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• W2024395172 abstract "Hormonal therapy of prostate cancer, by inhibiting androgen production and/or androgen function, is the treatment of choice for advanced prostate cancer. Although most patients respond initially, the effect is only temporary, and the tumor cells will resume proliferation in an androgen-deprived environment. The mechanism for androgen-independent proliferation of cancer cells is unclear. Hormonal therapy induces neuroendocrine differentiation of prostate cancer cells, which is hypothesized to contribute to tumor recurrence by a paracrine mechanism. We studied signal transduction pathways of neuroendocrine differentiation in LNCaP cells after androgen withdrawal, and we showed that both the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway and ERK are activated, but only the former is required for neuroendocrine differentiation. A constitutively active AKT promotes neuroendocrine differentiation and a dominant negative AKT inhibits it. Activation of AKT by IGF-1 leads to neuroendocrine differentiation, and neuroendocrine differentiation induced by epinephrine requires AKT activation. We also show that the AKT pathway is likely responsible for neuroendocrine differentiation in DU145, an androgen-independent prostate cancer cell line. Therefore, our study demonstrated a novel function of the AKT pathway in prostate cancer progression and identified potential targets that may be explored for the treatment of androgen-independent cancer. Hormonal therapy of prostate cancer, by inhibiting androgen production and/or androgen function, is the treatment of choice for advanced prostate cancer. Although most patients respond initially, the effect is only temporary, and the tumor cells will resume proliferation in an androgen-deprived environment. The mechanism for androgen-independent proliferation of cancer cells is unclear. Hormonal therapy induces neuroendocrine differentiation of prostate cancer cells, which is hypothesized to contribute to tumor recurrence by a paracrine mechanism. We studied signal transduction pathways of neuroendocrine differentiation in LNCaP cells after androgen withdrawal, and we showed that both the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway and ERK are activated, but only the former is required for neuroendocrine differentiation. A constitutively active AKT promotes neuroendocrine differentiation and a dominant negative AKT inhibits it. Activation of AKT by IGF-1 leads to neuroendocrine differentiation, and neuroendocrine differentiation induced by epinephrine requires AKT activation. We also show that the AKT pathway is likely responsible for neuroendocrine differentiation in DU145, an androgen-independent prostate cancer cell line. Therefore, our study demonstrated a novel function of the AKT pathway in prostate cancer progression and identified potential targets that may be explored for the treatment of androgen-independent cancer. Prostate cancer (PC) 2The abbreviations used are: PC, prostate cancer; AI, androgen-independent; NE, neuroendocrine; NED, neuroendocrine differentiation; AR, androgen receptor; S6K, S6 kinase; IGF-1, insulin-like growth factor-1; mTOR, mammalian target of rapamycin; FBS, fetal bovine serum; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; NSE, neuron-specific enolase; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HB-EGF, heparin-binding epidermal growth factor; C-FBS, charcoal-treated (androgen-deprived) FBS. 2The abbreviations used are: PC, prostate cancer; AI, androgen-independent; NE, neuroendocrine; NED, neuroendocrine differentiation; AR, androgen receptor; S6K, S6 kinase; IGF-1, insulin-like growth factor-1; mTOR, mammalian target of rapamycin; FBS, fetal bovine serum; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; NSE, neuron-specific enolase; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HB-EGF, heparin-binding epidermal growth factor; C-FBS, charcoal-treated (androgen-deprived) FBS. is the most common malignancy among men in western countries (1Quinn M. Babb P. BJU Int. 2002; 90: 162-173Crossref PubMed Scopus (334) Google Scholar). There are multiple treatment options for PC in early stages. For advanced and metastatic PC, hormonal therapy, consisting of androgen ablation and/or inhibition of androgen action by anti-androgen, is the treatment of choice (2Sharifi N. Gulley J.L. Dahut W.L. J. Am. Med. Assoc. 2005; 294: 238-244Crossref PubMed Scopus (771) Google Scholar). Although an initial response is seen in most patients receiving hormonal therapy, the effect is temporary, and the tumor eventually recurs and enters the androgen-independent (AI) stage in which the tumor cells proliferate in an androgen-deprived environment. There are no effective therapies for AI PC (3Balk S.P. Urology. 2002; 60: 132-139Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar).The mechanism of AI proliferation of PC is poorly understood, and many hypotheses have been proposed, such as androgen receptor (AR) amplification (4Visakorpi T. Hyytinen E. Koivisto P. Tanner M. Keinanen R. Palmberg C. Palotie A. Tammela T. Isola J. Kallioniemi O.P. Nat. Genet. 1995; 9: 401-406Crossref PubMed Scopus (1229) Google Scholar), AR mutation (3Balk S.P. Urology. 2002; 60: 132-139Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar), aberrant activation of AR (5Tan J. Sharief Y. Hamil K.G. Gregory C.W. Zang D.Y. Sar M. Gumerlock P.H. DeVere White R.W. Pretlow T.G. Harris S.E. Wilson E.M. Mohler J.L. French F.S. Mol. Endocrinol. 1997; 11: 450-459Crossref PubMed Scopus (266) Google Scholar), or increased AR sensitivity to low levels of androgen in the prostate (6Gregory C.W. Johnson Jr., R.T. Mohler J.L. French F.S. Wilson E.M. Cancer Res. 2001; 61: 2892-2898PubMed Google Scholar, 7Culig Z. Steiner H. Bartsch G. Hobisch A. Endocr.-Relat. Cancer. 2005; 12: 229-244Crossref PubMed Scopus (81) Google Scholar). In addition, many studies have shown that neuroendocrine (NE) differentiation (NED) may contribute to AI growth of PC (8Vashchenko N. Abrahamsson P.A. Eur. Urol. 2005; 47: 147-155Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Huang J. Di Sant'Agnese P.A. Lamberts S. Dogliotti L. Advances in Oncology: The Expanding Role of Octreotide I. BioScientifica Ltd., Bristol, UK2002: 243-262Google Scholar, 10Evangelou A.I. Winter S.F. Huss W.J. Bok R.A. Greenberg N.M. J. Cell. Biochem. 2004; 91: 671-683Crossref PubMed Scopus (49) Google Scholar).The epithelial compartment of benign prostate consists of luminal secretory cells, basal cells, and a minor component of NE cells that have neuron-like morphology and secret biogenic amines and neuropeptides (11di Sant'Agnese P.A. de Mesy Jensen K.L. Churukian C.J. Agarwal M.M. Arch. Pathol. Lab. Med. 1985; 109: 607-612PubMed Google Scholar). NE cells are also present in PC as scattered individual cells or small nests among the more abundant secretory type cancer cells. The number of NE cells increases in high grade and high stage tumors and particularly in hormonally treated and AI tumors (9Huang J. Di Sant'Agnese P.A. Lamberts S. Dogliotti L. Advances in Oncology: The Expanding Role of Octreotide I. BioScientifica Ltd., Bristol, UK2002: 243-262Google Scholar). It is hypothesized that hormonal therapy induces NED and the NE cells contribute to AI growth of PC in the androgen-deprived environment by secreting their products to act on the adjacent non-NE tumor cells in a paracrine fashion (8Vashchenko N. Abrahamsson P.A. Eur. Urol. 2005; 47: 147-155Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Huang J. Di Sant'Agnese P.A. Lamberts S. Dogliotti L. Advances in Oncology: The Expanding Role of Octreotide I. BioScientifica Ltd., Bristol, UK2002: 243-262Google Scholar, 10Evangelou A.I. Winter S.F. Huss W.J. Bok R.A. Greenberg N.M. J. Cell. Biochem. 2004; 91: 671-683Crossref PubMed Scopus (49) Google Scholar).Androgen withdrawal of the culture media leads to NED of LNCaP cells, a PC cell line (13Burchardt T. Burchardt M. Chen M.W. Cao Y. de la Taille A. Shabsigh A. Hayek O. Dorai T. Buttyan R. J. Urol. 1999; 162: 1800-1805Crossref PubMed Scopus (150) Google Scholar), mimicking in vivo observation in PC patients treated hormonally. This finding supports the trans-differentiation model and suggests that NE cells may be derived from the non-NE secretory-type cancer cells (13Burchardt T. Burchardt M. Chen M.W. Cao Y. de la Taille A. Shabsigh A. Hayek O. Dorai T. Buttyan R. J. Urol. 1999; 162: 1800-1805Crossref PubMed Scopus (150) Google Scholar, 14Cox M.E. Deeble P.D. Lakhani S. Parsons S.J. Cancer Res. 1999; 59: 3821-3830PubMed Google Scholar). However, the signaling pathway involved in the differentiation process is unclear. Here we report our study demonstrating that the PI3K-AKT-mTOR pathway is critically involved in NED.EXPERIMENTAL PROCEDURESMaterials—LNCaP and DU145 cells were obtained from the American Type Culture Collection (Manassas, VA); FBS, RPMI medium 1640, sodium pyruvate, penicillin, and streptomycin were purchased from Invitrogen; charcoal/dextran-treated FBS was purchased from Hyclone (Logan, UT); GeneJuice® transfection reagent was from Novagen; RNeasy® mini kit was from Qiagen (Valencia, CA); transcriptor reverse transcriptase and homogeneous protein A were from Roche Applied Science; random hexamers was from Promega (Madison, WI); iQ™ SYBR® Green Supermix and Bio-Rad Protein assay kit were from Bio-Rad; monoclonal anti-NSE antibody was from DAKO (Carpinteria, CA); polyclonal anti-IGF-1 receptorβ antibody and monoclonal anti-GAPDH antibody were from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal anti-AKT, anti-phospho-AKT, anti-ERK, anti-phospho-ERK, anti-S6K, anti-phospho-S6K antibodies, and monoclonal anti-Tyr(P)-100 antibody were from Cell Signaling (Dancers, MA); RIPA lysis buffer was from Upstate Cell Signaling Solutions (Lake Placid, NY); protease inhibitor mixture and epinephrine (used at 5 μm) were from Sigma; IGF-1 (used at 100 ng/ml) was from R&D Systems (Minneapolis, MN). U0126 (used at 10 μm) and AKT inhibitor IV (used at 20 μm) were from Calbiochem. LY294002 (used at 20 μm) was from Cayman Chemical (Ann Arbor, MI), and rapamycin (used at 10 nm) was from Biomol (Plymouth Meeting, PA).Cell Culture, Plasmid DNA, and Transfection—LNCaP cells were maintained in RPMI 1640 medium supplemented with 10% FBS. For androgen deprivation, cells were cultured in RPMI 1640 medium with 10% charcoal/dextran-treated FBS. When chemical inhibitors or stimulating agents (IGF-1, epinephrine) were used, the culture media were changed daily with fresh inhibitors.pCDNA3-cAkt (a constitutively active Akt with a deletion at amino acids 4–129 replaced with a consensus myristoylation domain) (15Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar) and pcDNA-dnAKT (kinase-deficient mutant, K179A) were kindly provided by Dr. Freeman of the University of Rochester. The liposome-mediated plasmid transfection was performed using GeneJuice® transfection reagent (Novagen). The cells were plated and maintained to 70–80% density in 50-mm plates and then transfected with the plasmid DNAs according to the protocol suggested by the manufacturer. For transient transfection, the cells were harvested 48 h after transfection. For stable transfection, the transfected cells were diluted at 1:5–1:10 and selected with 300 μg/ml G418.Real Time Reverse Transcription-PCR—Total RNA was isolated from cells with the RNeasy® kit. RNA was reverse-transcribed by transcriptor reverse transcriptase with random hexamers. The following specific forward and reverse primers were used for NSE, 5′-AGCTGCCCCTGCCTTAC-3′ and 5′-GAGACAAACAGCGTTACTTAG-3′, and for chromogranin A, 5′-GCGGTGGAAGAGCCATCAT-3′ and 5′-TCTGTGGCTTCACCACTTTTCTC-3′.Real time PCR was performed with iQ™ SYBR® Green Supermix in an iCycler iQ System (Bio-Rad) using the SYBR Green Detection protocol. Total reaction volume was 20 μl, and a cycle consists of 95 °C for 5 min, 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, for a total of 45 cycles followed by 72 °C for 5 min.Western Blotting—Cells were washed twice with cold phosphate-buffered saline and lysed in RIPA lysis buffer for 30 min on ice. The cells were sheared twice through a 20-gauge needle and centrifuged at 14,000 rpm for 15 min at 4 °C. The protein concentration in the supernatant was determined with the Bio-Rad protein assay kit. Equal amounts of protein were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membrane with Semi-Dry Transfer Cell (Bio-Rad). The membrane was blocked with TBS containing 5% w/v nonfat dry milk, hybridized with primary antibody in 2% w/v nonfat dry milk, followed by incubation with secondary antibody and detected with an ECL kit (Bio-Rad).Statistical Analysis—Statistical significance was determined by t test. The results are expressed as mean ± S.D. from three separate (replicate) experiments.RESULTSAndrogen Deprivation of LNCaP Cells Induces NED and the Activation of ERK and the PI3K-AKT-mTOR Signaling Pathways—As has been shown previously, androgen withdrawal in the culture media of LNCaP cells induced NED, characterized by changes in cell morphology (elongated cellular processes; Fig. 1A) and increased expression of NE cell markers chromogranin A (Fig. 1B) and NSE (13Burchardt T. Burchardt M. Chen M.W. Cao Y. de la Taille A. Shabsigh A. Hayek O. Dorai T. Buttyan R. J. Urol. 1999; 162: 1800-1805Crossref PubMed Scopus (150) Google Scholar) (Fig. 1C). Androgen withdrawal also induced phosphorylation of ERK (Fig. 1D) and the serine/threonine kinase AKT (Fig. 1E). Because AKT is a key player in the PI3K-AKT-mTOR signaling pathway and its activity is increased by phosphorylation, our observation suggests that the PI3K-AKT-mTOR pathway may be activated during NED of LNCaP cells. Consistent with this hypothesis, androgen deprivation of LNCaP cells significantly increased the levels of phosphorylation of S6 kinase (S6K) (Fig. 1F), an important downstream effector of mTOR whose level of phosphorylation directly correlates with the activity of the PI3K-AKT-mTOR pathway in PC (16Thomas G.V. Horvath S. Smith B.L. Crosby K. Lebel L.A. Schrage M. Said J. De Kernion J. Reiter R.E. Sawyers C.L. Clin. Cancer Res. 2004; 10: 8351-8356Crossref PubMed Scopus (56) Google Scholar).The PI3K-AKT-mTOR Pathway, Not ERK Pathway, Is Required for NED of PC Induced by Androgen Withdrawal—Because androgen withdrawal activates both ERK and PI3K-AKT-mTOR pathways in LNCaP cells, we studied whether one or both of them may be required for NED induced by androgen withdrawal. Addition of U0126, an inhibitor of MEK that inhibits the activation of ERK, had no effect on the increased NSE level in androgen-deprived LNCaP cells (Fig. 2A). The relative specificity of this compound was demonstrated by showing that U0126 blocked the phosphorylation of ERK (Fig. 2B) but not that of AKT (Fig. 2C), suggesting that activation of ERK is not required for NED of PC.FIGURE 2NED induced by androgen withdrawal is not suppressed by inhibition of ERK. A, LNCaP cells were cultured in FBS or C-FBS for 6 days in the absence or presence of the MEK inhibitor U0126. Equal amounts of cellular proteins were immunoblotted with anti-NSE and anti-GAPDH antibodies to show that U0126 did not inhibit NED. B and C, Western blots show that U0126 inhibited phosphorylation of ERK but increased the phosphorylation of AKT. Results are plotted as mean ± S.D. from three replicates for each treatment group. Significant changes (compared with control) are indicated by an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To study whether PI3K-AKT-mTOR pathway is required for NED of LNCaP cells, inhibitors that target key molecules of this pathway were added to LNCaP cells cultured in androgen-deprived media. LY294002, a PI3K inhibitor, and rapamycin, an inhibitor of mTOR, significantly inhibited the expression of NSE (Fig. 3A and Fig. 4A). The specificity of LY294002 was demonstrated by its ability to inhibit the phosphorylation of AKT (Fig. 3B) but not ERK phosphorylation (Fig. 3C). Similarly, we demonstrated that rapamycin inhibited the phosphorylation of S6K, a downstream molecule of mTOR (Fig. 4B), but not ERK (Fig. 4C). Additionally, we studied whether inhibition of AKT by a chemical inhibitor (AKT inhibitor IV) may inhibit NED. Because treatment of LNCaP cells with this compound for longer than 12 h causes significant cell apoptosis, we performed our study for 12 h (complete NED occurs in 6 days). LNCaP cells cultured in charcoal-treated FBS for 12 h showed increased NSE mRNA but not its protein (data not shown). We showed that AKT inhibitor IV abolished the increased expression of NSE mRNA after androgen withdrawal (Fig. 4D). Taken together, these results suggest that the PI3K-AKT-mTOR pathway, but not the Ras-MEK-ERK pathway, is required for androgen withdrawal-induced NED of PC.FIGURE 3NED induced by androgen withdrawal is suppressed by inhibition of PI3K activity. A–C, LNCaP cells were cultured in FBS or C-FBS for 6 days in the absence or presence of the PI3K inhibitor LY294002. Equal amounts of cellular proteins were immunoblotted with anti-NSE, anti-GAPDH, anti-AKT, anti-phospho-AKT, anti-ERK, and anti-phospho-ERK antibodies. LY294002 inhibited NED and the phosphorylation of AKT, but not that of ERK. Results are plotted as mean ± S.D. from three replicates for each treatment group. Significant changes (compared with control) are indicated by an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4NED induced by androgen withdrawal is suppressed by inhibition of mTOR or AKT. A–C, LNCaP cells were cultured in FBS or C-FBS for 6 days in the absence or presence of the mTOR inhibitor rapamycin. Equal amounts of cellular proteins were resolved by SDS-PAGE and immunoblotted with anti-NSE, anti-GAPDH, anti-S6K, anti-phospho-S6K, anti-ERK, and anti-phospho-ERK antibodies. Rapamycin inhibited NED and the phosphorylation of S6K, but not that of ERK. D, LNCaP cells were cultured in FBS, C-FBS, or C-FBS plus AKT inhibitor IV for 12 h, and a real time PCR assay was performed to measure the mRNA levels of NSE in the cells. Results are plotted as mean ± S.D. from three replicates for each treatment group. Significant changes (compared with control) are indicated by an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Activated AKT Leads to NED of LNCaP Cells—Results from the above experiments suggest that the PI3K-AKT-mTOR pathway is activated in LNCaP cells after androgen withdrawal and is required for NED. We next studied whether activation of AKT by other means also promoted NED. We transiently transfected LNCaP cells with pcDNA3-cAkt, which expresses a constitutively active AKT with a deletion of amino acids 4–129 replaced with a consensus myristoylation domain, or pcDNA3-dnAKT, which expresses a dominant negative (kinase-deficient mutant) AKT (K179A). A real time PCR assay 48 h after transfection showed that expression of c-AKT, not dnAKT, induced the expression of NSE mRNA in LNCaP cells (Fig. 5A), suggesting that activated AKT induces NED, and the kinase activity of AKT is required for this function.FIGURE 5A constitutively active AKT promotes NED while a dominant negative AKT inhibits androgen withdrawal-induced NED. A, LNCaP cells were transiently transfected with pcDNA3-cAKT (expressing a constitutively active AKT) or pcDNA3-dnAKT (expressing a dominant negative AKT) or cultured in c-FBS (positive control of NED). The cells were harvested 48 h after transfection, and a real time PCR assay was performed to study the expression of NSE mRNA and showed that cAKT, not dnAKT, induced NED. B, equal amounts of protein from control LNCaP (transfected with empty vector), LNCaP-cAKT, and LNCaP-dnAKT cells were immunoblotted with anti-AKT antibody and anti-GAPDH antibodies. C and D, LNCaP, LNCaP-cAKT, and LNCaP-dnAKT cells were cultured in FBS or in C-FBS for 6 days. Equal amounts of proteins were immunoblotted with anti-NSE, anti-GAPDH, anti-S6K, and anti-phospho-S6K antibodies to show that cAKT induced NED and dnAKT inhibited androgen withdrawal-induced NED. E, LNCaP or LNCaP-cAKT cells were cultured in FBS or C-FBS for 6 days in the absence or presence of rapamycin. Equal amounts of proteins were immunoblotted with anti-NSE and anti-GAPDH antibodies to show that cAKT-induced NED was inhibited by rapamycin. Results are plotted as mean ± S.D. from three replicates for each treatment group. Significant changes (compared with control) are indicated by an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5A constitutively active AKT promotes NED while a dominant negative AKT inhibits androgen withdrawal-induced NED. A, LNCaP cells were transiently transfected with pcDNA3-cAKT (expressing a constitutively active AKT) or pcDNA3-dnAKT (expressing a dominant negative AKT) or cultured in c-FBS (positive control of NED). The cells were harvested 48 h after transfection, and a real time PCR assay was performed to study the expression of NSE mRNA and showed that cAKT, not dnAKT, induced NED. B, equal amounts of protein from control LNCaP (transfected with empty vector), LNCaP-cAKT, and LNCaP-dnAKT cells were immunoblotted with anti-AKT antibody and anti-GAPDH antibodies. C and D, LNCaP, LNCaP-cAKT, and LNCaP-dnAKT cells were cultured in FBS or in C-FBS for 6 days. Equal amounts of proteins were immunoblotted with anti-NSE, anti-GAPDH, anti-S6K, and anti-phospho-S6K antibodies to show that cAKT induced NED and dnAKT inhibited androgen withdrawal-induced NED. E, LNCaP or LNCaP-cAKT cells were cultured in FBS or C-FBS for 6 days in the absence or presence of rapamycin. Equal amounts of proteins were immunoblotted with anti-NSE and anti-GAPDH antibodies to show that cAKT-induced NED was inhibited by rapamycin. Results are plotted as mean ± S.D. from three replicates for each treatment group. Significant changes (compared with control) are indicated by an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm the results obtained with transient transfection experiments, we established stable cell lines of LNCaP cells. The LNCaP cells were transfected with pcDNA3-cAKT and pcDNA3dnAKT, respectively and selected with G-418. Cells that survived the selection were pooled and designated as LNCaP-cAKT and LNCaP-dnAKT cells, respectively. Western blot showed significant overexpression of AKT in these cells in comparison to the parental LNCaP cells (Fig. 5B).LNCaP-cAKT cells had a significantly increased level of NSE in comparison with that in the parental LNCaP cells when cultured in normal FBS (Fig. 5C), suggesting that activation of the AKT pathway promotes NED of LNCaP cells. mTOR mediates the function of AKT in inducing NED because a downstream effector of mTOR, S6K, was constitutively activated in the LNCaP-cAKT cells cultured in normal FBS (Fig. 5D). Additionally, rapamycin, an mTOR inhibitor, inhibited NED of the LNCaP-cAKT cells (Fig. 5E). Expression of the dominant negative AKT significantly inhibited androgen withdrawal-induced NED (Fig. 5C) as well as phosphorylation of S6K (Fig. 5D), confirming that the PI3K-AKT-mTOR pathway is required for NED of PC.IGF-1 Activates AKT and Induces NED of LNCaP Cells—We next studied whether activation of endogenous AKT by a stimulus other than androgen withdrawal may induce NED. We chose to study the effects of IGF-1, which activates AKT and has been implicated in the progression of PC in many studies (17Chan J.M. Stampfer M.J. Ma J. Gann P. Gaziano J.M. Pollak M. Giovannucci E. J. Natl. Cancer Inst. 2002; 94: 1099-1106Crossref PubMed Scopus (391) Google Scholar). IGF-1 induced tyrosine phosphorylation of IGF-1 receptor β and the phosphorylation of AKT as expected (Fig. 6, A and B). Interestingly, addition of IGF-1 to LNCaP cells cultured in normal media induced NE morphologic changes similar to what is observed in cells cultured in androgen-deprived media (Fig. 6C) and also significantly increased the expression of chromogranin A and NSE (Fig. 6, D and E), suggesting that it induces NED. Importantly, NED induced by IGF-1 was inhibited by rapamycin (Fig. 6F), suggesting that IGF-1-induced NED is also mediated by the PI3K-AKT-mTOR pathway.FIGURE 6IGF-1, an activator of AKT, induces NED of LNCaP cells. A, LNCaP cells were cultured in FBS in the absence or presence of IGF-1 for 6 days. Equal amounts of proteins were immunoprecipitated (IP) with an anti-IGF-1-Rβ antibody and immunoblotted (IB) with an anti-phosphotyrosine antibody. B, LNCaP cells were cultured in FBS in the absence or presence of IGF-1 or in C-FBS (positive control of NED) for 6 days. Equal amounts of protein were immunoblotted with anti-AKT and anti-phospho-AKT antibodies. C, LNCaP cells were cultured in the absence (left panel) or presence (right panel) of IGF-1 for 6 days to show that IGF-1 induced NE-like morphology. D, LNCaP cells were cultured in FBS, C-FBS, or in FBS with IGF-1 for 6 days. A real time PCR assay was performed to study the expression of chromogranin A mRNA. E, equal amounts of proteins were immunoblotted with anti-NSE and anti-GAPDH antibodies to show that IGF-1 induces NED. F, LNCaP cells were cultured in FBS, C-FBS, or FBS plus IGF-1 in the absence or presence of rapamycin for 6 days. Equal amounts of proteins were immunoblotted with anti-NSE and anti-GAPDH antibodies to show that rapamycin inhibited IGF-1-induced NED. Results are plotted as mean ± S.D. from three replicates for each treatment group. Significant changes (compared with control) are indicated by an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Epinephrine Activates AKT and Induces NED—Results from the previous experiments support the hypothesis that AKT is essential for the induction of NED of PC by androgen withdrawal. However, other agents, such as β-adrenergic receptor agonist epinephrine, can also stimulate NED of LNCaP cells (14Cox M.E. Deeble P.D. Lakhani S. Parsons S.J. Cancer Res. 1999; 59: 3821-3830PubMed Google Scholar, 18Cox M.E. Deeble P.D. Bissonette E.A. Parsons S.J. J. Biol. Chem. 2000; 275: 13812-13818Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), but it was unclear whether AKT is involved under such conditions. Thus, we tested if NED of LNCaP cells induced by epinephrine also requires activation of AKT. Addition of epinephrine to LNCaP cells induced morphologic changes of NED and significant increases in the expression of chromogranin A and NSE as reported previously (Fig. 7, A–C) (14Cox M.E. Deeble P.D. Lakhani S. Parsons S.J. Cancer Res. 1999; 59: 3821-3830PubMed Google Scholar). Additionally, it also induced phosphorylation of AKT (Fig. 7D). Epinephrine-induced NED was significantly inhibited by the mTOR inhibitor rapamycin (Fig. 7E), suggesting that AKT also plays an essential role in this process.FIGURE 7NED induced by epinephrine (Epi) requires AKT signaling. A, LNCaP cells were cultured in the absence (left panel) or presence (right panel) of epinephrine for 6 days to show that epinephrine induced NE-like morphology. B, LNCaP cells were cultured in FBS, C-FBS, or FBS with epinephrine for 6 days. A real time PCR assay was performed to study the expression of chromogranin A mRNA. C and D, Western blots show that epinephrine induced NED and also induced phosphorylation of AKT. E, LNCaP cells were treated with epinephrine for 6 days in the absence or presence of rapamycin. Untreated LNCaP cells cultured in FBS or C-FBS were used as negative and positive controls, respectively. Equal amounts of proteins were immunoblotted with an anti-NSE and an anti-GAPDH antibody to show that rapamycin inhibits NED induced by epinephrine. Results are plotted as mean ± S.D. from three replicates for each treatment group. Significant changes (compared with control) are indicated by an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)NED in DU145 PC Cells Requires PI3K-AKT-mTOR Pathway—To confirm that the function of AKT signaling in NED is not limited to LNCaP cells only, we studied NED in DU145 PC cells. Unlike LNCaP cells that require androgen for proliferation, DU145 is an androgen-independent cell line that proliferates in the absence of androgen (19Webber M.M. Bello D. Quader S. Prostate. 1997; 30: 136-142Crossref PubMed Scopus (49) Google Sch" @default.
• W2024395172 created "2016-06-24" @default.
• W2024395172 creator A5029912396 @default.
• W2024395172 creator A5037470603 @default.
• W2024395172 date "2007-02-01" @default.
• W2024395172 modified "2023-10-01" @default.
• W2024395172 title "Phosphatidylinositol 3-Kinase-AKT-Mammalian Target of Rapamycin Pathway Is Essential for Neuroendocrine Differentiation of Prostate Cancer" @default.
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