Advertisement

World Journal of Urology

, Volume 37, Issue 2, pp 351–358 | Cite as

Inhibition of autophagy significantly increases the antitumor effect of Abiraterone in prostate cancer

  • Ashkan Mortezavi
  • Souzan Salemi
  • Benedikt Kranzbühler
  • Oliver Gross
  • Tullio Sulser
  • Hans-Uwe Simon
  • Daniel EberliEmail author
Original Article

Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusions

AA activates autophagy as a cytoprotective mechanism in LNCaP prostate cancer cells and targeting of autophagy enhances the antitumor effect of the compound.

Keywords

Autophagy PCa Abiraterone 

Notes

Acknowledgements

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.

Authors’ contributions

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.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent was obtained from all the individual participants included in the study.

Supplementary material

345_2018_2385_MOESM1_ESM.docx (39 kb)
Supplementary material 1 (DOCX 38 kb)
345_2018_2385_MOESM2_ESM.tif (7.7 mb)
Fig. 1S: A-E: A: WST cell proliferation assay: PC3 cells treated with AA 10 µM, 3MA 5 mM, Chl 20 µM and combinations for 2 and 4 days. Absorbance was measured daily at 450 nm. Bars represent mean ± SEM (n = 3). B: Cell death assay. PC3 cells were cultured in the presence and absence of AA 10 µM, 3MA 5 mM, Chl 20 µM and in combination of AA and 3MA and AA and Chl for 4 days. Cell viability was assessed using ethidium bromide and measured by FACS. Values are mean ± SEM of three independent experiments. C: Immunoblotting analysis (day 4) of PC3 cells. No changes of 55 kDa ATG5-ATG12 expression indicating no autophagy activation in response to different treatments. Supplementary material 2 (TIFF 7906 kb)

References

  1. 1.
    Siegel R, Ma J, Zou Z, Jemal A (2014) Cancer statistics. CA Cancer J Clin 64(1):9–29.  https://doi.org/10.3322/caac.21208 CrossRefGoogle Scholar
  2. 2.
    Lam JS, Leppert JT, Vemulapalli SN, Shvarts O, Belldegrun AS (2006) Secondary hormonal therapy for advanced prostate cancer. J Urol 175(1):27–34.  https://doi.org/10.1016/S0022-5347(05)00034-0 CrossRefGoogle Scholar
  3. 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
  4. 4.
    Berthold DR, Pond GR, Soban F, de Wit R, Eisenberger M, Tannock IF (2008) Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer: updated survival in the TAX 327 study. J Clin Oncol 26(2):242–245.  https://doi.org/10.1200/JCO.2007.12.4008 CrossRefGoogle Scholar
  5. 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. 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. 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. 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
  9. 9.
    Attard G, Belldegrun AS, de Bono JS (2005) Selective blockade of androgenic steroid synthesis by novel lyase inhibitors as a therapeutic strategy for treating metastatic prostate cancer. BJU Int 96(9):1241–1246.  https://doi.org/10.1111/j.1464-410X.2005.05821.x CrossRefGoogle Scholar
  10. 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. 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
  12. 12.
    Zhu K, Dunner K Jr, McConkey DJ (2010) Proteasome inhibitors activate autophagy as a cytoprotective response in human prostate cancer cells. Oncogene 29(3):451–462.  https://doi.org/10.1038/onc.2009.343 CrossRefGoogle Scholar
  13. 13.
    Li M, Jiang X, Liu D, Na Y, Gao GF, Xi Z (2008) Autophagy protects LNCaP cells under androgen deprivation conditions. Autophagy 4(1):54–60CrossRefGoogle Scholar
  14. 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. 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. 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
  17. 17.
    Attard G, Parker C, Eeles RA, Schroder F, Tomlins SA, Tannock I, Drake CG, de Bono JS (2016) Prostate cancer. Lancet 387(10013):70–82.  https://doi.org/10.1016/S0140-6736(14)61947-4 CrossRefGoogle Scholar
  18. 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. 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
  20. 20.
    Apel A, Herr I, Schwarz H, Rodemann HP, Mayer A (2008) Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy. Cancer Res 68(5):1485–1494.  https://doi.org/10.1158/0008-5472.CAN-07-0562 CrossRefGoogle Scholar
  21. 21.
    White E, Mehnert JM, Chan CS (2015) Autophagy, metabolism, and cancer. Clin Cancer Res 21(22):5037–5046.  https://doi.org/10.1158/1078-0432.CCR-15-0490 CrossRefGoogle Scholar
  22. 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. 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
  24. 24.
    Hammond EM, Brunet CL, Johnson GD, Parkhill J, Milner AE, Brady G, Gregory CD, Grand RJ (1998) Homology between a human apoptosis specific protein and the product of APG5, a gene involved in autophagy in yeast. FEBS Lett 425(3):391–395CrossRefGoogle Scholar
  25. 25.
    Salemi S, Yousefi S, Constantinescu MA, Fey MF, Simon HU (2012) Autophagy is required for self-renewal and differentiation of adult human stem cells. Cell Res 22(2):432–435.  https://doi.org/10.1038/cr.2011.200 CrossRefGoogle Scholar
  26. 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. 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. 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

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratory for Tissue Engineering and Stem Cell Therapy, Department of UrologyUniversity Hospital ZürichZurichSwitzerland
  2. 2.Institute of PharmacologyUniversity of BernBernSwitzerland

Personalised recommendations