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

on May 2, 2018. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 22, 2016; DOI: 10.1158/0008-5472.CAN-15-2614

http://cancerres.aacrjournals.org/

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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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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-




19

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




20

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.




21

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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




26

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-




27

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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