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,
,1
* Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada;
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada;
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada; and the
Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada
1Correspondence: University Health Network, Canadian Blood Services Bldg., 67 College St., Rm. 406, Toronto, ON, M5G 2M1, Canada. E-mail: jmedin{at}uhnres.utoronto.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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H2) impaired its biological function as measured by cAMP signaling. In a competition assay, H2 exhibited antagonistic activity by blocking recombinant H2 relaxin from binding to receptors on THP-1 cells. In a flow cytometry-based binding assay, H2 demonstrated weak binding to 293T cells expressing the LGR7 receptor in the presence of biotinylated H2 relaxin. When human prostate cancer cell lines (PC-3 and LNCaP) were engineered to overexpress eGFP, wild-type (WT) H2, or H2, and subsequently implanted into NOD/SCID mice, tumor xenografts overexpressing H2 displayed smaller volumes compared to H2 and eGFP controls. Plasma osmolality readings and microvessel density and area assessment suggest that H2 modulates physiological parameters in vivo. In a second murine model, intratumoral injections of lentivectors engineered to express H2/eGFP led to suppressed tumor growth compared to controls. This study provides further evidence supporting a role for H2 relaxin in prostate tumor growth. More importantly, we report how mutation of the H2 relaxin RBD confers the hormone derivative with antagonistic properties, offering a novel reagent for relaxin research.—Silvertown, J. D., Symes, J. C., Neschadim, A., Nonaka, T., Kao, J. C. H., Summerlee, A. J. S., Medin, J. A. Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth.
Key Words: lentivirus • LGR7 • blood vessel • MMP-9
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Each mature peptide (
6–7 kDa) has an internal A-chain disulfide bond. The A and B chains are joined by two interchain disulfide bonds (2)
. Relaxin is classically known as a reproductive hormone with peak levels circulating during pregnancy (2)
. However, in the past 20 years, studies have broadened the scope for relaxin biology to a number of paradigms. It is now considered a pleiotropic hormone with functions in the cardiovascular and central nervous systems, in neovascularization, extracellular matrix (ECM) and connective tissue remodeling believed to be mediated by matrix metalloproteinases (MMPs), and as a factor associated with cancer (2
3
4
5)
.
The human H2 relaxin hormone has been reported as a peptide implicated in a number of neoplasias originating in the endometrium (6)
, breast (7)
, and thyroid (8)
. Moreover, there is growing evidence in the literature supporting a role for relaxin peptides in the prostate (the main source of relaxin in the male) (4
, 9
, 10)
, and incidentally, in prostate cancer (9
, 11)
. We recently reported that lentiviral (LV)-engineered overexpression of H2 relaxin from PC-3 prostate xenograft tumors exhibited increased growth compared to controls (12)
. We attributed this relaxin-induced growth to enhanced angiogenesis, which was evidenced by up-regulated vascular endothelial growth factor (VEGF) transcript and increased tumoral microvessel density (12)
.
Relaxin family members have a highly conserved receptor binding domain (RBD) located on the B-chain (2)
. H1, H2, H3 relaxin hormones and their orthologs sequenced to date have the Arg-X-X-X-Arg-X-X-Ile motif at positions B13, B17, and B20, with the exception of porcine relaxin, which has a valine residue at B20 (2)
. This RBD motif is essential for relaxin activity and receptor binding (13
, 14)
. Bullesbach et al. (1992) confirmed the importance of these amino acids by synthesizing H2 relaxin derivatives with B13 and B17 arginines replaced with other amino acid residues (13)
. When a dilysine H2 relaxin (at sites B13, B17) was tested in a receptor binding assay, it exhibited 2200-fold lower affinity than that of WT H2 relaxin (13)
. Later studies determined that substitution of the isoleucine residue in WT H2 at B20 with alanine reduced receptor binding by 3 orders of magnitude, which led to the model that relaxin binds to its receptor as a trivalent structure (14)
.
In the current report, we hypothesized that substitution of the B13 and B17 arginine residues within the RBD with lysine residues would allow the peptide to retain partial affinity for the receptor, while not inducing significant signal transduction. Therefore, this study reports the in vitro characterization of this H2 relaxin derivative (H2) and examines its function in vivo using a prostate tumor model. We observed for the first time here that H2 relaxin exhibits antagonistic properties both in vitro and in vivo by interfering with H2 relaxin-induced signaling and impairing prostate xenograft tumor growth.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 15)
. The human prostate cancer cell line, LNCaP, was cultured in RPMI 1640 medium. All culture media (Sigma, Oakville, ON, Canada) were supplemented with 10% fetal calf serum (FCS; Gemini Bio-Products, Woodland, CA, USA), 100 U/mL penicillin and 10 µg/mL streptomycin. Cells were cultured at 37°C in a humidified atmosphere with 5% CO2.
Engineering of mutant H2 relaxin (H2) and construction of lentiviral vectors
The H2 relaxin cDNA present in a cytomegalovirus (CMV)-H2-IRES-eGFP expression cassette (12
, 16)
was mutated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The nucleotides encoding the two arginine residues of the classic receptor binding motif were replaced with nucleotides that encode lysine residues (Fig. 1
). This cDNA was denoted H2. LVs were constructed to engineer expression of the CMV-H2-IRES-eGFP, CMV-H2-IRES-eGFP and CMV-IRES-eGFP cassettes in the pHR’ LV backbone (12)
. LV virions constructed with one of three gene transfer vectors (pHR-cPPT-CMV-H2-IRES-eGFP-WPRE, pHR-cPPT-CMV-H2-IRES-eGFP-WPRE or pHR-cPPT-CMV-IRES-eGFP-WPRE) were produced, concentrated, titered, and analyzed for eGFP expression as described previously (17)
. Concentrated LV titers ranged from 1 x 108 – 5 x 108 productive 293T infectious units/mL.
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Generation of engineered PC-3 and 293T cell lines
PC-3 cells were engineered and clonally selected to express luciferase (PC3-Luc; ref 12
). PC3-Luc and 293T cell lines were transduced with viral supernatants (LV-H2/eGFP, LV-H2/eGFP, or LV-eGFP) for 24 h, followed by a change in culture medium. The upper 10% of these eGFP-expressing cells were sorted using a MoFlo cytometer [Cytomation Inc., Princess Margaret Hospital (PMH), Toronto, Canada]. Populations of PC3-Luc cells expressing either eGFP alone, or in a bicistronic format with either H2 or H2 relaxin, were characterized to be 85% positive for fluorescent protein expression (data not shown). These cell populations were termed PC3-Luc-eGFP, PC3-Luc-H2/eGFP and PC3-Luc-H2/eGFP. LV-infected 293T cell populations were also selected that were 50% and 90% for eGFP-positive expression. Cell cultures were termed 293T-H2/eGFP(50%), 293T-H2/eGFP(50%), 293T-H2/eGFP(90%), and 293T-eGFP(50%). All plots were gated on live cells by exclusion of 7-AAD (Sigma) labeled dead cells.
Measurement of relaxin expression and bioactivity
H2 relaxin expression from the transduced PC3-Luc and 293T cells lines was confirmed by ELISA analysis. Transduced PC3-Luc and 293T cell lines were seeded in serum-free media at a density of 3.5 x 106 and 5 x 106 cells in 10-cm-diameter plates. Samples were harvested at the specified time, lyophilized, and resuspended in 1/10th the original volume. To determine the amount of H2 relaxin peptide released into the cell culture medium, a recombinant human H2 relaxin-specific direct sandwich ELISA was performed as described previously (12
, 16)
.
To confirm the biological activity of the H2 and H2 relaxins, the cAMP Biotrak E1A System was employed (Amersham Biosciences) as described (15
, 16)
. For measurement bioactivity from conditioned medium (CM), 20 µl from each concentrated sample was added to THP-1 cells. For measurement cAMP levels from the LV-infected PC3-Luc cells, 1 x 105 cells were seeded in a 96-well plate and equilibrated for 2 h before cAMP was measured. Levels of cAMP from 293T-LGR7 cell cultures (18
, 19)
were measured after incubation with 15 µL of unconcentrated CM from LV-infected 293T cell cultures.
The THP-1 cAMP assay was also adapted for an H2 relaxin competitive assay. All steps remained consistent with previous assays with the exception that THP-1 cells were coincubated with 0.4 ng recombinant H2 relaxin (rH2; gift from Elaine Unemori; BAS Medical, Inc, San Francisco, CA, USA) and varying volumes (1, 3, 6 µL) of concentrated CM from LV-infected 293T cell cultures. Total cAMP (intra- and extracellular) was measured for all experiments.
Preparation of biotin-X-X-rhH2 (bH2)
Human recombinant relaxin was labeled with 6-(Biotinamidocaproylamido)caproic acid N-hydroxysuccinimide ester (Biotin-X-X-NHS, MW 567.70; Sigma-Aldrich, Saint Louis, MO, USA). Briefly, 5 µg of rH2 was added at a 1:20 M ratio with Biotin-X-X-NHS (reconstituted in dimethylformamide at 25 mg/mL) in a total volume of 250 µl of PBS (pH 7.3) on ice for 1.5 h. The reaction was quenched by the addition of 1 µl of 1M Tris-HCl (pH 8.0). The product was then dialyzed in 500 mL of PBS at RT overnight to remove uncoupled biotinylation reagent in a 3500 Da MW cut-off dialysis chamber (Elutatube Dialysis Kit; Fermentas, Burlington, ON, Canada). The final product was aliquoted and stored at –20°C until use. To confirm that bH2 did not differ in biological activity from rH2, 0.4 ng of each was subjected to the THP-1 cAMP assay as described above. Bioactivities of bH2 and rH2 remained virtually the same with demonstrated cAMP levels of 316 ± 7.5 and 307 ± 9.2 fmol/well, respectively (data not shown).
Flow cytometry-based relaxin binding assay
To optimize collection of recombinant H2 peptide for this assay, single-cell clones of LV-infected 293T cell cultures were isolated. Clones were confirmed by flow cytometry measuring eGFP fluorescence (as above) and selected based on highest expression. 293T-eGFP#3, 293T-H2/eGFP#6, 293T-H2/eGFP#3 cell cultures were seeded in 15-cm plates in complete medium overnight, refreshed with Dulbecco’s Modified Eagle Medium (DMEM) medium containing 2% FCS after 24 h, and incubated for an additional 48 h. At this point, CM were collected for assays. H2 relaxin levels in unconcentrated CM from 293T-eGFP#3, 293T-H2/eGFP#6, 293T-H2/eGFP#3 cell cultures were approximated by the H2 ELISA to be 0, 1, and 4 ng/mL (data not shown).
293T-LGR7 cells (18
, 19)
were collected, washed with PBS supplemented with 1% FCS (PBS-1%), and resuspended at 1.67 x 107 cells/mL. For each sample, 30 µl (0.5x106 cells/mL) were added per 5 mL polystyrene round-bottom Falcon tube (Becton Dickinson Labware, Franklin Lakes, NJ, USA). Control samples contained 50 µL of either PBS-1% containing 5 ng of rH2 or 50 µL of PBS-1% alone. Each control sample was then supplemented with 20 µL of 0.25 ng/µL bH2 in PBS-1% or PBS-1% alone, giving a total volume of 100 µL. Samples were incubated for 15 min at RT. Each sample was then supplemented with either 70 µL of either media alone (DMEM, 2% FBS), or CM from 4-day cultures of LV-transduced 293T clones and incubated for 5 min at RT. Cells were collected by centrifugation at 350 g for 3 min, resuspended in 100 µL of additional media or CM as above, and incubated for 5 min at RT. Following two washes, 20 µL of 0.25 ng/µL bH2 in PBS-1% was added for a total volume of 100 µL and incubated for 15 min at RT. Samples were washed with 500 µL of PBS-1%, and cells were collected by centrifugation at 400 g for 3 min and resuspended in 100 µL of 1 µg/mL of streptavidin-phycoerithrin (PE) reagent (eBioscience, San Diego, CA, USA) in PBS-1% and incubated in the dark for 15 min at 4°C. Cells were washed twice with 500 µL of PBS-1% and analyzed on a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA; ref. 6
).
To determine whether mouse relaxins can bind to human LGR7, relative affinity of mouse relaxin and mouse relaxin-3 hormone were also measured in parallel. CM containing LV-engineered recombinant mouse relaxin and mouse relaxin-3 were prepared as described previously (19)
. The flow cytometry-based binding assay for mouse relaxins was performed as above with the following modification: 293T-LGR7 cells were preincubated with CM (diluted 2-fold, 5-fold, and 20-fold) for 5 min at RT before addition of 10 ng of bH2. As above, the addition of controls containing bH2, rH2, and CM containing human H2 relaxin were also included.
Cell proliferation assays
Two assays were employed to measure cell proliferation. PC3-Luc-eGFP, PC3-Luc-H2/eGFP, and PC-3-Luc-H2/eGFP cells were plated in triplicate at 1 x 103, 3 x 103, and 8 x 103 cells per well in a 96-well dish. For the thymidine incorporation assay, cells were serum-starved for 24 h to induce cell cycle synchronization. At 24, 48, and 72 h, 1 µCi/mL [methyl-3H]thymidine (Amersham Biosciences Inc., Quebec, Canada) was added. At each time point, cells were harvested with trypsin, collected, and samples analyzed by liquid scintillation counting (LS 1801, Beckman Coulter, Mississauga, ON, Canada). Cell proliferation was also measured using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay reagent (Promega; ref. 12
).
PC3-Luc human prostate tumor model in NOD/SCID mice
Six- to eight-week old male NOD/SCID mice (The Jackson Laboratory; Bar Harbor, ME, USA) were maintained under an approved UHN protocol. Groups consisted of NOD/SCID mice injected with 2 x 106 PC3-Luc-H2/eGFP, PC3-Luc-H2/eGFP, or PC3-Luc-eGFP cells suspended in 200 µl of Matrigel (BD; n=6 or 7 animals/ group). Injections were performed s.c. into the dorsal right flank of NOD/SCID mice. Tumors were measured weekly by calipers and tumor volume approximated (l x w x d), as described previously (12)
. At weeks 9 and 11, animals underwent whole-body noninvasive bioluminescence imaging (BLI) to examine PC3-Luc cell biodistribution (12)
. After sacrificing animals, blinded autopsies were performed to examine for metastatic events. To confirm that the metastatic tissue was of PC3-Luc origin, tissue was placed in a 6-well dish, incubated in luciferin, and imaged (12)
. Tumors were harvested and examined for eGFP expression (12)
. Following imaging, tumors were subsequently separated into equal halves. One half was sectioned, paraffin-embedded, and stained with anti-mouse CD31 (PECAM-1). The other half was homogenized in 1 mL of sterile PBS for collection of tumor cell lysates and total RNA for analysis of gelatinase expression by gelatin zymography and LGR7 and VEGF isoform expression by reverse transcriptase-polymerase chain reaction (RT-PCR), respectively (see Ref 12
for all methods). To study the effect of relaxin on microvessel dilation, microvessel area (MVA) was measured as described previously (20)
. Briefly, leveled sections (n=2 to 4) within each tumor were evaluated in a blinded fashion using a Carl Zeiss Axioskop 2 microscope. At x100 magnification, 9–12 of the largest CD31-stained MVs were identified, and subsequently photomicrographed at x400 magnification using the Axiocam MRc camera. Area, diameter and MV length (µm) were measured using the length measurement tool of the Axiovision 3.1 software (Carl Zeiss). For plasma osmolality readings, 100–200 µL of blood was collected in heparin (heparin sodium, 1000 USP units/mL; Hepalean) and sent for analysis (Animal Health Laboratory, Department of Pathobiology, University of Guelph).
LNCaP human prostate tumor model in NOD/SCID mice
LNCaP cells were transduced with LV supernatants (LV-H2-IRES-eGFP, LV-H2-IRES-eGFP, or LV-eGFP) to generate LV-infected LNCaP cell cultures, analogous to methods described for PC3-Luc cells. Cell lines (85% eGFP positive) were termed LNCaP-H2/eGFP, LNCaP-H2/eGFP, and LNCaP-eGFP. H2 and H2 relaxin expression and bioactivity were confirmed by employing the H2 relaxin ELISA and the THP-1 cAMP assay (as described above). Groups consisted of 5-wk-old female NOD/SCID mice injected with 6 x 106 LNCaP-H2/eGFP, LNCaP-H2/eGFP, and LNCaP-eGFP cells (n=8 animals/ group) suspended in 200 µL of Matrigel (BD) into the dorsal right flank. After 14 wk, blinded autopsies on mice were performed to examine for metastases. Tumors were harvested, imaged, and weighed as described above.
PC3-luc soft agarose colony assay
Powdered F12 Nutrient mixture (Life Technologies, Inc., Gaithersburg, MD, USA) was prepared as a sterile 2 x complete medium and buffered with sodium bicarbonate to pH 7.67. Six-well culture dishes were coated with 1.5 mL/well of 1:1 mixture of 0.8% sterile low melting agarose (NuSieve GTG agarose; BioWhittaker Molecular Applications, Rockland, ME, USA) mixed with 2 x F12 medium (supplemented with 20% FCS, 200 U/mL penicillin; 20 µg/mL streptomycin) and stored at 4°C to solidify. Then 1.5 mL of varying dilutions of PC3-Luc-H2/eGFP, PC3-Luc-H2/eGFP, or PC3-Luc-eGFP cells (ranging from 100 to 1000 cells/mL) suspended in F12/ 0.4% agarose solution were applied in triplicate to the coated 6-well plates. Wells were replenished with fresh F12/0.4% agarose solution every 3 or 4 days for 5 wk. Colony spheres were counted, measured, and imaged using a Nikon TE200 inverted microscope mounted with a Hamamatsu ORCA 100 camera and analyzed with SimplePCI version 3.1 software (Compix Inc., Sewickley, PA, USA).
Intratumoral delivery of lentiviral vectors to PC3-Luc tumors
NOD/SCID male mice (n=15) were injected in the dorsal right flank with 3 x 106 PC3-Luc cells suspended in 200 µl of Matrigel (BD). When tumors reached 1 cm3, animals were divided into three groups to equally distribute age and weight of the mice. Every 3 or 4 days for 18 days, tumors were injected in four random sites with 5 µl of concentrated LV suspension (LV-H2/eGFP, LV-H2/eGFP, LV-eGFP) per site using a 25 µl Hamilton microsyringe (total=4x106 infectious units). Tumors were measured on the day of each intratumoral LV delivery by calipers. At day 18, animals underwent whole-body noninvasive BLI and a complete autopsy to examine for PC3-Luc cell biodistribution (12)
. After euthanizing animals, tumors were harvested and examined for eGFP expression (12)
.
Statistical analysis
Differences between two treatment groups were statistically analyzed using a two-tailed, independent samples t test. Differences among three or more groups were analyzed using a one-way ANOVA test. To account for multiple comparisons testing of the tumor volume, a Bonferroni t test within each time point was performed to make adjustment to the P values. Plasma osmolality data were analyzed using a median two-sample test (SAS). Error bars indicate the SE, and significance is indicated by an asterisk when P 
0.05. Statistical analyses were performed under consultation with the Clinical Studies Resource Centre, UHN (Toronto, Canada).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The other amino acid (isoleucine at B20), critical for receptor binding, was left intact. To produce H2 relaxin, a novel recombinant LV was constructed (Fig. 1C
).
Concentrated CM from PC3-Luc-H2/eGFP, and 293T-H2/eGFP(50%) samples contained significant levels of H2 relaxin compared to basal or undetectable levels in the PC3-eGFP and 293T-eGFP(50%) samples, respectively (Fig. 2
A,B). H2 was detected at lower concentrations in these cell culture samples compared to H2-expressing cells but higher than eGFP-expressing controls. Since transduced cells had similar expression levels of the downstream eGFP marker, lower levels of H2 suggest a potential reduction of antibody (Ab) affinity in the ELISA. Ab affinity to H2 relaxin appears to be 33% of the affinity that the ELISA antibodies have for WT H2 (6 and 2 ng/mL of H2, and H2 were measured by ELISA, respectively; Fig. 2B
). Although the epitopes recognized by these Abs are not known (Elaine Unemori, BAS Medical, Inc., personal communication), these data suggest that the amino acid substitutions of H2 relaxin may have altered the epitope binding region. However, we also cannot discount the possibility that the H2 relaxin peptide has a reduced stability compared to H2 relaxin. As expected, 293T-H2/eGFP(90%) CM samples had greater H2 relaxin levels compared to 293T-H2/eGFP(50%) samples (Fig. 2B
).
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Characterization of bioactivity of mutated H2 relaxin (H2)
The in vitro THP-1 cAMP bioassay is a standard assay that measures bioactivity of relaxin-like peptides (15)
. This assay confirmed that mutation of the relaxin RBD impairs the stimulatory capacity of the hormone (Fig. 2C
). PC3-Luc-H2/eGFP cells themselves were found to contain 13,221 ± 4185 fmol/well total cAMP compared with PC3-Luc-H2/eGFP (3,667±330 fmol/well) and PC3-Luc-eGFP (2,961±115 fmol/well) cultures (Fig. 2C
). THP-1 cells incubated with CM from PC3-Luc-H2/eGFP cultures elicited a substantial increase in total cAMP from cultures (6,599±674 fmol/well) compared to basal levels elicited from CM samples of both PC3-Luc-H2/eGFP (1,018±45 fmol/well) and PC3-Luc-eGFP (1,218±25 fmol/well).
293T cells stably-expressing human LGR7 exhibit a magnitude greater sensitivity to H2 relaxin compared to THP-1 cells (19)
, likely due to the greater density of receptor presented on the cell surface (15)
. Therefore, when 293T-LGR7 cells were subjected to a cAMP ELISA after stimulation with CM from LV-H2/eGFP-transduced 293T cell cultures, a significant (P=0.03) increase in cAMP of 10% (1,074±46 fmol/well) was observed compared to CM from LV-eGFP-transduced 293T cell cultures (945±35 fmol/well; Fig. 2D
). This suggests that the H2 hormone retains modest signaling capacity for the LGR7 receptor.
Mutated H2 relaxin (H2) exhibits antagonistic properties
Since H2 relaxin function was observed to be severely reduced while retaining some affinity for receptor binding, it was hypothesized that H2 (at high levels) may act as an antagonist and competitively inhibit H2 relaxin binding. Therefore, a THP-1 cAMP competitive assay was developed to measure the antagonistic properties of H2 relaxin (Fig. 3
A). Concentrated CM from LV-transduced 293T cells was used because these cells secrete high levels of WT H2 or H2 relaxins (as above). THP-1 cells were coincubated in 96-well plates with 0.4 ng rH2 and increasing CM volumes from LV-infected 293T cell cultures (from Fig. 2B
). When normalized to 293T-eGFP CM samples, 293T-H2/eGFP CM samples had a pronounced effect of reducing cAMP levels compared to control 293T-eGFP CM samples (Fig. 3A
). The addition of increasing volumes of 293T-H2/eGFP CM samples (1, 3, and 6 µl) resulted in greater suppression of total cAMP release from THP-1 cells. As expected, stimulation of THP-1 cells with 293T-H2/eGFP CM samples resulted in a dose-dependent increase of cAMP release compared to the basal and reduced levels that were stimulated by 293T-eGFP and 293T-H2/eGFP samples, respectively (Fig. 3A
). Taken together, these results suggest that the H2 hormone competes with rH2 for relaxin receptor binding on THP-1 cells.
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Mutated H2 relaxin (H2) binds to LGR7
A flow cytometry-based binding assay using 293T cells expressing human LGR7 receptors was developed for this study to further demonstrate the binding capabilities of H2 relaxin. Five nanograms of bH2 was determined to be the least amount of bH2 required to permit maximal receptor binding on 293T-LGR7 cells (Fig. 3B,C
). Amounts of bH2 greater than 5 ng resulted in negligible differences in mean fluorescence intensity (Fig. 3B
). As controls, incubation with bH2 but not incubation with rH2 resulted in complete labeling of 293T-LGR7 cells (Fig. 3C
). Incubation with unconcentrated CM harvested from 293T-eGFP cells followed by addition of bH2 (5 ng) permitted 100% bH2 binding (Fig. 3C
). As expected, incubation with CM samples containing 1 or 4 ng/mL of WT H2 relaxin followed by competition with bH2 resulted in 93% and 98% retention (or 7% and 2% bH2 binding) of WT H2 relaxin. However, incubation with CM containing 1 ng/mL H2 relaxin followed by competition with bH2 resulted in the retention of 19% of H2 binding (or 81% bH2 binding) that was significantly different (P<0.0001) compared to both eGFP control and WT H2 relaxin CM samples (Fig. 3C
). Taken together, these results suggest that the H2 hormone retains the capacity to bind to LGR7 receptor on 293T-LGR7 cells.
Effect of overexpression of H2 relaxin in two human prostate tumor models
To determine the effect of the overexpression of H2 relaxin in vivo, a prostate tumor model was chosen. PC3-Luc xenograft tumors were generated in NOD/SCID mice. Compared with PC3-Luc-H2/eGFP tumors overexpressing WT H2 relaxin, PC3-Luc-H2/eGFP tumors exhibited a significantly smaller tumor volume starting at week 6 (0.61-fold; P<0.05) until termination of the study at week 11 (0.56-fold; P < 0.02; Fig. 4
A). At week 6, PC3-Luc-H2/eGFP tumors exhibited significantly greater tumor volumes compared to PC3-Luc-eGFP tumors, in agreement with previous findings (12)
. At weeks 10 and 11, PC3-Luc-H2/eGFP tumors exhibited a significantly smaller tumor compared to both PC3-Luc-H2/eGFP and PC3-Luc-eGFP tumors.
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One component of this study was to determine how long-term overexpression of H2 relaxin affects PC3-Luc tumor cell metastasis. In our previous study, we delivered 3.5 x 106 tumor cells per NOD/SCID mouse, monitored tumor growth for up to 6 wk and observed no evidence of metastasis (12)
. In the current study, we injected 2 x 106 cells to allow prolonged relaxin expression before tumor size reached terminal endpoints, thereby permitting an extended opportunity for observation. At weeks 9 and 11, all animals were imaged for luciferase expression to 1) confirm general size of tumors as measured with calipers, and 2) assist in identifying PC3-Luc metastatic events. Figure 4B
illustrates the location, size, and cell density proportionate to level of luciferase expression (12)
of a tumor from representative animals from each PC3-Luc tumor group. This method of BLI confirms that PC3-Luc-H2/eGFP, and PC3-Luc-eGFP tumors have significantly larger tumor volumes at weeks 9 and 11 compared to PC3-Luc-H2/eGFP tumors. On termination of the study at week 11, putative metastatic tumors were harvested and imaged by BLI to confirm that the tissue originated from the PC3-Luc tumors (Fig. 4C
). Although not statistically significant, in animals bearing PC3-Luc-H2/eGFP tumors, 3 of 7 mice were found to have metastatic events compared to only 1 of 7 mice and 1 of 6 mice in the PC3-Luc-H2/eGFP and PC3-Luc-eGFP groups, respectively. Tumors were also harvested and imaged for eGFP expression with a fluorescent stereomicroscope before tumor tissue processing (12)
. Imaged tumors were confirmed to fluoresce from eGFP expression, due to the bicistronic format of the LV expression cassette. This indicates that H2 and H2 relaxins were also likely expressed because they are located upstream to the IRES element (see Fig. 1C
).
To demonstrate whether H2 relaxin overexpression had similar effects on other human prostate cancer models, LNCaP xenograft tumors were grown in NOD/SCID mice. From pilot studies, it was observed that tumors grown from this cell line, whether implanted in male or female NOD/SCID mice, never grew large enough for caliper measurement. Nonetheless, LNCaP-H2/eGFP tumors (59.2±3.5 mg) exhibited a significantly lighter tumor weight after 14 wk in vivo compared to LNCaP-eGFP (72.9±5.2 mg; P=0.05) and LNCaP-H2/eGFP (73.9±8.7 mg) tumors (Table 1
). Similar to the PC3-Luc prostate tumor xenograft model, and although not statistically significant, mice bearing H2 relaxin-overexpressing tumors exhibited greater metastatic incidences (n=3/8) compared to mice bearing LNCaP-H2/eGFP (n=1/8) and LNCaP-eGFP (n=1/8) tumors.
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Mouse relaxins can bind to human LGR7
The flow cytometry-based binding assay we developed was employed to demonstrate that mouse relaxins can bind to human LGR7. CM containing either mouse relaxin or mouse relaxin-3 diluted either 1:2 or 1:5 (but not 1:20) resulted in LGR7 binding that was significantly greater (P<0.0001) than eGFP control samples (Fig. 5
). These results provide additional evidence to suggest that endogenous human H2 relaxin (secreted from PC3 tumor) and now mouse relaxins (secreted from host) have the capacity to contribute to LGR7-signaling and compete with recombinant H2 or H2 peptides for receptor binding in vivo.
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Mutated H2 relaxin (H2) modulates physiological parameters in vivo
In addition to measuring tumor growth, we investigated downstream pathways affected by H2 and H2 relaxin overexpression. The regulation of MMPs by relaxin in prostate tumors (12)
and in other systems (21)
has been reported. Gelatin zymography was used to detect the presence of gelatinase activity in tumor cell lysates. Gelatinase B (MMP-9) but not gelatinase A (MMP-2) activity was observed to be present in these samples by zymography. As shown in Fig. 6
A, cell lysates analyzed from PC3-Luc-H2/eGFP tumors consistently exhibited lower levels of MMP-9 activity compared to PC3-Luc-eGFP (3.8-fold, P=0.001). PC3-Luc-H2/eGFP cell lysate samples appeared to exhibit intermediate levels of MMP-9 enzyme activity between PC3-Luc-eGFP (P=0.066) and PC3-Luc-H2/eGFP (P=0.16) samples, suggesting that H2 relaxin is affecting PC3-Luc signaling pathways responsible for MMP-9 expression.
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In our previous study, the ability of H2 relaxin to promote angiogenesis was implicated as a potential mechanism for its role in facilitating tumor growth (12)
. As an indicator of H2 relaxin’s angiogenic role in the present tumors, VEGF isoform mRNA expression by RT-PCR, total tumoral vascularization measured by IHC staining of endothelial cells (ECs) with anti-mouse CD31 (PECAM-1), and MVA were examined (12)
. Levels of VEGF transcript isoforms were present in greater levels within PC3-Luc-H2/eGFP tumors as shown by assay of individual and pooled samples from cDNA derived from each tumor within each treatment group compared to PC3-Luc-H2/eGFP and PC3-Luc-eGFP samples (Fig. 6B
). Tumoral microvessel density (MVD) was also determined to be significantly greater (1.5-fold; P<0.05) in PC3-Luc-H2/eGFP tumors (24.3±4.4) compared to PC3-Luc-eGFP tumors (16.2±1.8) with an intermediate MVD (21.1±3) in PC3-Luc-H2/eGFP tumors (Fig. 6C
). In addition, total MVAs averaged from sections of PC3-Luc-H2/eGFP tumors were significantly greater compared to the MVAs of PC3-Luc-H2/eGFP (1.76-fold; P=0.018) and PC3-Luc-eGFP (2.2-fold; P=0.003) tumors (Fig. 6D
).
To determine whether H2 and H2 relaxins secreted from the PC3-Luc tumor xenografts elicited a systemic physiological response, plasma osmolality was measured from the animals at the termination of the study (Fig. 6E
). Plasma osmolality readings obtained from PC3-H2/eGFP, PC3-Luc-eGFP, and PC3-Luc-H2/eGFP tumor-bearing animals averaged 330 ± 4.6, 339 ± 5, and 342 ± 4.6 mosmol/kg, respectively. PC3-Luc-H2/eGFP animals exhibited significantly greater levels of plasma osmolality (12 mosmol/kg; P=0.04) compared to PC3-Luc-H2/eGFP animals and only slightly greater levels compared to PC3-Luc-eGFP animals. These findings demonstrate that locally expressed H2 relaxin can have systemic effects.
The influence that H2 and H2 relaxins have on PC3-Luc tumor xenografts may be achieved at the level of signaling pathways between tumor and host. Indeed, LGR7 receptor was detected by RT-PCR at equal levels from pooled cDNA samples taken from each treatment group (Fig. 6F
).
To determine whether the influence of H2 and H2 relaxins on PC3-Luc cell growth can occur in the absence of in vivo signals, an in vitro soft agarose colony assay was used. After 5 wk in culture, PC3-Luc colony spheres were counted, measured, and imaged (Fig. 6G
). No apparent size differences were observed between the PC3-H2/eGFP, PC3-Luc-H2/eGFP, and PC3-Luc-eGFP colony spheres (data not shown). This suggests that the differences in tumor growth in NOD/SCID mice are likely due to angiogenic or other in vivo signaling pathways, which are not assessed by this in vitro assay.
Intratumoral delivery of LV-H2/eGFP suppresses tumor growth
To continue our investigation into the biological nature of H2 relaxin, LVs were delivered intratumorally to mice over an 18-day period. During the first 7 days, LV-H2/eGFP-injected tumors exhibited a significant impairment of growth (131±25.3 mm3) compared to LV-H2/eGFP (199±20.3 mm3; P=0.04) and LV-eGFP-injected (195±20.7 mm3; P=0.05) tumors (Fig. 7
A). Thereafter, LV-H2/eGFP-injected tumors displayed moderately suppressed tumor volumes compared to tumors receiving LV-H2/eGFP or LV-eGFP. At the end of the study, animals were imaged (as above) to measure tumor size, density, and location. Figure 7B
shows an image of representative animals from each group, illustrating the apparent tumor suppression observed in mice bearing LV-H2/eGFP-injected tumors. To confirm that the tumor cells were LV-transduced (i.e., positive for eGFP expression), tumors were harvested and imaged under a fluorescent stereomicroscope (Fig. 7C
). Compared to PBS-injected controls, LV-treated mice had regions positive for eGFP expression within the tumor (x0.8 magnification). At x10 magnification, clusters of transduced cells present at the LV injection sites on the tumor can be distinguished.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Halls et al. (22)
proposed a model that relaxin binds in a two-stage process to both a primary high-affinity binding site located on the ectodomain of the LRR and a lower affinity binding site located within the transmembrane region. The high-affinity binding site couples to cAMP signaling with high efficiency, whereas the low-affinity site couples with lower efficiency (22)
. Further work in that study showed that relaxin-like peptides can bind to the low-affinity transmembrane site without binding to the primary high-affinity site. That said, peptide binding to the lower-affinity region likely results in null or suboptimal signal transduction without engagement of the high-affinity region (22)
.
On the basis of the above mentioned work (22)
and the evidence reported in the current study, it is likely that the partial mutation of the RBD of H2 relaxin (H2) permits the retention of some level of receptor affinity (possibly at the lower-affinity region), while exhibiting significantly impaired function. It is possible that at high concentrations, H2 relaxin is able to bind to LGR7 and LGR8 receptors without eliciting a significant cAMP-mediated signal transduction cascade. Varying affinities of relaxin-like peptides to LGR7 and LGR8 receptors have suggested that amino acid composition and ligand structure influences the degree of receptor binding and activation (22)
. Therefore, the substitution of the B13 and B17 arginines for lysine residues (while leaving the isoleucine at B20 intact) may be sufficient to render the mutated H2 form (H2) with antagonistic properties by inducing subtle conformational changes that compromise its full capacity for receptor binding and signaling.
Two assays measuring either peptide biological activity or binding capabilities demonstrated that H2 relaxin does retain reduced affinity for the receptor albeit with significantly impaired signaling function. In a competition assay measuring H2 relaxin-induced cAMP release from THP-1 cells, H2 secreted from LV-infected 293T cell cultures competed with rH2 hormone (0.4 ng) for receptor binding (Fig. 3A
). As the ratio of H2:rH2 increased in each sample (with increasing sample volume), greater suppression of secreted cAMP levels from THP-1 cells compared to eGFP controls was observed. A second assay using flow cytometry was designed to measure receptor binding affinity of H2. This assay permits a rapid, cost-effective, and sensitive characterization of the receptor binding properties of relaxin-like peptides. The assay relies on the abilities of conjugate-labeled rH2 to displace receptor-bound relaxin-like peptides. In the current study, H2 relaxin exhibited modest binding to the relaxin receptor on 293T-LGR7 cells compared to uninhibited binding of WT H2 relaxin. Because H2 relaxin retains receptor-binding affinity, while exhibiting significantly impaired signaling capacity, H2 relaxin can function as a relaxin receptor antagonist when present in molar excess.
The antagonistic properties of the H2 hormone were also demonstrated in vivo in three distinct animal models. We observed that LV-engineered overexpression of H2/eGFP results in impaired prostate tumor xenograft growth compared to tumors overexpressing WT H2/eGFP or control tumors overexpressing eGFP. It can be theorized that tumor-expressed H2 relaxin is neutralizing available receptors in an autocrine fashion, thereby competing with endogenous relaxins for receptor binding and blocking endogenous H2 signaling. Indeed, we showed that mouse relaxin and mouse relaxin-3 are capable of binding to human LGR7 (Fig. 5)
. Therefore, H2 relaxin may be competing with human H2 relaxin secreted from PC3 tumors and/or mouse relaxins circulating in the host. At the same time, however, we cannot discount the possibility that H2 relaxin may exhibit preferential and/or partial affinity for one receptor (i.e., LGR7, LGR8, or other), thereby selecting for a receptor-specific signaling cascade. The precise pathways that LGR7 and LGR8 may regulate to facilitate tumor growth are not known, but an antagonist may abrogate endogenous H2-mediated pathways altering cellular proliferation, gene expression, and neoangiogenesis (23)
.
Although H2 relaxin exhibited significant antagonistic function, it also displayed evidence of an ability to elicit modest signaling both in vitro and in vivo. This observation allows one to infer that in our systems, leaky relaxin signaling is observed because the H2 analog is likely not completely neutralizing all available relaxin receptors and is exhibiting modest signal transduction. For example, the putative antagonistic activity demonstrated by H2 relaxin was evidenced by intermediate levels of MMP-9 activity (an established downstream effector in H2 relaxin signaling) and MVD in H2 relaxin-expressing tumors compared to values derived from H2/eGFP and eGFP-expressing tumors (Fig. 6A,C
; ref. 12
). Taken together, the H2 relaxin analog appears to be partially interfering with the endogenous functions of H2 relaxin in PC3-Luc tumors by acting on relaxin receptors to alter cellular signaling pathways. Future studies will be required to dissect the differential aspects on a molecular level.
In the current study, changes in plasma osmolality were also investigated as an approach to measure the paracrine influence of tumor-secreted H2 relaxin. Relaxin knockout (RLX–/–) mice exhibit a phenotype with an increased plasma osmolality of 10 mosmol/kg water compared to WT mice (24)
. On the contrary, delivery of human H2 relaxin i.v. or i.c.v. in the rat causes reductions in plasma osmolality (25
, 26)
. We observed that plasma osmolality readings were 12 mosmol/kg higher in animals with H2-expressing tumors compared to WT H2-expressing tumors (P=0.04). This suggests that H2 may be blocking endogenous ligand binding to relaxin receptors in the mouse (i.e., pituitary gland, kidney, or other regions) and thereby affecting osmoregulation.
The mechanism behind H2-mediated suppression as evidenced in the current study, may operate by interfering with signals shared between host and tumor because no differences were observed in vitro between PC3-Luc soft agarose spheroid colony sizes (Fig. 6F
) and cell proliferation (data not shown).
However, distinct differences of MVD and MVA between treatment groups suggest that H2 relaxin may be affecting pathways of angiogenesis. PC3-Luc-H2/eGFP tumors had two-fold greater MVA compared to PC3-Luc-H2/eGFP and PC3-Luc-eGFP tumors (Fig. 6D
). This suggests that H2 relaxin may have a role in prostate tumor growth not just by enhancing vascularization (12)
, but also by vasodilation, thereby increasing tumoral blood flow. Therefore, overexpression of H2 relaxin in the tumor microenvironment may inhibit endogenous H2 relaxin progrowth signals to tumor cells or tumor-infiltrated ECs (5)
, impairing biochemical pathways involving angiogenesis (2
, 12)
, vasodilation (27)
, cell proliferation, and apoptosis (2
, 3
, 28)
.
We previously reported a study showing that overexpression of H2 relaxin from PC3 tumors leads to greater tumor growth up to 6 wk in NOD/SCID male mice (12)
. At week 6, an advanced angiogenic phenotype was observed in the H2/eGFP-overexpressing tumors compared to the eGFP control tumors (12)
. In the current study, while PC3-Luc-H2/eGFP and PC3-Luc/eGFP tumors did not differ in size after week 6, H2 relaxin-expressing tumors maintained a suppressed tumor volume for the 11 wk study period (Fig. 4A
). In addition, tumors treated by intratumoral delivery of LV-H2/eGFP exhibited impaired tumor growth only up to 1 wk. It is possible that after this time period in this model, tumor growth overcomes any H2-induced impairment. Therefore, in combination with our previous findings (12)
, a hypothesis can be proposed that H2 relaxin expression can provide prostate tumors with an advantage for early tumorigenesis (up to week 6) mediated by angiogenic and/or other pathways. After this time period, other tumoral compensatory mechanisms may play a role in facilitating tumor growth.
Tumor development is a multistep process that involves signaling from a number of pathways. Considering that H2 relaxin is a pleiotropic hormone (2
3
4
5)
with functions in angiogenesis, blood flow, and pressure, ECM remodeling, cellular proliferation, and apoptosis, it is a desirable target that, if blocked, could demonstrate moderate antagonistic efficacy in both in vitro and in vivo systems. To date, no H2 relaxin peptide antagonists have been described in the literature; however, the LGR7 relaxin receptor ectodomains were demonstrated to bind relaxin, inhibit receptor-mediated signaling of cAMP, and suppress nipple development in mice (18)
. Recently, the systematic truncation of amino acids from the N terminus of relaxin-like factor resulted in the discovery of peptides that exhibit antagonistic-like properties by suppressing cAMP signaling (29
, 30)
. The H2 relaxin analog described in the present study offers the potential to inhibit H2 relaxin signaling at the receptor presented on the cell surface.
Because H2 relaxin has not been attributed an essential role in the physiological function of humans (2)
, systemic delivery of recombinant H2 relaxin to potentially curb tumor growth can be conceivable, considering that H2/LGR7 or H2/LGR8 signaling axes may be temporarily dispensable. In addition, because relaxin has been implicated in several other types of neoplasias (6
7
8)
, further modifications of the RBD within H2 relaxin and other relaxin-like peptides may also offer utility for other cancers.
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ACKNOWLEDGMENTS |
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Received for publication July 19, 2006. Accepted for publication October 25, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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