calcium-sensing receptor (casr) promotes breast cancer...
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Calcium-Sensing Receptor (CaSR) Promotes Breast Cancer by Stimulating Intracrine Actions of Parathyroid Hormone-Related Protein
Wonnam Kim1, Farzin M. Takyar1, Karena Swan1, Jaekwang Jeong1, Joshua
VanHouten1, Catherine Sullivan1, Pamela Dann1, Herbert Yu3, Nathalie Fiaschi-Taesch2, Wenhan Chang4, John Wysolmerski1
1) Section of Endocrinology and Metabolism, Department of Internal Medicine, Yale
University School of Medicine, New Haven CT.
2) Diabetes, Obesity and Metabolism Institute, Section of Endocrinology, Diabetes and Bone Disease, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY.
3) Cancer Epidemiology Program, University of Hawaii Cancer Center, University of
Hawaii School of Medicine, Honolulu, HI
4) Endocrine Unit, University of California, San Francisco and Veteran Affairs Medical Center, San Francisco, CA
Running Title: CaSR and Breast Cancer Keywords: calcium-sensing receptor, parathyroid hormone-related protein, breast cancer, MMTV-PyMT, p27kip1, apoptosis-inducing factor, calcilytic Financial Support: NIH grant CA153702, NIH grant HD076248, NIH grant CA94175, and a pilot grant from the Lion Heart Foundation to J. J. Wysolmerski; American Diabetes Association Grant 7-12-BS-046 to N. M. Fiaschi-Taesch COI: There are no financial conflicts of interest to report. Corresponding Author: John Wysolmerski, MD Professor of Medicine Section of Endocrinology and Metabolism Department of Internal Medicine TAC S123A, Box 208020 New Haven, CT 06520-8020 Email:[email protected] Phone: 203-785-7447
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Abstract
Parathyroid hormone-related protein (PTHrP) contributes to the development and metastatic
progression of breast cancer by promoting hypercalcemia, tumor growth and osteolytic bone
metastases, but it is not known how PTHrP is upregulated in breast tumors. Here we report
a central role in this process for the calcium-sensing receptor, CaSR, which enables cellular
responses to changes in extracellular calcium, through studies of CaSR-PTHrP interactions
in the MMTV-PymT transgenic mouse model of breast cancer and in human breast cancer
cells. CaSR activation stimulated PTHrP production by breast cancer cells in vitro and in vivo.
Tissue-specific disruption of the casr gene in mammary epithelial cells in MMTV-PymT mice
reduced tumor PTHrP expression and inhibited tumor cell proliferation and tumor outgrowth.
CaSR signaling promoted the proliferation of human breast cancer cell lines and tumor cells
cultured from MMTV-PyMT mice. Further, CaSR activation inhibited cell death triggered by
high extracellular concentrations of calcium. The actions of the CaSR appeared to be
mediated by nuclear actions of PTHrP that decreased p27kip1 levels and prevented nuclear
accumulation of the pro-apoptotic factor AIF. Taken together, our findings suggest that
CaSR-PTHrP interactions might be a promising target for the development of therapeutic
agents to limit tumor cell growth in bone metastases and in other microenvironments in
which elevated calcium and/or PTHrP levels contribute to breast cancer progression.
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Introduction
The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that
signals in response to extracellular calcium and other organic and inorganic cations (1,2). It
is expressed in the parathyroid glands and kidneys, and regulates parathyroid hormone
(PTH) production and renal calcium handling to maintain circulating calcium levels within a
narrow range (2). The CaSR is also expressed in other tissues, where it modulates cell
proliferation and differentiation as well as the production of parathyroid hormone-related
protein (PTHrP) (1-3).
The CaSR is expressed on lactating mammary epithelial cells and coordinates the
flow of calcium into milk. Stimulating the CaSR on normal breast cells inhibits PTHrP
production, which circulates to mobilize skeletal calcium stores for milk production (3-5).
The CaSR is also expressed in breast cancer cells where it stimulates, rather than inhibits,
PTHrP secretion (6,7). Whether the CaSR regulates breast cancer growth remains
uncertain since prior studies have reported that it can stimulate, inhibit, or have no effect on
breast cancer cell proliferation (3,8). To date, no studies have attempted to manipulate the
CaSR in breast cancer models in vivo (8).
PTHrP (gene symbol, PTHLH) is secreted by cancer cells and causes humoral
hypercalcemia of malignancy by activating the Type 1 PTH/PTHrP receptor (PTH1R) in
kidney and bone (9). PTHrP also contains canonical nuclear localization sequences and
appears to traffic in and out of the nucleus to affect cellular function independent of the
PTH1R (10). PTHrP is required for embryonic development of the breast and, during
lactation, it is secreted from mammary cells to regulate maternal and neonatal calcium and
bone metabolism (9). PTHrP also affects breast cancer. Tumor production of PTHrP can
contribute to the expansion of osteolytic bone metastases by stimulating osteoclast activity
(11). The actions of PTHrP in primary tumors are less clear. Studies in breast cancer cell
lines, genetically altered mice and clinical series have reported conflicting results regarding
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its effects on cell turnover, tumor growth or clinical outcome (9,11-18). Nevertheless,
several GWAS studies have implicated the PTHLH gene as a breast cancer susceptibility
locus, suggesting that alterations in PTHrP expression or signaling contribute to breast
cancer initiation and/or progression (19-21).
We examined interactions between the CaSR and PTHrP in rodent and human
breast tumors, in breast cancer cell lines and in a transgenic model of breast cancer. Our
results confirm that CaSR signaling stimulates PTHrP production in vitro and in vivo. We
also demonstrate that the CaSR stimulates the growth of mammary tumors in MMTV-PyMT
mice. Finally, we demonstrate that activation of the CaSR promotes proliferation and
inhibits apoptosis in breast cancer cells, in part, through nuclear actions of PTHrP.
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Materials and Methods
Cell Culture
MCF-7 and BT474 cells were obtained from ATCC in 2011 and MDA-MB 231.1833
cells were a gift of the Massague Lab in 2013 (22). Cells were maintained in
DMEM+GlutaMAX-1 (Gibco-life Technologies) containing 10% FBS and pen/strep (Gibco-
Life Technologies). All cells were routinely screened for mycoplasma contamination at
least every 6 months and remained negative during the course of these studies.
Experimental results were stable across different batches of cryopreserved cells. Stable
cell lines expressing shRNA directed against CASR, PTHLH, and PTH1R were generated by
transducing cells with commercially prepared lentiviruses: control (sc-108080), CaSR (sc-
44373-V), PTHrP (sc-39695-V), and PTH1R (sc-39695) from Santa Cruz followed by
selection with puromycin. Tumor cells were cultured from dissected mammary tumors.
Details of tumor cell preparation and preparation of stable knockdown cell lines can be found
in Supplemental Methods. To examine the effects of different calcium concentrations, cells
were cultured without FBS for 24 hours and then treated with calcium-free DMEM (Gibco-life
Technologies) containing 0.2% bovine serum albumin (Americanbio) and differing doses of
calcium chloride (Avantor Performance Materials) for 16 or 40 hours. For adenoviral
transfection experiments, BT474 CaSRKD cells were treated with recombinant adenovirus
with a multiplicity of 1000 viral particles per cell in serum-free medium for 6 hours.
RNA Extraction and Real-Time RT-PCR
RNA was isolated using TRIzol (Invitrogen). Quantitative RT-PCR was performed
with the SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen) using a Step One Plus
Real-Time PCR System (Applied Biosystems) and the following TaqMan primer sets:
PTHLH, Hs00174969_m1; CASR, Hs01047795_m1; Pthlh, Mm00436057_m1; Casr,
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Mm00443375_m1. Human HPRT1 (4326321E) and mouse Actb (4352341E) were used as
reference genes (Invitrogen). Relative mRNA expression was determined using the Step
One Software v2.2.2 (Applied Biosystems).
Biochemical Measurements
Serum calcium concentrations were measured using the Quantichrom Calcium
Assay Kit (DICA-500, BioAssay Systems, Hayward, CA) according to manufacturer’s
instructions. Plasma PTHrP was measured as previously described (4) using an
immunoradiometric assay (DSL-8100; Beckman Coulter, Webster, Texas), in which we
substituted custom rabbit anti-PTHrP (1–36) antibody as capture antibody. This assay has
a sensitivity of 0.5 pM/L.
Cell Proliferation and Apoptosis
Cell proliferation was measured by assessing BrdU incorporation (Cell proliferation
ELISA kit 11647229001; Roche). Apoptosis was measured by TUNEL assay (In Situ Cell
Death Detection Kit 11684817910; Roche).
CaSR Conditional Knockout Mice
Floxed CaSR (CaSRflox/flox) mice (4) were backcrossed onto an FVB background for
5 generations and bred to MMTV-PyMT mice. MMTV-Cre (line D) and MMTV-PyMT were
purchased from Jackson Laboratories on a FVB background. MMTV-
Cre;CaSRlox/lox;MMTV-PyMT and control CaSRlox/lox;MMTV-PyMT mice were assessed for
tumor incidence, latency and metastases as detailed in Supplemental Methods.
Breast Cancer Tissue Microarray
YTMA49 contains 652 primary breast cancer specimens retrospectively obtained
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from 1953 to 1983. Cases were evenly divided between lymph node-positive and -
negative, with a median follow-up of 8.9 years (23,24). Details of staining, automated
image acquisition and analysis using the semi-automated AQUA system have also been
described (23) and are detailed further in Supplemental Methods. Histospots were excluded
from analysis if the tumor mask represented less than 5% of the area. Analyses of CaSR
staining were based on 377 histospots with adequate tumor representation and staining.
Immunofluorescence and Immunoblotting
Cells were grown on coverslips, fixed in 4% paraformaldehyde for 25 min,
permeabilized with 0.2% Triton X100 for 15 min, and stained for immunofluorescence using
standard techniques. Whole-cell lysates were prepared using standard methods and
cytoplasmic and nuclear proteins were extracted using Thermo Scientific Pierce NE-PER
Nuclear and Cytoplasmic Extraction Kit (78833, Pierce Technology, Rockford, IL). Protein
samples were subjected to SDS-PAGE and transferred to a nitrocellulose membrane by wet
western blot transfer (Bio-Rad). Membranes were incubated with primary antibodies
overnight at 4˚C and staining was analyzed using an infrared imaging system (LI-COR).
Primary antibodies included those against: HA-Tag (2367 or 3724), PARP (9542), p27Kip1
(3686 or 3698), CDK2 (2546), HSP70 (4872), AIF (5318), and Caspase-3 (9665) from Cell
Signaling (Danvers, MA); and PTH1R (sc-12722) from Santa Cruz (Dallas, Texas). All
experiments were performed at least 3 times and representative blots are shown in the
figures.
Kinase Assay
Cdk2 kinase activity was measured by assessing phosphorylation of histone H1 by
immunoprecipitated cdk2 using standard techniques. Immunoprecipitates were
resuspended in 50 μL of kinase reaction buffer containing 50 μM ATP and 2 μg of Histone
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H1 (382150) from EMD Millipore (Billerica, MA) and incubated for 30 min at 30 ℃. The
samples were subjected to SDS-PAGE and transferred to a nitrocellulose membrane and
assessed with phospho-histone H1 (05-1324) from EMD Millipore (Billerica, MA). Further
details are provided in Supplemental Methods. All assays were performed at least 3 times
and representative blots are shown in the figures.
Statistics
Analyses were performed with Prism 6.0 (GraphPad Software, La Jolla, CA). Error
bars represent SEM except in Fig3E, where they represent SD. Significance for all
comparisons between 2 conditions were calculated using paired t-tests and significance for
multiple comparisons were performed using one-way Anova with Turkey post-test
corrections. Significance for Kaplan Meier analyses were calculated using the log-rank test
and correlations between CaSR and PTHrP were calculated using Pierson’s Correlation
Coefficient.
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Results
CaSR and PTHrP Expression in Breast Cancers
We examined Casr and Pthlh gene expression in mammary tumors in a microarray
study of rats treated with the chemical carcinogen, N-Nitroso-N-methylurea (NMU) (25).
Casr and Pthlh mRNA levels were significantly higher in tumors as compared to normal
mammary tissue, although the increase for Pthlh was more striking than that for Casr (Fig.
1A&B). In addition, there was a significant positive correlation between Casr and Pthlh
mRNA levels in the tumors (Fig. 1C). We also examined CASR and PTHLH gene
expression in 204 human breast tumors (26) and again found a positive correlation between
their mRNA levels in breast cancers (Fig. 1D). Next, we examined CaSR protein
expression using the semi-automated immunofluorescence AQUA platform in a tissue
microarray consisting of 652 breast tumors with a median clinical follow up of 8.9 years
(YTMA49) (23,24). In YTMA49, CaSR levels correlated inversely with pathological
progesterone receptor positive status and positively with node-positive status. CaSR levels
above the median value were associated with significantly shorter survival than those below
the median (Fig. 1E), although when considered as a continuous variable, they did not
significantly predict survival. We have also stained YTMA49 for PTHrP (manuscript in
preparation) and as shown in Fig. 1F, there was a positive correlation between CaSR and
PTHrP expression in this cohort. Together, these data document direct correlations
between CaSR and PTHrP mRNA and protein expression in breast cancers.
CaSR Activation Stimulates PTHrP Production by Breast Cancer Cell Lines
Next, we examined CaSR and PTHrP expression in 3 human breast cancer cell
lines: MCF7, MDA MB231.1833 and BT474 cells. Figs. 2A&B show baseline mRNA levels
for each transcript in the cell lines cultured in growth media. Cells were also serum-starved
for 24-hours and then treated with 0.1mM or 2.5mM calcium in serum-free media for 16
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hours. In each cell line, increasing the extracellular calcium concentration stimulated
PTHLH mRNA expression (Fig. 2C). MDA-MB 231.1833 cells and BT474 cells had higher
baseline levels of CaSR and PTHrP expression and a more robust PTHrP response to
calcium, so we used these cell lines for subsequent studies. Figs. 2D&G demonstrate
dose-dependent increases in PTHLH mRNA levels in both cell lines in response to
extracellular calcium or gadolinium, which also activates the CaSR. Exposing the cells to
increasing doses of extracellular calcium also increased intracellular cAMP levels, and
treatment with the PKA inhibitor, H89, blocked the increase in PTHLH mRNA in response to
calcium (Figs. 2E,F,H,I) (6). Increased PTHLH expression was mediated by CaSR
activation because it could be inhibited by treating the cells with a selective CaSR
antagonist, NPS2143 (Figs. 2J&L) or by knocking down CaSR expression using shRNA
(Figs. 2K&M). These data confirm that, in breast cancer cells, CaSR activation stimulates
cAMP/PKA signaling, which, in turn, increases PTHLH gene expression (6).
The CaSR Regulates PTHrP Production and Tumor Growth in MMTV-PyMT Mice.
In order to test whether the CaSR regulates PTHrP expression and/or tumor growth
in vivo, we generated MMTV-Cre; CaSRlox/lox; MMTV-PyMT (CaSR-CKO-PyMT) mice to
ablate the Casr gene in mammary tumor cells. MMTV-PyMT mice recapitulate the
progression from hyperplasia to ductal carcinoma-in-situ to invasive ductal carcinomas, and
invasive tumors often loose estrogen receptor expression (27). These mice develop
palpable mammary carcinomas within 60-70 days (27) and a previous study had
demonstrated that PTHrP promotes tumor growth in this model (15). Although the MMTV
promoter is expressed heterogeneously in mammary epithelial cells, using the MMTV-Cre
transgene together with the MMTV-PyMT transgene allowed us to disrupt CaSR expression
in the same cells expressing the PyMT oncogene.
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Ablating the Casr gene did not impair normal mammary development during puberty or
pregnancy (Fig. S1). As compared to control MMTV-PyMT tumors, mammary tumors from
CaSR-CKO-PyMT mice had an 85.6±5.6% reduction in CaSR mRNA (Fig 3A). This was
associated with a concomitant 48.0±3.9% reduction in tumor PTHrP mRNA, demonstrating
that, in mammary tumors, PTHrP production is regulated by CaSR signaling at physiological
calcium levels (Fig 3B). As expected, circulating PTHrP and calcium levels in CaSR-CKO-
PyMT mice were unchanged (Fig. S1).
Reducing CaSR expression did not alter the incidence or latency of tumor formation
in MMTV-PyMT mice; 100% of both control and CaSR-CKO-PyMT mice developed tumors,
and the time to 50% tumor incidence was identical for both genotypes (Fig.3C). However,
CaSR-CKO-PyMT mice survived longer (Fig. 3D) and had a slower rate of tumor growth and
an overall reduction in tumor burden (Fig. 3E&F). There were no differences in the
incidence of lung metastases (60% of mice in both groups) or the number of lung
metastases per mouse in CaSR-CKO-PyMT mice compared to controls (Fig, 3G). Because
ablation of the CaSR slowed tumor growth, we examined proliferation and apoptosis in
tumors by measuring TUNEL staining and BrdU incorporation. There was a significant
reduction in BrdU incorporation in CaSR-CKO-PyMT tumors as compared to controls (Fig
3H&I). These results reflected a direct effect on tumor cell proliferation since we saw
similar effects in tumor cells cultured ex vivo. As shown in Fig. 3J control PyMT tumor cells,
but not CaSR-CKO-PyMT tumor cells, demonstrated increased BrdU incorporation in
response to increasing doses of extracellular calcium (within and above the physiologic
range) or gadolinium. Finally, rates of apoptosis, as assessed by TUNEL staining, were
insignificant in both types of tumors in vivo (not shown).
Knocking Down CaSR or PTHrP Expression Inhibits Proliferation in Breast Cancer
Cells.
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The effects of ablating the CaSR on the growth of mammary tumors in MMTV-PyMT
mice mirrored those previously described for ablating PTHrP expression (15). Therefore,
we examined proliferation in BT474 and MDA-MB 231.1833 cells stably transfected with
shRNAs to knockdown either CaSR (CaSRKD, Fig 4A&D) or PTHrP (PTHrPKD, Fig 4B&E)
expression. As assessed by BrdU incorporation, treatment of control cells (transfected with
a non-specific, control shRNA) with increasing doses of calcium or gadolinium stimulated
proliferation in a dose responsive fashion (249±19% over baseline for BT474 cells and
225±6% over baseline for MDA-MB-231.1833 cells at 5mM calcium, Fig. 4C&F). The
CaSR antagonist, NPS2143, decreased BrdU incorporation in response to extracellular
calcium or gadolinium (Fig. 4G&J) and knocking down CaSR expression reduced
proliferation at baseline and blunted the increase in proliferation in response to calcium or
gadolinium (Fig 3H&K). Knocking down PTHrP expression reproduced the effects of
knocking down CaSR expression on proliferation (Fig. 3I&L). In vascular smooth muscle
cells, PTHrP has been shown to regulate cell proliferation at the G1-S checkpoint by altering
p27kip1 levels and CDK2 activity (28,29). Therefore, we examined p27Kip1 and CDK2 in
response to high calcium in CaSRKD and PTHrPKD cells. In control BT474 and MDA-MB-
231.1833 cells, activation of the CaSR with 5mM calcium was associated with a clear
decrease in p27Kip1 levels (Fig. 5A-F). By contrast, control cells treated with NPS2143,
CaSRKD cells and PTHrPKD cells all demonstrated either a blunting of the reduction in
p27Kip1 levels (MDA-MB-231 cells) or an actual increase in p27Kip1 levels (BT474 cells) when
treated with 5mM calcium (Fig. 5A-F). We also examined CDK2 levels by immunoblotting
and CDK2 activity by assessment of histone H1 phosphorylation by immunprecipitated
CDK2. As shown in Fig. S2, neither treatment with NPS2143 nor knockdown of the CaSR
or PTHrP affected total levels of CDK2. However, inhibition of the CaSR or PTHrP did
reduce baseline levels of CDK2 activity and prevented the usual increase in CDK2 activity
seen in response to increased extracellular calcium (Fig. 5G-J). These data suggest that
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activation of the CaSR promotes proliferation in breast cancer cells in vitro, in part, by
stimulating PTHrP production, which, in turn, decreases expression of the cell cycle inhibitor,
p27Kip1. In order to determine whether this was also true in PyMT tumors in vivo, we
examined p27Kip1 in extracts of control PyMT tumors and CaSR-CKO-PyMT tumors. As
shown in Fig. 5K&L, p27Kip1 levels were significantly higher in tumors lacking the CaSR,
correlating with reduced PTHrP expression and decreased proliferation. .
Knocking Down CaSR or PTHrP Expression Sensitizes Breast Cancer Cells to
Calcium-Induced Cell Death.
Although ablation of the CaSR did not affect apoptosis in PyMT tumors in vivo, both
the CaSR and PTHrP have been shown to regulate cell death in other settings (1,2,9).
Therefore, we exposed control and CaSR-CKO PyMT tumor cells to increased extracellular
calcium ex vivo. When exposed to 5mM calcium for 40 hours, tumor cells from CaSR-
CKO-PyMT mice underwent apoptosis whereas cells from control PyMT tumors did not (Fig
6A&B). However, control tumor cells underwent apoptosis when treated with NPS2143 at
5mM calcium (Fig. 6A&B). Likewise, control BT474 and MDA-MB231.1833 cells remained
viable when exposed to 5mM calcium for 40 hours (Fig. 6C&D and Fig. S3). In stark
contrast, when treated with NPS2143, 28±2.4% of control BT474 cells and 38±3.8% of
control MDA-MB231.1833 cells became TUNEL positive (Fig. 6C&D and Fig. S3). Similar
results were noted for both BT474 and MDA-MB231.1833 CaSRKD and PTHrPKD cells (Fig.
6C&D and Fig. S3).
To characterize the nature of the cell death caused by inhibiting the CaSR and
PTHrP, we assessed caspase 3 activation. Interestingly, we observed no cleaved caspase
3 in BT474 CaSRKD or BT474 PTHrPKD cells despite the fact that they were TUNEL
positive (Fig 6E). Therefore, we examined nuclear translocation of apoptosis inducing
factor (AIF). In response to DNA damage or cellular calcium stress, AIF is cleaved by
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calpain, released from mitochondria and enters the nucleus, where it can trigger caspase-
independent chromatin condensation and DNA fragmentation (30,31). Treatment of control
BT474 or MDA-MB-231.1833 cells with 5mM calcium resulted in a slight increase of AIF in
the nucleus (Figs. 6F-I and S3). While NPS2143 had little effect on AIF at low or
physiologic extracellular calcium, it markedly increased levels of nuclear AIF at 5mM calcium
(Fig. 6F&G and S3). Likewise, there was little difference in nuclear AIF levels between cell
lines when control, CaSRKD and PTHrPKD cells were cultured in 0.05 or 1 mM calcium.
However, when exposed to 5mM calcium, significantly more AIF accumulated in the nucleus
of the knockdown cell lines compared to controls (Fig. 6H&I and S3). We also observed a
marked increase in nuclear AIF expression in control PyMT tumor cells treated with
NPS2143 at 5mM calcium or in CaSR-CKO-PyMT cells exposed to 5mM calcium (Fig. S4).
To determine the functional consequences of nuclear AIF, we treated control, CaSRKD and
PTHrPKD cells with the AIF inhibitor, N-phenylmaleimide. As shown in Figs. 6J&K and
S3G&H, N-phenylmaleimide had no effect on apoptosis in control cells but significantly
reduced the percentage of TUNEL-positive CaSRKD and PTHrPKD cells after they were
treated with 5mM calcium. These data suggest that activation of the CaSR-PTHrP axis
allows breast cancer cells to resist apoptosis by inhibiting nuclear accumulation of AIF,
which otherwise, can be triggered by high extracellular calcium levels.
Nuclear PTHrP Mediates the Actions of the CaSR
To determine whether secreted PTHrP acts on the PTH1R to mediate the actions of
the CaSR, we treated BT474 and MDA-MB-231.1833 CaSRKD and PTHrPKD cells with
10nM or 100nM soluble PTHrP(1-86), which failed to stimulate BrdU incorporation at
baseline or after exposure to 5mM calcium (Fig 7A&B, S5). Similarly, adding PTHrP to the
media did not prevent apoptosis in CaSRKD or PTHrPKD cells treated with 5mM calcium
(Fig 7C, S5). Stable knockdown of PTH1R expression in BT474 cells reduced receptor
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expression by 89.3±4.50% (Fig 7D), which failed to alter proliferation or cell death in
PTH1RKD cells exposed to high extracellular calcium and/or gadolinium (Fig. 7E&F). The
inability of soluble PTHrP to rescue proliferation and/or cell death in CaSRKD or PTHrPKD
cells together with the failure of PTH1RKD cells to recapitulate the phenotype of CaSRKD
and PTHrPKD cells, suggest that neither secreted PTHrP nor the PTH1R mediate the
CaSR’s actions in breast cancer cells.
We next examined whether PTHrP might act in an intracrine fashion by utilizing
adenoviruses to express either wild-type PTHrP (WT-PTHrP) or mutant PTHrP with
disrupted nuclear localization sequences (ΔNLS-PTHrP) (28,29). As shown in Fig. 7G,
both wild-type and mutant PTHrP constructs included an HA-tag that allowed us to ensure
that we expressed equivalent amounts of WT-PTHrP or ΔNLS-PTHrP in BT474 CaSRKD
cells (Fig.7H). WT-PTHrP increased BrdU incorporation in CaSRKD cells at low calcium
concentrations but expression of ΔNLS-PTHrP did not. As noted previously, 5mM calcium
increased BrdU incorporation in control BT474 cells but this response was blunted in BT474
CaSRKD cells (Fig. 7I). Expression of WT-PTHrP in CaSRKD cells significantly increased
BrdU incorporation partly back to the level of control cells at 5mM calcium, whereas the
expression of ΔNLS-PTHrP failed to increase proliferation (Fig. 7I). As before, 5mM
calcium reduced p27Kip1 levels in control cells, but increased p27Kip1 levels in CaSRKD cells.
In CaSRKD cells transduced with WT-PTHrP, treatment with 5mM calcium decreased
p27Kip1 levels but in cells transduced with ΔNLS-PTHrP, 5mM calcium increased p27Kip1
levels (Figs. 7I&S5). These data suggest that PTHrP mediates the proliferative effects of
CaSR activation in these cells through an intracrine/nuclear pathway.
Examining cell death responses produced similar results. As noted in Figs. 7K&S5,
transduction with WT-PTHrP almost completely prevented apoptosis in BT474 CaSRKD
cells exposed to 5mM calcium for 40 hours. In contrast, ΔNLS-PTHrP had little effect on
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16
cell death. As before, increased cell death correlated with increased levels of nuclear AIF.
CaSRKD cells and CaSRKD cells transduced with ΔNLS-PTHrP demonstrated increased
nuclear AIF in response to 5mM calcium, whereas nuclear AIF levels in CaSRKD cells
transduced with WT-PTHrP were no different than in control BT474 cells (Figs. 7L&S5).
These data suggest that nuclear PTHrP inhibits nuclear accumulation of AIF and thus
prevents cell death in response to high extracellular calcium in vitro.
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Discussion
Despite the fact that 75% of breast cancers express the CaSR (32), relatively few
studies have examined its functional role in breast tumors. Whereas some studies have
reported that the CaSR increases proliferation in breast cancer cells (33-38), others have
suggested that it has either no effect on cell proliferation or actually suppresses proliferation
and increases apoptosis (39,40). In this study, we found that activation of the CaSR
upregulates PTHrP production, stimulates breast cancer cell proliferation and protects
against apoptosis. Furthermore, we find that the actions of CaSR signaling on cell
proliferation and apoptosis in vitro are mediated by PTHrP acting in the nucleus. Reducing
CaSR expression in transgenic mammary tumors and in breast cancer cell lines inhibited
PTHrP production and slowed tumor cell growth. In the aggregate, these data suggest that
activation of the CaSR promotes breast cancer growth, at least in part, through intracrine
actions of PTHrP.
Our data are consistent with prior reports that extracellular calcium stimulates
PTHrP expression by breast cancer cells in vitro, similar to findings in normal or malignant
keratinocytes (6,7,41,42). We also show that ablation of the CaSR in vivo inhibits PTHrP
production by mammary tumors from MMTV-PyMT mice. Furthermore, we documented
positive correlations between Casr and Pthlh mRNA levels in NMU-generated mammary
tumors in rats, and CASR and PTHLH mRNA and/or protein levels in combined cohorts of
over 800 individual breast cancers from human patients. These findings convincingly show
that CaSR activation stimulates PTHrP production by breast cancers at physiological
calcium concentrations. By contrast, CaSR activation reduces PTHrP production by normal
mammary epithelial cells in culture (4,6,43,44) and conditional ablation of the Casr gene in
the lactating mammary gland increases PTHrP production (4). Thus, malignant
transformation of breast epithelial cells rewires the relationship between the CaSR and
PTHrP such that extracellular calcium, which suppresses PTHrP production in normal cells
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becomes, instead, a stimulus for PTHrP production by malignant cells. Since PTHrP
activates peri-tumoral osteolysis, this change in PTHrP production may contribute to the
pathophysiology of bone metastases. In fact, Mihai and colleagues observed a positive
correlation between CaSR expression in primary breast cancers and the presence of bone
metastases (32).
Stimulation of PTHrP production by the CaSR contributes to the growth of breast
cancer cells in response to extracellular calcium in vitro. Knocking down either CaSR or
PTHrP expression reduced proliferation and increased cell death when BT474 and MDA-
MB-231.1833 cells were exposed to increasing concentrations of extracellular calcium.
Likewise, whereas targeting the CaSR in MMTV-PyMT mice with normal systemic calcium
concentrations did not change the incidence or latency of tumor development, it did
significantly reduce the rate of cell proliferation and slowed the overall growth of mammary
tumors. CaSR ablation also decreased PTHrP production by tumors and our results are
similar to data from Li et al demonstrating that targeting PTHrP expression inhibited the
growth of mammary tumors in MMTV-PyMT mice (15). Although there was no significant
change in apoptosis rates at physiological calcium concentrations in vivo, exposure of the
CaSR-CKO-PyMT tumor cells to 5mM calcium ex vivo led to significant cell death.
Likewise, using NPS2143 to pharmacologically inhibit CaSR function also killed control
MMTV-PyMT cells exposed to elevated extracellular calcium ex vivo. Therefore, inhibiting
the CaSR-PTHrP axis appears to convert the response of breast cancer cells to extracellular
calcium from supportive of tumor growth to detrimental to tumor growth.
Several observations suggest that the CaSR may activate an intracrine mode of
nuclear PTHrP action in breast cancer cells. First, adding PTHrP to the culture media could
not rescue the effects of knocking down CaSR or PTHrP expression. Second, knocking
down the PTH1R had no effect on the responses to extracellular calcium. Third, re-
expressing wild-type PTHrP rescued the effects of knocking down the CaSR but re-
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expressing mutant PTHrP, lacking the ability to translocate into the nucleus (ΔNLS-PTHrP),
could not. While we cannot exclude additional contributions of autocrine/paracrine PTHrP
signaling in vivo, our data in vitro are consistent with previous reports that nuclear PTHrP
stimulates proliferation and/or inhibits cell death in breast cancer cell lines, chondrocytes and
vascular smooth muscle cells (10,45). In particular, our data are similar to prior findings
that nuclear PTHrP acts to stimulate proliferation of vascular smooth muscle cells by
reducing p27kip1 levels and stimulating cdk2 activity (28,29). Ongoing experiments targeting
the PTH1R in MMTV-PyMT tumors will address the relative contribution of
autocrine/paracrine versus intracrine PTHrP signaling to tumor growth in vivo.
Activation of the CaSR also protected breast cancer cells from caspase-independent
cell death mediated by apoptosis-inducing factor (AIF). AIF is a mitochondrial protein
released into the cytoplasm in response to cellular calcium overload or DNA damage and
PARP-1 activation (30,31). From the cytoplasm, AIF can be transported into the nucleus
and activate chromatin condensation, DNA fragmentation and cell death. Knocking down
the CaSR or PTHrP augments the accumulation of nuclear AIF when cells are exposed to
high extracellular calcium. Furthermore, pharmacological inhibition of AIF blocks calcium-
induced apoptosis in CaSRKD or PTHrPKD cells. Therefore, activation of a CaSR-PTHrP
pathway protects these cells from apoptosis by either preventing the release of AIF from
mitochondria and/or by preventing its nuclear accumulation. AIF is an important mediator
of intracellular calcium-mediated “necroptotic” cell death triggered by excitotoxicity in
neurons, and PTHrP has previously been shown to protect hippocampal neurons from
excitotoxic cell death (46-48). The CaSR also modulates neuronal cell death in the setting
of reperfusion injury, which involves cellular calcium overload as well (46,49). While much
work will be needed to fully understand how the CaSR and PTHrP regulate AIF in breast
cancer, given the examples above, it is interesting to speculate whether interactions
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between the CaSR, PTHrP and AIF might have a wider role in regulating cell death
stimulated by intracellular calcium.
In summary, we find that the CaSR stimulates proliferative and survival responses in
breast cancer cells exposed to increasing doses of extracellular calcium. Both appear to be
mediated, in part, by the induction of PTHrP, which acts in the nucleus to stimulate
proliferation by suppressing p27kip1 levels and increasing CDK2 activity, and to prevent
apoptosis by inhibiting nuclear accumulation of AIF. As in cell lines in vitro, the CaSR
contributes to the secretion of PTHrP and the growth of mammary tumors in MMTV-PyMT
mice in vivo. Based on our findings in vitro and the fact that targeting either the CaSR or
PTHrP in MMTV-PyMT mice slows tumor growth (15), we suspect that PTHrP may mediate
the effects of the CaSR in tumor cells in vivo as well. However, the CaSR could also affect
tumor growth independent from changes in PTHrP. These findings may be particularly
relevant to bone metastases, where breast cancer cells are likely exposed to high levels of
extracellular calcium near active sites of osteoclast-mediated osteolysis. In that setting, the
CaSR may help breast cancer cells grow by triggering PTHrP expression, which might, in
turn, act through intracrine pathways to stimulate proliferation and prevent calcium-mediated
cell death. This hypothesis needs to be tested in vivo but, if this is the case, then
pharmacological targeting of the CaSR-PTHrP axis may offer new possibilities for breast
cancer treatment.
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Figure Legends
Figure 1. Casr (A) and Pthlh (B) mRNA levels in normal mammary glands vs. mammary
tumors in NMU-treated rats. C) Casr vs Pthlh mRNA levels in NMU-induced tumors. D)
CASR vs PTHLH mRNA levels in human breast cancers. E) Survival curves for patients
with CaSR AQUA scores above (triangles) and below (circles) the median cutoff. F)
Correlation between CaSR and PTHrP AQUA values in YTMA49 tumors.
Figure 2. CASR (A) and PTHLH (B) mRNA levels in breast cancer cell lines (relative to
baseline levels in MCF7 cells). C) PTHLH expression in response to extracellular calcium.
D) Response of PTHLH mRNA to increasing calcium or gadolinium (100μM) in BT474 cells.
E) cAMP response to increasing levels of calcium in BT474 cells. F) Response of PTHLH
mRNA to 5mM calcium ± H89 in BT474 cells. G) Response of PTHLH mRNA to increasing
calcium or gadolinium (100μM) in MDA-MB-231.1833 cells. H) cAMP response to
increasing levels of calcium in MDA-MB-231.1833 cells. I) Response of PTHLH mRNA to
5mM calcium ± H89 in MDA-MB-231.1833 cells. J) Response of PTHLH mRNA to calcium
± NPS2143 in BT474 cells. K) Response of PTHLH mRNA to calcium in control or
CaSRKD BT474 cells. L) Response of PTHLH mRNA to calcium ± NPS2143 in MDA-MB-
231.1833 cells. M) Response of PTHLH mRNA to calcium in control or CaSRKD MDA-MB-
231.1833 cells. Bars represent mean±SEM, n=3, * p<0.05, ** p<0.01, *** p<0.001.
Figure 3. Casr (A) and Pthlh (B) mRNA levels in MMTV-PyMT (control) or CaSR-CKO-
PyMT tumors. n=5. C) Tumor incidence and latency in MMTV-PyMT versus CaSR-CKO-
PyMT mice. n=10. D) Survival in MMTV-PyMT versus CaSR-CKO-PyMT mice. n=10. E)
Tumor growth (first tumor noted in each mouse) in MMTV-PyMT versus CaSR-CKO-PyMT
mice. n=10. F) Tumor weight (sum of all tumors from each mouse) in MMTV-PyMT versus
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CaSR-CKO-PyMT mice at sacrifice. n=8. G) Lung metastases in MMTV-PyMT versus
CaSR-CKO-PyMT mice. n=10. H) Typical staining for BrdU in MMTV-PyMT versus CaSR-
CKO-PyMT tumors. I) Quantitation of BrdU incorporation in MMTV-PyMT versus CaSR-
CKO-PyMT tumors. n=7. J) Proliferation in MMTV-PyMT versus CaSR-CKO-PyMT tumor
cells cultured ex vivo exposed to different calcium concentrations or gadolinium. N=3. Bars
represent mean±SEM, Curve in E shows mean±SD, * p<0.05, ** p<0.01, *** p<0.001.
Figure 4. CASR and PTHLH mRNA levels in control and CaSRKD or PTHrPKD BT474
(A&B) or MDA-MB-231.1833 (D&E) cells. C&F) BrdU incorporation in response to
increasing calcium or gadolinium (100μM) in control BT474 (C) and MDA-MB-231.1833 (F)
cells. G&J) BrdU incorporation in control BT474 (G) or MDA-MB-231.1833 (J) cells ±
treatment with NPS2143. H&K) BrdU incorporation in control vs CaSRKD BT474 (H) or
MDA-MB-231.1833 (K) cells. I&L) BrdU incorporation in control vs PTHrPKD BT474 (I) or
MDA-MB-231.1833 (L) cells. Bars represent mean±SEM, n=3. * p<0.05, ** p<0.01, ***
p<0.001.
Figure 5. A-C) Representative immunoblots and quantitation of p27kip1 levels in control
BT474 and MDA-MB-231.1833 cells ± NPS2143. D-F) Representative immunoblots and
quantitation of p27kip1 levels in control, CaSRKD and PTHrPKD BT474 and MDA-MB-
231.1833 cells. G) Quantitation of cdk2 activity as assessed by phosphorylation of histone
H1 in BT474 cells at low and high calcium ± NPS2143. H) Cdk2 activity in control versus
CaSRKD and PTHrPKD BT474 cells. I) Cdk2 activity in MDA-MB-231.1833 cells ±
NPS2143. J) Cdk2 activity in control versus CaSRKD and PTHrPKD MDA-MB-231.1833
cells. K) Representative immunoblots showing p27kip1 levels in MMTV-PyMT versus CaSR-
CKO-PyMT tumors. L) Quantitation of p27kip1 levels in MMTV-PyMT versus CaSR-CKO-
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PyMT tumors. Bars represent mean±SEM, For L, n=5, all others n=3 * p<0.05, ** p<0.01,
*** p<0.001.
Figure 6. A) Representative TUNEL staining and (B) quantitation of apoptosis in MMTV-
PyMT tumor cells ± NPS2143 and CaSR-CKO-PyMT tumor cells ex vivo. n=5. C)
Representative TUNEL staining of indicated BT474 cells at different calcium concentrations.
D) Quantitation of TUNEL-positive cells at high calcium. n=5. E) Representative immunoblot
of caspase 3. F) Representative immunoblot showing cytoplasmic and nuclear AIF in
BT474 cells treated with NPS2143. G) Quantitation of nuclear AIF accumulation in
response to NPS2143. n=3. H) Representative immunoblot showing cytoplasmic and
nuclear AIF in control and knockdown BT474 cells. I) Quantitation of change in nuclear AIF
accumulation in knockdown relative to control BT474 cells. n=3. J) Representative TUNEL
staining of BT474 cells ± treatment with N-phenylmaleimide. K) Quantitation of TUNEL
staining in control and knockdown BT474 cells at 5mM calcium ± N-phenylmaleimide. n=5.
Bars represent mean±SEM, * p<0.05, ** p<0.01, *** p<0.001.
Figure 7. BrdU incorporation in CaSRKD (A) or PTHrPKD (B) BT474 cells ± PTHrP n=3.
C) Quantitation of apoptosis in control or knockdown BT474 cells ± PTHrP n=5. D) Typical
immunoblot and quantitation of PTH1R protein expression in PTH1RKD BT474 cells. n=3. E)
BrdU incorporation in PTH1RKD BT474 cells. n=3. F) Representative TUNEL staining in
PTH1RKD BT474 cells. G) WT-PTHrP and ΔNLS-PTHrP constructs used in adenoviral
transduction experiments. H) Quantitation of immunoblotting or immunocytochemistry for
HA tag. n=3. I) BrdU incorporation in CaSRKD BT474 cells ± WT-PTHrP or ΔNLS-PTHrP.
n=3. J) p27kip1 expression in CaSRKD BT474 cells ± WT-PTHrP or ΔNLS-PTHrP. Results
expressed as change between 0.05 and 5mM calcium. n=3. K) Percent TUNEL-positive
CaSRKD BT474 cells ± WT-PTHrP or ΔNLS-PTHrP at 5mM calcium. n=5. L) Nuclear AIF
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accumulation in CaSRKD BT474 cells ± WT-PTHrP or ΔNLS-PTHrP at 5mM calcium.
Results expressed relative to control cells. n=3. Bars represent mean±SEM, * p<0.05, **
p<0.01, *** p<0.001.
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Published OnlineFirst July 22, 2016.Cancer Res Wonnam Kim, Farzin M Takyar, Karena Swan, et al. proteinstimulating intracrine actions of parathyroid hormone-related Calcium-sensing receptor (CaSR) promotes breast cancer by
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