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Endocrine-Hypertension Division and Membrane Biology Program, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Jennifer L. Sanders, Ph.D., Endocrine-Hypertension Division, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115. E-mail: jsanders{at}rics.bwh.harvard.edu
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Introduction |
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PTH-related protein (PTHrP) is thought to be an important mediator of malignant osteolysis with or without concomitant hypercalcemia, particularly that caused by breast cancers (4, 7). PTHrP increases the differentiation of osteoclasts from their precursors by enhancing the expression of osteoclast differentiation factor (ODF) on osteoblasts (8) and perhaps by increasing the production of soluble factors (i.e. cytokines) by osteoblasts and other cells (i.e. stromal cells) within the bone marrow microenvironment (4). By stimulating osteoclastogenesis as well as the activity of mature, preformed osteoclasts (8), PTHrP contributes importantly to the skeletal invasiveness, bone pain, and/or hypercalcemia caused by breast cancers metastatic to bone. Indeed, several lines of evidence suggest that PTHrP plays a central role in the development of breast cancer metastases to bone and resultant osteolysis. Skeletal metastases of breast cancers express PTHrP more frequently and at higher levels than normal breast epithelial cells, primary breast cancers, or nonskeletal metastases (3, 4, 9, 10), suggesting that PTHrP could contribute to the increased bone resorption in breast cancers metastatic to bone. Moreover, there is a positive correlation between the level of PTHrP expression in primary breast cancers and the subsequent risk of developing skeletal metastases and/or hypercalcemia (11). The work of Guise and co-workers in experimental mouse models strongly supports a central role for PTHrP in breast cancer-induced osteolysis (for reviews, see Refs. 4 and 8). Nude mice injected intraarterially with the parental MDA-MB-231 breast cancer cell line (12) reliably develop osteolytic metastases (8, 13, 14), the severity of which increases when the injected cells stably overexpress PTHrP (8, 13). Conversely, injection of the mice with anti-PTHrP monoclonal antibody reduces the severity of the skeletal metastases (13, 14). This body of data strongly implicates PTHrP as a central mediator of the osteolysis produced by breast cancer cells metastatic to bone.
Several growth factors, most notably transforming growth factor-ß (TGFß) and insulin-like growth factor I (IGF-I), are laid down in the bone matrix during bone formation and are later released when that bone is resorbed by osteoclasts, either during normal bone remodeling or because of malignant osteolysis (15). The released growth factors, by stimulating osteoblast recruitment and activity, are thought to contribute to the normal coupling of bone resorption to subsequent bone formation. TGFß up-regulates PTHrP production by some breast cancer cells (13). Moreover, when mice are injected with MDA-MB-231 breast cancer cells transfected with a dominant negative, type II TGFß receptor (e.g. which inhibits the endogenously expressed normal receptor), the severity of malignant osteolysis is reduced compared with that in mice injected with control cells (8, 16). Furthermore, introduction of a constitutively active TGFß type I receptor into the MDA-MB-231 clone expressing the dominant negative TGFß receptor increased PTHrP production in vitro and osteolysis in vivo. Therefore, TGFß may participate in the mechanism(s) underlying breast cancer cell-induced malignant osteolysis, in that PTHrP-evoked bone resorption releases TGFß stored in bone matrix, which then stimulates further PTHrP production by the cancer cells (16).
Not only is bone a repository for TGFß and other growth factors, but it is also a storehouse for calcium. Ca2+, released during the resorptive process into the bony microenvironment, could potentially modulate the normal remodeling process as well as metastatic osteolysis. The levels of Cao2+ in the vicinity of resorbing osteoclasts (i.e. as high as 8–40 mM) (17) are manyfold higher than the level of systemic Cao2+. Furthermore, high Cao2+ stimulates PTHrP production by both normal (18, 19) and malignant cells (20, 21, 22). This action of Cao2+ on PTHrP secretion could be mediated by the extracellular calcium (Cao2+)-sensing receptor (CaR); the G protein-coupled receptor originally cloned from bovine parathyroid that plays a central role in systemic Cao2+ homeostasis (23). Indeed, we previously showed that both normal breast ductal epithelial cells and primary breast cancers express the CaR (24). In the case of breast cancers metastatic to bone, CaR-mediated stimulation of PTHrP secretion could create a vicious cycle in which the release of Ca2+ from bone stimulates further PTHrP secretion and begets worsening osteolysis.
The goals of the current study, therefore, were 1) to determine whether the CaR is expressed in MCF-7 and MDA-MB-231 cells, two commonly used human breast cancer cell lines; 2) to ascertain whether polycationic CaR agonists, including Cao2+, neomycin, and spermine, modulate PTHrP secretion from these two cell lines; and 3) to investigate whether there is an interaction between TGFß and Cao2+ in modulating PTHrP secretion. Our results suggest that the CaR is expressed in and could, therefore, mediate the high Cao2+-induced PTHrP secretion from MCF-7 and MDA-MB-231 cells. Furthermore, TGFß stimulates PTHrP secretion synergistically with high Cao2+ from MDA-MB-231 cells. Thus, release of this growth factor along with Ca2+ during PTHrP-induced bone resorption could contribute to the postulated vicious cycle in which PTHrP-mediated osteolysis associated with breast cancers metastatic to bone promotes worsening osteolysis and, in some cases, hypercalcemia.
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Materials and Methods |
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MEM supplemented with 10% FBS, 10
µg/ml bovine insulin, and 100 U/ml penicillin-100 µg/ml
streptomycin, whereas MDA-MB-231 cells were cultured in DMEM
supplemented with 10% FBS and 100 U/ml penicillin-100 µg/ml
streptomycin. The cells were grown at 37 C in a humidified 5%
CO2 atmosphere and were passaged every 5–7 days
using 0.05% trypsin-0.53 mM EDTA. All cell culture
reagents were purchased from Life Technologies, Inc.
(Grand Island, NY) with the exception of FBS, which was obtained from
Gemini Bio-Products, Inc. (Calabasas, CA).
Northern blotting
Total RNA was prepared from the breast cancer cell lines using
the TRIzol reagent (Life Technologies, Inc.). Northern
blot analysis was performed using 7.5 µg polyadenylated
[poly(A)+] RNA obtained by
oligo(deoxythymidine) cellulose chromatography of total RNA.
Poly(A)+-enriched RNA samples were denatured and
electrophoresed in 2.2 M formaldehyde-1% agarose gels
along with an 0.2- to 9.5-kb RNA ladder (Life Technologies, Inc.) and transferred electrophoretically overnight to nylon
membranes (Duralon, Stratagene, La Jolla, CA). A 486-bp
KpnI/XbaI fragment corresponding to nucleotides
1745–2230 of the human parathyroid CaR complementary DNA (cDNA) was
subcloned into the pBluescript(SK+) vector. The
plasmid was then linearized with KpnI, and a
32P-labeled riboprobe was synthesized with the
MAXIscript T3 kit (Pharmacia Biotech, Piscataway, NJ) using T3 RNA
polymerase and [32P]UTP. Nylon membranes were
prehybridized for 2 h at 65 C in a solution containing 50%
formamide, 4 x Denhardt’s solution (50 x Denhardt’s
= 5 g Ficoll, 5 g polyvinylpyrrolidone, and 5 g BSA/50
ml), 5 x SSPE (20 x SSPE = 2.98
M NaCl and 0.02 M EDTA in
0.2 M phosphate buffer, pH 7.0), 0.5% SDS, 10%
dextran sulfate, 250 µg/ml yeast transfer RNA, and 200 µg/ml calf
thymus DNA. The labeled complementary RNA probe (2 x
106 cpm/ml) was then added, and the membranes
were hybridized overnight at the same temperature. Washing was carried
out at high stringency (0.1 x SSC-0.1% SDS at 65 C) for 30 min.
Membranes were sealed in plastic and exposed to a PhosphorImager
screen. The screens were analyzed on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) using the ImageQuant program.
RT-PCR
One-step RT-PCR (kit from QIAGEN, Santa Clarita,
CA) was used for analyzing the CaR-related transcript(s) in MCF-7 and
MDA-MB-231 cell lines using a pair of intron-spanning primers that
would yield a 480-bp product extending from nucleotides 1760–2240 of
the human CaR cDNA (25). The sequences of the primers
were: forward, 5'-CGGGGTACCTTAAGCACCTACGGCATCTAA-3'; and reverse,
5'-GCTCTAGAGTTAACGCGATCCCAAAGGGCTC-3'. The procedure used for RT-PCR
was the following. In brief, 2 µg total RNA were mixed with a master
cocktail containing RT-PCR buffer, sense and antisense CaR primers,
deoxy-NTP, ribonuclease inhibitor, and an enzyme mixture containing
reverse transcriptase (Omniscript and Sensiscript) and HotStart
Taq DNA polymerase in concentrations recommended by the
manufacturer in a final volume of 50 ml. The temperature cycle protocol
was as follows: room temperature, 30 min at 50 C, one cycle;
denaturation and activation of HotStart DNA polymerase, 15 min at 95 C;
and PCR amplification, 30 sec at 94 C, 30 sec at 55 C, and 1 min at 72
C for 40 cycles, followed by a final extension for 10 min at 72 C. The
set of primers employed was designed to span an intron of the human CaR
gene, thereby avoiding confusion arising from amplification of a
similarly sized product from contaminating genomic DNA.
PCR products were fractionated on 1.5% agarose gels. The presence of a 480-bp amplified product was indicative of a positive PCR reaction arising from CaR-related sequence within the cDNA. The PCR products in the reaction mixture were purified using the QIAquick PCR purification kit (QIAGEN, Santa Clarita, CA) and subjected to bidirectional sequencing employing the same primer pair used for PCR by means of an automated sequencer (AB377, PE Applied Biosystems, Foster City, CA) in the DNA Sequencing Facility of the University of Maine (Orono, ME), using dideoxy terminator Taq technology.
Immunocytochemistry
A CaR-specific polyclonal antiserum (no. 4637) was provided by
Drs. Forrest Fuller and Karen Krapcho of NPS Pharmaceuticals, Inc. (Salt Lake City, UT). This antiserum was raised against a
peptide (FF-7) corresponding to amino acids 345–359 of the bovine CaR
and 344–358 of the human and rat CaRs (26). The sequence
of the FF-7 peptide is identical in all three receptors and resides
within the CaR’s amino-terminal extracellular domain. The antiserum
was subjected to further purification using an affinity column
conjugated with the FF-7 peptide, and the affinity-purified antiserum
was used for immunocytochemistry and Western blot analysis as described
below. This antiserum is specific for the CaR, as assessed by the
abolition of immunoreactivity on immunocytochemistry after
preabsorption with the peptide against which it was raised
(26) and Western analysis of CaR-expressing and
nonexpressing cells (27). That is, Western analysis of
extracts of bovine parathyroid and CaR-transfected HEK293 cells, but
not of nontransfected HEK293 cells (which do not endogenously express
the CaR), performed with this antiserum revealed the characteristic
doublet of bands at 140–160 kDa corresponding to various glycosylated
forms of CaR monomers and dimers, respectively (27).
For immunocytochemistry, breast cancer cells were grown on glass coverslips. The cells were fixed for 5 min with 4% formaldehyde and then treated for 10 min with peroxidase-blocking reagent (DAKO Corp., Carpenteria, CA) to inhibit endogenous peroxidase activity (26). After washing once with PBS, the cells were blocked for 30 min with PBS containing 1% BSA. The cells were then incubated overnight at 4 C with the 4637 antiserum (5 µg/ml in blocking solution). Negative controls were carried out by incubating the cells with 4637 antiserum that had been preabsorbed with 10 µg/ml of the FF-7 peptide. After washing the cells three times for 10 min each time with PBS containing 0.5% BSA, peroxidase-conjugated, goat antirabbit IgG (1:100; Sigma, St. Louis, MO) was added and incubated for 1 h at room temperature. The cells were then washed three times for 10 min each time with PBS, and the color reaction was developed using the DAKO Corp. AEC substrate system (DAKO Corp.) for 5–15 min. The color reaction was stopped by washing the cells three times with PBS and once with water. The cells were then examined by light microscopy and photographed at x400.
Western blotting
For performing Western blots, MCF-7 or MDA-MB231 cells that had
been cultured in six-well plastic cluster plates were rinsed with
ice-cold PBS and scraped on ice into lysis buffer containing 10
mM Tris-HCl (pH 7.4), 1 mM EGTA, 1
mM EDTA, 0.25 M sucrose, 1% Triton X-100, 1
mM dithiothreitol, and a cocktail of protease inhibitors
(10 µg/ml each of aprotinin, leupeptin, and calpain inhibitor as well
as 100 µg/ml Pefabloc) (27). The cells were then passed
though a 22-gauge needle 10 times. Nuclei and cell debris were removed
by low speed centrifugation (1,000 x g for 10 min), and the
resultant total cellular lysate in the supernatant was either used
directly for SDS-PAGE or stored at -80 C. CaR-transfected HEK-293
cells (designated HEKCaR), included as a positive control, were
harvested using the same protocol.
Immunoblot analyses were performed essentially as described previously (26, 27). Aliquots of supernatant fractions containing the total cellular lysate (20–40 µg protein) were mixed with an equal volume of 2 x SDS-Laemmli gel loading buffer containing 100 mM dithiothreitol, incubated at 37 C for 15 min, and resolved electrophoretically on linear 3–10% gradient gels. The separated proteins were then transferred to nitrocellulose filters (Schleicher & Schuell, Inc., Keene, NH) and incubated with blocking solution (PBS with 0.25% Triton X-100 and 5% dry milk) for 1 h at room temperature. The blots were subsequently incubated overnight at 4 C with affinity-purified, polyclonal anti-CaR antiserum 4637 at 1 µg/ml with or without preincubation with 2 µg/ml of the FF-7 peptide (as a control for nonspecific binding) in blocking solution with 1% dry milk. The blots were subsequently washed five times with PBS containing 0.25% Triton X-100 and 0.1% dry milk (washing solution) at room temperature for 10 min each time. The blots were further incubated with a 1:2000 dilution of horseradish peroxidase-coupled, goat antirabbit IgG (Sigma) in blocking solution with 1% dry milk for 1 h at room temperature. The blots were finally washed five times with the washing solution, and protein bands were detected using an enhanced chemiluminescence system (Renaissance kit, NEN Life Science Products, Boston, MA).
PTHrP secretion studies
For studying the effects of high Cao2+ and
other polycationic CaR agonists on PTHrP secretion, MCF-7 or MDA-MB-231
cells were seeded in 24-well plates (105
cells/well) in 1 ml of their respective growth media. After
approximately 48 h, the growth media were carefully removed from
each well, and 1 ml medium A [Ca2+-free DMEM
(Life Technologies, Inc.) supplemented with 4
mM L-glutamine, 0.2% BSA, 100 U/ml
penicillin-100 µg/ml streptomycin, and 0.5 mM
CaCl2] was added to each well. Twenty-four hours
later, medium A was carefully removed from each well, and 0.35 ml
medium B (Ca2+-free DMEM supplemented with 4
mM L-glutamine, 2% FBS, 100 U/ml
penicillin-100 µg/ml streptomycin, and 0.5 mM
CaCl2) alone or supplemented with either
additional CaCl2 (to a final concentration of 1,
3, 5, 7.5, or 10 mM) or the polycationic CaR agonist,
neomycin (300 µM) or spermine (2 mM), was
added to each well. Six hour later, the conditioned medium was removed
for determination of PTHrP release.
For studies on the effect of pretreatment with TGFß1 on PTHrP secretion, MDA-MB-231 cells were seeded as described above. After approximately 48 h, the growth media were carefully removed from each well, and 0.5 ml medium A containing 0, 0.2, 1, or 5 ng/ml TGFß1 (R & D Systems, Minneapolis, MN) was added to each well. Twenty-four hours later, the pretreatment medium was removed from the wells, the cells were rinsed once with 0.5 ml medium B, and medium B alone or supplemented with additional CaCl2 (to a final concentration of 3, 5, 7.5, or 10 mM) was added to each well (0.35 ml/well). Six hours later, the conditioned medium was removed for determination of PTHrP content.
PTHrP was measured in conditioned medium using a two-site immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA) that detects PTHrP-(1–72) and has a sensitivity of about 0.3 pmol/liter. PTHrP assays were initiated immediately after removing the conditioned medium from the cell cultures to minimize degradation of the peptide from freeze-thawing and other manipulations. PTHrP concentrations were calculated from a standard curve generated by adding recombinant PTHrP-(1–86) to the treatment medium employed in this study (i.e. unconditioned medium B). CaR agonists alone had no effect in the PTHrP assay.
MTT assay
PTHrP in each well was normalized using the MTT assay, a
colorimetric assay used to determine cell number and/or assess cellular
viability (28). After removing medium for the PTHrP assay,
150 µl DMEM with 4 mM L-glutamine, 1% FBS,
100 U/ml penicillin-100 µg/ml streptomycin, and 0.5 mM
Cao2+ were added to each well with 10 µl MTT
(10.4 mg/ml). The plates were incubated at 37 C in humidified 5%
CO2 for 4 h to convert water-soluble MTT to
insoluble formazan, which occurs only in viable cells. After 4 h,
100 µl 50% dimethylformamide with 20% SDS was added to each well to
solubilize formazan, and the plates were incubated at 37 C overnight.
Absorbance was then measured at 595 nm with background subtraction at
655 nm.
Statistical analysis
A minimum of two independent experiments were performed for each
of the PTHrP secretion studies. Results are presented as the mean
± SEM for three determinations. Most data were analyzed by
one-way ANOVA followed by Dunnett’s multiple comparison test. To
determine whether there was any interaction between the effects of
Cao2+ and TGFß1 on PTHrP secretion, we employed
two-way ANOVA. For all statistical tests, P < 0.05 was
considered to indicate a statistically significant result.
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). The larger transcript was similar in
size to the predominant CaR transcript in human parathyroid gland
(25). RT-PCR, performed with CaR-specific human primers,
amplified a product of the expected size (480 bp) for a CaR-derived
product (Fig. 2, lanes A and B). No
product was observed when water was used instead of RNA during the RT
reaction (Fig. 2, lane C). We performed DNA sequence analysis only on
the PCR product isolated from MDA-MB-231 cells, which revealed more
than 99% identity with the corresponding region of the human
parathyroid CaR cDNA (data not shown). This result indicates that the
PCR product was amplified from authentic CaR transcript(s).
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) and MDA-MB-231 (Fig. 3B) breast
cancer cells. Staining was specific, as it was eliminated by
preincubating the anti-CaR antiserum with the specific peptide (FF-7)
against which it was raised (Fig. 3, C and D). Western blots of
proteins isolated from total cellular lysates of MCF-7 and MDA-MB-231
cells using antiserum 4637 were compared with those obtained using a
similar protein preparation from HEKCaR cells as a positive control
(Fig. 4, left
panel). Although the levels of CaR protein expression in
HEKCaR cells were substantially higher than those in MCF-7 and
MDA-MB-231 cells, the immunoreactive bands in both breast cancer cell
lines between about 120 and 200 kDa probably comprise CaR monomers
varying in their extents of glycosylation and are of sizes similar to
bands that are present in the positive control. The specificities of
these CaR-immunoreactive bands were confirmed by the marked reductions
in their intensities after preabsorption of antiserum 4637 with the
specific peptide (FF-7) against which it was raised (Fig. 4, right panel).
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). At
the end of the treatment period, conditioned media were removed for
determination of PTHrP content, and the cells remaining in the culture
wells were used for the MTT assay (Fig. 6). There were no substantive effects of
the various agonists on the MTT assay in either cell line (Fig. 6).
This result suggested that there were no nonspecific effects on
cellular viability and/or number; therefore, the results of the MTT
assays were used as a means of normalizing the results of PTHrP
secretion to the number of viable cells in each well (Fig. 5).
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). At 3, 5, 7.5, and 10 mM,
PTHrP secretion was increased 1.7-, 1.4-, 2-, and 2.3-fold,
respectively, compared with that observed at 0.5 mM
Cao2+. The polycationic CaR agonists, neomycin and
spermine, also elicited a statistically significant secretory response;
300 µM neomycin increased PTHrP secretion 1.9-fold over
that present at 0.5 mM Cao2+, whereas
2 mM spermine elicited a 2.1-fold increase in PTHrP
secretion (Fig. 5A). Like MCF-7 cells, MDA-MB-231 cells secreted a
small, but measurable, amount of PTHrP at 0.5 mM
Cao2+ (Fig. 5B). There was no significant increase
in PTHrP secretion at 1 mM Cao2+, but
higher levels of Cao2+ stimulated PTHrP secretion
in a dose-dependent manner (Fig. 5B). At 3 and 5 mM
Cao2+, PTHrP secretion was increased by 1.7- and
1.8-fold compared with that at 0.5 mM
Cao2+, respectively, whereas 7.5 and 10
mM Cao2+ each elicited a 2.2-fold
increase in secretion. Neomycin and spermine also elicited PTHrP
secretion from MDA-MB-231 cells. Neomycin (300 µM)
elicited a nonsignificant, 1.3-fold increase in PTHrP secretion over
that observed at 0.5 mM Cao2+, whereas
2 mM spermine elicited a significant, 1.5-fold increase in
PTHrP secretion (Fig. 5B).
Because TGFß stimulates PTHrP secretion from MDA-MB-231 cells, we
examined the possibility that there might be an interaction between
TGFß and Cao2+ in stimulating PTHrP secretion
from breast cancer cells. When MDA-MB-231 cells were pretreated for
24 h with TGFß1, there were marked, dose-dependent increases in
both basal (i.e. at 0.5 mM
Cao2+) and Cao2+-stimulated
PTHrP secretion (Fig. 7). As noted
above, TGFß1 had no effect on the MTT assay, independent of the level
of Cao2+ that was present (not shown).
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). This
transcript is similar in size to the predominant CaR transcript
observed in human parathyroid cells (26). The significance
of the approximately 2.5-kb transcript detected in MCF-7 cells is not
clear. Amplified products of a size consistent with those derived from
authentic CaR transcripts were also detected by RT-PCR in both cell
lines (Fig. 2), and sequence analysis of the stronger PCR product
obtained from MDA-MB-231 cells revealed essentially 100% identity with
the known sequence of the human parathyroid CaR (data not shown).
These breast cancer cell lines also express CaR protein, as assessed by
immunocytochemistry (Fig. 3, A and B) and Western blot analysis (Fig. 4, left panel) performed using an affinity-purified,
anti-CaR antiserum (no. 4637). In both cases, the immunoreactivity was
specific for the CaR, as it was eliminated by preincubating antiserum
4637 with the specific peptide against which it was raised (Fig. 3, C
and D; Fig. 4, right panel). Although the levels of CaR
protein expression observed in MCF-7 and MDA-MB-231 cells were
substantially lower than that present in the positive control,
CaR-transfected HEK293 cells, they were similar in quantity and
identical in size to those present in several other types of cells
(including HEK293 cells stably transfected with the CaR used as a
positive control here) in which we have shown that the CaR is expressed
and in which it modulates various biological responses, such as
regulating Ca2+-activated
K+ channels (29).
Cao2+ and the polycationic CaR agonists, neomycin
and spermine, each stimulated PTHrP secretion from MCF-7 and MDA-MB-231
cells in a dose-dependent manner, although the effect of neomycin on
PTHrP secretion from MDA-MB-231 cells was not significant (Fig. 5).
Neither Cao2+, neomycin, nor spermine had any
significant effect on the MTT values obtained from either cell line
used in these studies, indicating that CaR agonist-stimulated PTHrP
secretion was not simply the result of these agonists modifying cell
number and/or viability (Fig. 6). The levels of
Cao2+ in the vicinity of resorbing osteoclasts are
thought to be manyfold higher than the level of systemic
Cao2+ (i.e. as high as 8–40
mM) (17). Therefore, in the bony
microenvironment, metastatic breast cancer cells will probably
encounter levels of Cao2+ at least as high as
those used in the present studies.
Our results are consistent with studies in several other cell types, including normal keratinocytes (18), normal cervical epithelial cells (19), oral squamous cancer cells (20), JEG-3 cells (21), and H-500 rat Leydig cells, a model of humoral hypercalcemia of malignancy (22), which also exhibit Cao2+-stimulated PTHrP secretion. The molecular mechanism(s) underlying Cao2+-stimulated PTHrP secretion in these cell types, however, is not clear. Our data are consistent with a role for the CaR as a mediator of this effect in MCF-7 and MDA-MB-231 cells, as the receptor is clearly expressed in these cells, and PTHrP secretion is stimulated not only by elevated levels of Cao2+ but also by the polycationic CaR agonists, neomycin and spermine. Nevertheless, additional studies using stable transfection with dominant negative (30) and/or antisense CaR constructs (31) are necessary to establish definitively the CaR’s involvement in these secretory responses. An additional approach to studying further the role of the receptor in mediating high Cao2+-stimulated PTHrP secretion would be the use of the calcimimetic CaR activators, which are exemplified by NPS R-467 and R-568 and their less active stereoisomers, NPS S-467 and S-568 (32). The effects of these agents on PTHrP secretion have not been previously examined to our knowledge. Furthermore, the stimulation of insulin secretion by NPS R-467 and S-467 that was documented recently (33) failed to show the expected stereoselectivity for the relative potencies of these two agents. Thus, further work is needed to examine the effects of the calcimimetics on the full range of biological responses to Cao2+ in the numerous tissues now known to express the CaR, to characterize more fully the use of these agents for documenting which of these biological responses are actually mediated by this receptor.
We have recently carried out preliminary studies in CaR-transfected and nontransfected human embryonic kidney (HEK293) cells that indicate that the CaR is capable of mediating high Cao2+-stimulated PTHrP secretion from epithelial cells (MacLeod, R. J. N. Chattopadhyay, and E. M. Brown, manuscript in preparation). In CaR-transfected HEK293 cells, high Cao2+ (3 mM), neomycin (300 µM), and Gdo3+ (50 µM) each stimulated PTHrP secretion, by 5.8-, 3.4-, and 2.0-fold, respectively, whereas the same three agents had no effect on PTHrP release by the nontransfected HEK293 cells. Thus, although these data do not prove the CaR’s role in mediating high Cao2+-elicited PTHrP secretion in the breast cancer cell lines studied here, they do document that the CaR can mediate this biological action in cells originating from a tissue also known to produce PTHrP-mediated, malignant hypercalcemia in vivo (e.g. in the setting of renal cell cancers).
Regardless of the molecular mechanism(s) responsible for the high Cao2+-stimulated PTHrP secretion from MCF-7 and MDA-MB-231 breast cancer cells observed here, the implications of our findings for the existence of a vicious cycle involving breast cancer cells metastatic to bone are clear. When breast, and possibly other, cancers metastasize to the skeleton and induce PTHrP-mediated osteolysis, this will lead to high local levels of Cao2+ within the bony microenvironment due to PTHrP-stimulated bone resorption, with or without associated systemic hypercalcemia. Such high levels of Cao2+ elicit further PTHrP secretion from the cancer cells, thereby exacerbating the osteolytic disease. Guise et al. (8, 34) provided strong evidence for the existence of a similar vicious cycle involving TGFß released from bone. We confirmed that TGFß1 increases PTHrP secretion from MDA-MB-231 cells and have also demonstrated that the combination of TGFß1 and high Cao2+ produces a greater than additive stimulation of PTHrP secretion. The mechanism for this effect is not clear, but might involve TGFß1-induced up-regulation of CaR expression or its signaling pathways and/or increased expression of the PTHrP gene, thereby enhancing the amount of PTHrP available for secretion in response to elevated levels of Cao2+. Cao2+ and TGFß are both released from the bone matrix during bone resorption induced by PTHrP. Therefore, they are likewise both available to elicit further PTHrP secretion, in effect cooperating to generate a vicious cycle of tumor-induced bone resorption, begetting further bone resorption in the setting of skeletal metastases of breast cancers. The beneficial actions of bisphosphonates on the skeletal complications of metastatic breast cancer and on new metastases (35, 36, 37, 38) could result at least in part from reductions in the local concentrations of both Cao2+ and TGFß as a result of decreased bone resorption.
Mice injected with MDA-MB-231 clones expressing the dominant negative type II TGFß receptor exhibit a substantial reduction in osseous metastases. Blocking the effect of TGFß in this manner, however, does not completely prevent metastases and delays, but does not prevent, the eventual death of the injected mice (8, 16). One possible explanation for these observations is the persistence of a CaR-mediated feedforward mechanism that enhances PTHrP secretion, thereby enabling eventual accumulation of a lethal tumor burden. Further understanding of the CaR’s role in osteolytic skeletal metastases that secrete PTHrP and its interaction(s) with TGFß-mediated stimulation of PTHrP secretion may permit novel and more effective therapies for this complication based on the use of CaR antagonists.
In addition to its potential role in stimulating PTHrP secretion from breast cancer cells metastatic to bone, the CaR could also impact on tumor progression, osteolysis, and in some cases, hypercalcemia by modulating the proliferation and/or apoptosis of tumor cells. Recent studies have shown that CaR activation stimulates proliferation in several cell types, including ovarian surface cells and Rat-1 fibroblasts (20, 39). In PTHrP-producing tumors, the CaR could potentially increase proliferation directly and/or indirectly by enhancing PTHrP secretion. Indeed, PTHrP has been shown to stimulate the proliferation of H-500 rat Leydig cells in vitro and to increase the rate of tumor growth in vivo when H-500 cells are implanted subcutaneously in rats (40). The CaR also protects some cells against apoptosis, as demonstrated in AT-3 rat prostate cancer cells and CaR-transfected, but not nontransfected, HEK-293 cells (41). Therefore, high Cao2+-evoked, CaR- mediated stimulation of proliferation and/or inhibition of apoptosis of breast cancer cells metastatic to bone could clearly contribute to the progression of tumor growth and potentially render the tumor cells resistant to therapy.
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Received April 4, 2000.
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