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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2010 Feb 1;107(7):2995–3000. doi: 10.1073/pnas.1000101107

Disruption of the Lcn2 gene in mice suppresses primary mammary tumor formation but does not decrease lung metastasis

Thorsten Berger a, Carol C Cheung a,b, Andrew J Elia a, Tak W Mak a,1
PMCID: PMC2840296  PMID: 20133630

Abstract

Based largely on studies in xenograft models, lipocalin-2 (Lcn2) has been implicated in the progression of multiple types of human tumors, including breast cancer. Here we examine the role of Lcn2 in mammary tumorigenesis and lung metastasis using an in vivo molecular genetics approach. We crossed a well-characterized transgenic mouse model of breast cancer, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) mouse, with two independent gene-targeted Lcn2−/− mouse strains of the 129/Ola or C57BL/6 genetic background. The onset and progression of mammary tumor development and lung metastasis in the female progeny of these crosses were monitored over a 20-week period. Female Lcn2−/−MMTV-PyMT mice of the 129/Ola background (Lcn2−/−PyMT129) showed delayed onset of mammary tumors, and both Lcn2−/−PyMT129 mice and Lcn2−/−MMTV-PyMT mice of the C57BL/6 background (Lcn2−/−PyMTB6) exhibited significant decreases in multiplicity and tumor burden (∼2- to 3-fold), as measured by total tumor weight and volume. At the molecular level, mammary tumors derived from Lcn2−/−PyMTB6 females showed reduced matrix metalloproteinase-9 (MMP-9) activity and a lack of high molecular weight MMP activity. However, although increased MMP-9 activity has been linked to tumor progression, neither Lcn2−/−PyMTB6 nor Lcn2−/−PyMT129 female mice showed a reduction in lung metastases compared to Lcn2+/+PyMT controls. Our results demonstrate, using an in vivo animal model approach, that Lcn2 is a potent inducer of mammary tumor growth but not a significant promoter of lung metastasis.

Keywords: breast cancer, lipocalin-2, matrix metalloproteinase-9, mouse mammary tumor virus-polyoma middle T antigen

Lipocalin-2 is a member of the lipocalin protein family. Lipocalins comprise a group of over 20 diverse proteins that exhibit only limited amino acid sequence similarity but share a common tertiary structure (1). This three-dimensional structure consists of a single eight-stranded, antiparallel β-barrel that forms an enclosing calyx capable of flexible binding. This unique structure allows lipocalins to form covalent and noncovalent complexes with a wide range of soluble macromolecules, including retinoids, fatty acids, prostaglandins, steroids, and certain enzymes (2). Initially characterized as shuttle and transporter proteins, it is now well-established that the lipocalins perform a variety of important functions.

Lipocalin-2 was first isolated as a 25-kDa glycoprotein based on its covalent binding to matrix metalloproteinase-9 (MMP-9) in human neutrophils (3, 4). Another important Lcn2 ligand is enterochelin, a bacterial siderophore that binds iron with extremely high affinity (5). Bacteria use siderophores to capture the essential element iron from the host environment. We have previously shown in vitro and in vivo that Lcn2 functions in mammalian innate immunity by using its siderophore-chelating property to inactivate bacterial siderophores and thereby sequester iron from bacterial reach (6). Bacterial growth is thus inhibited and the host protected.

A surprising finding has been that Lcn2 expression is induced in a number of human cancers. Heterogeneous expression of Lcn2 was first documented at both the mRNA and protein levels in a subset of patients with primary breast cancers (7). The Lcn2 protein was found within the breast carcinoma cells of these patients but not in normal ductal epithelium. The highest levels of Lcn2 protein were detected in the lumens of normal ducts that were located in the vicinity of Lcn2-expressing tumors. In vitro experimentation has since supported a role for Lcn2 in breast tumor progression. For example, engineered overexpression of Lcn2 in MCF-7 human breast cancer cells results in increased growth rate, proliferation, angiogenesis, and MMP-9 levels (8). The complexing of Lcn2 to MMP-9 is thought to prevent MMP-9 autodegradation and thereby increase MMP-9’s enzymatic activity both in vitro (9) and in breast cancer patients (8).

MMPs are up-regulated in almost every type of human cancer, and their expression is often associated with poor survival. Some MMPs (e.g., MMP-7) are expressed by the tumor cells themselves, whereas others (e.g., MMP-9) are synthesized by the surrounding tumor stromal cells, including fibroblasts, myofibroblasts, inflammatory cells, and endothelial cells (10). MMPs promote cancer progression by enhancing tumor cell growth, migration, invasion, metastasis, and angiogenesis. These diverse effects are achieved through the cleavage of a broad range of substrates, including not only structural components of the extracellular matrix (ECM) but also growth-factor-binding proteins, growth-factor precursors, receptor tyrosine kinases, cell-adhesion molecules, and other proteases (10).

In vivo studies of Lcn2’s involvement in tumor progression have been limited to date to xenograft models in which Lcn2 levels have been increased through overexpression (8), or partially ablated by short-hairpin-induced gene silencing (11). Although these types of models can be useful, they do not reflect the complex multistage nature of human tumorigenesis. To obtain a more faithful representation of human cancer, we employed the well-established transgenic mouse mammary tumor virus (MMTV) polyoma middle T antigen (PyMT) model (12) to analyze the role of Lcn2 in tumor initiation, progression, and metastasis. Female MMTV-PyMT mice develop multifocal dysplastic atypia in the mammary epithelium as early as 3 weeks after birth. These lesions are thought to be the origin of the primary mammary adenocarcinomas that appear in these animals with an average latency of about 35–40 days (12). Thereafter, these primary tumors spontaneously metastasize to the lungs with a relatively high penetrance. The aggressive phenotype of these tumors is due to the multiple signaling pathways activated by the PyMT oncogene, including those involving Src kinase and phosphatidylinositol 3-kinase (13 –15). The relevance of the MMTV-PyMT model to human breast cancer has been validated by both genetic and histological studies (16, 17), which have demonstrated a striking similarity between MMTV-PyMT tumorigenesis and various stages of human ductal adenocarcinomagenesis. In particular, both ErbB2/Neu and cyclin D1 expression are up-regulated in MMTV-PyMT tumor cells, just as in human ductal adenocarcinoma cells (17, 18).

In this study, we demonstrate that female MMTV-PyMT mice lacking Lcn2 show a significant delay in the onset of palpable primary tumors and a greatly reduced primary tumor burden. However, in contrast to previous reports (11, 19), we find no evidence supporting a prometastatic function for Lcn2.

Results

Loss of Lcn2 Reduces Primary Tumor Formation in MMTV-PyMT Mice.

To test whether Lcn2 is an oncogene contributing to the formation of mammary tumors in the MMTV-PyMT model, we generated female Lcn2+/+PyMT and Lcn2−/−PyMT mice of the 120/Ola (PyMT129) and C57BL/6 (PyMTB6) backgrounds (as described in Materials and Methods), and analyzed tumor formation in the PyMT129 mice in detail. Lcn2+/+PyMT129 mice developed palpable mammary tumors starting at around 40 days after birth and, by 80 days after birth, 50% of these animals had palpable mammary tumors (Fig. 1A). In contrast, Lcn2−/−PyMT129 mice did not start to develop palpable tumors until 60 days after birth. Overall tumor incidence was significantly delayed in Lcn2−/−PyMT129 mice (60–125 days, with T50 = 100 days) compared to Lcn2+/+PyMT129 mice (40–120 days, with T50 = 80 days).

Fig. 1.

Fig. 1.

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Lcn2 ablation delays tumor onset and reduces tumor burden in MMTV-PyMT129 mice. (A) Longer latency. Starting at 35 days of age, female Lcn2+/+PyMT129 (n = 12) and Lcn2−/−PyMT129 (n = 9) mice were palpated on the indicated days to detect tumors. Data shown are the percentage of mice remaining tumor-free on the indicated day. (B) Decreased number of tumor-bearing mammary glands. Numbers of mammary glands with grossly visible tumors were counted for each Lcn2+/+PyMT129 (n = 11) and Lcn2−/−PyMT129 (n = 11) mouse. Data shown are the number of tumor-bearing glands per mouse. Horizontal bars indicate mean values. (C) Decreased tumor weight. The total wet weight in grams of all mammary tumors present in each Lcn2+/+PyMT129 (n = 11) and Lcn2−/−PyMT129 (n = 11) mouse was measured. Horizontal bars indicate mean values. (D) Decreased tumor volume. The total volume in cm3 of all mammary tumors present in each Lcn2+/+PyMT129 (n = 11) and Lcn2−/−PyMT129 (n = 11) mouse was measured. Horizontal bars indicate mean values. (E) Elevated plasma Lcn2 protein levels. Levels of Lcn2 in the plasma of tumor-bearing Lcn2+/+PyMTB6 and Lcn2−/−PyMTB6 mice (n = 3/group), as well as nontransgenic Lcn2+/+B6 and Lcn2−/−B6 controls (n = 2/group), were analyzed by immunoblotting. (F) Elevated Lcn2 in tumors. Mammary tumors from Lcn2+/+PyMTB6 mice (n = 4) and normal mammary glands from Lcn2+/+B6 mice (n = 3) were immunoblotted to detect Lcn2. Actin, loading control. For E and F, results shown are representative of three trials of randomly selected mice for each group.

We next compared the total tumor burden in Lcn2−/−PyMT129 and Lcn2+/+PyMT129 mice at 130 days after birth, a time point shortly before the tumors in the Lcn2+/+PyMT129 mice reached the humane end point. Mice from both groups were killed at 130 days and numbers of tumor-affected mammary glands (as assessed by gross histological analysis) were counted. Lcn2−/−PyMT129 mice had significantly fewer tumor-affected mammary glands than did Lcn2+/+PyMT129 controls (4.8 ± 1.5 vs. 9.1 ± 0.5 tumor-affected mammary glands/mouse; Fig. 1B). In addition, the total wet weight of all mammary tumors per animal was much lower in Lcn2−/−PyMT129 mice than in Lcn2+/+PyMT129 controls (1.2 ± 0.9 vs. 2.8 ± 1.8 g total wet weight of tumors/mouse; Fig. 1C). Finally, the total volume of all mammary tumors per animal was also significantly lower in Lcn2−/−PyMT129 mice than in Lcn2+/+PyMT129 controls (1.9 ± 1.6 vs. 5.6 ± 4.4 cm3 total volume of tumors/mouse; Fig. 1D).

To confirm that Lcn2 has an important role in PyMT-induced tumorigenesis, we used western blotting to analyze Lcn2 protein levels in Lcn2+/+PyMTB6 animals as well as in nontransgenic controls. Lcn2+/+PyMTB6 mice expressed high levels of secreted Lcn2 in plasma as compared to Lcn2+/+B6 nontransgenic mice (Fig. 1E). Suspecting that the tumors themselves might be the source of plasma Lcn2, we used western blotting to demonstrate that Lcn2 protein was highly expressed in the cancerous mammary glands of Lcn2+/+PyMTB6 mice but considerably less in the normal mammary glands of nontransgenic Lcn2+/+B6 mice (Fig. 1F). Immunohistochemical analysis confirmed the expression of Lcn2 in tumor tissue from Lcn2+/+PyMTB6 mice (Fig. S1). Taken together, these results suggest that Lcn2 has a significant oncogenic role in primary mammary tumor formation, at least in the MMTV-PyMT model.

Genetic Background Does Not Change Lcn2’s Effect on Tumor Formation in the MMTV-PyMT Model.

It has been previously shown that genetic background can have a significant impact on tumor formation and metastasis in the MMTV-PyMT model (20). To examine this issue, we analyzed tumor initiation in our Lcn2+/+PyMTB6 and Lcn2+/−PyMTB6 mice at an early time point, using whole-mount examination of mammary gland preparations at 30 days after birth. At this point in mammary gland development, the growth of the mammary ducts increases significantly, the system of ducts begins to elongate around the lymph node, and the highly proliferative terminal end buds at the tips of the ductal branches expand greatly (21). In MMTV-PyMT mice, premalignant multifocal dysplastic lesions of the mammary epithelium can be identified by whole-mount examination in a majority of mammary glands by 3 weeks of age (22). These lesions then spread throughout the entire mammary fat pad by 7–10 weeks of age and develop into adenocarcinomas (18).

To determine whether loss of Lcn2 in our PyMTB6 system affected the development of multifocal dysplastic lesions, we harvested the right fourth (inguinal) mammary glands from female Lcn2+/+PyMTB6 and Lcn2−/−PyMTB6 mice at exactly 30 days of age, fixed these tissues in Carnoy’s solution, and stained them overnight with carmine dye. A parallel set of experiments was carried out using 50-day-old virgin female Lcn2+/+PyMTB6 and Lcn2−/−PyMTB6 mice. At 30 days of age, Lcn2+/+PyMTB6 and Lcn2−/−PyMTB6 mice exhibited small hyperplastic focal lesions in the older portions of the mammary ductal tree. However, there was no significant difference in the frequency or size of these lesions in the absence of Lcn2 either at this point (Fig. 2A) or at 50 days after birth (Fig. 2B).

Fig. 2.

Fig. 2.

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Ablation of Lcn2 has no effect on early mammary tumorigenesis in MMTV-PyMTB6 mice. Mammary glands (inguinal) were harvested from virgin female Lcn2+/+PyMTB6, Lcn2−/−PyMTB6, and nontransgenic Lcn2+/+B6 mice at 30 days (A) or 50 days (B) of age. Glands were fixed in Carnoy’s solution, stained overnight with carmine dye, and examined microscopically for multifocal dysplastic lesions. Arrowheads, dysplastic foci; LN, subiliac lymph node; TEB, terminal end buds. No differences in tumor histology were observed among the groups. Results shown are representative of five randomly selected mice/group.

To examine a later stage of tumorigenesis in a more quantitative fashion, we analyzed the total tumor burden in female Lcn2+/+PyMTB6 and Lcn2−/−PyMTB6 mice at 150 days after birth, a time point shortly before the humane end point. As was performed for PyMT129 mice, numbers of tumor-affected mammary glands (as assessed by gross histological analysis) were counted. We found that the number of tumor-free mammary glands was significantly lower in Lcn2−/−PyMTB6 mice than in Lcn2+/+PyMTB6 controls (Fig. 3A). In addition, Lcn2−/−PyMTB6 mice had an average total wet weight of all mammary tumors of 2.0 ± 0.9 g compared to 6.0 ± 2.3 g in Lcn2+/+PyMTB6 mice (Fig. 3B). The total volume of all mammary tumors per animal was also significantly decreased in Lcn2−/−PyMTB6 mice as opposed to Lcn2+/+PyMTB6 mice (3.4 ± 1.8 cm3 vs. 10.5 ± 4.5 cm3; Fig. 3C). Finally, Lcn2 plasma levels as measured by direct ELISA correlated positively with total tumor weight (r = 0.83, P < 0.01; Fig. S2). These results confirm that Lcn2 promotes primary mammary tumor formation independent of the two genetic backgrounds analyzed here, and demonstrate that Lcn2 is more important for later (rather than earlier) stages of mammary tumor development.

Fig. 3.

Fig. 3.

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Lcn2 ablation delays tumor onset and reduces tumor burden in MMTV-PyMTB6 mice. (A) Decreased number of tumor-bearing mammary glands. Numbers of mammary glands with grossly visible tumors were counted for each Lcn2+/+PyMTB6 (n = 14) and Lcn2−/−PyMTB6 (n = 16) mouse. Data shown are the number of tumor-bearing glands per mouse. Horizontal bars indicate mean values. (B) Decreased tumor weight. The total wet weight in grams of all mammary tumors present in each Lcn2+/+PyMTB6 (n = 14) and Lcn2−/−PyMTB6 (n = 16) mouse was measured. Horizontal bars indicate mean values. (C) Decreased tumor volume. The total volume in cm3 of all mammary tumors present in each Lcn2+/+PyMTB6 (n = 14) and Lcn2−/−PyMTB6 (n = 16) mouse was measured. Horizontal bars indicate mean values.

MMP-9 Activity Is Reduced in Lcn2−/−PyMTB6 Mice.

Several groups have reported that the binding of Lcn2 to MMP-9 protects this ECM-remodeling enzyme from autodegradation (9, 23). In addition, complexes of Lcn2 bound to MMP-9 have been identified in cancer patients but not in healthy volunteers (8). We therefore compared relative levels of MMP-9 activity in the plasma of Lcn2−/−PyMTB6, Lcn2+/+PyMTB6, and nontransgenic control mice (where the value for Lcn2+/+ nontransgenic mice was taken as 100%). Compared to Lcn2+/+PyMTB6 mice (139% ± 10% relative activity; Fig. 4 and Fig. S3), MMP-9 activity was slightly reduced in the plasma of Lcn2−/−PyMTB6 mice (110% ± 6% relative activity; P = 0.035). Furthermore, we were able to detect the activity of a higher molecular weight MMP complex in the plasma of two out of three Lcn2+/+PyMTB6 animals but not in the plasma of any Lcn2−/−PyMTB6 mice (Fig. 4, HMW MMP). Interestingly, and in contrast to previous reports (8), we found no evidence for the formation of the putative Lcn2-MMP-9 complex in either our PyMTB6 mice or the nontransgenic controls. In any case, our data suggest that an absence of Lcn2 decreases MMP-9 activity, consistent with the dramatically reduced mean tumor size seen in our Lcn2−/−PyMTB6 mice.

Fig. 4.

Fig. 4.

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Reduced MMP-9 activity in the plasma of tumor-bearing Lcn2−/−PyMTB6 mice. MMP-9 activity in the plasma of tumor-bearing Lcn2+/+PyMTB6 and Lcn2−/−PyMTB6 mice (n = 3/group), as well as in healthy Lcn2+/+ and Lcn2−/− control mice (C57BL/6; n = 2/group), was measured by zymography as described in Materials and Methods. Numbers below the zymogram represent densitometric quantification of MMP-9 activity relative to the mean Lcn2+/+B6 value (100 arbitrary units). A band representing high molecular weight MMP activity (HMW MMP) was detectable only in Lcn2+/+PyMTB6 mice. Results shown are representative of three trials of randomly selected mice for each group.

Genetic Ablation of Lcn2 Does Not Reduce Mammary Tumor-Derived Metastatic Lung Disease.

A great advantage of the MMTV-PyMT mouse model is that it can be seamlessly used to study both primary mammary tumor development and metastasis. A high percentage of MMTV-PyMT mice develop metastases in the lungs, and these lesions can be readily observed macroscopically and microscopically (12). To determine the influence of Lcn2 on metastasis formation, we examined the lungs of Lcn2+/+PyMT and Lcn2−/−PyMT mice of both genetic backgrounds at the time of sacrifice (130 days for PyMT129 mice, 150 days for PyMTB6 mice). Lungs were first subjected to gross observation to detect surface lesions. However, in contrast to previous reports (20, 24), we were unable to identify any surface metastases on the lungs of mice of the C57BL/6 background, and only one Lcn2+/+PyMT129 mouse showed one grossly visible lung lesion. We then prepared lung tissue sections (200 μm apart), stained them with hematoxylin-eosin (H&E), and counted numbers of visible metastases. Again to our surprise, we detected many fewer lesions than previously reported, with <50% of our mice showing lung metastases, compared to >85% in previous studies (20, 25). Indeed, there was no statistically significant difference in lung metastasis formation between our Lcn2+/+PyMT and Lcn2−/−PyMT mice in either genetic background (Fig. 5A). About 36% of Lcn2+/+PyMT129 mice showed lung metastases, as opposed to 39% of Lcn2−/−PyMT129 mice. Similarly, 60% of Lcn2+/+PyMTB6 mice showed lung metastases compared to 50% of Lcn2−/−PyMTB6 mice. In quantitative terms, Lcn2+/+PyMT129 mice had (on average) 0.7 lung metastases compared to 2.2 metastases in Lcn2−/−PyMT129 mice (Fig. 5B), and 0.6 lung metastases occurred in Lcn2+/+PyMTB6 mice compared to 3.2 metastases in Lcn2−/−PyMTB6 mice (Fig. 5C). However, this apparent difference did not reach statistical significance. These data suggest that, although Lcn2 affects primary mammary tumor formation, it does not have a strong influence on the metastasis of these lesions to the lung.

Fig. 5.

Fig. 5.

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Lcn2 deficiency has no effect on the frequency or number of mammary gland tumor-derived lung metastases. (A) The percentage of mice with detectable lung tumors did not differ between Lcn2+/+PyMT and Lcn2−/−PyMT mice of either genetic background (Lcn2+/+PyMT129, n = 10 mice; Lcn2−/−PyMT129, n = 12; Lcn2+/+PyMTB6, n = 11; Lcn2−/−PyMTB6, n = 18). (B and C) The number of lung metastases/mouse did not differ between Lcn2+/+PyMT129 (n = 10 mice) and Lcn2−/−PyMT129 (n = 12) mice (B), or between Lcn2+/+PyMTB6 (n = 11) and Lcn2−/−PyMTB6 (n = 18) mice (C). For B and C, each data point is the total number of unique metastases counted in three H&E-stained sections/lung (taken 200 μm apart). Horizontal bars, mean number of total unique metastases for all mice/group.

Discussion

Although several reports have suggested that increased Lcn2 expression in breast cancer may be detrimental (8, 11, 26), the precise in vivo role of Lcn2 in breast cancer initiation and progression has been challenging to define. In this study, we have shown that Lcn2 is important for PyMT-induced mammary tumor initiation and growth. Previous studies employed xenograft models to elucidate the role of Lcn2 in breast cancer, a methodology that cannot emulate the multistage nature of human breast cancer progression. We have used the well-characterized MMTV-PyMT mouse model (12) to show that genetic ablation of Lcn2 leads to a significant reduction in palpable mammary tumors in both the 129/Ola and C57/B6 backgrounds. Interestingly, the development of multifocal dysplastic lesions, the earliest event in PyMT-induced mammary cancer initiation, was not affected by a lack of Lcn2. These results point to the involvement of Lcn2 only in the later stages of primary mammary tumor progression.

The most striking result arising from our study was the dramatic drop in mammary tumor burden in virgin female Lcn2-deficient mice at ∼20 weeks of age. Although almost all Lcn2+/+PyMT mice had reached the maximum permitted tumor burden by this age, no Lcn2−/−PyMT mouse had accumulated sufficient tumors to warrant sacrifice. These data point to a role for Lcn2 in primary tumor development that is consistent with published reports, and confirm Lcn2 as an important oncogenic factor in PyMT-induced mammary tumorigenesis.

Lcn2 reportedly forms a complex with MMP-9 that prevents autodegradation of this enzyme and thus sustains its activity. MMP-9 promotes cancer progression by perforating cellular basement membranes, degrading the ECM, and liberating vascular endothelial growth factor. Angiogenesis, invasion, and metastasis are thus all indirectly enabled by Lcn2. In our study, we detected a statistically significant but slight reduction in MMP-9 activity in the plasma of tumor-bearing Lcn2−/−PyMTB6 mice compared with Lcn2+/+PyMTB6 mice. However, this reduction represents only a small decrease in total MMP-9 activity and likely does not explain the markedly decreased tumor burden in Lcn2-deficient mice. Indeed, Martin et al. showed that MMP-9−/−MMTV-PyMT mice do not show any difference in primary mammary tumor burden compared to MMP-9+/+MMTV-PyMT controls (20), indicating that MMP-9 is not critical for PyMT-induced mammary tumor formation. Consequently, Lcn2’s oncogenic role is at best only partly dependent on its ability to stabilize MMP-9. Intriguingly, MMP-9−/−MMTV-PyMT mice do show a decrease in mammary tumor-derived lung metastases but only in mice of the C57BL/6 background (20). In our study, we were unable to detect a statistical difference in metastasis formation in the absence of Lcn2, regardless of genetic background. These findings reinforce the notion that Lcn2 acts as an oncogene independently of MMP-9.

Several groups have advanced other hypotheses as to how Lcn2 might exert its oncogenic function. These theories include induction of the epithelial-to-mesenchymal transition (11) and inhibition of the PI3K/Akt pathway (19). We are currently investigating these potential mechanisms in our Lcn2−/−PyMT mice.

In conclusion, our in vivo analyses of genetically modified tumor-prone mice have identified Lcn2 as an oncogene important for the later stages of primary mammary tumor development but not for the formation of lung metastases. The precise mechanism underlying this oncogenic function of Lcn2 appears to differ from its bacteriostatic function but remains to be fully elucidated. Future studies may pinpoint Lcn2 as a candidate therapeutic target for human breast cancer.

Materials and Methods

Animals.

Mice were housed in a specific pathogen-free environment, and experiments were conducted following approval by the Institutional Animal Use and Care Committee of the University Health Network as regulated by the Canadian Council on Animal Care. Lcn2−/− mice were generated previously (6) and were backcrossed into the C57BL/6 or 129/Ola backgrounds for at least 10 generations. MMTV-PyMT mice (the kind gift of Dr. W. Muller, Montreal, Canada) were originally derived from FVB mice (12) and were backcrossed at least 10 times into the C57BL/6 or 129/Ola backgrounds. All MMTV-PyMT mice used in this study were heterozygous for the PyMT transgene.

Only virgin female Lcn2+/+MMTV-PyMT and Lcn2−/−MMTV-PyMT mice were used for experiments. Because female MMTV-PyMT mice develop mammary tumors before they can breed, we took a stepwise strategy to generating female Lcn2−/−MMTV-PyMT mice. First, Lcn2−/− mice of the 129/Ola genetic background were bred to MMTV-PyMT mice of the same genetic background. Lcn2+/−PyMT male mice were generated by crossing Lcn2+/+PyMT males with Lcn2−/− females. Lcn2+/−PyMT males were then interbred with Lcn2−/− females, yielding Lcn2−/−PyMT males. These males were mated with Lcn2−/− females to generate Lcn2−/−PyMT females. This procedure was repeated to generate Lcn2−/−PyMT females of the C57BL/6 background. This breeding strategy did not affect the level of PyMT expression in Lcn2+/+PyMT or Lcn2−/−PyMT mice (Fig. S4). Offspring were genotyped by PCR of genomic DNA derived from tail clippings, and 100% of PyMT+ mice developed mammary carcinomas. Tumor-bearing mice were killed at 130 days of age for the 129/Ola background and at 150 days of age for the C57BL/6 background and analyzed for tumor burdens. Young mice between the ages of 30 and 50 days (before the development of palpable tumors) were used for the assessment of multifocal dysplastic lesions. For the evaluation of palpable tumor onset, animals were palpated twice weekly. At necropsy, numbers of tumor-positive mammary glands were counted before excision and determination of wet weight and size of each malignancy. The volume of an individual tumor was determined by measuring its length (L), width (W), and height (H) using a vernier caliper and calculating (L × W × H) as described (27). For histologic analysis, sections of tumors and inflated lungs were fixed in 10% buffered formalin.

Western Blot Analysis.

Protein extracts were prepared in lysis buffer [0.1% Nonidet P-40, 20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM EGTA, 150 mM NaCl, protease inhibitor mixture (Roche Diagnostics)]. Extracts were boiled in SDS/PAGE loading buffer for 10 min, resolved by SDS/PAGE under reducing conditions, and transferred to a polyvinylidene difluoride membrane (Roche Diagnostics). Blots were probed with primary rat polyclonal anti-Lcn2 antibody (R&D Systems) or rabbit anti-actin antibody (Sigma-Aldrich) followed by horseradish peroxidase-conjugated goat anti-rat or anti-rabbit secondary antibody. Immune complexes were visualized using enhanced chemiluminescence (ECL kit; Amersham Biosciences) according to the manufacturer’s recommendations.

MMP Zymography.

Zymography of MPP-9 activity in mouse plasma was performed using standard protocols and 10% gelatin zymogram gels (Invitrogen). Briefly, 2–5 μl plasma was prepared in standard SDS/PAGE buffer (no boiling or reducing agent) and resolved by SDS/PAGE. The SDS was removed from the zymogram gel by incubation in unbuffered Triton X-100, and the gel was incubated in digestion buffer at room temperature overnight. The zymogram was subsequently stained with Coomassie Brilliant Blue to detect MMP activity. Densitometry of the zymogram was performed using a LI-COR Odyssey infrared imaging system.

Histology.

Mouse lung and mammary tumor tissues were embedded in paraffin, and sections (4-μm thick) were cut and stained with H&E. For assessment of lung metastasis, three sections 200 μm apart in depth were analyzed. Whole-mount carmine alum staining was performed according to standard protocols (http://mammary.nih.gov/index.html). Briefly, mammary glands were excised and flattened on a microscope slide, followed by air drying for 5 min, fixation overnight in Carnoy’s solution (75% EtOH plus 25% glacial acetic acid), washing in 70% EtOH for 30 min, and staining overnight in carmine alum. Slides were destained in 2% HCl in 70% EtOH for about 2 h and dehydrated in increasing concentrations of EtOH (70%, 80%, 95%, and 100%) for 30 min at each step. Dehydrated mammary glands were defatted in toluene and examined using a stereomicroscope (Leica MZ16F). Whole mounts were digitally photographed on a stereomicroscope using the same magnification and lighting conditions for all samples.

Statistics.

The Kaplan–Meier method (log-rank test) was used to construct the survival curves of Lcn2+/+PyMT and Lcn2−/−PyMT mice as they developed tumors. The Student’s t test was used for intergroup comparisons. For the regression analysis the following formula was used:

graphic file with name pnas.1000101107uneq1.jpg

Note Added in Proof.

During the preparation of this manuscript, Leng et al. (28) published a report using a spontaneous mammary tumor mouse model, MMTV-ErbB2(V664E), crossed with Lcn2-deficient mice. These transgenic mice lacking mouse Lcn2 had significantly delayed mammary tumor formation and metastasis with reduced MMP-9 activity in the blood. These data nicely conform to our report, although lipocalin-2’s antimetastatic role as postulated by Leng et al. remains controversial in light of our results and needs to be further analyzed.

Supplementary Material

Supporting Information
supp_107_7_2995__index.html (799B, html)

Acknowledgments

We thank Lily Zhou for expert technical assistance, and Mary E. Saunders for scientific editing. C.C.C. is supported by the Canadian Institute of Health Research.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/1000101107/DCSupplemental.

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