudin E, Lombes M, Hescot S (2019) Potentiation of mitotane action by rosuvastatin: new insights for adrenocortical carcinoma management. Int J Oncol 54(6):2149–2156
Lehmann T, Wrzesinski T (2012) The molecular basis of adrenocortical cancer. Cancer Genet 205(4):131–137
Armignacco R, Cantini G, Canu L, Poli G, Ercolino T, Mannelli M, Luconi M (2018) Adrenocortical carcinoma: the dawn of a new era of genomic and molecular biology analysis. J Endocrinol Investig 41(5):499–507
El Wakil A, Lalli E (2011) The Wnt/beta-catenin pathway in adrenocortical development and cancer. Mol Cell Endocrinol 332(1–2):32–37
Berthon A, Martinez A, Bertherat J, Val P (2012) Wnt/β-catenin signalling in adrenal physiology and tumour development. Mol Cell Endocrinol 351(1):87–95
Heaton JH, Wood MA, Kim AC, Lima LO, Barlaskar FM, Almeida MQ, Fragoso MC, Kuick R, Lerario AM, Simon DP, Soares IC, Starnes E, Thomas DG, Latronico AC, Giordano TJ, Hammer GD (2012) Progression to adrenocortical tumorigenesis in mice and humans through insulin-like growth factor 2 and β-catenin. Am J Pathol 181(3):1017–1033
Gaujoux S, Tissier F, Groussin L, Libé R, Ragazzon B, Launay P, Audebourg A, Dousset B, Bertagna X, Bertherat J (2008) Wnt/beta-catenin and 3′,5′-cyclic adenosine 5′-monophosphate/protein kinase A signaling pathways alterations and somatic beta-catenin gene mutations in the progression of adrenocortical tumors. J Clin Endocrinol Metab 93(10):4135–4140
Tissier F, Cavard C, Groussin L, Perlemoine K, Fumey G, Hagneré AM, René-Corail F, Jullian E, Gicquel C, Bertagna X, Vacher-Lavenu MC, Perret C, Bertherat J (2005) Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors. Cancer Res 65(17):7622–7627
Tadjine M, Lampron A, Ouadi L, Bourdeau I (2008) Frequent mutations of β-catenin gene in sporadic secreting adrenocortical adenomas. Clin Endocrinol (Oxf) 68(2):264–270
Doghman M, Cazareth J, Lalli E (2008) The T cell factor/beta-catenin antagonist PKF115-584 inhibits proliferation of adrenocortical carcinoma cells. J Clin Endocrinol Metab 93(8):3222–3225
Gaujoux S, Hantel C, Launay P, Bonnet S, Perlemoine K, Lefèvre L, Guillaud-Bataille M, Beuschlein F, Tissier F, Bertherat J, Rizk-Rabin M, Ragazzon B (2013) Silencing mutated β-catenin inhibits cell proliferation and stimulates apoptosis in the adrenocortical cancer cell line H295R. PLoS One 8(2):e55743
Larriba MJ, González-Sancho JM, Barbáchano A, Niell N, Ferrer-Mayorga G, Muñoz A (2013) Vitamin D is a multilevel repressor of Wnt/beta-catenin signaling in cancer cells. Cancers (Basel) 5(4):1242–1260
Pálmer HG, González-Sancho JM, Espada J, Berciano MT, Puig I, Baulida J, Quintanilla M, Cano A, de Herreros AG, Lafarga M, Muñoz A (2001) Vitamin D3 promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of beta-catenin signaling. J Cell Biol 154(2):369–387
Pendás-Franco N, Aguilera O, Pereira F, González-Sancho JM, Muñoz A (2008) Vitamin D and Wnt/beta-catenin pathway in colon cancer: role and regulation of DICKKOPF genes. Anticancer Res 28(5A):2613–2623
Lehmann TP, Wrzesiński T, Jagodziński PP (2013) The effect of mitotane on viability, steroidogenesis and gene expression in NCI-H295R adrenocortical cells. Mol Med Rep 7(3):893–900
Cerquetti L, Sampaoli C, De Salvo M, Bucci B, Argese N, Chimento A, Vottari S, Marchese R, Pezzi V, Toscano V, Stigliano A (2015) C-MYC modulation induces responsiveness to paclitaxel in adrenocortical cancer cell lines. Int J Oncol 46(5):2231–2240
Ng K, Nimeiri HS, McCleary NJ, Abrams TA, Yurgelun MB, Cleary JM, Rubinson DA, Schrag D, Miksad R, Bullock AJ, Allen J, Zuckerman D, Chan E, Chan JA, Wolpin BM, Constantine M, Weckstein DJ, Faggen MA, Thomas CA, Kournioti C, Yuan C, Ganser C, Wilkinson B, Mackintosh C, Zheng H, Hollis BW, Meyerhardt JA, Fuchs CS (2019) Effect of high-dose vs standard-dose vitamin D3 supplementation on progression-free survival among patients with advanced or metastatic colorectal cancer: the SUNSHINE Randomized Clinical Trial. JAMA 321(14):1370–1379
Duffy MJ, Murray A, Synnott NC, O’Donovan N, Crown J (2017) Vitamin D analogues: potential use in cancer treatment. Crit Rev Oncol Hematol 112:190–197
Maestro MA, Molnár F, Carlberg C (2019) Vitamin D and its synthetic analogs. J Med Chem 62(15):6854–6875
Kroiss M, Quinkler M, Lutz WK, Allolio B, Fassnacht M (2011) Drug interactions with mitotane by induction of CYP3A4 metabolism in the clinical management of adrenocortical carcinoma. Clin Endocrinol (Oxf) 75(5):585–591
Acknowledgements
This study was partially supported by HRA Pharma (Grant no. 266) (Paris, France).
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Rubin, B., Pilon, C., Pezzani, R. et al. The effects of mitotane and 1α,25-dihydroxyvitamin D3 on Wnt/beta-catenin signaling in human adrenocortical carcinoma cells. J Endocrinol Invest 43, 357–367 (2020). https://doi.org/10.1007/s40618-019-01127-1
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DOI: https://doi.org/10.1007/s40618-019-01127-1