The Role of Prolactin in Bone Metastasis and Breast Cancer Cell-Mediated Osteoclast Differentiation

Abstract

Background: Metastasis to the bone is a deleterious aspect of breast cancer and is a preferred site that results in bone loss. Hormones such as prolactin (PRL) have not yet been studied for their role in modulating the secondary tumor bone microenvironment.

Methods: We used quantitative immunohistochemistry with 134 samples of human primary breast cancer and 17 matched primary breast cancers and bone metastases. A Cox proportional hazards regression model was fitted to evaluate the associations between high prolactin receptor (PRLR) expression and time to bone metastasis, adjusting for estrogen receptor status, lymph node status, and chemotherapy status. We assessed osteoclast differentiation, osteoclast size, and measured pit formation in dentine slices. Statistical tests were two-sided.

Results: High PRLR expression in the primary breast tumor was associated with a shorter time to metastasis that includes bone (PRLRAQUA Max-per 100 unit hazard ratio = 1.04, 95% confidence interval = 1.00 to 1.07, P = .03). We observed the PRLR in rare samples of bone metastases and matched primary breast cancer. PRL treatment of breast cancer cells induced osteoclast differentiation and bone lysis via secreted factors and was abrogated by a PRLR antagonist (delta1-9-G129R-hPRL). We demonstrated that sonic hedgehog is a PRL-regulated cytokine in breast cancer cells and part of the mechanism that induces osteoclast differentiation.

Conclusions: Our evidence indicates that PRL-PRLR can escalate the impact of breast cancer on bone metastasis and that the presence of the PRLR in the tumor microenvironment of breast cancer bone metastasis has the potential to modulate the microenvironment to induce lytic osteoclast formation.

Figure 1.

Prolactin receptor levels were tested in primary breast tumors and in bone metastases. A ) Prolactin receptor (PRLR) protein level quantification in breast cancer patient samples by AQUA scoring. The left panel indicates the specificity of the PRLR antibody used by demonstrating staining on the breast cancer cell line MDA-MB-231, which has low PRLR, and the higher PRLR breast cancer cell line BT 483. The right panel demonstrates the presence of PRLR in normal breast ducts ( top row ) as well as in breast cancer with low ( middle row ) or high ( bottom row ) expression. B ) The area under the curve for the PRLR cutpoint is presented. C ) Kaplan Meier plot of time to metastasis is stratified by site of metastasis for the metastasis TMA series. The PRLR AQUA score was used to dichotomize patients into low PRLR or high PRLR groups to analyze time to any bone metastasis. Patients whose maximal tumor AQUA score for PRLR was above 4800 were considered to be high expressors. Survival rate was calculated using the Kaplan-Meier method, and a log-rank test was used to compare statistical differences in survival among subgroups. D ) Isolation and biomarker characterization of circulating tumor cells (CTCs) from a breast cancer patient. Representative images of immunofluorescently stained CTCs mixed with peripheral blood mononucleated cells (PBMCs) show CTCs identified as CK-positive, CD45-negative, and nuclear intact cells. PBMCs are CK-negative and CD45-positive cells. The arrowhead points to a PRLR-negative CTC, and the arrow points to a PRLR ^- positive CTC. The scale bars represent 20 µM. E ) PRLR immunohistochemistry on matched primary breast cancer and bone metastases samples. The left panel panels shows staining of the PRLR on MDA-MB-231 cells (HTB26) and BT483 cells (HTB121). AQUA = automated quantitative analysis; PCK = pan cytokeratin PRLR = prolactin receptor.

Figure 2.

The effect of prolactin (PRL)–PRL receptor (PRLR) signaling on breast cancer–mediated osteoclastogenesis. A ) Primary bone marrow–derived osteoclasts (BMDOs) were plated in medium with RANKL (50ng/mL), M-CSF (75ng/mL), and hPRL (negative control) or stimulated with conditioned medium (CM) from SKBR3 cells treated with vehicle (CM) or 150ng/mL hPRL (CM+PRL). Tartrate-resistant acid-phosphatase (TRAP)+/multinucleate cells (MNC) were quantified from three replicates. B ) The surface area of each mature primary osteoclast within 10 random fields per well of panel 2A was calculated and averaged. C) (a ) Undifferentiated BMDOs, (b) a large TRAP+/multinucleate cell indicated by a black arrow . D ) Effect of a PRLR antagonist on TRAP+/multinucleate cell induction. RAW264.7 pre-osteoclasts were also cultured in growth media ± ovine PRL as negative controls or cultured in conditioned media from SKBR3 breast cancer cells treated with vehicle (CM - PRL), 5 µg/mL PRLR-antagonist ∆1-9-G129R-hPRL (CM + antag), 5 µg/mL ovine PRL (SKBR3 CM + PRL), or a combination. TRAP+/multinucleate cells were quantified for each condition. One of three experimental replicates with six internal replicates per experiment. E ) The effect of PRL-treated breast cancer CM on osteoclastogenesis. RAW264.7 pre-osteoclasts were cultured in conditioned media from SKBR3 breast cancer cells stimulated with increasing concentrations of ovine PRL or human recombinant PRL, and TRAP+/multinucleate cells were quantified. Sum of three experimental replicates. F ) Quantification of TRAP+/multinucleate cells treated with conditioned media from BT-483 breast cancer cells or MDA-MB-435S cells. Breast cancer cells were stimulated with vehicle or with 5 µg/mL oPRL for five days for preparation of conditioned media. RAW264.7 pre-osteoclasts were cultured in 20% conditioned media or growth media ± oPRL. TRAP-positive, multinucleate cells were quantified for each condition. One of three experimental replicates with six internal replicates per experiment. G ) Quantification of TRAP+/multinucleate cells after heat inactivation of SKBR3 breast cancer cell conditioned media. Ovine PRL-stimulated and -unstimulated SKBR3-conditioned media was heat-inactivated by incubation at 95°C for 10 minutes before addition to RAW264.7 cultures. One of three experimental replicates with four internal replicates per experiment. Statistical significance (* P < .05, † P < .01) was tested with the paired Student’s t test as indicated. Bars indicate standard deviation. CM = conditioned medium; MNC = multinucleate cell; PRL = prolactin; TRAP = tartrate-resistant acid-phosphatase.

Figure 3.

Assessment of dentine resorption as a measure of mature osteoclasts. RAW264.7 pre-osteoclasts were cultured on dentine discs in (A) growth medium or in (B) conditioned media from SKBR3 breast cancer cells treated with vehicle (CM-PRL) or (C) 5 µg/mL oPRL (CM +PRL). Scale bar = 100 µm. D ) Nuclei were alternately stained with DAPI and quantified to show equal loading. Five random fields of cells were counted in each sample. E ) Surface area of each pit was quantified using ImageJ software. Bars indicate standard deviation of three experimental replicates. Statistical significance ( P < .05) was tested with the paired Student’s t test. CM = conditioned media; PRL = prolactin.

Figure 4.

Detection of sonic hedgehog (SHH), osteoprotegerin (OPG), and receptor activator of nuclear factor-Κappa B ligand (RANKL) secretion from breast cancer cells. A ) Direct coculture of MCF-7 and SKBR3 breast cancer cells with RAW264.7 pre-osteoclasts -/+ oPRL induces formation of tartrate-resistant acid-phosphatase (TRAP)–positive multinucleate cells. B ) Conditioned media (CM) from MCF-7 and SKBR3 breast cancer cells -/+ hPRL with RAW264.7 pre-osteoclasts induces formation of TRAP-positive multinucleate cells. C ) RANKL and OPG were detected using Luminex technology from unconditioned medium (media blank) or CM from three experimental samples of SKBR3 cells (2% FBS) that were untreated or treated with 5 ug/mL of oPRL or ∆1-9-G129R-hPRL. Three experiments pooled (A-C) , showing interexperimental variation. SHH is detected by enzyme-linked immunosorbent assay from (D) two samples of SKBR3 cells treated with a hPRL dose response, (E) two samples of T47D cells, or (F) three independent samples of SKBR3 or MCF7 cells treated with vehicle or 25ng/mL hPRL. Concentration of SHH was calculated using a linear regression standard curve. Error bars represent standard deviation. Statistical significance ( P < .05) with the paired Student’s t test as indicated. CM = conditioned medium; MNC = multinucleate cell; OPG = osteoprotegerin; PRL = prolactin; RANKL = receptor activator of nuclear factor-Κappa B ligand; SHH = sonic hedgehog; TRAP = tartrate-resistant acid-phosphatase.

Figure 5.

The role of the hedgehog (HH) pathway in osteoclastogenesis. RAW264.7 cells were cultured in the presence of 5ng/mL M-CSF and 7.5ng/mL RANKL in control media -/+ hPRL as negative controls or in conditioned media from SKBR3 cells treated with vehicle (-CM), 100 ng/mL hPRL (+CM), 10 µM cyclopamine, or a combination thereof. A ) Alamar Blue cell survival assay ( upper ) and quantification of tartrate-resistant acid-phosphatase (TRAP)+/multinucleate cells (MNC) in absence of CM ( lower ) (six replicates of negative controls). Student’s t test. B ) Quantification of TRAP+/MNC from three replicates. C ) Surface area of TRAP+ multinucleate cells. All osteoclasts in each well ( above ) were measured and divided into size ranges, and the percentage within that group was calculated and multiplied by the ratio of the group relative to control medium. Error bars represent standard deviation. A two-way analysis of variance was performed, followed by Tukey post-testing, and lines between pairs or groups of pairs indicate a P value of less than .05 for each paired bar between different treatments. CM = conditioned medium; MNC = multinucleate cell; PRL = prolactin; TRAP = tartrate-resistant acid-phosphatase.