BACKGROUND: Tumor heterogeneity and the plasticity of cancer cells present challenges for effective clinical diagnosis and therapy. Such challenges are epitomized by neuroendocrine transdifferentiation (NED) and the emergence of neuroendocrine-like cancer cells in prostate tumors. This phenomenon frequently arises from androgen-depleted prostate adenocarcinoma and is associated with the development of castration-resistant prostate cancer and poor prognosis. RESULTS: In this study, we showed that NED was evoked in both androgen receptor (AR)-positive and AR-negative prostate epithelial cell lines by growing the cells to a high density. Androgen depletion and high-density cultivation were both associated with cell cycle arrest and deregulated expression of several cell cycle regulators, such as p27Kip1, members of the cyclin D protein family, and Cdk2. Dual inhibition of Cdk1 and Cdk2 using pharmacological inhibitor or RNAi led to modulation of the cell cycle and promotion of NED. We further demonstrated that the cyclic adenosine 3', 5'-monophosphate (cAMP)-mediated pathway is activated in the high-density conditions. Importantly, inhibition of cAMP signaling using a specific inhibitor of adenylate cyclase, MDL-12330A, abolished the promotion of NED by high cell density. CONCLUSIONS: Taken together, our results imply a new relationship between cell cycle attenuation and promotion of NED and suggest high cell density as a trigger for cAMP signaling that can mediate reversible NED in prostate cancer cells.

Department of Cytokinetics Institute of Biophysics Academy of Sciences of the Czech Republic v v i Královopolská 135 CZ 612 65 Brno Czech Republic

Zobrazit více v PubMed

Nelson WG, De Marzo AM, Isaacs WB. Prostate cancer. N Engl J Med. 2003;349:366–381. doi: 10.1056/NEJMra021562. PubMed DOI

Cindolo L, Cantile M, Vacherot F, Terry S, de la Taille A. Neuroendocrine differentiation in prostate cancer: from lab to bedside. Urol Int. 2007;79:287–296. doi: 10.1159/000109711. PubMed DOI

Bhatia S, Frangioni JV, Hoffman RM, Iafrate AJ, Polyak K. The challenges posed by cancer heterogeneity. Nat Biotech. 2012;30:604–610. doi: 10.1038/nbt.2294. PubMed DOI

Abrahamsson PA. Neuroendocrine cells in tumour growth of the prostate. Endocr Relat Cancer. 1999;6:503–519. doi: 10.1677/erc.0.0060503. PubMed DOI

Yuan TC, Veeramani S, Lin MF. Neuroendocrine-like prostate cancer cells: neuroendocrine transdifferentiation of prostate adenocarcinoma cells. Endocr Relat Cancer. 2007;14:531–547. doi: 10.1677/ERC-07-0061. PubMed DOI

Yuan TC, Veeramani S, Lin FF, Kondrikou D, Zelivianski S, Igawa T, Karan D, Batra SK, Lin MF. Androgen deprivation induces human prostate epithelial neuroendocrine differentiation of androgen-sensitive LNCaP cells. Endocr Relat Cancer. 2006;13:151–167. doi: 10.1677/erc.1.01043. PubMed DOI

Ismail AH, Landry F, Aprikian AG, Chevalier S. Androgen ablation promotes neuroendocrine cell differentiation in dog and human prostate. Prostate. 2002;51:117–125. doi: 10.1002/pros.10066. PubMed DOI

Deeble PD, Murphy DJ, Parsons SJ, Cox ME. Interleukin-6- and cyclic AMP-mediated signaling potentiates neuroendocrine differentiation of LNCaP prostate tumor cells. Mol Cell Biol. 2001;21:8471–8482. doi: 10.1128/MCB.21.24.8471-8482.2001. PubMed DOI PMC

Yang X, Chen MW, Terry S, Vacherot F, Chopin DK, Bemis DL, Kitajewski J, Benson MC, Guo Y, Buttyan R. A human- and male-specific protocadherin that acts through the wnt signaling pathway to induce neuroendocrine transdifferentiation of prostate cancer cells. Cancer Res. 2005;65:5263–5271. doi: 10.1158/0008-5472.CAN-05-0162. PubMed DOI

Cortes MA, Cariaga-Martinez AE, Lobo MV, Martin Orozco RM, Motino O, Rodriguez-Ubreva FJ, Angulo J, Lopez-Ruiz P, Colas B. EGF promotes neuroendocrine-like differentiation of prostate cancer cells in the presence of LY294002 through increased ErbB2 expression independent of the phosphatidylinositol 3-kinase-AKT pathway. Carcinogenesis. 2012;33:1169–1177. doi: 10.1093/carcin/bgs139. PubMed DOI

Cox ME, Deeble PD, Bissonette EA, Parsons SJ. Activated 3′,5′-cyclic AMP-dependent protein kinase is sufficient to induce neuroendocrine-like differentiation of the LNCaP prostate tumor cell line. J Biol Chem. 2000;275:13812–13818. doi: 10.1074/jbc.275.18.13812. PubMed DOI

Zelivianski S, Verni M, Moore C, Kondrikov D, Taylor R, Lin MF. Multipathways for transdifferentiation of human prostate cancer cells into neuroendocrine-like phenotype. Biochim Biophys Acta. 2001;1539:28–43. doi: 10.1016/S0167-4889(01)00087-8. PubMed DOI

Cantile M, Kisslinger A, Cindolo L, Schiavo G, D’Anto V, Franco R, Altieri V, Gallo A, Villacci A, Tramontano D, Cillo C. cAMP induced modifications of HOX D gene expression in prostate cells allow the identification of a chromosomal area involved in vivo with neuroendocrine differentiation of human advanced prostate cancers. J Cell Physiol. 2005;205:202–210. doi: 10.1002/jcp.20384. PubMed DOI

Deng X, Liu H, Huang J, Cheng L, Keller ET, Parsons SJ, Hu CD. Ionizing radiation induces prostate cancer neuroendocrine differentiation through interplay of CREB and ATF2: implications for disease progression. Cancer Res. 2008;68:9663–9670. doi: 10.1158/0008-5472.CAN-08-2229. PubMed DOI PMC

Deng X, Elzey BD, Poulson JM, Morrison WB, Ko SC, Hahn NM, Ratliff TL, Hu CD. Ionizing radiation induces neuroendocrine differentiation of prostate cancer cells in vitro, in vivo and in prostate cancer patients. Am J Cancer Res. 2011;1:834–844. PubMed PMC

Knudsen KE, Arden KC, Cavenee WK. Multiple G1 regulatory elements control the androgen-dependent proliferation of prostatic carcinoma cells. J Biol Chem. 1998;273:20213–20222. doi: 10.1074/jbc.273.32.20213. PubMed DOI

Balk SP, Knudsen KE. AR, the cell cycle, and prostate cancer. Nucl Recept Signal. 2008;6:e001. PubMed PMC

Fribourg AF, Knudsen KE, Strobeck MW, Lindhorst CM, Knudsen ES. Differential requirements for ras and the retinoblastoma tumor suppressor protein in the androgen dependence of prostatic adenocarcinoma cells. Cell Growth Differ. 2000;11:361–372. PubMed

Orbo A, Jaeger R, Sager G. Cell density dependence of cAMP and cGMP levels in four human cell lines derived from carcinomas of the uterine cervix. Gynecol Oncol. 1994;52:320–325. doi: 10.1006/gyno.1994.1056. PubMed DOI

Piast M, Kustrzeba-Wojcicka I, Matusiewicz M, Banas T. Molecular evolution of enolase. Acta Biochim Pol. 2005;52:507–513. PubMed

Easter SS Jr, Ross LS, Frankfurter A. Initial tract formation in the mouse brain. J Neurosci. 1993;13:285–299. PubMed PMC

Koshimizu H, Kim T, Cawley NX, Loh YP. Chromogranin A: a new proposal for trafficking, processing and induction of granule biogenesis. Regul Pept. 2010;160:153–159. doi: 10.1016/j.regpep.2009.12.007. PubMed DOI PMC

Wafa LA, Cheng H, Rao MA, Nelson CC, Cox M, Hirst M, Sadowski I, Rennie PS. Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system. Biochem J. 2003;375:373–383. doi: 10.1042/BJ20030689. PubMed DOI PMC

Marchiani S, Tamburrino L, Nesi G, Paglierani M, Gelmini S, Orlando C, Maggi M, Forti G, Baldi E. Androgen-responsive and -unresponsive prostate cancer cell lines respond differently to stimuli inducing neuroendocrine differentiation. Int J Androl. 2010;33:784–793. doi: 10.1111/j.1365-2605.2009.01030.x. PubMed DOI

Terry S, Ploussard G, Allory Y, Nicolaiew N, Boissiere-Michot F, Maille P, Kheuang L, Coppolani E, Ali A, Bibeau F, Culine S, Buttyan R, de la Taille A, Vacherot F. Increased expression of class III beta-tubulin in castration-resistant human prostate cancer. Br J Cancer. 2009;101:951–956. doi: 10.1038/sj.bjc.6605245. PubMed DOI PMC

Wafa LA, Palmer J, Fazli L, Hurtado-Coll A, Bell RH, Nelson CC, Gleave ME, Cox ME, Rennie PS. Comprehensive expression analysis of L-dopa decarboxylase and established neuroendocrine markers in neoadjuvant hormone-treated versus varying Gleason grade prostate tumors. Hum Pathol. 2007;38:161–170. doi: 10.1016/j.humpath.2006.07.003. PubMed DOI

Cox ME, Deeble PD, Lakhani S, Parsons SJ. Acquisition of neuroendocrine characteristics by prostate tumor cells is reversible: implications for prostate cancer progression. Cancer Res. 1999;59:3821–3830. PubMed

Vindrieux D, Reveiller M, Florin A, Blanchard C, Ruffion A, Devonec M, Benahmed M, Grataroli R. TNF-alpha-related apoptosis-inducing ligand decoy receptor DcR2 is targeted by androgen action in the rat ventral prostate. J Cell Physiol. 2006;206:709–717. doi: 10.1002/jcp.20520. PubMed DOI

Vindrieux D, Reveiller M, Chantepie J, Yakoub S, Deschildre C, Ruffion A, Devonec M, Benahmed M, Grataroli R. Down-regulation of DcR2 sensitizes androgen-dependent prostate cancer LNCaP cells to TRAIL-induced apoptosis. Cancer Cell Int. 2011;11:42. doi: 10.1186/1475-2867-11-42. PubMed DOI PMC

Neuwirt H, Puhr M, Cavarretta IT, Mitterberger M, Hobisch A, Culig Z. Suppressor of cytokine signalling-3 is up-regulated by androgen in prostate cancer cell lines and inhibits androgen-mediated proliferation and secretion. Endocr Relat Cancer. 2007;14:1007–1019. doi: 10.1677/ERC-07-0172. PubMed DOI

Evans MJ, Smith-Jones PM, Wongvipat J, Navarro V, Kim S, Bander NH, Larson SM, Sawyers CL. Noninvasive measurement of androgen receptor signaling with a positron-emitting radiopharmaceutical that targets prostate-specific membrane antigen. Proc Natl Acad Sci U S A. 2011;108:9578–9582. doi: 10.1073/pnas.1106383108. PubMed DOI PMC

Macdonald JI, Dick FA. Posttranslational modifications of the retinoblastoma tumor suppressor protein as determinants of function. Genes Cancer. 2012;3:619–633. doi: 10.1177/1947601912473305. PubMed DOI PMC

Lincova E, Hampl A, Pernicova Z, Starsichova A, Krcmar P, Machala M, Kozubik A, Soucek K. Multiple defects in negative regulation of the PKB/Akt pathway sensitise human cancer cells to the antiproliferative effect of non-steroidal anti-inflammatory drugs. Biochem Pharmacol. 2009;78:561–572. doi: 10.1016/j.bcp.2009.05.001. PubMed DOI

Brooks EE, Gray NS, Joly A, Kerwar SS, Lum R, Mackman RL, Norman TC, Rosete J, Rowe M, Schow SR, Schultz PG, Wang X, Wick MM, Shiffman D. CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal proliferation. J Biol Chem. 1997;272:29207–29211. doi: 10.1074/jbc.272.46.29207. PubMed DOI

Bang YJ, Pirnia F, Fang WG, Kang WK, Sartor O, Whitesell L, Ha MJ, Tsokos M, Sheahan MD, Nguyen P, Niklinski WT, Myers CE, Trepel JB. Terminal neuroendocrine differentiation of human prostate carcinoma cells in response to increased intracellular cyclic AMP. Proc Natl Acad Sci U S A. 1994;91:5330–5334. doi: 10.1073/pnas.91.12.5330. PubMed DOI PMC

Sharifi N, Gulley JL, Dahut WL. An update on androgen deprivation therapy for prostate cancer. Endocr Relat Cancer. 2010;17:R305–R315. doi: 10.1677/ERC-10-0187. PubMed DOI PMC

Xu Y, Chen SY, Ross KN, Balk SP. Androgens induce prostate cancer cell proliferation through mammalian target of rapamycin activation and post-transcriptional increases in cyclin D proteins. Cancer Res. 2006;66:7783–7792. doi: 10.1158/0008-5472.CAN-05-4472. PubMed DOI

Pernicova Z, Slabakova E, Kharaishvili G, Bouchal J, Kral M, Kunicka Z, Machala M, Kozubik A, Soucek K. Androgen depletion induces senescence in prostate cancer cells through down-regulation of Skp2. Neoplasia. 2011;13:526–536. PubMed PMC

Shariff AH, Ather MH. Neuroendocrine differentiation in prostate cancer. Urology. 2006;68:2–8. PubMed

Cindolo L, Cantile M, Franco R, Chiodini P, Schiavo G, Forte I, Zlobec I, Salzano L, Botti G, Gidaro S, Terracciano L, Cillo C. Parallel determination of NeuroD1, chromogranin-A, KI67 and androgen receptor expression in surgically treated prostate cancers. Int Braz J Urol. 2011;37:57–66. doi: 10.1590/S1677-55382011000100008. PubMed DOI

Cindolo L, Franco R, Cantile M, Schiavo G, Liguori G, Chiodini P, Salzano L, Autorino R, Di Blasi A, Falsaperla M, Feudale E, Botti G, Gallo A, Cillo C. NeuroD1 expression in human prostate cancer: can it contribute to neuroendocrine differentiation comprehension? Eur Urol. 2007;52:1365–1373. doi: 10.1016/j.eururo.2006.11.030. PubMed DOI

Bubendorf L, Sauter G, Moch H, Schmid H-P, Gasser TC, Jordan P, Mihatsch MJ. Ki-67 labeling index: an independent predictor of progression in prostate cancer treated by radical prostatectomy. J Pathol. 1996;178:437–441. doi: 10.1002/(SICI)1096-9896(199604)178:4<437::AID-PATH484>3.0.CO;2-4. PubMed DOI

Grobholz R, Griebe M, Sauer CG, Michel MS, Trojan L, Bleyl U. Influence of neuroendocrine tumor cells on proliferation in prostatic carcinoma. Hum Pathol. 2005;36:562–570. doi: 10.1016/j.humpath.2005.02.019. PubMed DOI

Mori S, Murakami-Mori K, Bonavida B. Interleukin-6 induces G1 arrest through induction of p27(Kip1), a cyclin-dependent kinase inhibitor, and neuron-like morphology in LNCaP prostate tumor cells. Biochem Biophys Res Commun. 1999;257:609–614. doi: 10.1006/bbrc.1999.0515. PubMed DOI

Taneja SS, Ha S, Garabedian MJ. Androgen stimulated cellular proliferation in the human prostate cancer cell line LNCaP is associated with reduced retinoblastoma protein expression. J Cell Biochem. 2001;84:188–199. PubMed

Tyagi A, Agarwal C, Agarwal R. Inhibition of retinoblastoma protein (Rb) phosphorylation at serine sites and an increase in Rb-E2F complex formation by silibinin in androgen-dependent human prostate carcinoma LNCaP cells: role in prostate cancer prevention. Mol Cancer Ther. 2002;1:525–532. PubMed

Vassilev LT, Tovar C, Chen S, Knezevic D, Zhao X, Sun H, Heimbrook DC, Chen L. Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci U S A. 2006;103:10660–10665. doi: 10.1073/pnas.0600447103. PubMed DOI PMC

Pitts TM, Davis SL, Eckhardt SG, Bradshaw-Pierce EL. Targeting nuclear kinases in cancer: Development of cell cycle kinase inhibitors. Pharmacol Ther. 2014;142:258–269. doi: 10.1016/j.pharmthera.2013.12.010. PubMed DOI

Santore TA, Chen Y, Smit MJ, Iyengar R. Adenovirus-directed expression of Q227L-G alpha(s) inhibits growth of established tumors of later-stage human breast cancer cells in athymic mice. Proc Natl Acad Sci U S A. 2002;99:1671–1676. doi: 10.1073/pnas.032661999. PubMed DOI PMC

Ugland H, Boquest AC, Naderi S, Collas P, Blomhoff HK. cAMP-mediated induction of cyclin E sensitizes growth-arrested adipose stem cells to DNA damage-induced apoptosis. Mol Biol Cell. 2008;19:5082–5092. doi: 10.1091/mbc.E08-01-0094. PubMed DOI PMC

Horoszewicz JS, Leong SS, Chu TM, Wajsman ZL, Friedman M, Papsidero L, Kim U, Chai LS, Kakati S, Arya SK, Sandberg AA. The LNCaP cell line–a new model for studies on human prostatic carcinoma. Prog Clin Biol Res. 1980;37:115–132. PubMed

Veldscholte J, Berrevoets CA, Ris-Stalpers C, Kuiper GG, Jenster G, Trapman J, Brinkmann AO, Mulder E. The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens. J Steroid Biochem Mol Biol. 1992;41:665–669. doi: 10.1016/0960-0760(92)90401-4. PubMed DOI

Klein KA, Reiter RE, Redula J, Moradi H, Zhu XL, Brothman AR, Lamb DJ, Marcelli M, Belldegrun A, Witte ON, Sawyers CL. Progression of metastatic human prostate cancer to androgen independence in immunodeficient SCID mice. Nat Med. 1997;3:402–408. doi: 10.1038/nm0497-402. PubMed DOI

Wu HC, Hsieh JT, Gleave ME, Brown NM, Pathak S, Chung LW. Derivation of androgen-independent human LNCaP prostatic cancer cell sublines: role of bone stromal cells. Int J Cancer. 1994;57:406–412. doi: 10.1002/ijc.2910570319. PubMed DOI

Hayward SW, Dahiya R, Cunha GR, Bartek J, Deshpande N, Narayan P. Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell line: BPH-1. In Vitro Cell Dev Biol Anim. 1995;31:14–24. doi: 10.1007/BF02631333. PubMed DOI

Hayward SW, Wang Y, Cao M, Hom YK, Zhang B, Grossfeld GD, Sudilovsky D, Cunha GR. Malignant transformation in a nontumorigenic human prostatic epithelial cell line. Cancer Res. 2001;61:8135–8142. PubMed

Slabakova E, Pernicova Z, Slavickova E, Starsichova A, Kozubik A, Soucek K. TGF-beta1-induced EMT of non-transformed prostate hyperplasia cells is characterized by early induction of SNAI2/Slug. Prostate. 2011;71:1332–1343. PubMed

Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3:1101–1108. doi: 10.1038/nprot.2008.73. PubMed DOI

Castration-resistant prostate cancer monitoring by cell-free circulating biomarkers

Single-cell profiling of surface glycosphingolipids opens a new dimension for deconvolution of breast cancer intratumoral heterogeneity and phenotypic plasticity

Variability of fluorescence intensity distribution measured by flow cytometry is influenced by cell size and cell cycle progression

Regulation of Neuroendocrine-like Differentiation in Prostate Cancer by Non-Coding RNAs

Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells