Inhibition of autophagy significantly increases the antitumor effect of Abiraterone in prostate cancer
Abiraterone acetate (AA) plus prednisone is an approved treatment of advanced prostate cancer (PCa). Autophagy is linked to drug resistance in numerous types of cancers. We hypothesized, that upregulation of autophagy is one of the mechanisms by which PCa cells survive AA anti-tumor treatment and therefore evaluated the potential effect of a combination with autophagy inhibition.
Human PCa LNCaP cell lines were cultured in steroid-free medium and treated with AA. Autophagy was inhibited by 3-methyladenine, chloroquine and ATG5 siRNA knock-down. Cell viability and apoptosis was assessed by flow cytometry and fluorescence microscopy, and autophagy was monitored by immunohistochemistry, AUTOdot and Western blotting.
Western blot revealed upregulation of ATG5 and LC3 II with a reduction of p62 protein expression in AA-treated cells, indicating upregulation of autophagy. These data were supported by results obtained with immunocytochemistry and AUTOdot assays. Using flow cytometry, we showed that combining AA with autophagy inhibition significantly impaired cell viability (1.3–1.6-fold, p < 0.001) and increased apoptosis (1.4–1.5-fold, p < 0.001) compared to AA treatment alone.
AA activates autophagy as a cytoprotective mechanism in LNCaP prostate cancer cells and targeting of autophagy enhances the antitumor effect of the compound.
KeywordsAutophagy PCa Abiraterone
This study was supported by Janssen Pharmaceutica NV and Fond zur Förderung des akademischen Nachwuchses (FAN). Special thanks to Damina Balmer for the critical assessment of this manuscript.
AM and SS contributed equally to this work: project development, carried out all the experiments, data collection, interpretation and manuscript writing. BK and OG: data collection and revising the manuscript. TS: revising the manuscript. HUS: data interpretation and revising the manuscript and DE: project development, data interpretation and manuscript writing.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent was obtained from all the individual participants included in the study.
- 3.de Bono JS, Oudard S, Ozguroglu M, Hansen S, Machiels JP, Kocak I, Gravis G, Bodrogi I, Mackenzie MJ, Shen L, Roessner M, Gupta S, Sartor AO, Investigators T (2010) Prednisone plus cabazitaxel or mitoxantrone for metastatic castration-resistant prostate cancer progressing after docetaxel treatment: a randomised open-label trial. Lancet 376(9747):1147–1154. https://doi.org/10.1016/S0140-6736(10)61389-X CrossRefGoogle Scholar
- 5.Montgomery RB, Mostaghel EA, Vessella R, Hess DL, Kalhorn TF, Higano CS, True LD, Nelson PS (2008) Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth. Cancer Res 68(11):4447–4454. https://doi.org/10.1158/0008-5472.CAN-08-0249 CrossRefGoogle Scholar
- 6.de Bono JS, Logothetis CJ, Molina A, Fizazi K, North S, Chu L, Chi KN, Jones RJ, Goodman OB Jr, Saad F, Staffurth JN, Mainwaring P, Harland S, Flaig TW, Hutson TE, Cheng T, Patterson H, Hainsworth JD, Ryan CJ, Sternberg CN, Ellard SL, Flechon A, Saleh M, Scholz M, Efstathiou E, Zivi A, Bianchini D, Loriot Y, Chieffo N, Kheoh T, Haqq CM, Scher HI, Investigators C-A (2011) Abiraterone and increased survival in metastatic prostate cancer. N Engl J Med 364(21):1995–2005. https://doi.org/10.1056/NEJMoa1014618 CrossRefGoogle Scholar
- 7.Ryan CJ, Smith MR, de Bono JS, Molina A, Logothetis CJ, de Souza P, Fizazi K, Mainwaring P, Piulats JM, Ng S, Carles J, Mulders PF, Basch E, Small EJ, Saad F, Schrijvers D, Van Poppel H, Mukherjee SD, Suttmann H, Gerritsen WR, Flaig TW, George DJ, Yu EY, Efstathiou E, Pantuck A, Winquist E, Higano CS, Taplin ME, Park Y, Kheoh T, Griffin T, Scher HI, Rathkopf DE, Investigators C-A (2013) Abiraterone in metastatic prostate cancer without previous chemotherapy. N Engl J Med 368(2):138–148. https://doi.org/10.1056/NEJMoa1209096 CrossRefGoogle Scholar
- 8.Jarman M, Barrie SE, Llera JM (1998) The 16,17-double bond is needed for irreversible inhibition of human cytochrome p45017alpha by Abiraterone (17-(3-pyridyl)androsta-5, 16-dien-3beta-ol) and related steroidal inhibitors. J Med Chem 41(27):5375–5381. https://doi.org/10.1021/jm981017j CrossRefGoogle Scholar
- 10.Mostaghel EA, Marck BT, Plymate SR, Vessella RL, Balk S, Matsumoto AM, Nelson PS, Montgomery RB (2011) Resistance to CYP17A1 inhibition with Abiraterone in castration-resistant prostate cancer: induction of steroidogenesis and androgen receptor splice variants. Clin Cancer Res 17(18):5913–5925. https://doi.org/10.1158/1078-0432.CCR-11-0728 CrossRefGoogle Scholar
- 11.Li R, Evaul K, Sharma KK, Chang KH, Yoshimoto J, Liu J, Auchus RJ, Sharifi N (2012) Abiraterone inhibits 3beta-hydroxysteroid dehydrogenase: a rationale for increasing drug exposure in castration-resistant prostate cancer. Clin Cancer Res 18(13):3571–3579. https://doi.org/10.1158/1078-0432.CCR-12-0908 CrossRefGoogle Scholar
- 14.Mortezavi A, Salemi S, Rupp NJ, Ruschoff JH, Hermanns T, Poyet C, Randazzo M, Simon HU, Moch H, Sulser T, Wild P, Eberli D (2017) Negative LC3b immunoreactivity in cancer cells is an independent prognostic predictor of prostate cancer specific death. Oncotarget 8(19):31765–31774. https://doi.org/10.18632/oncotarget.15986 CrossRefGoogle Scholar
- 15.Fizazi K, Tran N, Fein L, Matsubara N, Rodriguez-Antolin A, Alekseev BY, Ozguroglu M, Ye D, Feyerabend S, Protheroe A, De Porre P, Kheoh T, Park YC, Todd MB, Chi KN, Investigators L (2017) Abiraterone plus prednisone in metastatic, castration-sensitive prostate cancer. N Engl J Med 377(4):352–360. https://doi.org/10.1056/NEJMoa1704174 CrossRefGoogle Scholar
- 16.James ND, de Bono JS, Spears MR, Clarke NW, Mason MD, Dearnaley DP, Ritchie AWS, Amos CL, Gilson C, Jones RJ, Matheson D, Millman R, Attard G, Chowdhury S, Cross WR, Gillessen S, Parker CC, Russell JM, Berthold DR, Brawley C, Adab F, Aung S, Birtle AJ, Bowen J, Brock S, Chakraborti P, Ferguson C, Gale J, Gray E, Hingorani M, Hoskin PJ, Lester JF, Malik ZI, McKinna F, McPhail N, Money-Kyrle J, O’Sullivan J, Parikh O, Protheroe A, Robinson A, Srihari NN, Thomas C, Wagstaff J, Wylie J, Zarkar A, Parmar MKB, Sydes MR, Investigators S (2017) Abiraterone for prostate cancer not previously treated with hormone therapy. N Engl J Med 377(4):338–351. https://doi.org/10.1056/NEJMoa1702900 CrossRefGoogle Scholar
- 18.Attard G, Reid AH, Yap TA, Raynaud F, Dowsett M, Settatree S, Barrett M, Parker C, Martins V, Folkerd E, Clark J, Cooper CS, Kaye SB, Dearnaley D, Lee G, de Bono JS (2008) Phase I clinical trial of a selective inhibitor of CYP17, Abiraterone acetate, confirms that castration-resistant prostate cancer commonly remains hormone driven. J Clin Oncol 26(28):4563–4571. https://doi.org/10.1200/JCO.2007.15.9749 CrossRefGoogle Scholar
- 19.Li H, Wang Z, Tang K, Zhou H, Liu H, Yan L, Guan W, Chen K, Xu H, Ye Z (2017) Prognostic value of androgen receptor splice variant 7 in the treatment of castration-resistant prostate cancer with next generation androgen receptor signal inhibition: a systematic review and meta-analysis. Eur Urol Focus. https://doi.org/10.1016/j.euf.2017.01.004 Google Scholar
- 22.Gubko AA, Bespal’chuk PI, Volkov VF (1990) Surgical treatment of injuries of the long flexor tendon of the 1st finger. Ortop Travmatol Protez 7:16–18Google Scholar
- 23.Richards J, Lim AC, Hay CW, Taylor AE, Wingate A, Nowakowska K, Pezaro C, Carreira S, Goodall J, Arlt W, McEwan IJ, de Bono JS, Attard G (2012) Interactions of Abiraterone, eplerenone, and prednisolone with wild-type and mutant androgen receptor: a rationale for increasing Abiraterone exposure or combining with MDV3100. Cancer Res 72(9):2176–2182. https://doi.org/10.1158/0008-5472.CAN-11-3980 CrossRefGoogle Scholar
- 26.Rosenfeld MR, Ye X, Supko JG, Desideri S, Grossman SA, Brem S, Mikkelson T, Wang D, Chang YC, Hu J, McAfee Q, Fisher J, Troxel AB, Piao S, Heitjan DF, Tan KS, Pontiggia L, O’Dwyer PJ, Davis LE, Amaravadi RK (2014) A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme. Autophagy 10(8):1359–1368. https://doi.org/10.4161/auto.28984 CrossRefGoogle Scholar
- 27.Rangwala R, Chang YC, Hu J, Algazy KM, Evans TL, Fecher LA, Schuchter LM, Torigian DA, Panosian JT, Troxel AB, Tan KS, Heitjan DF, DeMichele AM, Vaughn DJ, Redlinger M, Alavi A, Kaiser J, Pontiggia L, Davis LE, O’Dwyer PJ, Amaravadi RK (2014) Combined MTOR and autophagy inhibition: phase I trial of hydroxychloroquine and temsirolimus in patients with advanced solid tumors and melanoma. Autophagy 10(8):1391–1402. https://doi.org/10.4161/auto.29119 CrossRefGoogle Scholar
- 28.Barnard RA, Wittenburg LA, Amaravadi RK, Gustafson DL, Thorburn A, Thamm DH (2014) Phase I clinical trial and pharmacodynamic evaluation of combination hydroxychloroquine and doxorubicin treatment in pet dogs treated for spontaneously occurring lymphoma. Autophagy 10(8):1415–1425. https://doi.org/10.4161/auto.29165 CrossRefGoogle Scholar