Growth hormone enhances LNCaP prostate cancer cell motility.

http://informahealthcare.com/erc ISSN: 0743-5800 (print), 1532-4206 (electronic) Endocr Res, Early Online: 1–9 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07435800.2014.966383

ORIGINAL ARTICLE

Growth hormone enhances LNCaP prostate cancer cell motility Alona O. Nakonechnaya* and Brian M. Shewchuk

Endocr Res Downloaded from informahealthcare.com by Korea University on 01/02/15 For personal use only.

Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC, USA

Abstract

Keywords

Purpose: Prostate cancer cells are responsive to multiple hormones and growth factors that can affect cell function. These effects may include modulating cell proliferation and apoptosis, but the ability to impinge on the metastatic potential of prostate cancer cells by affecting cell motility should also be considered, as prostate tumor metastasis correlates with limited therapeutic options and poor prognosis. Human growth hormone (hGH) can affect the growth and survival of prostate cancer cells, but the effect of hGH on prostate cancer cell motility is unknown. In the present study, the potential for exogenous and autocrine hGH to directly affect prostate cancer cell motility was addressed. Materials and methods: The effects of exogenous and autocrine hGH on the chemokinesis and chemotaxis of LNCaP prostate cancer cells were tested using cell monolayer wound healing and Boyden chamber invasion assays. The signaling pathways underlying these effects were resolved with chemical inhibitors and the correlation with cytoskeletal actin reorganization evaluated microscopically by staining cells with fluor-conjugated phalloidin. Results: Both exogenous and autocrine hGH augmented the migration and invasion of LNCaP cells, and hGH itself acted as a chemoattractant. This activity was dependent upon the STAT5, MEK1/2 and PI3K signaling pathways, and was accompanied by an alteration in cellular actin organization. Conclusions: hGH may enhance the metastatic potential of prostate cancer cells, both as a stimulant of cellular motility and invasiveness and as a chemoattractant.

Growth hormone, prostate cancer, metastasis, motility, signal transduction

Introduction Prostate cancer is a leading cause of cancer mortality among males, illuminating the need for a greater understanding of the mechanisms underlying the initiation and progression of the disease (1). An important aspect of this goal is resolving the effects of the multiple growth factors and hormones that regulate prostate development and function and thus have the potential to affect prostate cancer. Androgens play a major role in these processes, and androgen ablation is a common treatment for early-stage androgendependent prostate cancer (2,3). However, the common recurrence of castration-resistant cancer cells following antiandrogen therapy can lead to a more virulent and disseminated cancer (4), indicating that the actions of androgens alone are insufficient to explain all aspects of prostate cancer progression. Primary prostate tumor cells migrate into blood vessels and ultimately into distal tissues to form secondary metastatic tumors, principally in bone, which is associated with limited therapeutic options and poor prognosis (5). Thus, in addition to characterizing factors that can affect the proliferation and survival of prostate cancer cells, identifying

*Present address: Department of Pediatrics, McGill University, Montreal, QC, Canada Correspondence: Brian M. Shewchuk, 5W-52 Brody Medical Sciences Bldg., 600 Moye Blvd., Greenville, NC 27834, USA. Tel: +252 744 5096. E-mail: [email protected]

History Received 20 January 2014 Revised 27 August 2014 Accepted 9 September 2014 Published online 20 October 2014

the agents that stimulate their motility and invasiveness is also important for understanding prostate cancer progression. Multiple hormones and growth factors in addition to androgens affect prostate cell function and may influence prostate cancer progression (6), including the pituitaryderived endocrine peptide hormones prolactin (PRL), luteinizing hormone (LH), follicle stimulating hormone (FSH), and growth hormone (GH) (7–9). GH in particular is required for male reproductive system development, including normal prostate organogenesis and function (8–10), and the extensive distribution of the GH receptor (GHR) throughout the male reproductive system suggests a direct effect of GH on the prostate (9,11,12). Current evidence also indicates the potential for GH to influence prostate tumorigenesis in addition to normal prostate development. An increase in the incidence of benign prostate hyperplasia (BPH) is associated with elevated GH levels in acromegaly (13,14), and increased endogenous GH expression has been observed in prostate tissue biopsies taken from individuals with BPH that ultimately progressed to prostate cancer (15). An inverse correlation between serum GH concentration and prostate cancer incidence has been reported (16), and the absence of GH signaling due to a targeted GHR deficiency abrogated tumor progression in rodent prostate cancer models (17,18). Prostate cancer cells have been shown to express GHR, consistent with the potential for hGH responsiveness (19–22), but the direct effects of GH on prostate cancer cell function and the underlying molecular mechanisms are not fully understood.


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A. O. Nakonechnaya & B. M. Shewchuk

The ectopic expression of GH in prostate cancer cell lines and tissue indicate that autocrine GH signaling could also affect tumor progression (15,23), but the functional significance of autocrine GH in prostate cancer cells and the possible distinction from the effects of endocrine GH have not been fully resolved. We recently compared the effects of exogenous and autocrine human GH (hGH) on the growth and survival of the model hGH-responsive prostate cancer cell line LNCaP. This study surprisingly revealed that while exogenous (endocrine) hGH increased LNCaP cell proliferation, autocrine hGH had a negative effect on proliferation and survival (24). However, the potential for hGH to affect prostate cancer cell motility was not addressed. Previous studies in other cell types have shown that hGH can stimulate chemokinesis and chemotaxis to affect cell migration (25), and autocrine hGH in particular has been associated with cell spreading and invasion (26,27), supporting the potential for hGH to affect the motility and invasion of hGH-responsive prostate cancer cells. In the present study, we test the effects of both exogenous and autocrine hGH on the motility and invasiveness of LNCaP prostate cancer cells, and show results consistent with a potential contribution of hGH to prostate cancer metastasis.

Materials and methods Cell culture LNCaP cells were purchased from American Type Culture Collection and maintained in RPMI 1640 (Cellgro) supplemented with 10% fetal bovine serum (FBS, Gemini Bio Products, West Sacramento, CA) and 1% penicillin/streptomycin/amphotericin B solution (Cellgro, Herndon, VA) in a humidified incubator at 37 C in 5% CO2 (Napco 8000DH, Thermo Scientific, Waltham, MA). The generation and validation of LNCaP cells stably transfected with an expression vector encoding hGH (LNCaP/hGH cells) and empty vector-transfected control cells (LNCaP/EV) was previously described in detail (24). Briefly, the LNCaP/hGH cells express and secrete a physiological level of hGH as determined by hGH ELISA and represent a model for autocrine hGH. The LNCaP/EV cells, which do not express hGH but control for stable transfection and drug selection, represent a parallel model for the effects of exogenous hGH and were confirmed to respond to exogenous hGH identically to wildtype LNCaP cells with respect to signal transduction pathway activation and proliferation (24). Stably transfected cells were maintained under neomycin selection with 500 mg/ml G418 (Gemini BioProducts). Bromodeoxyuridine incorporation assay Cell proliferation assays to confirm the action of aphidicolin were performed using a commercial ELISA-based BrdU incorporation assay (Calbiochem) according to the manufacturer’s protocol. Cells were plated in 96-well plates at 1 104 cells/well in supplemented RPMI. The next day, media was replaced with fresh media containing 10 mg/ml aphidicolin (Sigma) or an equal volume of diluent, and the cells incubated at 37 C for 30 min. BrdU reagent was added and the cells

Endocr Res, Early Online: 1–9

incubated at 37 C for 6, 24, or 48 h, followed by analysis of BrDU incorporation levels. The chromogenic readout from the BrDU incorporation assay was measured with a plate spectrophotometer (Multiskan FC, Fisher Scientific, Waltham, MA). Western blot analysis of signaling pathway activation Cells were seeded at 2 106 cells/10 cm plate in supplemented RPMI. The next day, media was removed, cells were washed with PBS, and cells were incubated in serum free RPMI for 24 h. The media was replaced with fresh serum free media containing 500 ng/ml of purified hGH (provided by A.F. Parlow, National Hormone and Peptide Program) added from a 1 mg/ml stock prepared in phosphate-buffered saline (PBS) and the cells incubated at 37 C for 15 min. Cells were scraped into denaturing lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2% SDS) supplemented with protease and phosphatase inhibitor cocktails (Sigma). Lysates were sonicated with a cup horn in an ice bath (Sonicator 3000, Misonix, Farmingdale, NY) and cleared by centrifugation at 14 000 rpm for 15 min. at 4 C. Western blot of lysates was performed as previously described (24). Activated AKT was detected with a rabbit antiphospho-AKT (Ser473) antibody. A rabbit anti-AKT antibody was used as a control. Activated STAT5 was detected with a rabbit anti-phospho-STAT5 (Tyr694) antibody. A rabbit antiSTAT5 antibody was used as a control. Activated ERK1/2 (p44/42 MAPK) was detected with a rabbit anti-phosphoERK1/2 (Thr202/Tyr204) antibody. A rabbit anti-ERK1/2 antibody was used as a control. Antibodies were purchased from Cell Signaling (Danvers, MA) and were used at a 1:1000 dilution. Cell monolayer wound healing assay LNCaP cells were plated in a 6 cm plate for each condition and grown until highly confluent in supplemented RPMI 1640. In order to precisely index the wound closure measurement points, three X-shaped scratches were made in each cell monolayer plate using a 2 ml micropipette tip. Cells were washed with PBS to remove scraped cells and serum-free RPMI containing 10 mg/ml aphidicolin (Sigma) and either 10% FBS, 500 ng/ml purified hGH or an equal volume of diluent (PBS). The cross-shaped scratch wounds in the cell monolayers were photographed at the beginning (Day 0) and end (Day 2) of the experiment at 40x magnification using an Olympus IX50 microscope with a mounted CCD camera. Digital images were captured and the distances between opposite points at the edges of each of the three scratched wounds per plate were measured (six measurements total for each condition) using Photoshop software (Adobe, San Jose, CA) as illustrated in Figure 1(C) and converted to the absolute distance. The change in distance at each axis from Day 0 to Day 2 was calculated as the distance migrated. For the experiment with pathway inhibitors, cells were treated as above with the addition of either 0.1 mM U0126 (MEK1/2 inhibitor, Selleck Chemicals, Houston, TX), 5 mM LY294002 (PI3K inhibitor, Selleck Chemicals, Houston, TX), or 50 mM STAT5 inhibitor (N0 -((4-Oxo-4H-chromen3-yl)methylene)nicotinohydrazide, Santa Cruz Biotechnology, Santa Cruz, CA) to the media.

GH and cell motility

DOI: 10.3109/07435800.2014.966383

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in RPMI containing 10 mg/ml aphidicolin were added to the upper well. For cells treated with exogenous hGH, 500 ng/ml purified hGH was included in the media in the upper chamber. Three replicate wells were prepared for each condition. After incubation for 30 h at 37 C, cells that had migrated to the bottom surface of the membrane were fixed in 70% ethanol, stained with 0.1% crystal violet, and destained with dH2O. The membranes were mounted on a slide with a grid-etched cover slip and the cells visualized by light microscopy and counted.


Phalloidin staining of F-actin Cells were seeded on a poly-D-lysine-coated multi-chambered slide (BD Falcon) in serum-free RPMI and incubated for 24 h. Purified hGH was added at a final concentration of 500 ng/ml and the cells incubated for the indicated times at 37 C in 5% CO2. For the experiment testing STAT5 inhibition, cells were treated with 50 mM STAT5 inhibitor or DMSO for 24 h. The cells were washed with PBS and fixed with 3.7% paraformaldehyde for 10 min at room temperature. After fixation, cells were washed with PBS and permeabilized with 0.1% Triton for 5 min at room temperature. Cells were blocked with 1% BSA in PBS for 1 h at room temperature. Five units/ml of Alexa Fluor 488-conjugated phalloidin (Invitrogen, Carlsbad, CA), 1% BSA in PBS was added to the cells and incubated for 30 min. Cells were washed five times with PBS and visualized by confocal fluorescence microscopy (LSM 510, Carl Zeiss, Jena, Germany). Statistical analysis Statistical significance of observed results was evaluated by ANOVA and a post-hoc Tukey-Kramer multiple comparison test (a ¼ 0.05) using SPSS Statistics software (IBM, Armonk, NY). Figure 1. hGH increases LNCaP cell migration. (A) Immunoblot of hGH in LNCaP/hGH and LNCaP/EV cell lysates. The somatotrope cell line GH3 is included as a positive control (PC). b-tubulin serves as a loading control. Performed as previously described (24). (B) LNCaP cell BrdU incorporation assay. The data are normalized to the -BrdU sample and represent the mean ± SD (n ¼ 3). Levels not connected by the same letter are significantly different. (C) Image of LNCaP monolayer wound healing assay (40). The arrows indicate the indexed measurement reference points. (D) Quantification of wound healing assays. The data are normalized to vehicle (PBS) treated LNCaP/EV cells (migration distance 0.078 ± 0.013 mm) and represent the mean ± SD from three experiments (n ¼ 6 for each experiment). Levels not connected by the same letter are significantly different.

Boyden chamber Matrigel invasion assay The Boyden chamber transwell migration assay was performed using BD BioCoat Growth Factor Reduced Matrigel Invasion Chambers (BD Biosciences, San Jose, CA) according to the manufacturer’s instructions. Briefly, inserts were pre-incubated in RPMI containing 10 mg/ml aphidicolin for 2 h at 37 C. RPMI containing 10 mg/ml aphidicolin and either 1 mg/ml purified hGH, 1 mg/ml bovine serum albumin (BSA, negative control) or 10% FBS (positive control) as a chemoattractant was added to the wells of a 24-well tissue culture plate. The Boyden chambers were placed into the wells containing the chemoattractant media, and 1 105 cells

Results Exogenous and autocrine hGH increase LNCaP cell motility The LNCaP cell line has been established in multiple studies as a model for investigating hGH action in prostate cancer cells, in which hGHR expression, ligand binding, and signal transduction pathway activation were validated (20,22,24,28). We recently employed LNCaP cells to test the effects of exogenous and autocrine hGH on prostate cancer cell proliferation and survival (24). In that study, we developed a clonal LNCaP cell line stably transfected with an hGH expression vector (LNCaP/hGH) that expresses and secretes a physiological concentration of hGH as a model for autocrine hGH, and a parallel control cell line stably transfected with the empty vector (LNCaP/EV) that behaves identically to untransfected wild type LNCaP cells in response to exogenous hGH (24). These validated cell lines were used in the present investigation of the effects of autocrine and exogenous hGH on LNCaP cell motility (Figure 1A). The evaluation of cell motility in the present study encompasses two distinct phenomena: chemokinesis and chemotaxis. Chemokinesis describes the scanning of the environment by cells through non-directional random


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movement, while chemotaxis defines directional migration toward the source of a diffusible chemoattractant (29). The type of assay used to measure cell migration thus determines which aspect of cell motility is observed. We first tested the ability of hGH to stimulate the chemokinesis of LNCaP/EV and LNCaP/hGH cells by utilizing the established cell monolayer wound healing assay (30,31). We modified this assay to include the treatment of cells with aphidicolin, a DNA polymerase inhibitor that blocks cell proliferation, to ensure that cell migration was observed and not the effect of cell proliferation within the monolayer. The ability of aphidicolin to inhibit LNCaP cell proliferation was confirmed by a BrdU incorporation assay (Figure 1B). An example of the imaged cell monolayer scratches and measurements in the wound healing assay is shown in Figure 1(C). Treatment of LNCaP/EV cells with exogenous hGH resulted in a significant increase in cell migration in the wound healing assay relative to the PBS negative control (control mean migration distance 0.078 ± 0.013 mm), to a level comparable to that stimulated by the addition of serum (FBS). The expression of autocrine hGH in the LNCaP/hGH cell line resulted in a similar increase in cell migration that was not augmented by additional exogenous hGH or serum (Figure 1D). Thus, both exogenous and autocrine hGH increased the chemokinesis of LNCaP cells, but cells expressing autocrine hGH were refractory to additional exogenous hGH. While the monolayer wound healing assay is able to identify changes in chemokinesis, it does not allow observation of chemotaxis or cell invasion, which are also relevant to the characterization of cancer cell motility with respect to metastatic potential. To address these properties, LNCaP/EV and LNCaP/hGH cell migration was assayed in a Boyden chamber with Matrigel-coated filters to model cell migration in the extracellular matrix and to test whether hGH affected chemotaxis or acted as a chemoattractant. As in the wound healing assay, cells were treated with aphidicolin to inhibit proliferation. Figure 2(A) shows example images of cells on the bottom surface of the Boyden chamber membrane stained with crystal violet indicating cell migration and invasion. As expected, BSA in the lower chamber as a chemoattractant negative control resulted in minimal LNCaP/EV cell invasion (238 ± 48 invading cells), while FBS stimulated cell invasion as a positive control chemoattractant (Figure 2A, B). Remarkably, the addition of hGH alone to the lower chamber also resulted in a significant increase in LNCaP/EV cell migration compared to the BSA control, indicating that hGH can act as a chemoattractant for LNCaP cells. When LNCaP/ EV cells were treated with hGH in the upper wells of the Boyden chamber (+hGH), migration toward the FBS chemoattractant was increased, indicating that hGH also enhanced the chemotaxis of LNCaP/EV cells toward other chemoattractants present in serum (Figure 2B). When LNCaP/hGH cells expressing autocrine hGH were assayed in this manner with BSA in the lower chamber, increased cell migration across the membrane was observed compared to the LNCaP/EV control cells, reinforcing the increased chemokinesis associated with autocrine hGH observed in the wound healing assay. The addition of hGH to the lower chamber did not increase LNCaP/EV cell migration, further indicating that these cells are refractory to


Figure 2. hGH augments LNCaP cell chemotaxis and basement membrane invasion. (A) Image of LNCaP cells stained with crystal violet in the bottom face of the Matrigel-coated Boyden chamber membrane (100). The small homogenous speckles in the background are the 8 mm pores in the membrane. The chemoattractant present in each sample is indicated to the left of the image. (B) Quantification of transwell migration/invasion assays. The cell counts were normalized to the number of transwell migrating LNCaP/EV cells with BSA as a chemoattractant (negative control; 238 ± 48 migrating cells) and represent the mean ± SD from two experiments (n ¼ 3 for each experiment). Levels not connected by the same letter are significantly different. CHT, chemoattractant.

exogenous hGH. The migration of the LNCaP/hGH cells toward the FBS chemoattractant was also greater than that of the LNCaP/EV control cells and similar to that stimulated by exogenous hGH, indicating that autocrine hGH could augment the chemotaxis and invasion of LNCaP cells in the Boyden chamber assay (Figure 2B).

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Multiple signaling pathways mediate the effects of hGH on LNCaP cell motility GHR signaling is proximately mediated by the activation of Janus kinase 2 (JAK2) associated with the GHR intracellular domain, which in turn can activate at least three downstream signaling pathways — signal transducer and activator of transcription-5 (STAT5), mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K) — resulting in a range of functional effects (32). We and others have confirmed the activation of these canonical GHRlinked signal transduction pathways by hGH in LNCaP cells (22,24). It was thus of interest to determine whether these pathways are involved in the observed effects of hGH on LNCaP cell motility. To this end, STAT5, MEK1/2 (MAPK kinase) and PI3K activities were blocked in LNCaP/EV and LNCaP/hGH cells using chemical inhibitors as previously established (24), and the effects on exogenous and autocrine hGH-stimulated cell motility measured in the wound healing assay. Immunoblot analysis confirmed the activity of the inhibitors in GH-activated PI3K, STAT5 and MAPK pathways (Figure 3A). STAT5 inhibition in LNCaP/EV cells in the absence of exogenous hGH resulted in an increase in cell migration while MEK1/2 and PI3K inhibition had no effect (control mean migration distance 0.081 ± 0.018 mm; Figure 3B). This suggests that STAT5 is involved in modulating the basal motility of LNCaP cells. Stimulation of LNCaP/EV cells with exogenous hGH alone increased cell migration as expected, and the addition of each pathway inhibitor resulted in attenuated cell migration. Inhibition of each pathway also reduced the migration of LNCaP/hGH cells expressing autocrine hGH (control mean migration distance 0.214 ± 0.021 mm; Figure 3C). These results indicate that the STAT5, MAPK, and PI3K pathways each contribute to the effects of both exogenous and autocrine hGH on LNCaP cell motility. Exogenous and autocrine hGH alter F-actin localization in LNCaP cells Cell migration is a multi-step process involving the reorganization of cell adhesion complexes and components of the cytoskeleton. One of the rate-limiting steps in this mechanism is the polymerization of actin filaments. The formation of filamentous or F-actin is necessary for the establishment of cell polarization and plasma membrane structures associated with directed cell migration (29,33). In light of the effects of exogenous and autocrine hGH on LNCaP cell motility, we determined whether hGH was associated with an observable alteration in the structure or localization of F-actin in LNCaP cells by staining cells with fluor-conjugated phalloidin, which binds specifically to F-actin (Figure 4). Prior to treatment with exogenous hGH, F-actin was located primarily in filamentous cytoplasmic structures in LNCaP/EV cells (0 h, arrows). The addition of exogenous hGH resulted in the increased formation of dense foci rich in F-actin at the cell perimeter by 1 h, which became more pronounced at 4 h. (arrows and inset). After 24 h, there was an additional appearance of increased F-actin at the plasma membrane and F-actin-rich microspikes protruding from the cell

Figure 3. Multiple signal transduction pathways are involved in the effect of hGH on LNCaP cell migration. Quantification of wound healing assays after treatment with pathway inhibitors or vehicle (DMSO) as indicated on the x-axes. The data represents the mean ± SD from three experiments (n ¼ 6 for each experiment). Levels not connected by the same letter are significantly different. (A) Repression of GH-mediated pathway activation by the indicated inhibitors (in.). (B) LNCaP/EV cells in serum-free media containing aphidicolin treated with vehicle (PBS) or 500 ng/ml hGH in combination with the indicated pathway inhibitor or vehicle (DMSO). Migration distances normalized to the LNCaP/EV (PBS, DMSO) sample (migration distance 0.081 ± 0.018 mm) and represent the mean ± SD from three experiments (n ¼ 6 for each experiment). (C) LNCaP/hGH cells expressing autocrine hGH in serum-free media containing aphidicolin treated with the indicated pathway inhibitor or vehicle (DMSO). Migration distances were normalized to DMSO-treated cells (migration distance 0.214 ± 0.021 mm) and represent the mean ± SD from three experiments (n ¼ 6 for each experiment).

periphery (arrows and inset). Staining of LNCaP/hGH cells expressing autocrine hGH showed a pattern of F-actin localization similar to the LNCaP/EV cells treated with exogenous hGH for 24 h. These results indicate that both exogenous and autocrine hGH were capable of affecting F-actin localization, which may be related to the observed hGH-mediated increases in cell migration. Notably, the treatment of LNCaP/EV cells with the STAT5 inhibitor resulted in a reorganization of F-actin from intracellular filaments (DMSO, arrows) to dense foci at the cell periphery (STAT5 inhibitor, arrows; Figure 5) similar to that observed with GH treatment, which may also be related to the observed increase in cell migration upon treatment of LNCaP/EV cells with the STAT5 inhibitor alone (Figure 3B).

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Figure 4. hGH mediates actin reorganization in LNCaP cells. Fluorescence confocal microscopy of LNCaP/EV cells treated with 500 ng/ml hGH for the indicated times (left) or LNCaP/hGH cells expressing autocrine hGH (right) stained with Alexafluor 488conjugated phalloidin (200). Examples of the noted structures are indicated by white arrows and magnified insets (boxes).

Discussion Cell migration is governed by multiple coordinated mechanisms that can influence the metastatic potential of prostate cancer cells. This process involves the dynamic reorganization of the actin cytoskeleton and cell contacts with the surrounding substrate in order to allow movement of the cell

and responsiveness to extracellular chemotactic signals. The findings presented here indicate that exogenous (endocrine) hGH can function at multiple points in this process, stimulating both the chemokinesis and chemotaxis of LNCaP cells in association with actin reorganization, and acting as a chemoattractant itself. The latter observation suggests that hGH in circulation or from paracrine cellular

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Figure 5. Inhibition of STAT5 alters actin localization in LNCaP cells. Fluorescence confocal microscopy of LNCaP/EV cells treated with DMSO (vehicle, left) or 50 mM STAT5 inhibitor (right) for 24 h. stained with Alexafluor 488-conjugated phalloidin (200). Examples of the noted structures are indicated by white arrows.

sources has the potential to direct the migration of prostate tumor cells. Interestingly, hGH has previously been shown to act as a chemoattractant for human peripheral blood monocytes, which the authors propose may direct their recruitment to sites of tissue damage and inflammation as a product of lymphocytes. An enhancement of monocyte chemokinesis by hGH was also observed, similar to our observations in LNCaP cells (25), suggesting that hGH may influence cell motility in a range of cell types. The question of whether autocrine hGH expression, a phenomenon that has been observed in multiple prostate cancer cells lines and hyperplastic prostate tissue (15,23,24), could affect motility was also addressed. Autocrine hGH increased the chemokinesis and chemotaxis of LNCaP cells, indicating that ectopic hGH expression observed in prostate cancer cells may affect cell migration. This is consistent with previous observations of the ability of autocrine hGH to affect the morphology (spreading) of breast cancer cells attached to a collagen substrate, which involves the same mechanisms underlying cell migration (26). LNCaP cells expressing autocrine hGH (LNCaP/hGH cells) were refractory to exogenous hGH, a phenomenon that we observed previously in the context of LNCaP cell proliferation and apoptosis. This appeared to be due to an alteration in hGHR trafficking and localization that was paralleled by changes in hGHR-mediated signaling pathways that suggested a shift to an intracrine signaling mode. Specifically, endogenously expressed hGH in the LNCaP/hGH cell line sequestered hGHR in the endoplasmic reticulum and Golgi with a corresponding loss of plasma membrane associated hGHR (24). Thus, the loss of the ability of LNCaP/hGH cells to respond to extracellular hGH in the wound healing and Boyden chamber assays likely reflects this phenomenon. The effect of autocrine GH on LNCaP cell motility observed in the present study was notably distinct from the effect on cell proliferation and survival observed previously. While exogenous GH increased LNCaP cell proliferation, autocrine GH reduced proliferation and increased apoptosis (24), in contrast to the positive effects of both exogenous and autocrine GH on cell motility. While the mechanism underlying this distinction is unclear, the selective role of MAPK signaling in GH-mediated motility (present study) but

not proliferation (24), may be significant in this regard. In addition, autocrine GH has been proposed in recent work by others to be a cellular senescence signal that plays a role in the suppression of cell proliferation and the induction of apoptosis in non-pituitary cells that contrasts with the proliferative effects of endocrine GH (34). This distinction may also relate to the cellular relocalization of GHR and its interaction with GH from the plasma membrane to intracellular organelles mediated by autocrine GH (24), which may result in distinct modes of signal transduction that differentially affect proliferation and motility compared to exogenous GH. Along these lines, tumor progression has been described as involving the transition from a highly proliferative phenotype to a more motile and invasive phenotype, which may be related to contrasting effects of specific signaling pathways on proliferation and motility (35,36). The GHR interacts with multiple intracellular signaling pathways that have the potential to directly influence motility mechanisms (32). Of the canonical GHR-associated signaling pathways, PI3K is known to be involved in growth factormediated activation of Rac (37,38), one of the Rho family GTPases that control the cytoskeletal changes required for cell movement (39,40). PI3K-dependent pathways also activate focal adhesion kinase (FAK), which localizes with integrins in focal adhesions and at the cell periphery to coordinate the actin reorganization and turnover of focal adhesions involved in cell migration (41,42), and hGH has also been shown to stimulate FAK phosphorylation (43,44). Consistent with the potential contribution of PI3K-mediated mechanisms in the action of GH on cell motility, inhibition of PI3K abrogated the stimulation of LNCaP cell migration in the wound healing assay (Figure 3). However, the PI3K pathway does not appear to be the sole mediator of hGHstimulated LNCaP cell migration, as inhibition of the STAT5 and MAPK pathways also blocked hGH-induced migration, indicating the involvement of these pathways at some level as well. This contrasts somewhat with the role of these pathways in the effects of exogenous and autocrine hGH on LNCaP cell proliferation, in which only PI3K and STAT5 pathways appeared to be involved (24). This disparity in the roles of the individual signaling pathways may also underlie the converse effects of autocrine GH on cell proliferation and migration.


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Since cell motility is linked to reorganization of the actin cytoskeleton, we looked for any observable changes in F-actin localization that paralleled the GH-mediated induction of LNCaP cell motility. While it is premature to ascribe specific structural changes induced by exogenous or autocrine GH, the preliminary results presented here appear to show the increased concentration of F-actin at discrete foci at the periphery of cells and the tips of cell extensions in response to GH exposure that may relate to morphological changes associated with increased motility. Interestingly, the increased transition of prostate cancer cells, and specifically LNCaP cells, from an epithelial to a neuroendocrine phenotype upon culturing in androgen-depleted conditions has been described, and results in morphological changes such as increased cellular extensions and processes, changes in the expression of neuroendocrine cell markers, and the association with an increasingly invasive phenotype (45). Thus, some of the observed changes in LNCaP cell motility and F-actin distribution observed in the experiments presented here may also involve such phenomena. One surprising observation from the present pathway analysis was that inhibition of STAT5 increased basal LNCaP cell chemokinesis in the absence of exogenous hGH stimulation (Figure 3A, PBS-treated cells), with a parallel change in F-actin localization (Figure 5), while it blocked the stimulation of cell migration by hGH. In contrast, the MAPK and PI3K pathway inhibitors had no effect in the absence of hGH compared to their negative effects on hGH-stimulated migration. This observation suggests that STAT5 inhibits cell motility in the absence of ligand activation, perhaps by competing with the related factor STAT3 in the regulation of Rho-mediated cell motility (46). Indeed, STAT3 has been implicated in stimulating prostate cancer cell metastasis (47,48). Thus, STAT5 activation by hGH may abrogate this competition and cause a switch to a pro-motility effect of STAT5, which has also been correlated with the metastatic behavior of prostate cancer cells (49). This dynamic contrasts with the effects of STAT5 inhibition on LNCaP cell growth in which STAT5 inhibitor had no effect on basal cell proliferation but blocked hGH-stimulated proliferation (24). Interestingly, while we have shown that hGH can induce STAT5 phosphorylation in LNCaP cells (24), these cells possess a degree of constitutive STAT5 activation (Figure 5) (50), consistent with the effect of the STAT5 inhibitor observed in the absence of ligand. Taken together, the present study and previous reports indicate that STAT5 may be involved in different aspects of prostate cancer cell-function depending on the specific upstream ligand-dependent activation pathways in action. Significantly, recent work by others has corroborated the role of STAT5 signaling in mediating the effects of GH on prostate tumorigenesis (51).

Conclusions Numerous studies have established a correlation between cell motility and prostate cancer metastasis. Thus, agents that can increase the motility of prostate cancer cells have the potential to augment metastasis. The results of the present study indicate that endocrine hGH may increase prostate cancer cell


motility and function as a chemoattractant for prostate cancer cells which could contribute to stimulating and directing their migration. The findings presented also suggest that the ectopic activation of hGH gene expression that has been observed in prostate cancer cells may also enhance cell motility. These effects involve multiple signaling pathways, and culminate at least in part in the reorganization of F-actin localization. We conclude from these observations that hGH may influence prostate cancer metastasis in addition to its established effects on cell proliferation and survival, supporting the hypothesis that hGH is a potential contributor to prostate cancer progression.

Acknowledgements The authors wish to thank Holly Jefferson for technical assistance.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by grants from the Leo W. Jenkins Cancer Center and the Brody Brothers Medical Foundation (#MT7749) to BMS.

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Induction of Apoptosis and Growth Suppression by Homeobox Gene TGIFLX in Prostate Cancer Cell Line Lncap.

Combination of Quercetin and 2-Methoxyestradiol Enhances Inhibition of Human Prostate Cancer LNCaP and PC-3 Cells Xenograft Tumor Growth.

v-rasH expression confers hormone-independent in vitro growth to LNCaP prostate carcinoma cells.

Production of epidermal growth factor and transforming growth factor-alpha by the androgen-responsive LNCaP human prostate cancer cell line.

EphA2 enhances the proliferation and invasion ability of LNCaP prostate cancer cells.

Effect of pentosan, a novel cancer chemotherapeutic agent, on prostate cancer cell growth and motility.

Antiproliferative effects of luteinizing hormone-releasing hormone agonists on the human prostatic cancer cell line LNCaP.

Effect of Saw Palmetto Supplements on Androgen-Sensitive LNCaP Human Prostate Cancer Cell Number and Syrian Hamster Flank Organ Growth.

KIBRA promotes prostate cancer cell proliferation and motility.

The effects of basic fibroblast growth factor and suramin on cell motility and growth of rat prostate cancer cells.

Induction of caspase cascade pathway by kaempferol-3-O-rhamnoside in LNCaP prostate cancer cell lines.

Comparative genomic and transcriptomic analyses of LNCaP and C4-2B prostate cancer cell lines.

Rapamycin inhibits prostate cancer cell growth through cyclin D1 and enhances the cytotoxic efficacy of cisplatin.

Characterization of a novel metastatic prostate cancer cell line of LNCaP origin.

Crab Shell Extract Induces Prostate Cancer Cell Line (LNcap) Apoptosis and Decreases Nitric Oxide Secretion.

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones.

Divergent responses to epidermal growth factor in hormone sensitive and insensitive human prostate cancer cell lines.

Effects of 14 frequently used drugs on prostate-specific antigen expression in prostate cancer LNCaP cells.

Heparin co-factor II enhances cell motility and promotes metastasis in non-small cell lung cancer.

Regulation of fibroblast growth factor-like protein(s) in the androgen-responsive human prostate carcinoma cell line LNCaP.

Stimulatory effects of antiandrogens on LNCaP human prostate tumor cell growth, EGF-receptor level and acid phosphatase secretion.

[Metastatic hormone-sensitive prostate cancer].

AZD1152-HQPA induces growth arrest and apoptosis in androgen-dependent prostate cancer cell line (LNCaP) via producing aneugenic micronuclei and polyploidy.

Increased hydrostatic pressure enhances motility of lung cancer cells.