Parathyroid Hormone–Related Protein Protects Against Mammary Tumor Emergence and Is Associated with Monocyte Infiltration in Ductal Carcinoma in Situ

Published OnlineFirst September 1, 2009; DOI: 10.1158/0008-5472.CAN-09-0194

Tumor Microenvironment

Parathyroid Hormone–Related Protein Protects against Mammary Tumor Emergence and Is Associated with Monocyte Infiltration in Ductal Carcinoma In situ

Nicholas I. Fleming,1,4 Melanie K. Trivett,2 Joshy George,1 John L. Slavin,3 William K. Murray,2 Jane M. Moseley,4 Robin L. Anderson,1 and David M. Thomas1

1Research Division and 2Department of Anatomical Pathology, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia and Departments of 3Pathology and 4Medicine, St. Vincent’s Hospital, Fitzroy, Victoria, Australia

Abstract presence of bone metastases (8–11), to increase the growth of Parathyroid hormone–related protein (PTHrP) is required for tumor cells in bone (12–14), and to correlate with reduced survival mammary gland development and promotes the growth of of breast cancer patients (11, 15). However, other studies have breast cancer metastases within bone. However, there are reported that PTHrP expression in breast cancers is associated conflicting reports of the prognostic significance of its with improved survival (2, 16) and is inversely associated with expression in primary breast cancers. To study the role of lymph node metastasis (17). To establish the significance of PTHrP PTHrP in early breast cancer, the effect of conditional deletion expression in primary mammary tumors, Pthrp was conditionally of PTHrP was examined in the context of neu-induced deleted in the MMTV-neu mammary tumorigenesis model, which mammary tumorigenesis. Loss of PTHrP resulted in a higher facilitates the delineation of primary and metastatic tumor biology tumor incidence. Transcriptional profiling of the tumors (18–21). Deletion of Pthrp led to an increase in the incidence of mammary tumors but had a limited effect on lung metastasis. revealed that PTHrP influenced genes relevant to heterotypic cell signaling, including regulators of monocyte recruitment. Gene expression profiling on the tumors indicated that PTHrP Immunohistochemical analysis of human breast cancers influences the signaling of tumor cells to cells of the immune revealed that PTHrP expression was associated with both system. Immunohistochemical analysis of PTHrP in human ductal HER-2/neu expression and macrophage infiltration in pre- carcinoma in situ (DCIS) revealed that PTHrP expression is invasive ductal carcinoma in situ. The gene expression associated with monocyte infiltration. The gene expression signature associated with loss of PTHrP expression in vivo signature associated with lack of PTHrP expression in the mouse correlated with poorer outcome in human breast cancer. mammary tumors correlated with reduced survival in human Together, these data indicate that loss of PTHrP accelerates breast cancer. mammary tumorigenesis possibly by a non–cell-autonomous tumor suppressor pathway. [Cancer Res 2009;69(18):7473–9] Materials and Methods Generation and monitoring of mice. MMTV-neu (strain D) mice (18) Introduction and MMTV-cre mice (22) were obtained from The Jackson Laboratory. They were crossed to generate MMTV-neu/MMTV-cre mice and further crossed Parathyroid hormone–related protein (PTHrP) is the principal to Pthrpflox/flox mice obtained from John Wysolmerski (23). The MMTV-neu mediator of humoral hypercalcemia of malignancy (1) and is and MMTV-cre alleles were genotyped by PCR using published primers expressed in many tumor types, including 60% of breast cancers (22, 24). The Pthrpflox allele was genotyped by PCR with the primers (2). It has homology to parathyroid hormone with which it shares a 5¶-AGGCCACACCAGTCCTAGTC-3¶ and 5¶-ATGTGGGAATGGCTCAGAAG-3¶ common receptor, parathyroid hormone receptor 1 (PTHR1). Mice that were designed to discriminate between heterozygote and homozygote null for Pthrp, Pth,orPthr1 have related phenotypes, involving animals. The breeding strategy ensured that all mice positive for MMTV- defective skeletal development, cardiac abnormalities, and defects neu or MMTV-cre only carried a single allele of those transgenes. Given that the genetic backgrounds of the three starting strains were varied, including in ectodermal appendage development, including that of the teeth flox/flox and the mammary glands (3–5). In bone, parathyroid hormone/ FVB/N, BALB/c, and, in the case of the Pthrp mice, the outbred strain CD1, the deleting and control genotypes were generated as siblings and PTHrP signaling controls bone density and calcium storage by retained from a single generation. From age 12 weeks, each animal influencing the recruitment of circulating monocyte precursors to underwent three rounds of pregnancy and pup rearing and were monitored form osteoclasts (1). Although the function of PTHrP in mammary for the emergence mammary tumors thereafter. tissues is unclear, restricted nonoverlapping expression of PTHrP Analysis of tumor emergence. The ages of the animals was recorded and Pthr1 in relevant tissues indicates a role in epithelial-stromal when a mammary tumor was first detected by palpation. A right-censored interactions (6, 7). Kaplan-Meier plot of tumor emergence, according to Pthrp status, was Consistent with its role in bone development, PTHrP expression generated using the software package Minitab (version 14), and the in primary breast cancers has been reported to correlate with the difference in emergence was tested using the log-rank test. Animals that had tumors in more than one gland at time of sacrifice were counted twice as a means of combining the tumor multiplicity data with the latency data. Note: Supplementary data for this article are available at Cancer Research Online Processing and histology of tumors and lung tissues. Once the mice (http://cancerres.aacrjournals.org/). developed tumors of 10 mm in diameter, they were culled and their Requests for reprints: David M. Thomas, Research Division, Peter MacCallum tumor(s) and lungs were harvested. Each tumor was cleaved into segments Cancer Centre, Locked Bag 1, A’Beckett Street, Melbourne, Victoria 8006, Australia. that were either fresh-frozen in OCT embedding compound (Tissue-Tek) or Phone: 61-396561238; Fax: 61-395946125; E-mail: [email protected]. I2009 American Association for Cancer Research. fixed in 10% formalin overnight and paraffin-embedded. Lung pairs were doi:10.1158/0008-5472.CAN-09-0194 formalin fixed and paraffin-embedded. www.aacrjournals.org 7473 Cancer Res 2009; 69: (18). September 15, 2009

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Analysis of lung metastasis. Sections (5 Am thick) were taken at 450 Am control mice carrying the cre allele but heterozygous for PTHrPflox, intervals through the paraffin-embedded lungs. They were stained with and 31 control mice negative for cre but homozygous for the floxed H&E and metastases were counted under light microscopy. Metastases that allele were generated for testing the effect of PTHrP deletion on clearly extended from one section to the next were only counted once. Each tumor development. The use of the MMTV-LTR promoter to drive pair of lungs was represented by eight tissue sections. expression of both neu and cre ensured that cells prompted to Whole-mount analysis of mammary glands. Whole mammary glands were harvested and transferred to glass slides. They were fixed overnight in establish tumors would also be those subject to deletion of the Carnoy’s fixative, rehydrated through an alcohol series, and stained Pthrp gene. The use of two control groups allowed us to test overnight in 0.2% carmine red stain. They were dehydrated, treated with whether cre expression itself influenced the data. Germ-line xylene until the fat tissue was clear, and then photographed. deletion of Pthrp results in failure of mammary gland development Computed tomography analysis. Before tumor harvest, culled animals (7); therefore, we employed a MMTV-cre allele from which cre is were scanned with a Discovery LS CT scanner (GE Medical Systems) and expressed progressively (22), thus avoiding significant deletion bones were examined for metastasis. during early development. We examined normal mammary gland Human tissues. A tissue microarray representing 192 invasive breast development of our mice at 6 weeks, 12 weeks, during lactation, cancers and 20 DCIS was obtained from the Cooperative Breast Cancer and 10 days post-involution using mammary gland whole mounts Tissue Resource (25) and an additional 25 DCIS samples were obtained in and histology of mammary glands from additional animals standard paraffin blocks from the Department of Anatomical Pathology, Peter MacCallum Cancer Centre. (Supplementary Fig. S1A). No alteration of mammary gland Immunohistochemistry. PTHrP levels were assessed by immunohisto- development was observed in mice of the deleting genotype. chemistry using a goat polyclonal antibody as described previously (2). The Dysfunction of the mammary gland could result in pups receiving staining was given a score of 0, 1, 2, or 3based on staining intensity. HER-2/ inadequate milk for growth; recently, it was reported that PTHrP neu staining was done and scored according to standard clinical diagnostic expressed in the lactating gland has an endocrine function, practice employing the rabbit polyclonal antibody A0485 (DAKO). mobilizing skeletal calcium (29, 30). We monitored the body Immunohistochemistry for CD68 was done using the PG-M1 antibody weights of pups suckled on the animals as a measure of mammary (DAKO) at a 1:200 dilution and scored according to the relative level of gland function; again, no difference was detected according to dam positive cell (monocyte) infiltration within the observed range. On the genotype (Supplementary Fig. S1B). advice of W.K.M., an experienced pathologist, samples were given a score of The mice were monitored for mammary tumor emergence by 0, 1, 2, or 3in increasing order of monocyte infiltration. Immunohisto- chemistry on mouse tissues for F4/80 and von Willebrand factor was done regular palpation and tumors were harvested for analysis once they reached 10 mm in diameter. Tumors developed with a similar on sections from frozen samples and pretreated with 3% H2O2 for 10 min using the antibodies BM8 (eBioscience; 1:50) and A0082 (DAKO; 1:3,000), latency and penetrance to those observed in a previous MMTV-neu respectively. studies involving comparable genetic backgrounds (31, 32). The Real-time reverse transcription-PCR. To confirm the deletion of expected ablation of PTHrP was confirmed in emerging tumors by PTHrP, frozen mouse mammary tumor tissue was homogenized, RNA was real-time reverse transcription-PCR. Tumors arising in animals of extracted with Trizol (Invitrogen), and real-time reverse transcription-PCR the deleting genotype had little or no expression, whereas those was done using primers that detect the floxed exon of PTHrP (which is with the gene intact had variable expression (P = 0.043, Student’s normally included in all transcript variants; refs. 23, 26). Real-time reverse t test; Fig. 1A). Within the two control groups, PTHrP expression transcription-PCR for the designated housekeeping gene h -globin was 2 did not correlate with the number of remaining intact alleles done, also using previously described primers (27). Global gene expression analysis of mouse tumors. Frozen tumor tissue (Fig. 1A). At the completion of the study, nearly twice as many was homogenized, and RNA was isolated with Trizol and further purified with animals carrying the deleting genotype (58%) had succumbed to 2 a RNeasy column (Qiagen). Labeled RNA was generated with the One-Cycle disease compared with littermate controls (30%; P = 0.01, m test; kit (Affymetrix) and hybridized to Mouse Genome 430v2 GeneChips Fig. 1B). This difference was not due to cre expression, as there was (Affymetrix). The GeneChips were scanned and the resulting expression no difference in tumor emergence between animals expressing cre data were normalized with the Robust Multi-Array algorithm (Genespring). but retaining a functional Pthrp allele, and the cre-negative control Differentially expressed genes were identified using the Student’s t test group alone (data not shown). These data therefore indicate that (class prediction/BRB Array Tools). Gene ontology (GO) analysis was done PTHrP expression was protective against the development of using the DAVID Gene Functional Annotation Clustering tool (28). mammary tumors. In addition, mice in which mammary gland Analysis of human breast cancer global gene expression data set. To Pthrp was deleted developed a slightly greater number of tumors analyze human breast cancer gene expression data using the differentially expressed genes from the murine tumors, human homologues were per mouse (1.47 versus 1.35). Kaplan-Meier survival analysis identified using the Ensembl BioMart toolset and genes for which a incorporating both tumor incidence and the number of tumors homologue could not be unambiguously identified were omitted (30%). indicated that the loss of PTHrP was associated with an increased Patients were ranked according to directional differential expression of the susceptibility to neu-driven mammary tumors (P = 0.023, log-rank gene set within their tumor gene expression data. They were divided into test; Fig. 1B). Interestingly, the difference in tumor emergence two equal-sized groups of those with gene expression across the set most between the genotypes was greater in the second half the animal’s similar to the PTHrP-deleted mouse tumors and those for which gene lives than in the first half (Fig. 1B). This may indicate that PTHrP expression was most dissimilar. Kaplan-Meier survival analysis was done expression is more relevant to tumors arising in older animals. and the difference between the genotypes was tested with the log-rank test. The growth rate of tumors was monitored once tumors were Statistical significance was validated by a Monte Carlo simulation method, detected (typically at 5-6 mm in diameter) until they reached where briefly differential expression of the detected gene set was compared with 10,000 randomly selected gene sets of the same size. 10 mm in diameter, but no significant difference corresponding to genotype was detected (Fig. 1C). Their histologic appearance was similar to previously reported MMTV-neu tumors (ref. 33; Results Supplementary Fig. S2A) with no differences in nuclear morphol- Effect of conditional deletion of Pthrp in neu-dependent ogy, tubule formation, extent of necrosis, or number of mitotic tumorigenesis. Thirty-three mice of the deleting genotype, 26 bodies (data not shown). Macrophage infiltration and tumor

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PTHrP, Monocytes, and Early Breast Cancer

MMTV-neu tumors are known to undergo metastasis to the lungs (18); therefore, metastases were counted in serial H&E- stained sections. There was a higher number of lung metastases in Pthrp-deleted animals, but this difference was not statistically significant (Table 1). Interestingly, the lung lesions varied widely in their number and size but were relatively uniform within any given animal (Table 1). Despite this, we were unable to show a significant relationship of their size or morphology with genotype. PTHrP has been shown to facilitate bone metastasis (8–11). Fine-slice computed tomography scans of 9 animals (3deleted, 3heterozy- gous for PTHrP, and 3homozygous) failed to reveal evidence of skeletal metastases (data not shown). This indicated, as expected from previous studies using the MMTV-neu model, that the experiment would be unlikely to provide meaningful information regarding bone metastasis, so further analyses were not pursued. Of relevance, none of the animals exhibited signs of impaired mobility, which is also an indication of bone metastasis. Draining lymph nodes of mammary glands harboring tumors were invariably disrupted by expansion of the tumor mass; therefore, it was not possible to examine these for colonization by cancer cells. In summary, the in vivo data indicate that PTHrP expression was protective against early stages of tumorigenesis but did not dramatically alter tumor biology or the propensity to metastasize to lung once a tumor was established.

Table 1. Analysis of lung metastasis on Pthrp deletion in MMTV-neu tumors

Genotype Pthrp status Size Count

À/À Deleted Large 1 Large 2 Large 2 Large 3 Large 4 Small 4 Micro 2 Micro 2 Micro 7 Mixed 5 Mixed 10 Mixed 17

Mean = 4.916

+/À, +/+ Present Large 1 Large 1 Large 1 Small 3 Micro 2 Figure 1. Validation and analysis of tumor emergence following conditional PTHrP deletion in the mammary gland using the MMTV-neu in vivo model. A, Micro 2 real-time reverse transcription-PCR analysis of PTHrP expression in mammary Micro 3 tumors from sample groups of the À/À animals (n = 11), +/À animals (n = 6), Micro 7 +/+ animals (n = 9), and the two control groups combined (n = 15). *, P = 0.043. Micro 7 Bars, SE. B, m2 and Kaplan-Meier analysis of tumor emergence (À/À, n = 33; +/À and +/+ combined, n = 57). Number above line, number of mice developing Mean = 3 tumors; number below line, number of mice not developing tumors. For Kaplan-Meier analysis, tumor emergence was weighted by tumor multiplicity P = 0.231 and tested by the log-rank test. C, tumor growth from detection to 10 mm in diameter. Bars, SE. B2M, h2-globin. NOTE: P value was calculated by Student’s t test on lesion count. vascularity were measured in a subset of the tumors by Large lesions were visible to the naked eye, small lesions were f50 immunohistochemistry using F4/80 and von Willebrand factor cells wide in section, and microscopic lesions were 5 to 6 cells wide in antibodies, respectively, but again no differences were detected section. (Supplementary Fig. S2B and C). www.aacrjournals.org 7475 Cancer Res 2009; 69: (18). September 15, 2009

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Table 2. GO analysis of differentially expressed genes on PTHrP ablation in MMTV-neu tumors

Rank Term Count Mean P Genes

1 Chemotaxis 23<0.001 Ccl2, Fcgr3, Itgb2, Ccr1, Itga1, Opn, Ccl12, Cxcl4, Ccl8, Cxcl12, Ear2, Ccl11, Ccl7, Ccl6, Ccl21b, Ccl9, Lsp1, Ccr2, Cxcl13, Slit2, Dock2 2 Chemokine activity 14 <0.001 Cxcl14, Ccl2, Cxcl9, Ccl12, Cxcl4, Ccl8, Cxcl12, Ccl11, Ccl6, Ccl7, Ccl21b, Ccl9, Cxcl13, Ccl21a 3Regulation of apoptosis 36 <0.001 Cidec, Fcgr3, Naip4, Mapk8ip1, Hells, Rarb, Pmaip1, Clec2d, Bcl2l11, Apip, Igf1, Thy1, 9030416C10Rik, Ifi204, Igh-4, Ptprc, Stk17b, Fcgr1, Gdf5, Btg2, Myc, Fgfr3, Opn, 4933405K19Rik, Zbtb16, Snai2, Aldh1a1, Plekhf1, S100b, Casp7, Traf4, Angptl4, Cebpb, Ripk3 4 Regulation of cell migration 10 0.002 Egfl7, Thy1, Trip6, Pecam1, Tek, Lama2, Cxcl12, Ccr2, Map3k1, Pdpn 5 Ossification 12 0.003 Ahsg, Dmp1, Pthrp, Ptger4, Smo, Opn, Ifi204, Mgp, Ear1, Ear2, P2rx7, Foxc1 6 Cell growth 14 0.005 Socs2, 4432404P07Rik, Emp3, Igfbp4, Esr1, Fstl2, Ing3, Igfbp2, Nov, Chpt1, Ndn, Nedd9, Igfbp6, Cyr61 7 Acylglycerol metabolism 5 0.014 Pck1, Mttp, Hpic53, Lipe, Cav1 8 Regulation of ossification 5 0.028 Ahsg, Ptger4, Opn, Mgp, P2rx7

NOTE: All statistically significant functional groups included. Ranked by mean P value of GO terms. Count, number of genes detected.

Global gene expression analysis of MMTV-neu following PTHrP expression, HER-2/neu expression, and immune cell conditional deletion of Pthrp. To identify genes influenced by recruitment in human disease. Immunohistochemistry was done PTHrP ablation, tumors from 6 mice of the deleted genotype and 4 for PTHrP, HER-2/neu, and the human monocyte marker CD68 in wild-type tumors, which had exhibited robust PTHrP expression, 192 invasive breast cancers and 45 DCIS cases. Staining for PTHrP were subjected to global gene expression analysis. A total of 1,283 was scored in a blinded fashion by an experienced pathologist genes (of 22,000 represented) were differentially expressed accord- (J.L.S.) and was predominantly cytoplasmic, consistent with ing to PTHrP status, when defined as having at least a 1.5-fold previously published studies (Fig. 2A). Sixty-six percent of cases difference in their geometric mean expression and statistical were positive for PTHrP when defined by having a score of z1. significance at the 5% level (Student’s t test). On GO analysis, eight HER-2/neu immunostaining was scored according to routine significant functional GO groups were identified (Table 2). clinical practice and 16% of samples were positive. CD68 Remarkably, these included the groups of ossification and bone immunohistochemistry was scored by an experienced pathologist remodeling, which relate to the known role of PTHrP in bone (W.K.M.) and clearly identified solitary mononuclear cells, as development. Three of the top four groups are related to reported previously (36), in 72% of cases. It was surprising that 28% chemokine signaling and chemotaxis, including multiple genes of the samples lacked intratumoral infiltration, but these data were involved in immune cell and endothelial cell recruitment. The third validated in the majority of cases by the detection of macrophages ranking group is related to apoptosis and the remaining groups are in stromal tissues on the same tissue sections (Supplementary related to cell growth and to lipid metabolism. The highest ranking Fig. S3A and B). PTHrP positivity, HER-2/neu positivity, and CD68 gene was insulin growth factor binding protein 4 (Igfbp4), which is infiltration were more frequent in DCIS than in invasive carcinoma one of a handful of genes previously identified as a target of PTHrP (Fig. 2B). The intensity of PTHrP staining positively correlated with signaling by use of expression profiling (refs. 34, 35; Supplementary HER-2/neu positivity (Table 3) and with the extent of monocyte Table S1). The gene osteopontin (Opn) was included in both the infiltration in DCIS (P = 0.032; Fig. 2C), but no equivalent bone-related groups and the chemokine signaling groups and relationships were detected in the invasive carcinomas (P = differential expression of PTHrP itself was detected, which further 0.576; Fig. 2C). Interestingly, an association was not detected validated the study (Table 2; Supplementary Table S1). These data between HER-2/neu positivity and monocyte infiltration in either therefore indicate that expression of PTHrP in MMTV-neu tumors DCIS or the invasive set (Supplementary Fig. S3C), despite this is concerned primarily with the management of heterotypic cell being reported previously (36). These results therefore indicate that relationships and suggest that a functional overlap exists for PTHrP PTHrP expression is functionally relevant to both HER-2/neu signaling between mammary tumors and bone. expression and monocytic infiltration in the early stages of breast PTHrP, HER-2/neu, and CD68+ cell infiltration in human cancer development and that these relationships may be selected DCIS and invasive carcinomas. Given that PTHrP signaling in against as lesions progress to invasive disease. MMTV-neu murine mammary tumors was associated positively Stratification of human breast cancer survival by PTHrP with chemotactic signaling, we examined the relationships of influenced gene expression. The generation of global gene

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PTHrP, Monocytes, and Early Breast Cancer

Figure 2. Immunohistochemical analysis of PTHrP, HER-2/neu, and CD68 expression in human DCIS and invasive breast cancers. A, example of PTHrP expression in a DCIS sample, with nonimmune serum (N.S.) control. Magnification, Â60. B, PTHrP, HER-2/neu, and CD68 positivity in DCIS and invasive carcinomas. C, monocyte infiltration (CD68) versus PTHrP staining in DCIS and invasive carcinomas. expression data on mammary tumors ablated for PTHrP provided and for which unambiguous human homologues could be an opportunity to determine whether PTHrP-influenced gene identified (903genes, 70%). We then tested whether patient expression is relevant to outcomes in human breast cancer. Several survival differed with respect to this stratification by Kaplan-Meier investigators have published large global gene expression data sets analysis and the log-rank test. Patients whose tumors expressed the for human breast cancer. Among the largest of these is that by set of genes at levels most similar to that in mammary tumors Miller and colleagues (37) who analyzed breast cancers from 251 retaining Pthrp had a longer survival (P = 0.013, log-rank test; patients and notably collated 12-year survival data. We stratified Fig. 3A). What is more, this difference is similar in magnitude and the patients in their study for directional differential expression of direction to that reported by Henderson and colleagues (16) for the genes identified as differentially expressed in our animal model PTHrP detected by immunohistochemistry. These data are therefore consistent with the hypothesis that PTHrP signaling in human breast tumors is associated with a better outcome. Table 3. m2 analysis of PTHrP expression compared with Interestingly, when the human tumors were ranked as either high HER-2/neu positivity or low expressors for PTHrP alone (probe ID: 211756_at), its expression correlated with the in vivo PTHrP influenced profile and HER-2/neu PTHrP P was directly associated with longer survival (P = 0.031, log-rank test; Fig. 3B). 0123X

DCIS Discussion À 489310.013 Our studies indicate that PTHrP expression acts to prevent the + 12971 emergence of mammary tumors, influences heterotypic cell Invasive signaling within tumors, and correlates with monocyte infiltration À 62 66 237 6 0.965 in human DCIS and that these aspects of its action may be relevant +109414 to its reported association with longer survival in breast cancer patients (2, 16). These findings appear relevant to the previously NOTE: X, samples with undetermined score for PTHrP immunohisto- established role for PTHrP in osteoblast-osteoclast signaling, where chemistry. PTHrP regulates the recruitment of monocytes in that setting (1); notably, relevant GO terms were detected in our analyses. We www.aacrjournals.org 7477 Cancer Res 2009; 69: (18). September 15, 2009

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Figure 3. Kaplan-Meier analysis of 251 breast cancer patients (37) stratified by (A) directional differential expression of genes (human homologues) identified following conditional Pthrp deletion in MMTV-neu tumors and (B) for expression of PTHrP alone (probe ID: 211756_at). Solid line, patients with gene expression most similar to that of MMTV-neu tumors with Pthrp deleted; broken line, the converse. propose that a functional overlap exists for PTHrP signaling less efficient. Finally, given the genes identified, it is possible that between breast and bone, relating to the management of PTHrP influences signals to other cell lineages including further heterotypic cell relationships, and that this may include the components of the immune system (e.g., T cells), endothelial cells, regulation of the well-characterized cytokine, Opn. We further fibroblasts, or other lineages that are also potentially relevant to suggest that this role of PTHrP in mammary tumors is relevant to the observed difference in tumor emergence. the reported association of PTHrP expression in primary breast Although we cannot rule out cell-autonomous functions for cancer with longer survival (16). PTHrP in breast cancer, we did not observe an effect of PTHrP on The role of innate immune effector cells in tumor immuno- the growth of established tumors or of mammary epithelial cells in surveillance is not well understood but is likely to be most culture (MCF-10A; Fig. 1C).5 We did, however, detect significant GO important early in tumorigenesis. PTHrP may therefore influence categories relating to growth and apoptosis in our gene expression this process, and consistent with this hypothesis, monocyte analysis (Table 2). Previous studies are conflicting in regard to the infiltration was dramatically decreased in the progression from influence of PTHrP on mammary epithelial proliferation, with DCIS to invasive disease (Fig. 2C). Previous studies were overexpression (44) and deletion (7) of PTHrP in the mammary inconclusive as to the significance of macrophage infiltration, but gland reported to inhibit epithelial proliferation. Treatment with few included samples of DCIS or clearly delineated between PTHrP peptide did not alter the growth of MCF7 cells (45) but intratumoral and stromal infiltration (36, 38, 39). In many of the increased proliferation of SV40-transformed cells (46), whereas invasive samples we examined, a marked dissociation of intra- small interfering RNA knockdown of PTHrP modestly reduced tumoral and stromal infiltration was evident (Supplementary proliferation (47). Collectively, it is difficult to conclude that PTHrP Fig. S3B) and we suggest that a stricter definition of macrophage has a consistent cell-autonomous effect on breast cancer cells. infiltration may clarify its prognostic significance. It will also be of The prognostic value of PTHrP expression in breast cancer has interest to test the correlation of macrophage infiltration with long- been contentious (2, 11, 15, 16). Despite excellent in vitro and term outcomes for DCIS specifically. Several recent reports have animal studies supporting a role for PTHrP in promoting growth of implicated monocytes in the dissemination of breast cancer cells breast cancer cells within bone, the largest and most comprehen- and therefore argue that their involvement would indicate a worse sive prospectively collected data set from Henderson and prognosis, but these studies did not evaluate levels of monocytic colleagues (16) showed that PTHrP expression was associated infiltration within primary tumors (40, 41). Recently, it was with better survival in human breast cancer. Our testing of a PTHrP reported that a partial disabling of monocyte function in vivo influenced gene set derived from the MMTV-neu model against resulted in an increased tumor incidence in the polyoma virus independent human breast cancer gene expression data supports middle T antigen tumor model (42), which is consistent with our its association with a better outcome. This is consistent with data. Interestingly, in our experiment, the difference in tumor PTHrP having a protective role in early breast cancer, which is emergence was greater during the latter half of the mouse lifespan. distinct from its role in established bone metastasis. This suggests that the influence of PTHrP may be more important These data taken together suggest that PTHrP expression in for preventing tumors when the animals are older. In human breast early breast cancer may act against its progression and has a direct cancer samples, PTHrP expression does not alter with age (16), but the competency of immunosurveillance in cancer prevention is thought to be age dependent (43). It is possible therefore that PTHrP expression is more important once immunosurveillance is 5 Unpublished data.

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PTHrP, Monocytes, and Early Breast Cancer relevance to the recruitment of immune cells into tumors. They Acknowledgments also corroborate previous reports of its expression in established Received 1/18/09; revised 6/15/09; accepted 7/23/09; published OnlineFirst 9/1/09. disease being associated with a better prognosis. They may go Grant support: National Cancer Institute/NIH grant RO1CA90291 (R.L. Anderson); some way toward resolving the controversy surrounding the GSK Australia and National Breast Cancer Foundation, Australia (N.I. Fleming); and Concern Foundation, National Health and Medical Research Council, and Victorian prognostic value of PTHrP expression in breast cancer. Cancer Agency, Australia (D.M. Thomas). 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. Disclosure of Potential Conflicts of Interest We thank Dr. Patrick Humbert for advice throughout this study, Dr. John Wysolmerski for providing the floxed PTHrP mice, and David Binns for performing No potential conflicts of interest were disclosed. computed tomography scans.

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www.aacrjournals.org 7479 Cancer Res 2009; 69: (18). September 15, 2009

Correction: Parathyroid Hormone-Related Protein Protects against Mammary Tumor Emergence and Is Associated with Monocyte Infiltration in Ductal Carcinoma In situ

In this article (Cancer Res 2009;69:7473–9), which was published in the September 15, 2009 issue of Cancer Research (1), two errors were made. A rabbit antiserum was used to detect PTHrP rather than a goat antiserumas stated, and the reference for the antiserumshould have been Henderson, et al. (ref. 16) rather than Southby, et al. (ref. 2).

Reference 1. Fleming NI, Trivett MK, George J, Slavin JL, Murray WK, Moseley JM, Anderson RL, Thomas DM. Parathyroid hormone- related protein protects against mammary tumor emergence and is associated with monocyte infiltration in ductal carcinoma in situ. Cancer Res 2009;69:7473–9.

Cancer Res 2009; 69: (22). November 15, 20098832 www.aacrjournals.org Published OnlineFirst September 1, 2009; DOI: 10.1158/0008-5472.CAN-09-0194

Parathyroid Hormone−Related Protein Protects against Mammary Tumor Emergence and Is Associated with Monocyte Infiltration in Ductal Carcinoma In situ

Nicholas I. Fleming, Melanie K. Trivett, Joshy George, et al.

Cancer Res 2009;69:7473-7479. Published OnlineFirst September 1, 2009.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-0194

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