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Prolactin Promotes Mammary Pathogenesis Independently from Cyclin D1

Open AccessPublished:May 31, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.03.041
      Epidemiological and experimental studies have revealed an important role for prolactin (PRL) in breast cancer. Cyclin D1 is a major downstream target of PRL in lobuloalveolar development during pregnancy and is amplified and/or overexpressed in many breast carcinomas. To examine the importance of cyclin D1 in PRL-induced pathogenesis, we generated transgenic mice (NRL-PRL) that overexpress PRL in mammary epithelial cells, with wild-type, heterozygous, or genetically ablated cyclin D1 in the FVB/N genetic background. Although loss of one cyclin D1 allele did not affect PRL-induced mammary lesions in nonparous females, the complete absence of cyclin D1 (D1−/−) markedly decreased tumor incidence. Nevertheless, NRL-PRL/D1−/− females developed significantly more preneoplastic lesions (eg, epithelial hyperplasias and mammary intraepithelial neoplasias) than D1−/− females. Moreover, although lack of cyclin D1 reduced proliferation of morphologically normal mammary epithelium, transgenic PRL restored it to rates of wild-type females. PRL posttranscriptionally increased nuclear cyclin D3 protein in D1−/− luminal cells, indicating one compensatory mechanism. Consistently, pregnancy induced extensive lobuloalveolar growth in the absence of cyclin D1. However, transcripts for milk proteins were reduced, and pups failed to survive, suggesting that mammary differentiation was inadequate. Together, these results indicate that cyclin D1 is an important, but not essential, mediator of PRL-induced mammary proliferation and pathology in FVB/N mice and is critical for differentiation and lactation.
      The hormone prolactin (PRL) is critical for mammary alveolar morphogenesis and differentiation.
      • Oakes S.R.
      • Rogers R.L.
      • Naylor M.J.
      • Ormandy C.J.
      Prolactin regulation of mammary gland development.
      Recent epidemiological studies have also implicated PRL in the risk of breast cancer, highlighting its importance in tumorigenesis. Elevated circulating PRL is associated with a higher risk of development of tumors that express estrogen receptor α (ERα+) and with poorer patient outcomes, and PRL receptors (PRLR) are expressed at high levels in many cancers.
      • Swaminathan G.
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      • Fuchs S.Y.
      Regulation of prolactin receptor levels and activity in breast cancer.
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      Prolactin and breast cancer etiology: an epidemiologic perspective.
      Moreover, particularly in women, the mammary gland is exposed to locally produced PRL, in addition to that from pituitary lactotrophs.
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      • Vonderhaar B.K.
      Prolactin synthesis and secretion by human breast cancer cells.
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      Prolactin as an autocrine/paracrine factor in breast cancer.
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      • Ben Jonathan N.
      Prolactin expression and secretion by human breast glandular and adipose tissue explants1.
      Murine transgenic experimental models with elevated mammary PRL have demonstrated the oncogenic potential of this hormone, and permit investigation of the mechanisms whereby PRL promotes breast cancer development and progression.
      • Arendt L.M.
      • Schuler L.A.
      Transgenic models to study actions of prolactin in mammary neoplasia.
      Epithelial proliferation is a key feature of PRL-driven lobuloalveolar development during pregnancy,
      • Oakes S.R.
      • Rogers R.L.
      • Naylor M.J.
      • Ormandy C.J.
      Prolactin regulation of mammary gland development.
      and the cell cycle regulator, cyclin D1, has been reported to be a critical mediator of this process.
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      • Weinberg R.A.
      IGF-2 is a mediator of prolactin-induced morphogenesis in the breast.
      However, the role of cyclin D1 in PRL-induced pathogenesis has not been examined. The classic function of the D cyclins (D1, D2, and D3) is promotion of the G1 to S phase of the cell cycle, via regulation of their cyclin-dependent kinase partners, CDK4 and CDK6.
      • Sherr C.J.
      G1 phase progression: cycling on cue.
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      • Sicinski P.
      Cell cycle in mouse development.
      Activation of these kinases by D cyclins results in phosphorylation of retinoblastoma protein, leading to increased transcription of E2F-responsive genes, and subsequent mitosis. In addition, cyclin D1 has been shown to regulate multiple other processes relevant to oncogenesis, including other actions in cell cycle progression, adhesion and migration, responses to DNA damage, protein synthesis, metabolism, and differentiation, in many cases, independently of CDK4/6 or its kinase activity.
      • Arnold A.
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      Cyclin D1 in breast cancer pathogenesis.
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      Linking cyclins to transcriptional control.
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      Cyclin D as a therapeutic target in cancer.
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      • Pestell R.G.
      Minireview: cyclin D1: normal and abnormal functions.
      The expression of individual D cyclins is tissue specific, but redundancy permits compensation in many tissues.
      • Ciemerych M.A.
      • Sicinski P.
      Cell cycle in mouse development.
      Mammary lobuloalveolar proliferation has appeared to be an exception; genetic ablation of cyclin D1 (D1−/−) in the C57BL/6 × 129SV genetic background prevented this event, culminating in lactation failure.
      • Fantl V.
      • Stamp A.
      • Andrews A.
      • Rosewell I.
      • Dickson C.
      Mice lacking cyclin D1 are small and show defects in eye and mammary gland development.
      • Sicinski P.
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      • Weinberg R.A.
      Cyclin D1 provides a link between development and oncogenesis in the retina and breast.
      This phenotype is very similar to that observed in Prlr−/− females in the same mixed strain background.
      • Oakes S.R.
      • Rogers R.L.
      • Naylor M.J.
      • Ormandy C.J.
      Prolactin regulation of mammary gland development.
      CCDN1 is amplified in a substantial subset of breast carcinomas, and cyclin D1 protein is overexpressed in many others (50% to 70%).
      • Musgrove E.A.
      • Caldon C.E.
      • Barraclough J.
      • Stone A.
      • Sutherland R.L.
      Cyclin D as a therapeutic target in cancer.
      • Barnes D.M.
      • Gillett C.E.
      Cyclin D1 in breast cancer.
      • Bartkova J.
      • Lukas J.
      • Muller H.
      • Lutzhoft D.
      • Strauss M.
      • Bartek J.
      Cyclin D1 protein expression and function in human breast cancer.
      Many hormones and growth factors, including PRL and estrogen, activate its promoter.
      • Pestell R.G.
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      • Reutens A.T.
      • Segall J.E.
      • Lee R.J.
      • Arnold A.
      The cyclins and cyclin-dependent kinase inhibitors in hormonal regulation of proliferation and differentiation.
      In MCF-7 breast cancer cells, PRL increases transcription of cyclin D1,
      • Brockman J.L.
      • Schroeder M.D.
      • Schuler L.A.
      Prolactin activates the cyclin D1 promoter via the JAK2-STAT pathway.
      which is required for the subsequent proliferative response.
      • Schroeder M.D.
      • Symowicz J.
      • Schuler L.A.
      Prolactin modulates cell cycle regulators in mammary tumor epithelial cells.
      PRL also induces nuclear accumulation of this cyclin in murine mammary epithelial cells.
      • Sakamoto K.
      • Creamer B.A.
      • Triplett A.A.
      • Wagner K.U.
      The Janus kinase 2 is required for expression and nuclear accumulation of cyclin D1 in proliferating mammary epithelial cells.
      The requirement for cyclin D1 in mammary tumorigenesis secondary to well-characterized oncogenes has been investigated in murine models in the C57BL/6 × 129SV genetic background. These experiments demonstrated that cyclin D1 was essential for some oncogenes, such as MMTV-driven neu (erbB2) and v-Ha-ras, but also that the cyclin D1 status of mice expressing Myc (alias c-myc) and Wnt-1 driven by the same promoter did not affect tumor incidence or latency.
      • Yu Q.Y.
      • Geng Y.
      • Sicinski P.
      Specific protection against breast cancers by cyclin D1 ablation.
      • Jeselsohn R.
      • Brown N.E.
      • Arendt L.
      • Klebba I.
      • Hu M.G.
      • Kuperwasser C.
      • Hinds P.W.
      Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTV-ErbB2 tumorigenesis.
      Experimental models have demonstrated that augmented proliferation also is an important contribution of PRL to mammary tumorigenesis.
      • Oakes S.R.
      • Robertson F.G.
      • Kench J.G.
      • Gardiner-Garden M.
      • Wand M.P.
      • Green J.E.
      • Ormandy C.J.
      Loss of mammary epithelial prolactin receptor delays tumor formation by reducing cell proliferation in low-grade preinvasive lesions.
      • Vomachka A.J.
      • Pratt S.L.
      • Lockefeer J.A.
      • Horseman N.D.
      Prolactin gene-disruption arrests mammary gland development and retards T-antigen-induced tumor growth.
      • Rose-Hellekant T.A.
      • Arendt L.M.
      • Schroeder M.D.
      • Gilchrist K.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin induces ERα-positive and ERα-negative mammary cancer in transgenic mice.
      • Arendt L.M.
      • Rose-Hellekant T.A.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin potentiates TGFα induction of mammary neoplasia in transgenic mice.
      To investigate the requirement for cyclin D1 in PRL-induced pathology, we used a murine transgenic model of elevated mammary PRL, NRL-PRL. In this model, local PRL overexpression driven by the estrogen- and PRL-independent promoter, NRL, results in preneoplastic lesions, including epithelial hyperplasias and mammary intraepithelial neoplasias, similar to ductal carcinoma in situ in women, and eventually, invasive carcinomas, which resemble the clinical luminal subtype.
      • Rose-Hellekant T.A.
      • Arendt L.M.
      • Schroeder M.D.
      • Gilchrist K.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin induces ERα-positive and ERα-negative mammary cancer in transgenic mice.
      • Arendt L.M.
      • Rugowski D.E.
      • Grafwallner-Huseth T.L.
      • Garcia-Barchino M.J.
      • Rui H.
      • Schuler L.A.
      Prolactin-induced mouse mammary carcinomas model estrogen resistant luminal breast cancer.
      We generated NRL-PRL mice in the context of genetic ablation of Ccdn1 in the FVB/N genetic background. We found that cyclin D1 was important, but not essential, for PRL-induced pathology. In nonparous females without cyclin D1, transgenic PRL was able to augment mammary epithelial proliferation, support alveolar development, and promote preneoplastic lesions and tumors, albeit at a lower level than with wild-type cyclin D1. PRL increased cyclin D3 expression posttranscriptionally, suggesting one compensatory mechanism. Cyclin D1−/− FVB/N females also displayed marked lobuloalveolar development during pregnancy, but expressed reduced levels of milk protein transcripts. Together, these observations indicate that cyclin D1 is not required for PRL-initiated mammogenic and tumorigenic signals in the FVB/N genetic background. Understanding the mediators of PRL actions in carcinogenesis will reveal potential sites for preventative and therapeutic interventions.

      Materials and Methods

      Reagents

      5-Bromo-2-deoxyridine (BrdU) was purchased from Sigma-Aldrich (St. Louis, MO), and 17β-estradiol was obtained from Steraloids (Newport, RI). Antibodies were purchased from the following sources: BrdU (MAS-250) from Accurate Scientific (Westbury, NY), estrogen receptor α (ERα; SC-542), cyclin D2 (SC-53637), cyclin D3 (SC-182), and Grb2 (SC-255) from Santa Cruz Biotechnology (Santa Cruz, CA); cyclin D1 (CP 236 B) from Biocare Medical (Concord, CA), and rat anti-cytokeratin 8 (Troma1) from Developmental Studies Hybridoma Bank, University of Iowa. Secondary antibodies, anti-rat and anti-rabbit, were obtained from BioGenex (San Ramon, CA) and signals detected with 3,3′ diaminobenzidine from Vector Laboratories (Burlingame, CA).

      Mice

      Cyclin D1 heterozygous mice that had been backcrossed into the FVB/NJ strain (FVB.129S2(B6)-Ccnd1tm1Wbg/J) were purchased from Jackson Laboratories (Bar Harbor, ME). NRL-PRL mice [line 1647-13, TgN (Nrl-Prl)23EPS; line 1655, TgN(Nrl-Prl)24EPS] were generated and maintained in the FVB/N strain as described.
      • Rose-Hellekant T.A.
      • Arendt L.M.
      • Schroeder M.D.
      • Gilchrist K.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin induces ERα-positive and ERα-negative mammary cancer in transgenic mice.
      Offspring were genotyped for the PRL transgene and cyclin D1 using the primers shown in Table 1. All mice were housed and handled in accordance with the Guide for Care and Use of Laboratory Animals in Association for the Assessment and Accreditation for Laboratory Animal Care–accredited facilities. All procedures were approved by the University of Wisconsin–Madison Institutional Animal Care and Use Committee.
      Table 1PCR Primers
      TargetPrimer
      Genetic manipulations
       Prolactin transgeneF, 5′-CCTCCTCATTCTCTGCTCTTC-3′
      R, 5′-CCAATCACCCTTGCTCTAAACCC-3′
       Cyclin D1 ablation5′-TAGCAGAGAGCTACAGACTTCG-3′
      5′-CTCCGTCTTGAGCATGGCTC-3′
      5′-CTAGTGAGACGTGCTACTTC-3′
      Real-time PCR
       Cyclin D1F, 5′-CGCCCTCCGTATCTTACTTCAA-3′
      R, 5′-CTCACAGACCTCCAGCATCCA-3′
       Cyclin D2F, 5′-GCTCTGTGCGCTACCGACTT-3′
      R, 5′-CCACGCTTCCAGTTGCAAT-3′
       Cyclin D3F, 5′-CGACTTCCTGGCCTTGATTC-3′
      R, 5′-CAAAGGTGTAATCTGTAGCACAGA-3′
       β-CaseinF, 5′-GCAGAAACTTCAGAAGGTGAATCTC-3′
      R, 3′-TGACTGGATGCTGGAGTGAACT-3′
       γ-CaseinF, 5′-GGTCAACCTAAACCAGCAGAAAA-3′
      R, 5′-TGTGCAACATTGGGAAAAGG-3′
       Whey acidic proteinF, 5′-CGCTCAGAACCTAGAGGAACAAG-3′
      R, 5′-TGATACACTCTGTGCCCTCAATG-3′
       Cytokeratin 8F, 5′-TGAACAACAAGTTCGCCTCCTT-3′
      R, 5′-TCCACTTGGTCTCCAGCATCT-3′
       18SF, 5′-CGCCGCTAGAGGTGAATTTCT-3′
      R, 5′-CGAACCTCCGACTTTCGTTCT-3′

      17β-Estradiol Treatment

      For some experiments, Silastic pellets containing 20 μg of 17β-estradiol were implanted subcutaneously in intact female mice starting at 8 weeks of age, and replaced every 6 weeks until the animal was euthanized at 1 year of age. This dose has been shown to supplement circulating 17β-estradiol levels to approximately those at estrus.
      • Gupta P.B.
      • Kuperwasser C.
      Contributions of estrogen to ER-negative breast tumor growth.
      Uteri were weighed at the time of collection to confirm the positive effect of the pellets. Uterine weight did not differ in untreated mice and was significantly increased in response to 17β-estradiol in all genotypes (see Supplemental Figure S1 at http://ajp.amjpathol.org).

      Examination of Mammary Tissue

      Histological assessments were performed on hematoxylin and eosin–stained sections. Sections of some genotypes and treatment groups were stained for BrdU, ERα, cyclin D1, and/or cyclin D3 (primary antibodies: BrdU, 1:40; ERα, 1:1000; cyclin D1, 1:200; cyclin D3, 1:200), and apoptosis was determined by morphological criteria as described.
      • Rose-Hellekant T.A.
      • Arendt L.M.
      • Schroeder M.D.
      • Gilchrist K.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin induces ERα-positive and ERα-negative mammary cancer in transgenic mice.
      The proportion of epithelial cells undergoing proliferation, apoptosis, and those expressing ERα and cyclin D3 was quantified in three to five mice of each genotype by counting 2000 cells in at least 10 different fields. One thousand cells from at least five distinct microscopic fields were counted in tumors to determine the proportion of cells expressing detectable cyclin D1. For gross evaluation of epithelial structures, mammary whole mounts were prepared as described.
      • Rose-Hellekant T.A.
      • Arendt L.M.
      • Schroeder M.D.
      • Gilchrist K.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin induces ERα-positive and ERα-negative mammary cancer in transgenic mice.

      Immunoblot Analysis

      Western blot analyses of mammary homogenates were performed as previously described
      • Arendt L.M.
      • Rose-Hellekant T.A.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin potentiates TGFα induction of mammary neoplasia in transgenic mice.
      Signals were quantified by densitometry (ImageQuant software, v.4.2a; Molecular Dynamics, Sunnyvale, CA).

      Real-Time PCR

      RNA was isolated from mammary lysates using RNeasy Mini Kit (Qiagen, Valencia, CA), and levels of transcripts determined by quantitative real-time PCR analysis as described previously.
      • Arendt L.M.
      • Evans L.C.
      • Rugowski D.E.
      • Garcia-Barchino M.J.
      • Rui H.
      • Schuler L.A.
      Ovarian hormones are not required for PRL-induced mammary tumorigenesis but estrogen enhances neoplastic processes.
      The primers used are shown in Table 1.

      Statistics

      Statistical analyses were performed as described using Prism version 4.03 (GraphPad Software, San Diego, CA).

      Results

      Absence of Cyclin D1 Reduces but Does Not Prevent PRL-Induced Lesions and PRL-Augmented Proliferation

      In light of the association between PRL and cyclin D1 in mammary epithelial proliferation revealed by several experimental approaches, we examined cyclin D1 expression in PRL-induced mammary carcinomas that developed in the context of wild-type cyclin D1. As shown in Figure 1A, the proportion of cells expressing cyclin D1 correlated moderately positively with the rate of proliferation (Spearman r = 0.5647; P < 0.0004), consistent with an important role for this cell cycle regulator in diverse, established PRL-induced primary tumors.
      Figure thumbnail gr1
      Figure 1A: Cyclin D1 expression correlates with the rate of proliferation in PRL-induced tumors. A panel of diverse primary mammary tumors that developed in NRL-PRL females in the presence of wild-type cyclin D1 was examined by IHC for cyclin D1 and BrdU incorporation as described in Materials and Methods (n = 35; panel as described
      • Arendt L.M.
      • Rugowski D.E.
      • Grafwallner-Huseth T.L.
      • Garcia-Barchino M.J.
      • Rui H.
      • Schuler L.A.
      Prolactin-induced mouse mammary carcinomas model estrogen resistant luminal breast cancer.
      ). Each symbol represents a single tumor. The correlation coefficient was determined using Spearman's nonparametric test (r = 0.57; P < 0.0004). B: The absence of cyclin D1 reduces the incidence and increases the latency of tumors in NRL-PRL females. Females of all genotypes were monitored until tumors reached 1.5 cm in diameter, the mice developed significant health problems, or they reached 24 months of age (end stage). Absence of one cyclin D1 allele did not alter the incidence or latency of PRL-induced tumors (P = 0.83). However, loss of both alleles significantly reduced tumor incidence (P = 0.001), and tended to increase tumor latency (P = 0.0528, one-tailed Student's t-test). Latencies were compared using the Kaplan-Meier test, and differences between groups were detected using the Mantel-Haenszel test.
      To evaluate the requirement for cyclin D1 in PRL-promoted tumor development, we generated NRL-PRL mice with wild-type, heterozygous, or genetically ablated cyclin D1 in the FVB/N genetic background. Nonparous females of all genotypes were evaluated at 24 months of age, when tumors reached 1.5 cm in diameter, or mice developed significant health problems (end stage). As shown in Figure 1B and Table 2, NRL-PRL females with wild-type cyclin D1 developed palpable mammary tumors with a high incidence, consistent with previous studies.
      • Rose-Hellekant T.A.
      • Arendt L.M.
      • Schroeder M.D.
      • Gilchrist K.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin induces ERα-positive and ERα-negative mammary cancer in transgenic mice.
      Heterozygosity for cyclin D1 did not alter the incidence or latency of PRL-induced tumors. However, ablation of cyclin D1 reduced the incidence to 12.5%, and the latency tended to be longer than those with wild-type cyclin D1 levels (P = 0.0528, one-tailed Student's t-test). Interestingly, although tumors that arose in NRL-PRL females with both or only a single cyclin D1 allele displayed varying histotypes (Table 2; Figure 2, A–C), tumors that developed in NRL-PRL/D1−/− females were adenosquamous carcinomas.
      Table 2Effect of Cyclin D1 Status on Mammary Carcinogenesis in Nonparous Females
      GenotypeTumor incidenceTumor latencyTumor histotypes
      at end stage
      End stage is defined as a tumor reaching 1.5-cm diameter in size or 2 years of age.
      mean ± SD (months)(%)
      Wild-type (FVB/N)0/10 (0%)N/AN/A
      NRL-PRL8/1 8/11 (72.7%)
      Incidences differ significantly (P < 0.001, chi-square test).
      18.0 ± 2.9
      Latencies trend toward a significant difference (P = 0.0528, one-tailed Student's t-test).
      Adenocarcinomas:
       Glandular (25)
       Papillary (25)
       Adenosquamous (12.5)
       Carcinosarcomas (37.5)
      Cyclin D1+/−0/7 (0%)N/AN/A
      NRL-PRL/D1+/−9/11 (81.8%)17.6 ± 2.3Adenocarcinomas:
       Glandular (22.2)
       Papillary (55.6)
       Adenosquamous (22.2)
      Cyclin D1−/−0/12 (0%)N/AN/A
      NRL-PRL/D1−/−2/1 2/16 (12.5%)
      Incidences differ significantly (P < 0.001, chi-square test).
      22.1 ± 2.4
      Latencies trend toward a significant difference (P = 0.0528, one-tailed Student's t-test).
      Adenocarcinomas:
       Adenosquamous (100)
      low asterisk End stage is defined as a tumor reaching 1.5-cm diameter in size or 2 years of age.
      Incidences differ significantly (P < 0.001, chi-square test).
      Latencies trend toward a significant difference (P = 0.0528, one-tailed Student's t-test).
      Figure thumbnail gr2
      Figure 2AH: Diverse mammary carcinomas and lesions develop in NRL-PRL and NRL-PRL/D1−/− females. A: Glandular adenocarcinoma with eosinophilic secretions from an NRL-PRL female. B: Papillary adenocarcinoma from an NRL-PRL female. C: Adenosquamous carcinoma from an NRL-PRL/D1−/− female. D: Irregular (degenerative) ductal epithelium and dilated ducts in NRL-PRL/D1−/− female. E: Mammary intraepithelial neoplasia (MIN) within an epithelial hyperplasia in NRL-PRL/D1−/− female. F: Epithelial hyperplasia in an NRL-PRL/D1−/− female. G: BrdU-labeled epithelial cells in a duct of an NRL-PRL/D1−/− female. H: ERα-labeled epithelial cells in a duct of an NRL-PRL/D1−/− female. IL: Immunohistochemical localization of cyclin D3 expression in mammary glands of end-stage females. I: Wild-type (WT); J: NRL-PRL; K: Cyclin D1−/−; L: NRL-PRL/D1−/−. Original magnification: ×200 (A, C, D, E, F, and IL); ×100 (B); ×400 (G and H).
      Despite the paucity of carcinomas, transgenic PRL induced many preneoplastic lesions in the absence of cyclin D1 that were readily apparent on histological examination (Figure 2, D–F, Table 3). NRL-PRL/D1−/− females displayed significantly more epithelial hyperplasias that were larger and more widespread than in cyclin D1−/− females (Table 3). Furthermore, although mammary intraepithelial neoplasias were scarce in D1−/− mammary glands, they were readily apparent in NRL-PRL/D1−/− females. These data indicate that the lack of D1 does not abrogate PRL-initiated pathogenesis, but rather suggest that it slows lesion progression. However, the limited healthy lifespan of these mice precludes analysis of longer-term effects on tumor development.
      Table 3PRL Increases Mammary Abnormalities in Cyclin D1−/− Glands
      Cyclin D1−/−NRL-PRL/D1−/−NRL-PRL/D1+/+
      Epithelial hyperplasias
      Topographical distribution of hyperplasias as defined.32
      4/12
      Indicates reduced frequency compared to NRL-PRL/D1−/− (P < 0.05).
      11/1611/11
      Focal: 2/12Focal: 2/16Focal: 0/11
      Multifocal: 2/12Multifocal: 2/16Multifocal: 0/11
      Diffuse: 0/12Diffuse: 7/16Diffuse 11/11
      Mammary intraepithelial neoplasias1/12
      Indicates reduced frequency compared to NRL-PRL/D1−/− (P < 0.05).
      11/1610/11
      Mammary glands were histologically examined at end stage.
      Analysis performed by one-tailed chi-square test.
      low asterisk Topographical distribution of hyperplasias as defined.
      • Cardiff R.D.
      • Anver M.R.
      • Gusterson B.A.
      • Hennighausen L.
      • Jensen R.A.
      • Merino M.J.
      • Rehm S.
      • Russo J.
      • Tavassoli F.A.
      • Wakefield L.M.
      • Ward J.M.
      • Green J.E.
      The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting.
      Indicates reduced frequency compared to NRL-PRL/D1−/− (P < 0.05).
      To better understand the underlying mechanism, the rates of proliferation and apoptosis of morphologically normal mammary epithelium were determined. BrdU-labeled and ERα-labeled epithelial cells were observed in ducts of NRL-PRL/D1−/− females (Figure 2, G and H, respectively). Transgenic PRL increased both proliferation and apoptosis in the presence of wild-type cyclin D1 compared to nontransgenic females (Figure 3, A and B), as previously reported.
      • Rose-Hellekant T.A.
      • Arendt L.M.
      • Schroeder M.D.
      • Gilchrist K.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin induces ERα-positive and ERα-negative mammary cancer in transgenic mice.
      • Arendt L.M.
      • Rose-Hellekant T.A.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin potentiates TGFα induction of mammary neoplasia in transgenic mice.
      As expected, rates of proliferation were very low in cyclin D1−/− glands. However, PRL in the context of cyclin D1 ablation was able to augment proliferation to levels of nontransgenic glands (Figure 3A). The absence of cyclin D1 significantly increased apoptosis, which strikingly was reduced by transgenic PRL to wild-type levels (Figure 3B). This opposite net effect of transgenic PRL on apoptosis, depending on cyclin D1 status, suggests crosstalk between these factors in otherwise distinct pathways. Similar patterns were observed in PRL-induced hyperplasias (Figure 3, A and B), although as expected,
      • Rose-Hellekant T.A.
      • Arendt L.M.
      • Schroeder M.D.
      • Gilchrist K.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin induces ERα-positive and ERα-negative mammary cancer in transgenic mice.
      levels of proliferation were higher in lesions, compared to morphologically normal structures. Together, these results indicate that cyclin D1 is not required for PRL-induced responses, but both proliferation and neoplastic processes are facilitated by its presence.
      Figure thumbnail gr3
      Figure 3A and B: Transgenic PRL induces proliferation of morphologically normal ductal and hyperplastic epithelia in end-stage glands in the absence of cyclin D1−/− (A), and differently affects apoptosis depending on cyclin D1 status (B). Rates of proliferation and apoptosis were determined as described in Materials and Methods. Data are expressed as the mean ± SEM (n = 3–5). Analyses were performed by the Kruskal-Wallis test with the Mann–Whitney posttest (P < 0.05). Different letters (a, b, c) indicate statistically significant differences among genotypes. C: NRL-PRL/D1−/− females exhibit significant lobuloalveolar development, albeit reduced compared to NRL-PRL females with wild-type cyclin D1. Glands from all genotypes, including cyclin D1−/−, displayed normal ductal development. Representative whole mounts at the end stage of each genotype as indicated. Scale bar = 2 mm. WT, wild type.
      The ability of PRL to increase epithelial growth in the absence of cyclin D1 was evident in mammary whole mounts and histological sections from these end-stage females. As expected from previous reports,
      • Fantl V.
      • Stamp A.
      • Andrews A.
      • Rosewell I.
      • Dickson C.
      Mice lacking cyclin D1 are small and show defects in eye and mammary gland development.
      • Sicinski P.
      • Donaher J.L.
      • Parker S.B.
      • Li T.
      • Fazell A.
      • Gardner H.
      • Haslam S.Z.
      • Bronson R.T.
      • Elledge S.J.
      • Weinberg R.A.
      Cyclin D1 provides a link between development and oncogenesis in the retina and breast.
      wild-type and cyclin D1−/− glands displayed normal ductal development and branching, as well as some alveolar budding, but developed alveoli were rare (Figure 3C). By contrast, glands of NRL-PRL females with wild-type cyclin D1 exhibited many lobuloalveoli and hyperplasias, as previously reported.
      • Rose-Hellekant T.A.
      • Arendt L.M.
      • Schroeder M.D.
      • Gilchrist K.
      • Sandgren E.P.
      • Schuler L.A.
      Prolactin induces ERα-positive and ERα-negative mammary cancer in transgenic mice.
      Consistent with the ability of PRL to augment proliferation in the absence of cyclin D1, transgenic PRL also induced limited lobuloalveolar development in NRL-PRL/D1−/− animals.

      PRL Further Increases Cyclin D3 Expression in the Absence of Cyclin D1

      Although cyclin D2 and cyclin D3 can compensate for the loss of cyclin D1 in many tissues,
      • Ciemerych M.A.
      • Kenney A.M.
      • Sicinska E.
      • Kalaszczynska I.
      • Bronson R.T.
      • Rowitch D.H.
      • Gardner H.
      • Sicinski P.
      Development of mice expressing a single D-type cyclin.
      the lack of lobuloalveoli in pregnant C57BL/6 × 129SV cyclin D1−/− mice
      • Fantl V.
      • Stamp A.
      • Andrews A.
      • Rosewell I.
      • Dickson C.
      Mice lacking cyclin D1 are small and show defects in eye and mammary gland development.
      • Sicinski P.
      • Donaher J.L.
      • Parker S.B.
      • Li T.
      • Fazell A.
      • Gardner H.
      • Haslam S.Z.
      • Bronson R.T.
      • Elledge S.J.
      • Weinberg R.A.
      Cyclin D1 provides a link between development and oncogenesis in the retina and breast.
      suggested that the mammary gland may be an exception. In light of the observed PRL-induced mammary pathology in the absence of cyclin D1 in our study, we examined the effect of elevated local PRL on expression of the other D cyclins. As shown in Figure 4, A–C, mammary transcripts for both cyclin D2 and D3 were increased in the absence of cyclin D1 as previously reported.
      • Fantl V.
      • Stamp A.
      • Andrews A.
      • Rosewell I.
      • Dickson C.
      Mice lacking cyclin D1 are small and show defects in eye and mammary gland development.
      • Sicinski P.
      • Donaher J.L.
      • Parker S.B.
      • Li T.
      • Fazell A.
      • Gardner H.
      • Haslam S.Z.
      • Bronson R.T.
      • Elledge S.J.
      • Weinberg R.A.
      Cyclin D1 provides a link between development and oncogenesis in the retina and breast.
      However, transgenic PRL did not further increase these mRNAs. Analysis of protein expression, however, revealed a more complex picture. PRL tended to raise total mammary cyclin D2 and D3 protein levels in D1−/− glands, but not in those expressing wild-type levels of cyclin D1 (Figure 4, D–F). Immunohistochemistry verified the low levels of cyclin D3 in mammary glands with wild-type cyclin D1 (Figures 2, I and J, and 4G), although it was readily detectable in uteri of these individuals (see Supplemental Figure S2 at http://ajp.amjpathol.org). In glands of D1−/− females, cyclin D3 expression was clearly evident in some epithelial as well as stromal cells (Figure 2K). Transgenic PRL strikingly elevated cyclin D3 protein levels in D1−/− glands, most notably in a subset of the cells lining the lumens of the epithelial structures, indicating posttranscriptional action (Figures 2L and 4G).
      Figure thumbnail gr4
      Figure 4Transgenic PRL does not further increase cyclin D2 and cyclin D3 mRNA in cyclin D1−/− glands, but tends to increase cyclin D3 protein. mRNA levels of cyclin D1 (A), cyclin D2 (B), and cyclin D3 (C) were quantitated by RT-PCR as described in Materials and Methods. D: Western blot analyses of mammary lysates from individual end-stage mice for cyclin D1 (D1), cyclin D2 (D2), cyclin D3 (D3), cytokeratin 8 (Krt8), and Grb2, as shown. Quantitation of cyclin D2 (E) and cyclin D3 (F) relative to keratin 8 levels from the Western blot analyses (D). G: Proportion of epithelial cells that are positive for cyclin D3 protein by immunohistochemistry (as described in Materials and Methods). EG: Data are expressed as the mean ± SEM (n = 3). Different letters (a, b, c) indicate statistically significant differences among genotypes, detected using the Kruskal-Wallis test, followed by the Mann–Whitney posttest (P < 0.05). WT, wild type.

      Cyclin D1−/− Glands Exhibit Elevated ERα Expression, and Respond to 17β-Estradiol Supplementation and the Hormonal Milieu of Pregnancy with Lobuloalveolar Development, but Lactation Failure

      To examine the effects of the absence of cyclin D1 on other indicators of hormonal responsiveness in the FVB/N genetic background, we examined ERα expression in glands of age-matched 1-year-old nonparous females. The absence of cyclin D1 elevated the proportion of cells in normal mammary structures that expressed detectable ERα to levels comparable to those induced by transgenic PRL in wild-type glands (Figure 5A), but this was not further elevated by PRL. Increased numbers of ERα+ cells in D1−/− glands may reflect the low proliferative activity (Figure 3A); segregation of proliferation and ERα expression is observed in normal mammary tissue.
      • Shoker B.S.
      • Jarvis C.
      • Davies M.P.A.
      • Iqbal M.
      • Sibson D.R.
      • Sloane J.P.
      Immunodetectable cyclin D 1 is associated with oestrogen receptor but not Ki67 in normal, cancerous and precancerous breast lesions.
      Mammary epithelial structures in all genotypes responded readily to elevated systemic estrogen. Supplementation with 17β-estradiol beginning after ductal elongation had occurred (8 weeks) elicited alveolar budding when examined at 1 year of age regardless of the presence of cyclin D1 (Figure 5, B and C). This treatment also increased uterine weight, although to a lesser extent in D1−/− females, compared to those with wild-type cyclin D1 (see Supplemental Figure S1 at http://ajp.amjpathol.org).
      Figure thumbnail gr5
      Figure 5A: ERα is elevated in cyclin D1−/− glands of 1-year-old nonparous females. Morphologically normal epithelial cells expressing detectable ERα by IHC were quantified as described in Materials and Methods (mean ± SEM, n = 5). *P < 0.05 compared to WT. B and C: Nonparous females of all genotypes respond to long-term 17β-estradiol treatment with enhanced lobuloalveolar development. Intact nonparous females of all genotypes were untreated (B), or supplemented with 17β-estradiol beginning at 8 weeks of age (C), and glands were collected at 1 year of age (see Materials and Methods). Representative whole-mounted glands from each genotype as indicated. Scale bar = 1 mm. WT, wild type.
      Likewise, pregnancy induced extensive lobuloalveolar development in nulliparous females of all genotypes. Mammary glands of cyclin D1−/− females were morphologically similar to those with wild-type D1 at 24 hours postpartum (Figure 6A). However, although apparently healthy pups were born in comparably sized litters to mothers of all genotypes, survival of pups born to cyclin D1−/− mothers regardless of transgenic PRL status was dramatically reduced (Table 4). Although secretions were apparent in alveolar lumens of D1−/− mothers, transcripts for milk proteins were significantly less than those of mothers with wild-type D1, regardless of transgenic PRL status (Figure 6, B–D). These data indicate that mammary glands of FVB/N females are able to robustly proliferate in response to physiological combinations of mammogenic hormones during pregnancy in the absence of cyclin D1. However, cyclin D1 itself is necessary for functional lactation, and compensatory mechanisms are not adequate.
      Figure thumbnail gr6
      Figure 6Cyclin D1 status does not affect mammary development during pregnancy, but is essential for optimal milk protein synthesis. Nulliparous females of the different genotypes were bred to wild-type FVB/N males, and pup health and mammary morphology were assessed 24 hours after parturition. A: Mammary glands at day 1 postpartum of each genotype as indicated. Original magnification, ×100. BD: Mammary glands of cyclin D1−/− mothers contain lower levels of transcripts for milk proteins β casein (B), γ casein (C), and WAP (D) compared to cytokeratin 8, regardless of PRL transgene status. Levels of specific RNAs in mammary lysates from mothers of different genotypes were determined by real-time PCR as described in Materials and Methods (mean ± SD, n = 3 to 5). Different letters (a, b, c) indicate statistically significant differences among genotypes by analysis of variance followed by the Tukey Multiple Comparison test (P < 0.05).
      Table 4Pups of cyclin D1−/− Mothers Exhibit Poor Survival
      GenotypeLitter size at birthPup survival (%)
      Mean ± s.d.Mean ± s.d.
      Wild type (FVB/N) n = 57.4 ± 1.584.0 ± 12.1
      NRL-PRL n = 49.0 ± 1.186.9 ± 12.5
      Cyclin D1−/− n = 77.0 ± 1.421.1 ± 17.7
      Indicates reduced frequency compared to wild-type and NRL-PRL mice.
      NRL-PRL/D1−/− n = 97.0 ± 1.435.7 ± 43.0
      Nulliparous females of all genotypes were bred to nontransgenic males. Litter size was observed on PND 0 and survival on PND 1, when tissues were collected for analysis.
      Analysis performed by Student's t-test.
      low asterisk Indicates reduced frequency compared to wild-type and NRL-PRL mice.

      Discussion

      Cyclin D1 is a physiological mediator of lobuloalveolar proliferation induced by PRL and cooperating hormones during pregnancy.
      • Sicinski P.
      • Weinberg R.A.
      A specific role for cyclin D1 in mammary gland development.
      Using a transgenic model of elevated local PRL exposure in combination with germline deletion of Ccnd1 in the FVB/N genetic background, we demonstrated herein that cyclin D1 is also required for maximal PRL-promoted mammary tumorigenesis. However, even in the absence of cyclin D1, transgenic PRL increased mammary epithelial proliferation, induced early lesions, and promoted carcinomas, albeit with a reduced incidence and longer latency. PRL posttranscriptionally increased epithelial cyclin D3 expression, revealing one underlying compensatory mechanism. Mammary epithelium of cyclin D1–deficient females also proliferated in response to other hormones, including supplemental estrogen and the complex endocrine milieu of pregnancy. Nonetheless, despite extensive lobuloalveolar development during gestation, pups failed to survive due to apparent lactational failure. Together, our data indicate that compensatory mechanisms, including cyclin D3, are able to partially replace cyclin D1 in PRL-induced proliferation, but cannot compensate for cyclin D1 in PRL-induced differentiation. These studies point to distinct actions of cyclin D1 in hormonal regulation of mammary function, as well as genetic differences among mouse strains.
      Circulating PRL is associated primarily with ERα+ breast cancer,
      • Tworoger S.S.
      • Hankinson S.E.
      Prolactin and breast cancer etiology: an epidemiologic perspective.
      modeled by experimental PRL-induced carcinomas.
      • Arendt L.M.
      • Rugowski D.E.
      • Grafwallner-Huseth T.L.
      • Garcia-Barchino M.J.
      • Rui H.
      • Schuler L.A.
      Prolactin-induced mouse mammary carcinomas model estrogen resistant luminal breast cancer.
      The significant correlation between proliferation and cyclin D1 expression in the tumors that develop in NRL-PRL females shown here is consistent with the observed high cyclin D1 expression in ERα+ clinical tumors.
      • Arnold A.
      • Papanikolaou A.
      Cyclin D1 in breast cancer pathogenesis.
      • Musgrove E.A.
      • Caldon C.E.
      • Barraclough J.
      • Stone A.
      • Sutherland R.L.
      Cyclin D as a therapeutic target in cancer.
      PRL increases expression of cyclin D1 via transcription,
      • Brockman J.L.
      • Schroeder M.D.
      • Schuler L.A.
      Prolactin activates the cyclin D1 promoter via the JAK2-STAT pathway.
      as well as nuclear accumulation and stabilization of the protein.
      • Sakamoto K.
      • Creamer B.A.
      • Triplett A.A.
      • Wagner K.U.
      The Janus kinase 2 is required for expression and nuclear accumulation of cyclin D1 in proliferating mammary epithelial cells.
      Although mammary transcripts for both cyclin D2 and D3 were elevated in D1−/− FVB/N females in the current study, similar to reports in other genetic backgrounds,
      • Fantl V.
      • Stamp A.
      • Andrews A.
      • Rosewell I.
      • Dickson C.
      Mice lacking cyclin D1 are small and show defects in eye and mammary gland development.
      • Sicinski P.
      • Donaher J.L.
      • Parker S.B.
      • Li T.
      • Fazell A.
      • Gardner H.
      • Haslam S.Z.
      • Bronson R.T.
      • Elledge S.J.
      • Weinberg R.A.
      Cyclin D1 provides a link between development and oncogenesis in the retina and breast.
      PRL did not further increase levels of these mRNAs. The apparent lack of PRL-stimulated transcription of these other D cyclins in the mammary gland in vivo suggests that regulatory signals to these genes are cell specific; Stat5, a prominent mediator of PRL in the mammary gland,
      • Wagner K.U.
      • Rui H.
      Jak2/Stat5 signaling in mammogenesis, breast cancer initiation and progression.
      mediates activation of both the cyclin D2 and D3 promoters in response to other cytokines in other cell types.
      • Malin S.
      • McManus S.
      • Busslinger M.
      STAT5 in B cell development and leukemia.
      • Martino A.
      • Holmes J.H.
      • Lord J.D.
      • Moon J.J.
      • Nelson B.H.
      Stat5 and Sp1 regulate transcription of the cyclin D2 gene in response to IL-2.
      However, our studies showed that PRL increased cyclin D3 protein in luminal epithelial cells of NRL-PRL/D1−/− females. Nuclear accumulation of cyclin D3, like cyclin D1, is regulated by glycogen synthase kinase-3β–dependent phosphorylation, subsequent nuclear export and proteasomal degradation,
      • Diehl J.A.
      • Cheng M.G.
      • Roussel M.F.
      • Sherr C.J.
      Glycogen synthase kinase 3 beta regulates cyclin D1 proteolysis and subcellular localization.
      • Naderi S.
      • Gutzkow K.B.
      • Låhne H.U.
      • Lefdal S.
      • Ryves W.J.
      • Harwood A.J.
      • Blomhoff H.K.
      cAMP-induced degradation of cyclin D3 through association with GSK-3β.
      a pathway inhibited by PRL in mammary epithelium.
      • Sakamoto K.
      • Creamer B.A.
      • Triplett A.A.
      • Wagner K.U.
      The Janus kinase 2 is required for expression and nuclear accumulation of cyclin D1 in proliferating mammary epithelial cells.
      This mechanism is likely to contribute to the ability of PRL to induce the mammary epithelial proliferation and pathology in the absence of cyclin D1 observed in our studies. In light of the incomplete compensation observed in our study, it is interesting to note that cyclin D3 was less effective than cyclin D1 in stimulating mitosis of hepatocytes.
      • Mullany L.K.
      • White P.
      • Hanse E.A.
      • Nelsen C.J.
      • Goggin M.M.
      • Mullany J.E.
      • Anttila C.K.
      • Greenbaum L.E.
      • Kaestner K.H.
      • Albrecht J.H.
      Distinct proliferative and transcriptional effects of the D-type cyclins in vivo.
      The role of cyclin D3 in breast cancer is less studied than that of cyclin D1. Cyclin D3 is elevated in some clinical breast cancers, frequently in conjunction with cyclin D1.
      • Russell A.
      • Thompson M.A.
      • Hendley J.
      • Trute L.
      • Armes J.
      • Germain D.
      Cyclin D1 and D3 associate with the SCF complex and are coordinately elevated in breast cancer.
      • Zhang Q.
      • Sakamoto K.
      • Liu C.
      • Triplett A.A.
      • Lin W.C.
      • Rui H.
      • Wagner K.U.
      Cyclin D3 compensates for the loss of Cyclin D1 during ErbB2-induced mammary tumor initiation and progression.
      • Wong S.C.C.
      • Chan J.K.C.
      • Lee K.C.
      • Hsiao W.L.W.
      Differential expression of p16/p21/p27 and cyclin D1/D3, and their relationships to cell proliferation, apoptosis, and tumour progression in invasive ductal carcinoma of the breast.
      Experimental overexpression of cyclin D3 results in squamous cell carcinomas in mice,
      • Pirkmaier A.
      • Dow R.
      • Ganiatsas S.
      • Waring P.
      • Warren K.
      • Thompson A.
      • Hendley J.
      • Germain D.
      Alternative mammary oncogenic pathways are induced by D-type cyclins; MMTV-cyclin D3 transgenic mice develop squamous cell carcinoma.
      demonstrating that this D cyclin also can contribute to mammary oncogenesis. In addition, cyclin D3 may confer distinct phenotypic features to mammary cancers. Like those tumors that developed in MMTV-cyclin D3 mice, both of the carcinomas that developed in NRL-PRL/D1−/− females exhibited a squamous histotype. This phenotype contrasts with the adenocarcinomas that develop in MMTV-cyclin D1 females,
      • Wang T.C.
      • Cardiff R.D.
      • Zukerberg L.
      • Lees M.
      • Arnold A.
      • Schmidt E.V.
      Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice.
      suggesting distinct actions of different D cyclins on either a subpopulation of epithelial cells or cellular functions. Indeed, limited studies have demonstrated that cyclin D family members determine different substrates of activated CDK4/6,
      • Sarcevic B.
      • Lilischkis R.
      • Sutherland R.L.
      Differential phosphorylation of T-47D human breast cancer cell substrates by D1-, D3-, E-, and A-type cyclin-CDK complexes.
      and associate with distinct transcriptional regulators in breast cancer cells in vitro (see below).
      In addition to effects on D cyclins, PRL alters expression of the cell cycle regulators p21Cip/WAF1 and cyclin B1 in breast cancer cells.
      • Schroeder M.D.
      • Symowicz J.
      • Schuler L.A.
      Prolactin modulates cell cycle regulators in mammary tumor epithelial cells.
      These targets of PRL, complemented by direct and indirect actions of other hormones in the complex environment of pregnancy, are likely to contribute to the extensive lobuloalveolar development in D1−/− periparturient females observed here.
      Compensation for the loss of cyclin D1 in lobuloalveolar growth was not evident in the first descriptions of cyclin D1−/− mice, which were carried out in the C57BL/6 × 129SV genetic background.
      • Fantl V.
      • Stamp A.
      • Andrews A.
      • Rosewell I.
      • Dickson C.
      Mice lacking cyclin D1 are small and show defects in eye and mammary gland development.
      • Sicinski P.
      • Donaher J.L.
      • Parker S.B.
      • Li T.
      • Fazell A.
      • Gardner H.
      • Haslam S.Z.
      • Bronson R.T.
      • Elledge S.J.
      • Weinberg R.A.
      Cyclin D1 provides a link between development and oncogenesis in the retina and breast.
      However, in addition to our study, Wagner and colleagues
      • Zhang Q.
      • Sakamoto K.
      • Liu C.
      • Triplett A.A.
      • Lin W.C.
      • Rui H.
      • Wagner K.U.
      Cyclin D3 compensates for the loss of Cyclin D1 during ErbB2-induced mammary tumor initiation and progression.
      also recently reported reduced mammary cyclin D1 dependence in the FVB/N strain, and Haslam and colleagues
      • Aupperlee M.D.
      • Drolet A.A.
      • Durairaj S.
      • Wang W.
      • Schwartz R.C.
      • Haslam S.Z.
      Strain-specific differences in the mechanisms of progesterone regulation of murine mammary gland development.
      reported extensive alveologenesis during pregnancy in BALB/c D1−/− females. Together, these studies suggest that the original strain background, which is known to display reduced hormonal responsiveness,
      • Aupperlee M.D.
      • Drolet A.A.
      • Durairaj S.
      • Wang W.
      • Schwartz R.C.
      • Haslam S.Z.
      Strain-specific differences in the mechanisms of progesterone regulation of murine mammary gland development.
      • Medina D.
      Mouse models for mammary cancer.
      may be unusual in this aspect. These reports underscore the importance of genetic background, which needs to be taken into account because mouse models are used to dissect complex biological processes, such as oncogenesis. This is particularly critical in light of the role of cyclins in hormonal actions in breast cancer,
      • Musgrove E.A.
      • Caldon C.E.
      • Barraclough J.
      • Stone A.
      • Sutherland R.L.
      Cyclin D as a therapeutic target in cancer.
      and the potential role of cyclin D1 in tumor progenitor populations.
      • Yu Q.Y.
      • Geng Y.
      • Sicinski P.
      Specific protection against breast cancers by cyclin D1 ablation.
      • Jeselsohn R.
      • Brown N.E.
      • Arendt L.
      • Klebba I.
      • Hu M.G.
      • Kuperwasser C.
      • Hinds P.W.
      Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTV-ErbB2 tumorigenesis.
      • Ling H.
      • Sylvestre J.R.
      • Jolicoeur P.
      Notch1-induced mammary tumor development is cyclin D1-dependent and correlates with expansion of pre-malignant multipotent duct-limited progenitors.
      Strain differences also present the opportunity to identify the genetic loci that dictate the distinct phenotypes; these studies also may elucidate factors underlying susceptibility and resistance to breast cancer in women.
      In contrast to the ability of compensatory mechanisms to support proliferation and support marked lobuloalveolar development in the absence of cyclin D1 in both FVB/N as well BALB/c females, pups failed to survive, indicating that cyclin D1 itself is essential for lactational competence. The reduced transcripts for milk proteins in D1−/− mammary glands shown here indicate that this may result from incomplete differentiation rather than inadequate alveolar capacity. Using a “knocked-in” cyclin D1 mutant that is unable to activate CDK4/6, Landis and colleagues
      • Landis M.W.
      • Pawlyk B.S.
      • Li T.
      • Sicinski P.
      • Hinds P.W.
      Cyclin D1-dependent kinase activity in murine development and mammary tumorigenesis.
      demonstrated that lobuloalveolar development is independent of kinase activity. Cyclin D family members have been shown to exert kinase-independent actions on transcription via multiple mechanisms in a variety of systems.
      • Arnold A.
      • Papanikolaou A.
      Cyclin D1 in breast cancer pathogenesis.
      • Musgrove E.A.
      • Caldon C.E.
      • Barraclough J.
      • Stone A.
      • Sutherland R.L.
      Cyclin D as a therapeutic target in cancer.
      • Fu M.
      • Wang C.
      • Li Z.
      • Sakamaki T.
      • Pestell R.G.
      Minireview: cyclin D1: normal and abnormal functions.
      An elegant study examining cyclin D1–associated proteins in mouse embryos determined that about one third of the identified proteins were transcription factors.
      • Bienvenu F.
      • Jirawatnotai S.
      • Elias J.E.
      • Meyer C.A.
      • Mizeracka K.
      • Marson A.
      • Frampton G.M.
      • Cole M.F.
      • Odom D.T.
      • Odajima J.
      • Geng Y.
      • Zagozdzon A.
      • Jecrois M.
      • Young R.A.
      • Liu X.S.
      • Cepko C.L.
      • Gygi S.P.
      • Sicinski P.
      Transcriptional role of cyclin D1 in development revealed by a genetic-proteomic screen.
      Our data suggest that these actions may be less readily compensated in the mammary gland. In support of this, Mullany and colleagues
      • Mullany L.K.
      • White P.
      • Hanse E.A.
      • Nelsen C.J.
      • Goggin M.M.
      • Mullany J.E.
      • Anttila C.K.
      • Greenbaum L.E.
      • Kaestner K.H.
      • Albrecht J.H.
      Distinct proliferative and transcriptional effects of the D-type cyclins in vivo.
      found that substantial subsets of transcripts were distinct in hepatocytes overexpressing individual D cyclins. Indeed, cyclin D1 is a strikingly stronger activator of the transcriptional activity of ERα than either D2 or D3.
      • Zwijsen R.M.L.
      • Weintjens E.
      • Klompmaker R.
      • Michalides R.J.A.M.
      CDK-independent activation of estrogen receptor by cyclin D1.
      • Neuman E.
      • Ladha M.H.
      • Lin N.
      • Upton T.M.
      • Miller S.J.
      • DiRenzo J.
      • Pestell R.G.
      • Hinds P.W.
      • Dowdy S.F.
      • Brown M.
      • Ewen M.E.
      Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4.
      Additional study will be required to dissect the specific targets of individual D cyclins in the mammary gland and genetic modulation of these responses.
      In light of the accumulating evidence implicating PRL in the development and progression of ERα+ tumors
      • Tworoger S.S.
      • Hankinson S.E.
      Prolactin and breast cancer etiology: an epidemiologic perspective.
      and increased expression of cyclin D1 in early lesions and carcinomas, especially luminal tumors,
      • Arnold A.
      • Papanikolaou A.
      Cyclin D1 in breast cancer pathogenesis.
      • Musgrove E.A.
      • Caldon C.E.
      • Barraclough J.
      • Stone A.
      • Sutherland R.L.
      Cyclin D as a therapeutic target in cancer.
      • Barnes D.M.
      • Gillett C.E.
      Cyclin D1 in breast cancer.
      it is important to understand the relationship between these factors in breast cancer. Our studies here demonstrate the importance of cyclin D1 in PRL-induced mammary proliferation and pathogenesis, but also reveal other mediators likely to include cyclin D3 in the murine FVB/N genetic background. Understanding the web of signals that generate the array of phenotypes and variation in therapeutic responsiveness of the luminal subtype of breast cancer will illuminate strategies to prevent and treat this disease.

      Acknowledgments

      We thank Courtney Blohm for valuable technical assistance and Dr. Kay-Uwe Wagner (University of Nebraska) and Dr. Ruth Sullivan (University of Wisconsin) for helpful discussions.

      Supplementary data

      • Supplemental Figure S1

        Effects of 17β-estradiol (E2) supplementation on uterine weights (P<0.0001). Nonparous females of all genotypes were treated with long-term 17β-estradiol beginning at 8 weeks of age, as described in Materials and Methods. Tissues were collected at 1 year of age. Different symbols indicate statistically significant differences among genotypes in E2-supplemented females (P<0.05). Graph is expressed as the mean + SD (n = 5), and analyses were performed by the Kruskal-Wallis and Mann-Whitney post tests.

      • Supplemental Figure S2

        Cyclin D3 expression in uterine cells and mammary glands of wild-type FVB/N females. Immunohistochemical detection of cyclin D3 was performed as described in Materials and Methods (Original magnification, ×200).

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