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Neurotensin Is an Autocrine Trophic Factor Stimulated by Androgen

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Neurotensin Is an Autocrine Trophic Factor Stimulated by Androgen Proc. Nat!. Acad. Sci. USA Vol. 91, pp. 4673-4677, May 1994 Medical Sciences Neurotensin is an autocrine trophic factor stimulated by androgen withdrawal in human prostate cancer (neuroendocrine/growth factor) INDER SEHGAL*, STEPHEN POWERSt*, BRENDA HUNTLEY*, GARTH POWIs§1, MARK PITrELKOWI, AND NITA J. MAIHLE*,** Departments of *Biochemistry and Molecular Biology, tGastroenterology, §Pharmacology, and I'Dermatology, Mayo Clinic, Rochester, MN 55905 Communicated by Susan E. Leeman, January 21, 1994 ABSTRACT After therapeutic hormone deprivation, pros- docrine cells have been reported to be a constituent of most tate cancer cells often develop androgen-insensitive growth prostatic adenocarcinomas (10), with incidences of up to through mechanisms thus far undefined. Neuropeptides have 100o (9). The prevalence of neuroendocrine cells in prostate been previously implicated as growth factors in some prostate cancer has been correlated with higher-grade malignancy and cancers. Here, we demonstrate that androgen-sensitive LNCaP poor prognosis (11), and prostatic neuroendocrine tumors are human prostate cancer cells produce and secrete neurotensin typically unresponsive to hormonal therapy (9). These ob- following androgen withdrawal. We show that while LNCaP servations led us to investigate the potential for neurotensin, cells express the neurotensin receptor, only androgen-deprived a tridecapeptide, to function as an androgen-independent cells exhibit a growth response to exogenous neurotensin. We trophic factor in prostate cancer. Neurotensin is a trophic further demonstrate that androgen-stimulated cells may be factor for normal intestinal cells (12) and has been speculated refractory to exogenous neurotensin due to androgen induction to play an autocrine role in small-cell lung cancer growth (13). of a metalloprotease active toward neurotensin. Thus, prostate Although neurotensin has been detected in human prostatic cancer cells deprived of androgen develop an alternative au- cancer tissues (14), this peptide is not usually expressed in tocrine growth mechanism involving neurotensin. normal prostate (15). The potential effects of sex steroids on the production and secretion of neuroendocrine peptides, Prostate cancer is the most commonly diagnosed cancer and including neurotensin, have not been studied in human ma- the second leading cause of cancer death among men in the lignancies, and the role that neuropeptides may play in United States (1). Androgens stimulate the growth of these promoting the growth of androgen-deprived cells is un- malignancies, and hormone deprivation is the primary treat- known. As clinical data suggest a possible role for small ment for advanced prostate cancer. While androgen with- peptides in prostate cancer growth, investigation of the drawal initially reduces the growth of metastatic prostatic regulatory events associated with peptide production is tumors, this clinical response is temporary and these cancers strongly warranted. ultimately recur. At this point, hormonal deprivation therapy To characterize the role of neurotensin in the process of fails and survival is usually <1 year (2, 3). To further the androgen withdrawal and subsequent hormone-independent understanding ofprostate tumor growth and progression, the cellular proliferation in human prostate tumors, the human growth-regulatory pathways associated with the develop- prostate cell line LNCaP (16) was used. ment of androgen-independent tumor cell -growth must be defined. MATERIALS AND METHODS Androgens promote the growth of prostate cancer cells, at least in part, through growth factor-mediated autocrine and Cell Culture and Growth Assays. LNCaP cells were rou- paracrine mechanisms (4). Androgen-regulated growth fac- tinely plated,- allowed to attach for 2 days and then fed tors implicated in such growth stimulatory pathways include serum-free defined medium [phenol-red free RPMI 1640 the epidermal growth factor and fibroblast growth factor supplemented with insulin (5 ,ag/ml), holo-transferrin (10 families (5, 6). Since it has been shown that androgen p.g/ml), 30 nM sodium selenite, penicillin G (100 units/ml), withdrawal leads to reduced proliferation and programmed streptomycin sulfate (100 Hg/ml), 2 mM L-glutamine, and cell death of androgen-dependent prostate cancer cells (7), it amphotericin B (1.25 pug/ml) at pH 7.4], with or without seems plausible that androgen depletion may also inhibit cell addition of 1 nM synthetic nonmetabolizable androgen, growth by decreasing the activity of autocrine/paracrine R1881 (17) (NEN/DuPont). Cells were routinely verified free growth factor loops. The evolution of androgen-insensitive of mycoplasma. For growth assays, cell were plated at low prostate cancer may, therefore, involve the selection for, or density (500 cells per cm2) in T-25 flasks, supplemented with development of, alternative growth-regulatory pathways. In 0.5, 1.0, 2.0, 5.0, 10.0, or 25 nM neurotensin in the presence this study, we have considered the potential role of neuro- or absence of androgen, and were grown with medium endocrine peptides as potential trophic factors in the devel- changes every 2 days (to minimize the accumulation of opment of such pathways. secreted growth factors) for 12 days. Cell number determi- One member ofthe family ofneuropeptides, bombesin, has nations were made with a Coulter Counter. been shown to exhibit autocrine growth activity in human Radloimmunoassays (RIAs). LNCaP cells, plated in 100- small-cell lung cancer (8). More recently, a variety of neu- mm Petri dishes (Coming) at an initial density 5 x 103 cells ropeptides have been shown to be expressed in prostatic carcinomas; these peptides are secreted by focal areas of tPresent address: Immunologic Pharmaceutical Corp., 610 Lincoln neuroendocrine differentiated cells (9). Clusters of neuroen- Street, Waltham, MA 02154. Present address: Arizona Cancer Center, University of Arizona, 1501 North Cambell Avenue, Tucson, AZ 85724. The publication costs ofthis article were defrayed in part by page charge **To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Biochemistry and Molecular Biology, Guggenheim 14, Mayo in accordance with 18 U.S.C. §1734 solely to indicate this fact. Clinic, Rochester, MN 55905. 4673 Downloaded by guest on October 1, 2021 4674 Medical Sciences: Sehgal et al. Proc. NatL Acad. Sci. USA 91 (1994) per cm2, were grown for various times, and RIA for neuro- their potential to inhibit the 24-hr degradation of neurotensin tensin was performed as described (18) on conditioned me- (1 ng/ml). Values are the mean ± SE of four replicates. dium and acid-extracted cell lysate. Neurotensin fragments Northern RNA Analysis. Samples of poly(A)+ RNA (6 pg) are biologically active in some neural assays; these include collected from LNCaP, DU-145, and PC-3 prostate cancer amino-terminal fragments containing the peptide's midpor- cells and NCI-HI345 small-cell lung carcinoma [positive con- tion (19, 20) and carboxyl-terminal fragments consisting of trol for neurotensin receptor (15)] were denatured and elec- amino acids 8-13 and 9-13 (21, 22). Therefore, immunoas- trophoresed in a formaldehyde/0.8% agarose gel, and the says were designed to identify whole neurotensin or poten- separated RNAs were transferred to a Zeta-Probe GT mem- tially active fragments: results were derived by using an brane (Bio-Rad) by capillary transfer (23, 24). Membranes antibody which recognized the intact midportion (data not were baked at 80'C for 2 hr, prehybridized, hybridized, and shown) and were verified by using a second antibody recog- washed as per the Zeta-Probe manual. By using degenerate nizing the intact carboxyl terminus (Affinity Research Prod- primers derived from the cloned rat neurotensin receptor ucts, Nottingham, U.K.). Data, therefore, reflect quantities (25), a 300-bp fragment of the human neurotensin receptor of either whole neurotensin or potentially active fragments. was generated by PCR of human substantia nigra cDNA The LNCaP cell-associated neurotensin immunoreactivity (construct generously provided by E. Richelson, Mayo detected by RIA was confirmed as neurotensin by HPLC Clinic, Jacksonville, FL) and was subcloned into pBluescript profiles which demonstrated coelution of the immunoreac- SK(+) (Stratagene). An antisense RNA probe for neuroten- tive material from androgen-deprived LNCaP cells with sin receptor was generated by in vitro transcription of this neurotensin-(1-13) standard. Neuromedin N, a neurotensin- plasmid subclone with phage T7 polymerase and [a-32P]UTP like peptide that is cotranscribed with neurotensin, was not (800 Ci/mmol; 1 Ci = 37 GBq). Final radionucleotide con- significantly recognized by either antibody (data not shown). centration in the hybridization solution was >1 x 107 cpm/ The limit ofdetection for whole neurotensin was 20 pg/ml for ml. Molecular sizes were determined by comparison with an both antibodies. ethidium bromide-stained RNA marker (0.24- to 9.5-kb RNA Cell Lysate Preparation. LNCaP cells from sample plates ladder; GIBCO/BRL). After hybridization, blots were were removed with acell lifter (Costar) in phosphate-buffered stripped with 15 mM NaCl/1.5 mM trisodium citrate, pH saline, washed once with phosphate-buffered saline, and 7/0.1% SDS at 900C and hybridized with a probe for glycer- pelleted by centrifugation. Peptides were extracted by the aldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. A addition of 0.5 ml of
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