Skip to main content
NCBI home page
Molecular Endocrinology logoLink to Molecular Endocrinology
. 2012 Jun 25;26(9):1521–1530. doi: 10.1210/me.2012-1006

Estrogen Promotes Prostate Cancer Cell Migration via Paracrine Release of ENO1 from Stromal Cells

Lin Yu 1, Jiandang Shi 1,✉, Sa Cheng 1, Yan Zhu 1, Xiulan Zhao 1, Kuo Yang 1, Xiaoling Du 1, Helmut Klocker 1, Xiaoli Yang 1, Ju Zhang 1,✉
PMCID: PMC5416971  PMID: 22734040

Abstract

As a key glycolytic enzyme, enolase 1 (ENO1) is critical for cellular energy metabolism. Recent studies have revealed its important role in growth and metastasis of lung, head and neck, and breast cancer. However, the regulatory mechanisms of ENO1 expression and secretion remain unclear. We observed that conditioned medium from estradiol-stimulated prostate stromal cells significantly promoted the migration of prostate cancer (PCa) cells. Two-dimensional protein electrophoresis, mass spectrometry, and immunodepletion assays identified one of the major active factors in the conditioned medium as α-type enolase (α-enolase, or ENO1). Moreover, in prostate stromal cells, estradiol not only enhanced the stability of ENO1 at the protein level in an estrogen receptor-α-dependent manner but also promoted its secretion to the extracellular matrix. Furthermore, recombinant ENO1 bound to the surface of PCa cells and promoted cell migration via their plasminogen receptor activity in a paracrine manner. Immunohistochemistry suggested that stromal ENO1 levels increased in PCa compared with those in normal tissue.

In recent years, accumulating evidence suggests that estradiol (E2) promotes the progression of prostate cancer (PCa) (1, 2). In aging males, the elevated ratio of circulating levels of estrogen to androgen correlates with PCa incidence (3). Moreover, the antiestrogen toremifene was shown to inhibit tumor progression in patients with early PCa (4). However, it has been demonstrated that although estrogen treatment temporarily inhibited PCa growth, the proportion of patients with metastasis was significantly higher (5). The mechanism underlying this phenomenon remains unclear.

Enolase, also known as pyruvate dehydrogenase phosphatase, catalyzes not only the transformation of 2-phosphate-d-glycerate to phosphoric acid-pyruvate during glycolysis but also the reverse conversion of phosphoric acid-pyruvate to 2-phosphate-d-glycerate during glycogen synthesis (6). Thus, enolase plays a critical role in anaerobic glycolysis. In mammalian cells, there are three enolase subunits: α-, β-, and γ-enolase, encoded by the genes ENO1, ENO3, and ENO2, respectively (7). Enolase 1 (ENO1) is also known as non-nerve-specific enolase and is expressed in liver, brain, kidney, spleen, and many other tissues (8). By participating in anaerobic glycolysis (Warburg effect) and providing ATP, ENO1 is thought to promote tumor development and progression (6). Indeed, ENO1 expression is frequently increased in diverse tumors, including brain, breast, colon, lung, kidney, and ovary (9).

ENO1 could also be observed on the cell surface of tumors, such as breast, lung, and pancreatic cancer (10–12). At the cell surface, ENO1 binds to plasminogen, which 1) enhances plasminogen hydrolysis to plasmin by tissue plasminogen activator and urokinase plasminogen activator and 2) protects plasminogen from inhibition by α2-plasmin inhibitor (13, 14). The pericellular proteolytic activities of plasminogen and plasmin (e.g. matrix metalloproteinase activation, extracellular matrix degradation, tissue remodeling, and angiogenesis) play an important role in cancer cell proliferation, invasion, and metastasis (15, 16). Thus, in addition to providing cellular energy, the pro-tumorigenic properties of ENO1 may also be attributed to its ability to act as a plasminogen receptor.

Despite the well-documented contribution of ENO1 in promoting growth, metastasis, and migration of lung, head and neck, and breast cancer cells (11, 17, 18), the role of ENO1 on the migration of PCa cells and the mechanisms underlying ENO1 regulation remains unknown. This study aimed to determine whether secretion of ENO1 by human prostate stromal cells could be modulated by E2 and whether stromal-derived secreted ENO1 acts in a paracrine manner to promote plasminogen activation and PCa cell migration.

Materials and Methods

Cell culture and agonist

Human prostate primary stromal cells (PrSC) were isolated from fresh surgical prostate specimens of individual patients with benign prostatic hyperplasia (BPH) and cultured as previously described (19). Informed consent was obtained from each patient. This study was approved by the Institutional Review Board of the First Central Hospital, Tianjin, China. The human prostate stromal cell line WPMY-1 was obtained from the American Type Culture Collection (Manassas, VA). WPMY-1 cells were routinely maintained in DMEM phenol red-free medium (Sigma-Aldrich, St. Louis, MO) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA) and 5% fetal bovine serum (FBS) (Invitrogen) at 37 C under 5% CO2. The human PCa cell lines PC3 and Du145 were obtained from American Type Culture Collection. All PCa cell lines were cultured in RPMI 1640 phenol red-free medium (Sigma) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin and 10% FBS (Invitrogen). The estrogen receptor-α (ERα) agonist 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) and G protein-coupled receptor 30 (GPR30) agonist G1 were ordered from Sigma.

Collection of conditioned medium (CM)

WPMY-1 cells and PrSC were cultured in 15-cm dishes in DMEM with 2.5% charcoal-dextran-treated FBS (Invitrogen) for 48 h. The medium was changed to serum-free DMEM/F12 with 5 ng/ml sodium selenite, 40 μg/ml l-proline, 1% nonessential amino acids, and 1% penicillin-streptomycin. After 24 h, media were replaced and supplemented with E2 or ethanol (vehicle) equivalent at the indicated concentration for 48 h. The CM were centrifuged at 200 × g for 10 min. The CM from cells treated with E2 and vehicle, in the text, are referred to CM-E2 and CM-con, respectively. Unconditioned medium (unCM) supplemented with E2 or ethanol equivalent were prepared as basal control and are referred to in the text as unCM-E2 and unCM-con, respectively.

Transwell migration assay

PCa cells were seeded in Transwell inserts with 8-μm pore size (BD Biosciences, San Jose, CA) at 500,000 cells per well in DMEM/F12 serum-free medium with 5 ng/ml mitomycin C to inhibit cell growth. CM were added to the bottom chamber. After 24 h, cells on the upper surface of the filter were removed using a cotton swab. Cells that had migrated through the filter to the lower surface were fixed with 4% paraformaldehyde and stained with 10% crystal violet. Cells were counted from five randomly selected fields per chamber.

Wound healing assay

PCa cells were seeded in equal numbers in six-well plates. Once 90% confluent, three vertical wounds per well were scratched using a 0.1-μl pipette tip and cells incubated with the indicated CM from prostate stromal cells supplemented with 5 ng/ml mitomycin C to inhibit cell growth. Images were collected at the designated time thereafter at ×100 magnification to assess wound closure.

Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The CM were concentrated by Amicon Ultra-15 centrifugal filter units with Ultracel-10 membrane according to the manufacturer's instructions (Milipore, Billerica, MA). Two-dimensional gel electrophoresis and mass spectrometry of concentrated CM were performed at the Tianjin Biochip Corp. (Tianjin, China) (20).

α-ENO1 immunodepletion of prostate stromal cell CM

One milliliter of CM was incubated with 10 μg α-ENO antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and Protein A/G Plus (Calbiochem, Hamburg, Germany) according to the manufacturer's instructions.

Small interfering RNA (siRNA) and plasmid transfection

ERα(ESR1-homo-1014), GPR30(GPER-homo-1163), and scrambled control siRNA were purchased from GenePharma (Shanghai, China). Prostatic stromal cells were transfected with siRNAs as described (21). The mammalian pcDNA3.1-ENO1 overexpression plasmid was obtained from the IMAGE consortium (IMAGE ID: 3447583) and subcloned to the plasmid pcDNA3.1. pcDNA-SE was constructed by inserting a signaling peptide from Ig κ-chain into the backbone of pcDNA3.1. Cells were transfected with 0.6 μg plasmid DNA using Effectene transfection reagent (QIAGEN, Hilden, Germany) according to the manufacturer's protocol.

RNA extraction and real-time RT-PCR

Total RNA extraction and real-time RT-PCR were performed as described (22). The housekeeping gene HPRT was used as internal reference. Real-time RT-PCR primers were obtained from Sangon (Shanghai, China): ENO1, 5′-CAGGCCAATGGTTGGGGCGT-3′ and 5′-GGCTTGCCTGCCCACAGCTT-3′, and HPRT, 5′-TGACACTGGCAAAACAATGCA-3′, 5′-GGTCCTTTTCACCAGCAAGCT-3′

Western blotting

Total cell lysates from prostate stromal cells were prepared using RIPA buffer. Lysates were cleared by centrifugation at 10,000 × g for 20 min at 4 C and the protein concentration determined using the bicinchoninic acid assay (Thermo, Rockford, IL). SDS-PAGE and Western blotting were performed as described (21). Anti-ENO1 and anti-glyceraldehyde-3-phosphate dehydrogenase antibodies were from Santa Cruz.

ELISA

Secreted ENO1 in CM was quantified using an α-ENO ELISA kit (LaiBio, Shanghai, China) according to the manufacturer's instructions.

Immunofluorescence

PC3 cells were seeded on poly-d-lysine-coated coverslips and grown until 85% confluent, and then the coverslips were cultured in medium with 30 μg/ml recombinant 6xHis-tagged ENO1 for 24 h. The monolayers were rinsed with PBS twice and fixed with 4% paraformaldehyde for 10 min. The cell monolayers were blocked with 3% BSA/PBS for 30 min. Bound 6xHis-tagged recombinant ENO1 was detected by fluorescent microscopy (Eclipse TE2000; Nikon, Shanghai, P.R. China) using a mouse monoclonal anti-6xHis Tag antibody (Santa Cruz) diluted 1:75 in PBS with 3% BSA followed by Alexa Fluor 594-conjugated donkey antimouse secondary antibody (Invitrogen).

Plasminogen activation assay

Plasminogen activation by CM of stromal cells treated with E2 was measured as described (18). Briefly, 40 μl CM was combined with 50 μl PBS (pH 7.4), 5 μl 10 mm Val-Leu-Lys-4-nitroanilide (VALY) (Sigma), and 0.1 U human plasminogen (Sigma) before incubation at 37 C for 4 h. Absorbance was measured at 405 nm on a Multiskan FC plate reader (Thermo).

Immunohistochemistry

Prostate cancer (seven patients who performed radical prostatectomy for prostate carcinoma), BPH (seven patients treated by surgery for BPH), and normal prostate (seven autopsy cases of patients who died due to illness other than malignancy) tissues were obtained from the Department of Pathology and the Department of Urology, the Second Affiliated Hospital of Tianjin Medical University, Tianjin, China. Informed consent was obtained from each patient. Use of the tissue samples in this study was approved by the Institutional Review Board. Fresh tissues were fixed in 4% formaldehyde and embedded in paraffin. Five-micrometer sections were deparaffinized in xylene and rehydrated in a graded series of alcohol. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide for 10 min, followed by incubation with 10% serum for 30 min at room temperature. Sections were incubated with primary antibody directed to ENO1 (1:300; Santa Cruz) at room temperature for 2 h. A biotinylated secondary antibody was added for 30 min at 37 C, followed by immunohistochemical staining using a diaminobenzidine kit (Zhongshan, Beijing, China).

Statistical analysis

Numerical data are presented as mean ± sd from at least three independent experiments. Statistical differences were analyzed using SPSS version 16.0 software (IBM, Chicago, IL). Differences among groups were assessed using ANOVA, and the least significant difference test was used to compare the differences between every combination of two groups. Differences between two groups were assessed using Student's t test. A P value <0.05 was considered statistically significant.

Results

E2 induces stromal cell paracrine effects that promote PCa cell migration

To investigate whether E2 induces changes in the stromal cell secretome that modulate PCa cell migration, the prostate stromal cell line WPMY-1 and human PrSC (19) were cultured in serum-free DMEM/F12 in the presence of 10 nm E2 for 48 h and in the CM applied to the PCa cell lines PC3 and Du145. Using the wound-healing assay, CM from E2-treated WPMY-1 cells and E2-treated PrSC were found to increase PC3 cell migration 1.8- and 2.2-fold, respectively (Fig. 1, A and B). In addition, CM from E2-treated WPMY-1 cells and PrSC increased Du145 migration by 1.9- and 2.0-fold, respectively (Fig. 1, C and D), the nonconditioned medium and mock-treated CM did not show such increased effects. Similar observations were found using the chemotactic Transwell assay (Fig. 1, E and F).

Fig. 1.

Fig. 1.

Open in a new tab

CM of E2-treated prostate stromal cells stimulate PCa cell migration. A–D, Wound healing assay of PC3 (A and B) or Du145 cells (C and D) incubated for 0 or 24 h with CM from WPMY-1 cells or PrSC treated for 48 h with 10 nm E2 or ethanol equivalent (con). B and D, Quantification of the relative distance of PC3 (B) or Du145 (D) cell migration from cells treated as in A or C, respectively. Bars represent mean distance migrated ± sd relative to cells treated with unconditioned media supplemented with ethanol equivalent (unCM-con). E and F, Chemotactic Transwell assay of PC3 (E) or Du145 (F) cells incubated for 24 h with CM from WPMY-1 cells or PrSC treated for 48 h with 10 nm E2 or ethanol equivalent (con) as indicated. Bars represent mean number of migrated cells ± sd. A and C, Images are representative of three independent experiments. Scale bar, 200 μm. C–F, Unconditioned media supplemented with 10 nm E2 or ethanol equivalent were used as control (unCM-E2 and unCM-con, respectively). Data are derived from three independent experiments. *, P < 0.05 vs. controls.

ENO1 is the activating factor in prostate stromal CM that promotes PCa cell migration

To identify the components in stromal cell CM that promoted PCa cell migration, CM were concentrated, separated by two-dimensional electrophoresis, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Supplemental Figs. 1 and 2, published on The Endocrine Society's Journals Online web site at http://mend.endojournals.org). Five secretory proteins between E2- and mock-treated CM were identified (Table 1). In particular, ENO1 was 36.3-fold enriched in CM from E2-treated cells.

Table 1.

Secreted proteins in prostate stromal cell CM

Dot number Protein name Fold change (E2/mock) P value
4604 Hypothetical protein TRIADDRAFT_63547 3.85 0.02871
4606 ENO1 36.34 0.00354
5303 Triosephosphate isomerase 1 −2.61 0.01796
2301 Ubiquitin carboxyl-terminal esterase L1 5.52 0.01241
8408 Heterogeneous nuclear ribonucleoprotein C 4.42 0.00856
Open in a new tab

The table shows five identified significantly different secretory proteins between E2- and mock-treated CM. A negative fold change means the protein was down-regulated by E2 in the CM.

α-Enolase-specific antibodies inhibit enolase function at the cell surface (23). To investigate whether the migration-promoting effect of stromal cell CM was mediated by secreted ENO1, a specific polyclonal antibody against the plasminogen-binding site of ENO1 was employed to immunodeplete ENO1 from CM, which was subsequently applied to PCa cells. The level of ENO1 in the PCa cell lysates and CM were also measured by Western blot and ELISA. E2 treatments had no effect on the ENO1 level of PCa cells, whereas the concentration of ENO1 secreted from PCa cells was nearly undetectable (Supplemental Fig. 3). In scratch wound assays, ENO1-immunodepleted CM from E2-treated prostatic stromal cells reduced PC3 and Du145 cell migration by 83 and 100%, respectively, relative to CM from E2-treated cells immunodepleted with nonspecific IgG control antibody (Fig. 2).

Fig. 2.

Fig. 2.

Open in a new tab

Immunodepletion of ENO1 from E2-treated prostatic stromal cell CM abrogates prostate cancer cell migration. A, ELISA quantification of ENO1 in CM from WPMY-1 cells and PrSC treated for 48 h with 10 nm E2 or ethanol equivalent (con) and subsequently immunodepleted with anti-ENO1 or IgG control antibodies. Data represent mean ENO1 concentration ± sd. B and C, Wound healing assays of PC3 (B) or Du145 cells (C) incubated for 24 h with CM as in A. Bars denote mean distance migrated ± sd relative to cells treated with control CM immunodepleted with IgG control antibodies. Data are derived from three independent experiments. *, P < 0.05 vs. controls.

E2 increases ENO1 protein accumulation and secretion of prostatic stromal cells via an ERα-dependent manner

To investigate whether E2 modulates ENO1, WPMY-1 cells and PrSCs were treated with different concentrations of E2 for 24 h. Although there was no change in ENO1 observed at the mRNA level (Fig. 3A), E2 treatment for 48 h significantly increased cytoplasmic and secreted ENO1 protein levels in both WPMY-1 cells and PrSC in a dose-dependent manner (Fig. 3, B and C). Moreover, ENO1 protein levels were not affected by pretreating stromal cells with the mRNA synthesis inhibitor actinomycin D (Fig. 3D), whereas E2-mediated up-regulation of ENO1 was significantly attenuated by the protein synthesis inhibitor cycloheximide (Fig. 3D). Furthermore, E2 significantly prolonged ENO1 protein half-life (Fig. 3E). Collectively, these data suggest that E2 may increase ENO1 protein accumulation in stromal cells via a posttranscriptional mechanism.

Fig. 3.

Fig. 3.

Open in a new tab

E2 regulates ENO1 protein levels in prostate stromal cells via a posttranscriptional mechanism. A, Real-time RT-PCR of ENO1 mRNA in WPMY-1 cells and PrSC treated with the indicated concentration of E2 for 24 h. Bars denote mean fold change in expression ± sd relative to mock-treated (con) cells. B, Western blot of ENO1 in WPMY-1 cells and PrSC treated with the indicated concentration of E2 for 48 h. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. C, ELISA quantification of ENO1 in CM from WPMY-1 cells and PrSC treated for 48 h with the indicated concentration of E2. Bars denote mean concentration of ENO1 ± sd. D, ELISA quantification of ENO1 in total cell lysates from WPMY-1 cells and PrSC treated for 48 h with the indicated concentration of E2 in the presence or absence of actinomycin D (ACTD; 10 μg/ml) or cycloheximide (CHD; 10 μg/ml) as depicted. Bars denote mean concentration of ENO1 ± sd. E, Western blot of ENO1 in WPMY-1 cells and PrSC treated with 10 nm E2 (+) or ethanol equivalent (−) in the presence of 10 μg/ml CHD for the duration stated. GAPDH served as loading control. B and E, Images are representative of three independent experiments; A, C, and D, data are derived from three independent experiments. *, P < 0.05 vs. controls.

To investigate whether cellular or secreted ENO1 was responsible for promoting PCa cell migration, the ENO1 open reading frame was cloned into the eukaryotic expression vector pcDNA3.1 (pcDNA-ENO1) and the secreted eukaryotic expression vector pcDNA-SE (pcDNA-SE-ENO1) as well, and the two plasmids were transfected into WPMY-1 cells. Of note, human ENO1 lacks a classic signal peptide sequence, and its secretion is thought to be mediated via exosomes (24–29). ENO1 expression was confirmed at the mRNA and protein level (Fig. 4, A and B). The ENO1 concentration of CM from WPMY-1 transfected with pcDNA-SE-ENO1 was much higher than that with pcDNA-ENO1 (Fig. 4C). The CM from WPMY-1 cells transfected with these two plasmids and treated with or without E2 was applied to PCa cells in scratch wound assays. The CM of WPMY-1 transfected with pcDNA-ENO1 did not promote Du145 and PC3 cell migration (Fig. 4, D and E). In contrast, The CM of WPMY-1 transfected with pcDNA-SE-ENO1 significantly promoted the migration of Du145 and PC3 cells (Fig. 4, D and E) to the levels comparable with E2-treated control cells (Fig. 4, D and E). These data suggest that E2 can promote stromal cell ENO1 secretion.

Fig. 4.

Fig. 4.

Open in a new tab

E2 promotes secretion of ENO1 by prostate stromal cells. A, Real-time RT-PCR of ENO1 mRNA levels from WPMY-1 cells transiently transfected to overexpress ENO1 from the basal expression vector pcDNA3.1 (pcDNA-ENO1) or from the signal peptide containing expression vector pcDNA-SE (pcDNA-SE-ENO1). Cells transfected with corresponding empty vectors (pcDNA and pcDNA-SE) served as control. Data represent mean fold change in ENO1 expression ± sd relative to empty vector control (pcDNA). B, Western blotting of ENO1 in total cell lysates from cells treated as in A. Images are representative of three independent experiments. C, ELISA quantification of ENO1 in CM from WPMY-1 cells transiently transfected to overexpress ENO1 from the basal expression vector pcDNA3.1 (pcDNA-ENO1) or from the signal peptide containing expression vector pcDNA-SE (pcDNA-SE-ENO1). Cells transfected with corresponding empty vectors (pcDNA and pcDNA-SE) served as control. Data represent mean fold change of ENO1 concentration ± sd relative to empty vector control (pcDNA). D and E, Wound healing assay of PC3 (D) or Du145 (E) cells incubated for 24 h with CM from WPMY-1 cells treated as in B in the presence or absence (con) of 10 nm E2 for 48 h. Bars represent mean distance migrated relative to cells incubated with CM from mock-treated (con) WPMY-1 cells transfected with empty pcDNA vector. Data are derived from three independent experiments. *, P < 0.05 vs. controls.

WPMY-1 cells express two types of estrogen receptor, namely ERα and the membrane-bound GPR30 (21, 22). WPMY-1 cells were transfected with receptor-specific siRNA to investigate via which receptor E2-mediated ENO1 up-regulation occurred (Supplemental Fig. 4). E2 significantly induced cellular and secreted levels of ENO1 in WPMY-1 cells transfected with scrambled control and GPR30-specific siRNA but not in cells transfected with ERα-specific siRNA (Fig. 5, A and B), and ERα agonist PPT but not GPR30 agonist G1 significantly induced cytoplasmic and secreted protein levels of ENO1 in WPMY-1 (Fig. 5, C and D), suggesting that E2-mediated induction of ENO1 occurs in an ERα-dependent manner.

Fig. 5.

Fig. 5.

Open in a new tab

E2-mediated induction of ENO1 in WPMY-1 cells is ERα dependent. A and B, WPMY-1 cells transfected for 48 h with scramble or ERα- or GPR30-specific siRNA before ELISA quantification of ENO1 in cell lysates (A) or CM (B). C and D, ELISA quantification of ENO1 in cell lysates (C) or CM (D) from WPMY-1 cells treated with E2, PPT, or G1 for 48 h. Bars denote mean ENO1 concentration ± sd from three independent experiments. *, P < 0.05 vs. controls.

Secreted ENO1 promotes PCa cell migration via its plasminogen-binding domain

The 13th lysine residue within the C-terminal plasminogen-binding domain of ENO1 is critical for interaction with plasminogen (13, 14). Thus, to assess the importance of plasminogen binding by secreted ENO1 in regulating PCa cell migration, wild-type ENO1 (pcDNA-SE-ENO1) or an ENO1 harboring a plasminogen-binding site mutation (pcDNA-SE-ENO1-ΔPlg) were subcloned into the eukaryotic secretory expression vector pcDNA-SE and transfected into WPMY-1 cells. In scratch wound assays, the CM of WPMY-1 cells transfected with pcDNA-SE-ENO1-ΔPlg no longer promoted PCa cell migration relative to the CM of control cells (Fig. 6A). The plasminogen-binding activity of ENO1 is inhibited by the lysine analog tranexamic acid (TXA) (30). To further confirm whether secreted ENO1 promotes PCa cell migration via an interaction with plasminogen, WPMY-1 cells were pretreated with TXA (Fig. 6B). TXA attenuated PCa cell migration induced by CM from E2-treated WPMY-1 cells (Fig. 6B).

Fig. 6.

Fig. 6.

Open in a new tab

Secreted ENO1 promotes prostate cancer migration via its plasminogen-activating ability. A, Wound healing assay was performed to evaluate the effect of different WPMY-1 CM on migration of PC3 and Du145 cells. The CM were obtained from WPMY-1 cells transfected with the following expression vectors for secreted ENO1: wild-type ENO1 (pcDNA-SE-ENO1), ENO1 with plasminogen-binding site mutation (pcDNA-SE-ENO1-ΔPlg), or control vector (pcDNA-SE). Data are shown as relative migration distance under the microscope. Results are presented as mean ± sd; n = 3. *, P < 0.05 vs. controls. B, Effect of WPMY-1 CM pretreated with 10 mm TXA on migration of PC3 and Du145 cells. Wound healing assay was performed to evaluate the effect of CM from WPMY-1 cells on migration of PC3 and Du145 cells. Data are shown as relative migration distance under the microscope. Results are presented as mean ± sd; n = 3. *, P < 0.05 vs. controls. C, Immunofluorescence analysis of the ENO1 localized on PC3 cells. Photographs were captured under ×200 fields. The length of the dash is 100 μm. D, E2 induced plasmin generation in the CM of prostate stromal cell in a dose-dependent manner. E–G, ENO1 immunodepletion (E), ENO1 plasminogen-binding site mutation (F), and TXA treatment (G) inhibited the plasmin activity in the CM.

To detect whether a direct interaction may exist between ENO1 protein and PCa cells, PC3 cells were incubated for 24 h with 5 μg/ml 6xHis-tagged recombinant ENO1 protein or 6xHis-tagged green fluorescent protein as mock control before immunostaining with an anti-His tag antibody. Immunofluorescent staining was apparent on the surface of PC3 cells incubated with His-tagged ENO1 but not with His-tagged green fluorescent protein, suggesting that recombinant ENO1 protein was located at the surface of PC3 cells (Fig. 6C).

The plasminogen-activating ability of E2-treated CM from WPMY-1 cells/PrSC was evaluated by measuring hydrolysis of VALY (18). E2 increased the ability of PrSC CM to hydrolyze VALY to plasmin in a dose-dependent manner (Fig. 6D). Notably, however, ENO1 immunodepletion, ENO1 plasminogen-binding site mutation, or TXA treatment significantly attenuated E2-induced plasmin generation in stromal cell CM (Fig. 6, E–G).

ENO1 is elevated in tumor-associated stroma of PCa

Immunohistochemistry staining revealed that ENO1 protein levels were higher in the stroma of human PCa tissue (Gleason score 4–8) than those in the stroma of normal prostate and BPH tissue (Fig. 7, A–C).

Fig. 7.

Fig. 7.

Open in a new tab

Protein levels of stromal ENO1 are elevated in prostate cancer. A–C, Immunohistochemical staining of ENO1 in biopsy specimens from normal prostate tissue (A), tissue BPH tissue (B), and prostate cancer tissue (C). Sections were counterstained with hematoxylin; D, quantification of the ENO1-positive stromal cell per total stromal cells; n = 7. *, P < 0.05.

Discussion

In addition to its well-documented enzymatic role in glycolysis, ENO1 at the cell surface acts as a plasminogen receptor serving to promote pericellular plasminogen activation, which plays a critical role in physiological and pathophysiological processes involving extracellular matrix remodeling, e.g. angiogenesis, tumor cell invasion, and migration (6, 13, 14). For example, ENO1 promotes lipopolysaccharide-driven chemokine-directed in vitro pulmonary monocyte migration and matrix invasion (31).

Expression of ENO1 is regulated at multiple levels via different mechanisms in distinct tissues. For example, miRNA17/20 inhibits the expression of ENO1 in breast cancer (18), retinoic acid reduces ENO1 at the protein level in thyroid carcinoma (32), whereas valproic acid inhibits histone deacetylase activity and increases ENO1 protein levels in PCa cells (33). We observed that E2 increases ENO1 protein stability in prostatic stromal cells and promotes ENO1 secretion from stromal cells in an ERα-dependent manner.

The prostate stromal compartment plays a critical role in the cancer metastasis process by secreting growth factors and extracellular matrix components. A recent report described up-regulation of prostatic stromal cell-derived matrix metalloproteinase 2 by E2 via an ERα-dependent pathway, which promoted PCa invasion in a paracrine manner (21). Data herein indicate that CM of E2-treated prostatic stromal cells promotes the migration of PCa cells. Proteomic analysis revealed significant enrichment of ENO1 in E2-treated stromal cell-derived CM, and immunodepletion of ENO1 was sufficient to abrogate the promigratory effects on PCa cells of CM from E2-treated cells. Human ENO1 lacks a signal peptide sequence; thus, its secretion may not be mediated by the classic endoplasmic reticulum-Golgi pathway. Rather, it is thought that ENO1 secretion to the extracellular space is mediated primarily via exosomes (24). Supportively, ENO1 has been identified in exosomes from different cells and tissues, including B cells (25), colon cancer cells (26, 27), melanoma cells (28), and PCa cell xenografts (29). In this study, we demonstrate that only ENO1 expressed from a vector encoding an exogenous signal peptide sequence recapitulated the effects of CM from E2-treated prostatic stromal cells. The immunohistochemistry results showed stromal ENO1 levels were significantly higher in PCa than in the stroma of normal and BPH, whereas ENO1 level in the epithelium had no significant difference during the malignant process. Given that PCa cells secrete low levels of ENO1 (Supplemental Fig. 3), these data strongly suggest that ENO1 secreted from prostate stromal cells is the primary paracrine factor that promotes PCa cell migration.

Being consistent with plasminogen recruitment by cell surface ENO1 (12, 14, 34, 35), we observed that mutation of the plasminogen-binding site within secreted ENO1 or treatment with the lysine analog TXA, which blocks the plasminogen receptor activity of ENO1 (30), was sufficient to attenuate the PCa cell migration-promoting effects induced by CM of E2-treated prostatic stromal cells. Furthermore, recombinant ENO1 was found to associate with the surface of PC3 cells. These results suggest that prostate stromal cell-derived ENO1 binds to the surface of PCa cells as a plasminogen receptor promoting PCa cell migration, revealing a novel role of key plasminogen receptor ENO1 in prostate stroma-epithelium interaction.

In summary, the data presented herein suggest that E2 promotes the secretion of stromal cell-derived ENO1 by enhancing the stability of ENO1 protein, which upon association with the surface of PCa cells recruits and activates plasminogen, thereby promoting extracellular matrix remodeling and cancer cell migration.

Acknowledgments

This research was funded by National Basic Research Programs (973 Programs, No. 2010CB945003), National Natural Science Foundation of China (Grants 81072111 and 81060214), Joint Research Fund for Overseas Chinese Scholars and Scholars in Hong Kong and Macao (30928027), Tianjin Municipal Science and Technology Commission (Grants 09ZCKFSF00800 and 10JCYBJC12800), and the 111Project (B08011).

Disclosure Summary: The authors have nothing to disclose.

NURSA Molecule Pages†:

  • Ligands: 17β-estradiol.

†

Annotations provided by Nuclear Receptor Signaling Atlas (NURSA) Bioinformatics Resource. Molecule Pages can be accessed on the NURSA website at www.nursa.org.

Abbreviations:
BPH
Benign prostatic hyperplasia
CM
conditioned medium
E2
estradiol
ENO1
enolase 1
ERα
estrogen receptor-α
FBS
fetal bovine serum
GPR30
G protein-coupled receptor 30
PCa
prostate cancer
PrSC
prostate primary stromal cells
PPT
1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole
siRNA
small interfering RNA
TXA
tranexamic acid
VALY
Val-Leu-Lys-4-nitroanilide.

References

  • 1. Prins GS , Korach KS. 2008. The role of estrogens and estrogen receptors in normal prostate growth and disease. Steroids 73:233–244 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Ricke WA , McPherson SJ , Bianco JJ , Cunha GR , Wang Y , Risbridger GP. 2008. Prostatic hormonal carcinogenesis is mediated by in situ estrogen production and estrogen receptor α signaling. FASEB J 22:1512–1520 [DOI] [PubMed] [Google Scholar]
  • 3. Ho SM , Tang WY , Belmonte de Frausto J , Prins GS. 2006. Developmental exposure to estradiol and bisphenol A increases susceptibility to prostate carcinogenesis and epigenetically regulates phosphodiesterase type 4 variant 4. Cancer Res 66:5624–5632 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Price D , Stein B , Sieber P , Tutrone R , Bailen J , Goluboff E , Burzon D , Bostwick D , Steiner M. 2006. Toremifene for the prevention of prostate cancer in men with high grade prostatic intraepithelial neoplasia: results of a double-blind, placebo controlled, phase IIB clinical trial. J Urol 176:965–970; discussion 970–971 [DOI] [PubMed] [Google Scholar]
  • 5. de la Monte SM , Moore GW , Hutchins GM. 1986. Metastatic behavior of prostate cancer. Cluster analysis of patterns with respect to estrogen treatment. Cancer 58:985–993 [DOI] [PubMed] [Google Scholar]
  • 6. Vander Heiden MG , Cantley LC , Thompson CB. 2009. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324:1029–1033 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Pancholi V. 2001. Multifunctional α-enolase: its role in diseases. Cell Mol Life Sci 58:902–920 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Piast M , Kustrzeba-Wójcicka I , Matusiewicz M , Bana T. 2005. Molecular evolution of enolase. Acta Biochim Polonica 52:507–513 [PubMed] [Google Scholar]
  • 9. Altenberg B , Greulich KO. 2004. Genes of glycolysis are ubiquitously overexpressed in 24 cancer classes. Genomics 84:1014–1020 [DOI] [PubMed] [Google Scholar]
  • 10. Cappello P , Tomaino B , Chiarle R , Ceruti P , Novarino A , Castagnoli C , Migliorini P , Perconti G , Giallongo A , Milella M , Monsurrò V , Barbi S , Scarpa A , Nisticò P , Giovarelli M , Novelli F. 2009. An integrated humoral and cellular response is elicited in pancreatic cancer by α-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen. Int J Cancer 125:639–648 [DOI] [PubMed] [Google Scholar]
  • 11. He P , Naka T , Serada S , Fujimoto M , Tanaka T , Hashimoto S , Shima Y , Yamadori T , Suzuki H , Hirashima T , Matsui K , Shiono H , Okumura M , Nishida T , Tachibana I , Norioka N , Norioka S , Kawase I. 2007. Proteomics-based identification of α-enolase as a tumor antigen in non-small lung cancer. Cancer Sci 98:1234–1240 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Seweryn E , Pietkiewicz J , Bednarz-Misa IS , Ceremuga I , Saczko J , Kulbacka J , Gamian A. 2009. Localization of enolase in the subfractions of a breast cancer cell line. Z Naturforsch C 64:754–758 [DOI] [PubMed] [Google Scholar]
  • 13. Miles LA , Dahlberg CM , Plescia J , Felez J , Kato K , Plow EF. 1991. Role of cell-surface lysines in plasminogen binding to cells: identification of α-enolase as a candidate plasminogen receptor. Biochemistry 30:1682–1691 [DOI] [PubMed] [Google Scholar]
  • 14. Redlitz A , Fowler BJ , Plow EF , Miles LA. 1995. The role of an enolase-related molecule in plasminogen binding to cells. Eur J Biochem 227:407–415 [DOI] [PubMed] [Google Scholar]
  • 15. Plow EF , Felez J , Miles LA. 1991. Cellular regulation of fibrinolysis. Thromb Haemost 66:32–36 [PubMed] [Google Scholar]
  • 16. Danø K , Behrendt N , Høyer-Hansen G , Johnsen M , Lund LR , Ploug M , Rømer J. 2005. Plasminogen activation and cancer. Thromb Haemost 93:676–681 [DOI] [PubMed] [Google Scholar]
  • 17. Ralhan R , Masui O , Desouza LV , Matta A , Macha M , Siu KW. 2011. Identification of proteins secreted by head and neck cancer cell lines using LC-MS/MS: strategy for discovery of candidate serological biomarkers. Proteomics 11:2363–2376 [DOI] [PubMed] [Google Scholar]
  • 18. Yu Z , Willmarth NE , Zhou J , Katiyar S , Wang M , Liu Y , McCue PA , Quong AA , Lisanti MP , Pestell RG. 2010. microRNA 17/20 inhibits cellular invasion and tumor metastasis in breast cancer by heterotypic signaling. Proc Natl Acad Sci USA 107:8231–8236 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Zhang J , Hess MW , Thurnher M , Hobisch A , Radmayr C , Cronauer MV , Hittmair A , Culig Z , Bartsch G , Klocker H. 1997. Human prostatic smooth muscle cells in culture: estradiol enhances expression of smooth muscle cell-specific markers. Prostate 30:117–129 [DOI] [PubMed] [Google Scholar]
  • 20. Feng L , Wang W , Cheng J , Ren Y , Zhao G , Gao C , Tang Y , Liu X , Han W , Peng X , Liu R , Wang L. 2007. Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir. Proc Natl Acad Sci USA 104:5602–5607 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Yu L , Wang CY , Shi J , Miao L , Du X , Mayer D , Zhang J. 2011. Estrogens promote invasion of prostate cancer cells in a paracrine manner through up-regulation of matrix metalloproteinase 2 in prostatic stromal cells. Endocrinology 152:773–781 [DOI] [PubMed] [Google Scholar]
  • 22. Zhang Z , Duan L , Du X , Ma H , Park I , Lee C , Zhang J , Shi J. 2008. The proliferative effect of estradiol on human prostate stromal cells is mediated through activation of ERK. Prostate 68:508–516 [DOI] [PubMed] [Google Scholar]
  • 23. López-Alemany R , Longstaff C , Hawley S , Mirshahi M , Fábregas P , Jardí M , Merton E , Miles LA , Félez J. 2003. Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against α-enolase. Am J Hematol 72:234–242 [DOI] [PubMed] [Google Scholar]
  • 24. van Niel G , Porto-Carreiro I , Simoes S , Raposo G. 2006. Exosomes: a common pathway for a specialized function. J Biochem 140:13–21 [DOI] [PubMed] [Google Scholar]
  • 25. Wubbolts R , Leckie RS , Veenhuizen PT , Schwarzmann G , Möbius W , Hoernschemeyer J , Slot JW , Geuze HJ , Stoorvogel W. 2003. Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation. J Biol Chem 278:10963–10972 [DOI] [PubMed] [Google Scholar]
  • 26. Choi DS , Lee JM , Park GW , Lim HW , Bang JY , Kim YK , Kwon KH , Kwon HJ , Kim KP , Gho YS. 2007. Proteomic analysis of microvesicles derived from human colorectal cancer cells. J Proteome Res 6:4646–4655 [DOI] [PubMed] [Google Scholar]
  • 27. Mathivanan S , Lim JW , Tauro BJ , Ji H , Moritz RL , Simpson RJ. 2010. Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature. Mol Cell Proteomics 9:197–208 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Mears R , Craven RA , Hanrahan S , Totty N , Upton C , Young SL , Patel P , Selby PJ , Banks RE. 2004. Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Proteomics 4:4019–4031 [DOI] [PubMed] [Google Scholar]
  • 29. Jansen FH , Krijgsveld J , van Rijswijk A , van den Bemd GJ , van den Berg MS , van Weerden WM , Willemsen R , Dekker LJ , Luider TM , Jenster G. 2009. Exosomal secretion of cytoplasmic prostate cancer xenograft-derived proteins. Mol Cell Proteomics 8:1192–1205 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Hawley SB , Green MA , Miles LA. 2000. Discriminating between cell surface and intracellular plasminogen-binding proteins: heterogeneity in profibrinolytic plasminogen-binding proteins on monocytoid cells. Thromb Haemost 84:882–890 [PubMed] [Google Scholar]
  • 31. Wygrecka M , Marsh LM , Morty RE , Henneke I , Guenther A , Lohmeyer J , Markart P , Preissner KT. 2009. Enolase-1 promotes plasminogen-mediated recruitment of monocytes to the acutely inflamed lung. Blood 113:5588–5598 [DOI] [PubMed] [Google Scholar]
  • 32. Trojanowicz B , Winkler A , Hammje K , Chen Z , Sekulla C , Glanz D , Schmutzler C , Mentrup B , Hombach-Klonisch S , Klonisch T , Finke R , Köhrle J , Dralle H , Hoang-Vu C. 2009. Retinoic acid-mediated down-regulation of ENO1/MBP-1 gene products caused decreased invasiveness of the follicular thyroid carcinoma cell lines. J Mol Endocrinol 42:249–260 [DOI] [PubMed] [Google Scholar]
  • 33. Valentini A , Biancolella M , Amati F , Gravina P , Miano R , Chillemi G , Farcomeni A , Bueno S , Vespasiani G , Desideri A , Federici G , Novelli G , Bernardini S. 2007. Valproic acid induces neuroendocrine differentiation and UGT2B7 up-regulation in human prostate carcinoma cell line. Drug Metab Dispos 35:968–972 [DOI] [PubMed] [Google Scholar]
  • 34. Nakajima K , Hamanoue M , Takemoto N , Hattori T , Kato K , Kohsaka S. 1994. Plasminogen binds specifically to α-enolase on rat neuronal plasma membrane. J Neurochem 63:2048–2057 [DOI] [PubMed] [Google Scholar]
  • 35. Dudani AK , Cummings C , Hashemi S , Ganz PR. 1993. Isolation of a novel 45 kDa plasminogen receptor from human endothelial cells. Thromb Res 69:185–196 [DOI] [PubMed] [Google Scholar]

Articles from Molecular Endocrinology are provided here courtesy of The Endocrine Society

RESOURCES