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Coexpression of Parathyroid Hormone Related Protein and Its Receptor in Early Breast Cancer Predicts Poor Patient Survival¹

Academic Department of Surgery, Division of Cancer Studies, Schools of Medicine [R. L., N. A., R. H., S. D., L. A., N. B.] and Biological Sciences [T. N., G. B.], University of Manchester, Manchester M23 9LT, United Kingdom

	ABSTRACT

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Purpose: Parathyroid hormone-related protein (PTHRP) is in part responsible for the clinical syndrome of hypercalcaemia of malignancy and has been implicated as an important factor in the development of bone metastases. The aim of this study was to determine the coexpression of PTHRP and its receptor in early breast cancer (EBC) and bone metastases (BM), and correlate these findings to clinical outcome.

Experimental Design: Samples of surgically excised EBC (n = 176) and BM (n = 43) were collected and stored in liquid nitrogen. PTHRP protein was determined using immunohistochemistry and receptor mRNA using in situ hybridization (n = 107) or semiquantitative reverse transcription-PCR (n = 69).

Results: PTHRP protein was expressed in 115 of 170 (68%) EBC compared with 100% of BM (P < 0.001), whereas its receptor mRNA was expressed in 88 of 176 (50%) EBC compared with 35 of 43 (81%) BM (P < 0.001). Coexpression of both PTHRP and its receptor was present in 62 EBC samples (37%) and in 35 BM samples (81%; P < 0.001). The PTHRP receptor correlated well with increasing patient age, but not with tumor size, grade, estrogen receptor, progesterone receptor, or lymph node status. Individually PTHRP and PTHRP receptor both correlated well with a reduced disease-free survival (P < 0.004) and receptor alone with reduced overall survival (P < 0.003). Coexpression of both PTHRP and receptor predicted the worst clinical outcome at 5 years, with a mortality rate of 20 of 62 (32%) compared with the ligand and receptor-negative group with 2 of 32 (6%; P < 0.004).

Conclusions: Overall these results show that the PTHRP receptor is expressed more frequently in BM than EBC, and is associated with poor clinical outcome and survival.

	INTRODUCTION

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Up to 70% of patients who die from breast cancer have bone metastases, of which 20% arise within 5 years of surgery (1) . The mechanisms responsible for the preferential spread of breast cancers to bone compared with other sites remain unclear, but accumulating evidence indicates that PTHRP³ plays a significant role (2 , 3) . PTHRP was first identified as the causative agent in the paraneoplastic syndrome hypercalcemia of malignancy (4) . The NH₂-terminal amino acid sequence of PTHRP is similar to that of PTH, and the two hormones share a common receptor known as the PTH/PTHRP receptor (5) , hereafter referred to as the PTHRP receptor. PTHRP is produced by normal mammary epithelial cells in response to stroma-derived growth factors (6 , 7) . In addition, overproduction of PTHRP occurs in 60% of primary breast cancers, most likely as a result of the up-regulation of stromal growth factor synthesis and/or their respective signaling mechanisms (8, 9, 10) .

PTHRP production by breast cancer cells may also facilitate the development of skeletal metastases, either by acting locally to enhance the survival of tumor cells in bone or by promoting the ability of tumor cells to invade bone as opposed to other sites. Immunohistochemical detection of PTHRP in primary breast cancers predicted for the subsequent development of bone metastases as opposed to metastases in other sites (2) . In addition, PTHRP mRNA has been shown to be expressed by 92% of skeletal metastases from breast cancer compared with 20% of metastases to nonskeletal sites (11) . Osteolysis may also occur in the absence of detectable serum PTHRP because of localized actions of PTHRP secreted by tumor cells within the bone matrix. Work by Guise et al. (12) showed that blockade of PTHRP secretion by breast tumor cells significantly reduced the formation of osteolytic lesions in a mouse model of BM. Although these studies indicate that positive PTHRP expression in primary breast cancers is linked to the subsequent development of bone metastases, the role played by the PTHRP receptor in this process has received less attention.

It is known from ISH studies that the expression of the receptor is up-regulated in primary breast cancers and that expression is localized predominantly in the epithelial cells (13, 14, 15) . Other studies have shown that the PTHRP receptor is expressed in breast cancer cell lines and that PTHRP acts as an autocrine growth factor in these lines (16 , 17) . Therefore, increased coexpression of PTHRP and its receptor in primary breast cancers is likely to be important in the development of skeletal metastases as this may increase tumor cell proliferation through autocrine activation in the metastatic site. The aim of this study, therefore, was to determine the extent of coexpression of PTHRP and the PTHRP receptor in primary breast cancers and in bone metastases, and relate these findings to follow-up data on patient survival.

	MATERIALS AND METHODS

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Collection of Samples.
After ethical approval, samples of malignant breast carcinoma tissue and bone metastases were collected from patients undergoing surgery at the South Manchester University Hospital. Patients presenting with an apparent EBC underwent conservative wide local excision with axillary clearance and radiotherapy, or mastectomy with axillary clearance. Routine adjuvant therapy and follow-up was performed in the surgical outpatient clinic in accordance with national guidelines.

After surgical excision, samples were removed from the tumor and snap frozen in liquid nitrogen. Bone metastases were collected during surgery on patients presenting with acute pathological fractured femurs because of breast cancer, who underwent open reduction and internal fixation of the fracture. Snap-frozen samples were used for RNA extraction, whereas the remainder of the specimens were fixed in 10% neutral buffered formalin and embedded in paraffin wax for IH and ISH. Specimens were assessed by consultant pathologists for tumor size, grade, and lymph node status using the system adopted by the National Health Service breast screening (18) . ER and PR status were determined as described previously (19) by immunohistochemistry using commercially available kits (Abbot Labs, Maidenhead, United Kingdom).

Assessment of PTHRP Receptor Expression by RT-PCR.
RNA was extracted from a 250-mg piece of each tissue sample using TRIzol (Life Technologies, Inc.) in accordance with the manufacturer’s protocol. RNA yield was quantitated by UV spectrophotometry and integrity verified on a 1% agarose gel. RNA was stored at -80°C in the presence of RNase inhibitor. PCR analysis of PTHRP receptor expression was carried out after global amplification of expressed genes by poly(A) PCR (20 , 21) . This procedure produces unbiased amplification of all of the expressed genes in a given sample and yields an effectively limitless supply of analyzable material. Direct comparison of mRNA expression levels measured using this technique gave identical results to those obtained using conventional RT-PCR or TaqMan real-time quantitative PCR.⁴ For gene-specific PCR, reactions were carried out in a total volume of 22 µl and contained 1 ng of poly(A) cDNA, 0.33 µM each oligonucleotide PCR primer, 0.5 units Taq polymerase (Roche Biochemicals), and 0.25 mM deoxynucleotide triphosphates in buffer supplied by the manufacturer. PCR primers were directed toward the mRNA sequence within 300 bp of the poly(A) addition site, as described previously (21) , and were as follows: PTHRP receptor: forward primer CCG CCT ACT GCC CAC TGC CAC CAC, reverse primer TCC ATC CAC TAT GTC AGC AGG TCC. Samples were also analyzed for expression of two housekeeping genes, ribosomal gene S29 [forward primer (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) : TTT TAC CTG GTT GCA CTG CT, reverse primer (169–188): ATG AAA CCG ATA TCC TTC GC] and GAPDH [forward primer (1088–1111): CCA GCA AGA GCA CAA GAG GAA GAG, reverse primer (1244–1267): AGC ACA GGG ATA CTT TAT TAG ATG], and for the luminal epithelial cell-specific protein cytokeratin 18 [forward primer (1207–1224): GAT GGC GAG GAC TTT AAT, reverse primer (1395–1412): GAC ATC CAA TGA ACT CTG]. The reactions were carried out in a programmable thermocycler (Techne PHC-3) using the following conditions: 1 cycle of 2 min at 94°C, then 25–35 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C, and finally 1 cycle of 5 min at 72°C.

To quantify gene expression, PCR reaction products were run on a 1% agarose gel alongside a series of standards containing known amounts of PCR product DNA. These were prepared by combining the gene-specific PCR product generated from several reactions and diluting these to produce a series containing a known amount of DNA molecules from 1.5 x 10¹⁰ to 1.5 x 10⁴/ml, as described (21) . Dilutions were made using a solution of sonicated carrier DNA.

After agarose gel electrophoresis in the presence of ethidium bromide and visualization of gels under UV light, gels were denatured in 1.5 M NaCl and 0.5M NaOH for 45 min, and then neutralized with 1 M NH₄OH before blotting on to Hybond-N membrane (Amersham) by capillary action. The membrane was then rinsed in sodium phosphate buffer and prehybridized for 2 h at 55°C with denatured salmon sperm DNA. Membranes were then hybridized at 55°C overnight with a ³²P-labeled oligonucleotide probe directed to a specific sequence within the PCR product. The probes used were as follows: PTHRP receptor: GAC GAT GGG TTC CTC AAC GGC TCC; S29: (72–91) CGG CCA GGG TTC TCG CTC TT; GAPDH: (1135–1158) CCC TGC CAC ACT CAG ACC CCC ACC; and CK-18: (1234–1251) GCC TTG GAC AGC AGC AAC. The membrane was then washed twice in (NaCl/sodium citrate solution), dried, and exposed to X-ray film for 1–3 days. In addition, the hybridized bands were quantified using a phosphorimager. The values for the DNA standards were used to construct a standard curve from which values for the samples were read. Samples falling outwith the linear range of the standard curve were resubjected to PCR using a modified number of amplification cycles. PTHRP receptor expression was normalized to that of total cell markers S29 and GAPDH, and to cytokeratin 18, an epithelial cell marker.

ISH.
A cDNA probe directed against the human PTHRP receptor (a kind gift from Dr. Ernestina Schipani, Harvard Medical School, Boston, MA) was used as described previously (13) . Briefly, sections of each paraffin-embedded sample were cut and mounted onto APES coated slides, and dried at 50°C overnight. After dewaxing and rehydration, the sections were treated with proteinase K and then postfixed in 0.4% (w/w) paraformaldehyde. After washing, the sections were incubated with PTHRP receptor probe (labeled using the megaprime DNA labeling system to specific activities of 10⁸ cpm/mg using [³⁵S]dCTP; Amersham) overnight at 37°C. After hybridization the tissue sections were washed with a series of high-stringency washes followed by dehydration in 95% ethanol and air drying. Autoradiography was performed with Ilford K5 emulsion melted at 40°C and diluted 1:1 with distilled water. The slides were exposed for approximately 7–14 days at 4°C before being developed and finally counterstained with Harris’s H&E. Controls consisted of randomized serial sections subjected to ISH after RNase pretreatment and the use of DNA radiolabelled to the same specific activity as the PTHRP receptor probe, in place of the specific probe. Grain densities over tumor cells and surrounding tissue were assessed using arbitrary scoring of nil (no staining), 1 (weak staining), 2 (moderate staining), and 3 (dense staining). The number of tumor cells showing expression of mRNA was scored in a similar manner with 0 = 0% cells positive, 1 = <20% cells positive, 2 = 20–80% cells positive, and 3 = >80% cells positive. Individual observer scores agreed in >90% of cases. Where individual scores disagreed, the sample was reassessed by a third observer. The agreed scores for staining density and proportion of cells stained were multiplied together to give a final score, which was representative of the total PTHRP receptor mRNA expressed in the area studied. Receptor expression was deemed positive if samples scored >3. To demonstrate a correlation between the RT-PCR and ISH receptor expression, a number of samples had expression determined both techniques.

PTHRP Ligand Expression by IH.
This was performed as described previously (22) . Briefly, sections (5 µM) of each paraffin-embedded sample were mounted on APES-coated slides, dewaxed, and rehydrated before background peroxidase activity and nonspecific binding sites were blocked with methanol-peroxide solution and swine serum, respectively. A polyclonal antibody (a kind gift from Dr. Wendy A. Ratcliffe, Wolfson Research Laboratories, Birmingham, United Kingdom), raised in rabbit to human PTHRP_1–34 was then applied for 1 h at room temperature at a concentration of 1 in 600 in 5% swine serum. Disclosure of immunoreactivity was performed using a biotinylated swine antirabbit antibody followed by horseradish peroxidase-labeled streptavidin. Peroxidase was localized with diaminobenzidine-hydrogen peroxides and nuclei counterstained with hematoxylin. Control slides for each run included omission of the primary antiserum for each section and in a known positive and negative section.

Statistical Methods.
Statistical analysis was performed using SPSS software (SPSS, Chicago, IL). Nonparametric tests were used throughout. Spearman’s rank correlation coefficient was calculated to examine the degree of correlation between groups of variables. Overall survival was evaluated by descriptive analysis using Kaplan-Meier estimates of the survival curves and the log rank test for comparisons of survival. The patient characteristics were compared using Pearson’s ² test. All of the significance tests were two-sided, using the conventional 5% significance level.

	RESULTS

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Frequency of PTHRP Receptor Expression in EBC and BM.
PTHRP receptor expression was determined in 177 EBC (70 by RT-PCR and 107 by ISH) and 43 BM (13 by RT-PCR and 30 by ISH). Using RT-PCR, PTHRP receptor expression was detectable in 36 of 70 (51%) EBC and 11 of 13 (84%) BM. Statistical analysis revealed that receptor expression was more frequent in bone metastases compared with primary breast cancers (P = 0.026, Pearson’s ² test). In addition Southern blotting of the RT-PCR reaction products demonstrated a significant increase in expression of the receptor in bone metastases compared with primary breast cancers.

PTHRP receptor expression was also measured by ISH in a series of archival paraffin-embedded tissue sections. Expression was detectable (score >3) in 52 of 107 (49%) EBC compared with 24 of 30 (80%) BM (P = 0.002, Pearson’s ² test). In addition the median intensity of the signal was higher in the BM samples compared with the EBC samples (median scores 6.0 and 4.0, respectively).

Because the patterns of PTHRP receptor expression measured by the two techniques gave very similar results, both in terms of frequency of expression and relative level of expression, we tested the validity of combining the data from the two series. Twenty randomly selected samples were analyzed by both RT-PCR and ISH. Comparison of PTHRP receptor expression measured by the two techniques revealed a significant correlation (R² = 0.688, P = 0.02; Fig. 1 ). Therefore, subsequent analysis was performed on combined data derived from both methods. Combining the data showed that the overall frequency of positive PTHRP receptor expression was 88 of 177 (50%) in EBC compared with 33 of 43 (81%) in BM (P < 0.001, Pearson’s ² test; Table 1 ).

View larger version (13K): [in this window] [in a new window]   Fig. 1. Relationship between PTH/PTHRP receptor expression measured by RT-PCR and ISH. Twenty randomly selected primary breast tumor samples were analyzed by RT-PCR and ISH as described in "Materials and Methods." Data were plotted as shown and the best straight line fit computed from Spearman’s correlation test. Because a number of samples showed identical scores for both RT-PCR and ISH not every data point can be seen.

Relationship of PTHRP Receptor Expression to Clinical and Prognostic Markers.
In the EBC samples PTHRP receptor expression correlated with positive coexpression of PTHRP ligand (Table 1) and with increasing patient age (Table 2) . No significant correlations were seen with other prognostic markers (tumor size, tumor grade, ER status, PR status, and lymph node status). Before testing for a relationship between PTHRP receptor status and patient survival we confirmed, using univariate and multivariate analysis, that the data on prognostic factors known to predict patient survival held for this series of patients. As shown in Table 3 , large tumor size, high tumor grade, positive lymph node status, negative ER status, and negative PR status all predicted for a reduction in disease-free and overall survival at 5 years of follow-up, as expected.

View larger version (14K): [in this window] [in a new window]   Fig. 2. PTHRP and PTH/PTHRP receptor expression in primary breast cancers and patient survival. Data on PTHRP ligand and PTH/PTHRP receptor expression frequency in primary breast cancers were divided into four groups: receptor (R) negative/ligand (L) negative (n = 33), R negative/L positive (n = 52), R positive/L negative (n = 24), and R positive/L positive (n = 70). Kaplan-Meier curves for overall survival were then plotted for each of these groups as a function of time from diagnosis. Survival in both receptor-positive groups was significantly worse than in receptor negative groups (P = 0.003; log rank test).

	DISCUSSION

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The present study suggests that it is the presence of the PTHRP receptor in the primary tumor, as opposed to PTHRP itself, that plays the dominant role in determining clinical outcome. We found that positive ligand expression in the absence of receptor expression predicted a far more favorable outcome than if the receptor was coexpressed. Furthermore, receptor expression in the absence of ligand by itself predicted a worse clinical outcome. The mechanisms underlying the poorer survival rates in patients with PTHRP receptor expression are unclear at this stage.

The increased frequency and level of receptor expression in BM compared with the primary tumors suggest that the receptor may play a role in the metastatic process. Furthermore, our data showing that patient survival rates are worse if the primary tumor expresses PTHRP ligand in addition to the receptor support the idea that autocrine or paracrine PTHRP action is an important factor in the poor prognosis associated with these tumors. There are a number of possible mechanisms that could drive a more aggressive behavior in tumors with PTHRP and PTHRP receptor expression. PTHRP may simply increase the proliferation of tumor cells by autocrine action, as suggested by studies in MCF-7 breast carcinoma cells (16 , 17) . On the other hand, PTHRP may promote invasiveness (25 , 26) or angiogenesis (27) . Finally, cells expressing the PTHRP receptor may preferentially survive and proliferate in the bone microenvironment because of endogenous PTHRP production by host cells such as osteoblasts (28) . Our data showing elevated PTHRP receptor expression in bone metastases compared with primary tumors support the idea that metastatic tumor cells with high PTHRP receptor expression selectively colonize the bone microenvironment. Alternatively, PTHRP receptor expression may be up-regulated as a result of bone-derived signals (29) .

In conclusion, coexpression of the PTHRP receptor and ligand is a predictor of poor clinical outcome in patients with EBC, and may play an important role in the development of BM. Treatments designed to inhibit PTHRP receptor function, such as a monoclonal antibodies or synthetic antagonists (30 , 31) , may therefore offer improved clinical outcome in patients with PTHRP receptor expression.

	ACKNOWLEDGMENTS

 
We thank Anthony Freemont, Judith Hoyland, and Ann Canfield (University of Manchester) for assistance received with various aspects of this work.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by grants from the Royal Society, the Royal College of Surgeons Edinburgh, and the South Manchester University Hospital Trust Gardner Endowment Fund.

² To whom requests for reprints should be addressed, at Academic Department of Surgery, Research and Education Building, University Hospital South Manchester, Southmoor Road, Manchester M23 9LT, United Kingdom. Phone: 161-291-5859; E-mail: bundredn{at}fs1.with.man.ac.uk

³ The abbreviations used are: PTHRP, parathyroid hormone-related protein; EBC, early breast cancer; BM, bone metastasis; IH, immunohistochemistry; ISH, in situ hybridization; RT-PCR, reverse transcription-PCR; ER, estrogen receptor; PR, progesterone receptor; PTH, parathyroid hormone; poly(A), polyadenylic acid; APES, 3'aminopropyltriethoxysilane.

⁴ T. Nolan and G. Brady, unpublished observations.

Received 12/ 6/01; revised 5/ 2/02; accepted 6/ 4/02.

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