The RhoGEF Net1 is required for normal mammary gland development.

ORIGINAL

RESEARCH

The RhoGEF Net1 Is Required for Normal Mammary Gland Development Yan Zuo, Rebecca Berdeaux, and Jeffrey A. Frost Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas 77030

Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily-specific guanine nucleotide exchange factor that is overexpressed in human breast cancer and is required for breast cancer cell migration and invasion. However, the role of Net1 in normal mammary gland development or function has never been assessed. To understand the role of Net1 in the mammary gland, we have created a conditional Net1 knockout mouse model. Whole-body deletion of Net1 results in delayed mammary gland development during puberty characterized by slowed of ductal extension and reduced ductal branching. Epithelial cells within the developing ducts show reduced proliferation that is accompanied by diminished estrogen receptor-␣ expression and activity. Net1deficient mammary glands also exhibit reduced phosphorylation of regulatory subunits of myosin light chain and myosin light-chain phosphatase, indicating that RhoA-dependent actomyosin contraction is compromised. Net1 deficiency also leads to disorganization of myoepithelial and ductal epithelial cells and increased periductal collagen deposition. Mammary epithelial cell transplantation experiments indicate that reduced ductal branching and disorganization are cell autonomous. These data identify for the first time a role for NET1 in vivo and indicate that NET1 expression is essential for the proliferation and differentiation of mammary epithelial cells in the developing mammary gland. (Molecular Endocrinology 28: 1948 –1960, 2014)

ostnatal mammary gland development in mice is a complex process controlled by ovarian hormones and local growth factors that begins at puberty and continues until adulthood. During this time mammary epithelial cells extend from a rudimentary ductal tree to invade the mammary fat pad through a process of ductal extension, bifurcation, and side branching (1–3). Ductal invasion of the fat pad is mediated by structures called terminal end buds (TEBs), which consist of highly proliferative and motile cells located at the ends of the ductal tree. Complexity of the extending ductal tree is derived by side branching that requires reciprocal interactions between the epithelial cells and their surrounding stromal cells (4). Rho family small GTPases are critical regulators of actin cytoskeletal organization and cell motility (5–7). Thus, it is not surprising that recent studies have impli-

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cated these proteins as important regulators of mammary gland development. For example, Rac1 and RhoA activations are required for mammary gland branch formation in vitro (8). Moreover, inducible overexpression of Cdc42 in vivo causes increased branching, stromal cell accumulation, and collagen deposition in the developing mammary gland (9). The best evidence for a role for RhoA in mammary gland development in vivo comes from work on the RhoA-inactivating protein p190-RhoGAP. Genetic deletion of the p190A-RhoGAP isoform alters TEB architecture, increases ductal expression of estrogen receptor (ER)-␣ and progesterone receptor, and inhibits stromal cell accumulation and collagen deposition (10). Deletion of p190B-RhoGAP causes a distinct phenotype characterized by a delay in mammary gland development and inhibition of proliferation within TEBs (11). Con-

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received April 23, 2014. Accepted October 8, 2014. First Published Online October 16, 2014

Abbreviations: Calca, calcitonin-related polypeptide-␣; EMT, epithelial-to-mesenchymal transition; ER, estrogen receptor; ES, embryonic stem; GAP, growth-associated protein; GEF, guanidine nucleotide exchange factor; MEC, mammary epithelial cell; MYPT1, myosin light-chain phosphatase; Net1, neuroepithelial transforming gene 1; PR, progesterone receptor; RANKL, receptor activator of nuclear factor-␬B ligand; TEB, terminal end bud.

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Mol Endocrinol, December 2014, 28(12):1948 –1960

doi: 10.1210/me.2014-1128

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doi: 10.1210/me.2014-1128

versely, p190B-RhoGAP overexpression also delays mammary gland development but does so through a separate mechanism that is characterized by increased ductal side branching, stromal cell accumulation, and collagen deposition (12). Taken together, these studies indicate that proper temporal and spatial activation of Rho proteins is crucial for normal mammary gland development. Thus, it is surprising that the role of specific Rho guanine nucleotide exchange factors, which are required to stimulate Rho GTPase signaling, have not been well documented in mammary gland development. The neuroepithelial transforming gene 1 (Net1) is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for breast cancer cell motility and extracellular matrix invasion (13, 14). Net1 is overexpressed in numerous human cancers including gastric adenocarcinoma, hepatocellular carcinoma, gliomas, and breast cancer (15–18), and coexpression of Net1 with ␤4-integrin is prognostic for reduced metastasis-free survival in ER␣-positive breast cancer patients (19). Two isoforms of Net1, Net1 and Net1A, exist in most cells, which are identical except for alternative amino-terminal regulatory domains. Net1 isoforms localize to the nucleus in resting cells, and redistribution of Net1 proteins outside the nucleus is required for RhoA activation and actin cytoskeletal reorganization (20, 21). In this regard, the Net1A isoform accumulates more readily in the cytoplasm when overexpressed (21) and is actively exported to the plasma membrane in response to Rac1 activation (22). Alternatively, the longer isoform of Net1 contributes to breast cancer cell proliferation and controls mitotic progression (23, 24). Although these data indicate diverse roles for Net1 in breast cancer cell migration, invasion and proliferation, the role of Net1 in normal mammary gland development has not been explored. To determine whether Net1 contributes to mammary gland development in vivo, we generated a Net1 knockout mouse model. We found that whole-body deletion of Net1 significantly delays mammary gland development by inhibiting proliferation within terminal end buds, which is correlated with a significant loss of ER␣ expression and transcriptional activity. Net1 deletion also impairs the activation of proteins associated with RhoAdependent actomyosin contractility. Surprisingly, the loss of Net1 results in a progressive disorganization of myoepithelial and luminal epithelial cells within mature ducts once they have formed. This is accompanied by a significant increase in stromal cell numbers surrounding the ducts as well as a dramatic increase in collagen deposition. Taken together, these data demonstrate a unique role for NET1 in vivo and identify for the first time a Rho


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guanine nucleotide exchange factor that is required for normal mammary gland development.

Materials and Methods Mouse husbandry and care Mice were housed in the Center for Laboratory Animal Medicine and Care within the Medical School at the University of Texas Health Science Center at Houston. All studies were approved by the Institutional Animal Care and Use Committee (protocol AWC 13– 073) and were conducted in accordance with the guidelines of the US Public Health Service Policy for Humane Care and Use of Laboratory Animals.

Generation of Net1 knockout mice To generate Net1 knockout mice, we obtained a Net1 conditional targeting vector with C57BL/6 strain background (PRPGS00153_A_F11) from the Knockout Mouse Project repository. After sequencing confirmation, the targeting vector was linearized by AsiSI digestion and electroporated into C57BL/6 embryonic stem (ES) cells by the Transgenic Mouse and Stem Cell Core facility at the University of Texas Health Science Center at Houston. Neomycin-resistant clones were screened by Southern blotting for correct targeting of the Net1 locus. For Southern blotting genomic DNA was digested using EcoRV or KpnI and probed with 5⬘-EcoRV or 3⬘-KpnI probes, respectively. The 5⬘-EcoRV and 3⬘-KpnI probes were amplified from the Net1 targeting vector using the following primers: 5⬘-EcoRV forward, 5⬘-TTGTAGGAAGGAGTCTGTCT-3⬘; 5⬘-EcoRV reverse, 5⬘-GTAAGCCGTGTAGTTAGCA-3⬘; 3⬘Kpn I forward, 5⬘-TGGCAATCAGTTCAAGTCA-3⬘; 3⬘-KpnI reverse, 5⬘-TGTATCCAGAAGATGAGCAT-3⬘. Correctly targeted ES cells were microinjected into BALB/c blastocysts to generate chimeric offspring by the Transgenic Mouse and Stem Cell Core facility. Chimeric mice were bred with wild-type C57BL/6 mice (The Jackson Laboratory) to achieve germline transmission. Offspring with a floxed Net1 allele were bred with Actin-Flpe mice [B6N.CgTg(ACTFLPe)9205Dym/CjDswJ; The Jackson Laboratory] to remove the Frt-flanked selection and LacZ cassettes and then subsequently bred with GDF9-Cre mice [STOCK Tg(Gdf9-cre)5092Coo/J, The Jackson laboratory] to delete the loxP-flanked region of Net1. Primer pairs for screening chimeric mice carrying the floxed Net1 allele were as follows: forward primer 5GF1, 5⬘-GTACACCACCTTGTCCAG-3⬘; reverse primer 3GR2, 5⬘-CTCTTATTGCTTGGCTCCT-3⬘, and reverse primer LAR3 5⬘-CACAACGGGTTCTTCTGTTAGTCC-3⬘. Primer pairs for screening Flpe-out mice were as follows: forward primer 5GF3, 5⬘-TGCTATGCTATTGCTGCTT-3⬘, and the reverse primer 3GR1, 5⬘-AGAACACCACCAAGTAACAA-3⬘. Primer pairs for screening Net1 deficient mice were as followsd: forward primer 5GF1, 5⬘TTGTTACTTGGTGGTGTTCT-3⬘ and reverse primers 3GR2, 5⬘-CTCTTATTGCTTGGCTCCT-3⬘, and TV3–1R, 5⬘-AAGTGCTAACCTTCCTGC-3⬘.


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Net1 Controls Mammary Gland Development

Antibodies The following antibodies were used: anti-Net1 (sc-271207), antivinculin (sc-25336), and anti-ER␣ (sc-543; Santa Cruz Biotechnology); anti-pSer19-MLC2 (3675), anti-pThr853-MYPT1 (4563), antimyosin light-chain phosphatase (MYPT1) (2634), anti-pT567-Ezrin (3141), antiezrin (3142), anti-Ki67 (12202), anticleaved caspase-3 (9661), and anti-E-cadherin (3195) (Cell Signaling Technology); anti-MLC2 (GTX62453) (GeneTex); antikeratin 5 (PRB-160P) (Covance); antikeratin 8 (TROMA-I; Developmental Studies Hybridoma Bank); antivimentin (550513) (BD Pharmingen); and anti-␤-actin (A5316) (Sigma-Aldrich).


diluted in blocking buffer, for 1 hour at room temperature. Sections were washed with PBS, stained with 4⬘,6-diamidino-2phenylindole to visualize DNA, and mounted on glass slides with Fluormount reagent (EMD4 Biosciences). Immunofluorescent images were visualized with a Zeiss Axiophot epifluorescence microscope and acquired using Axiovision software. Masson’s trichrome staining (Sigma-Aldrich) was performed following the manufacturer’s guidelines, and the collagen area surrounding the ducts was quantified using the ImageJ software (National Institutes of Health, Bethesda, Maryland. For the quantification of data from the images, at least five random fields per mouse per genotype were assessed.

Mammary gland staining and analysis After dissection, number four inguinal mammary glands were immediately fixed in Carnoy’s fixative (60% ethanol, 30% chloroform, and 10% glacial acetic acid) for 2– 4 hours at room temperature. Glands were stained in carmine alum solution (2 mg/mL carmine and 10.5 mM aluminum potassium sulfate dodecahydrate) overnight with gentle shaking followed by successive dehydration steps in 70%, 95%, and 100% ethanol for 1 hour each at room temperature. The glands were cleared in xylene overnight and mounted on glass slides with Permount (Thermo Fisher Scientific). The mammary glands were imaged with a SMZ-745T stereo microscope (Nikon) mounted with an Eclipse 80i digital camera (Nikon). Filled fat pat area was measured using the outline measurement tool in NIS-Elements Basic Research software (Nikon). The branch points per duct length were measured by counting the number of branch points along the length of the longest three ducts invading past the lymph node. The invasion distance was scored by measuring the distance between the lymph node and the farthest epithelial terminal end bud.

Tissue immunohistochemistry and immunofluorescence Number four inguinal mammary glands were immediately fixed in 4% paraformaldehyde overnight at 4°C and stored in 70% ethanol at 4°C until paraffin embedding. Five-micron sections were cut, deparaffinized in xylene, and rehydrated. Sections were boiled for 20 minutes in 10 mM sodium citrate for antigen retrieval, rinsed in PBS, and quenched for 30 minutes in 3% H2O2 at room temperature. Sections were blocked in 5% BSA/0.5% Tween 20 or mouse on mouse blocking buffer, (BMK2202; Vector Laboratories) for 1 hour at room temperature. Primary antibodies were diluted in blocking solution, and sections were incubated with primary antibodies overnight at 4°C. After washing five times in PBS, sections were incubated with secondary antibodies for 45 minutes at room temperature, washed in PBS, and incubated in avidin biotin complex solution (PK7100; Vector Laboratories) for 30 minutes. Sections were then developed in diaminobenzidine (K3468; Dako) and counterstained with hematoxylin (Thermo Fisher Scientific). Images were visualized with an Eclipse 80i microscope (Nikon) and acquired using NIS-Elements Basic Research software (Nikon). For immunofluorescence, after antigen retrieval, the sections were rinsed in PBS, blocked in 5% BSA/0.5% Tween 20 or M.O.M. blocking buffer for 1 hour at room temperature and incubated with the appropriate primary antibodies, diluted in blocking solution, overnight at 4°C. Sections were rinsed in PBS and incubated with Alexa Fluor 488 antimouse and Alex Fluor 594 antirabbit, or Alex Fluor 594 antirat (Life Technologies),

Mammary epithelial cell transplantation Numbers three, four, and five mammary gland pairs were dissected from five age-matched (12-week-old) wild-type and Net1 knockout mice, and the lymph nodes were removed from the number four glands. The glands were manually minced and incubated in DMEM/F12 (Hyclone) with 2 mg/mL collagenase A (10103578001; Agilent), and 1⫻ antibiotic-antimycotic (15240 – 062; Life Technologies) for 1.5 hours at 37°C with 150 rpm rotation at a 45° angle. The tissues were then shaken vigorously to loosen epithelial cells from the fat, and the cells were pelleted by centrifugation at 600 ⫻ g for 10 minutes at room temperature. Pelleted organoids were washed in washing buffer (DMEM/F12 with 5% fetal bovine serum and 1⫻ antibioticantimycotic) and centrifuged three times at 450 ⫻ g for 5 seconds. The organoids were washed once with PBS and incubated in 2 mL of 0.05% Trypsin-EDTA (Life Technologies) for 10 minutes at 37°C. The trypsin was neutralized with 8 mL of washing buffer, and the mammary epithelial cells (MECs) were passed through a 70-␮m cell strainer (22363548; Thermo Fisher Scientific). The MECs were washed once with the washing buffer and centrifuged at 450 ⫻ g for 5 minutes at room temperature. Single cells were resuspended in washing buffer, and trypan blue excluding cells were counted using a hemacytometer. MECs were diluted to a concentration of 1 ⫻ 106/mL and kept on ice. Immunocompromised 4-week-old Fox Chase SCID/Beige mice (CB17.Cg-PrkdcscidLystbg/Crl; Charles River Laboratories) were used as hosts to avoid graft rejection. Both of the number four inguinal mammary glands were cleared of endogenous epithelium by cauterization. Ten microliters of wild-type MECs (1 ⫻ 104 cells) were implanted into the cleared fat pad of one recipient gland, with an equal number of Net1-deficient MECs placed into the contralateral gland. Mammary gland outgrowths were analyzed 6 or 8 weeks after transplantation by whole-mount carmine staining. Alternatively, mammary glands were paraffin embedded, sectioned, and used for immunohistochemistry or immunofluorescence staining, as described above.

Isolation of tissues and Western blotting Mouse tissues were rinsed quickly in cold PBS, snap frozen in liquid nitrogen, and stored at ⫺80°C until use. For extraction of proteins and mRNA, frozen tissues were pulverized with a mortar and pestle under liquid nitrogen and homogenized on ice in sodium dodecyl sulfate lysis buffer for protein extraction (2% sodium dodecyl sulfate; 20 mM Tris-HCl, pH 8.0; 100 mM NaCl; 80 mM ␤-glycerophosphate; 50 mM NaF; 1 mM sodium orthovanadate; 10 ␮g/mL pepstatin A; 10 ␮g/mL leupeptin; and


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10 ␮g/mL aprotinin), or total RNA kit lysis buffer (E.Z.N.A. Total RNA Kit I; Omega Bio-Tek) for RNA extraction, using a rotor-stator homogenizer. For protein analysis, lysed tissue was sonicated and protein concentrations were determined by bicinchoninic acid assay (Pierce). Equal amounts of protein were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and analyzed by Western blotting (13).

Analysis of gene expression Total RNA was extracted from homogenized tissue with oncolumn deoxyribonuclease digestion (E.Z.N.A. Total RNA Kit I; Omega Bio-Tek) and cDNA synthesized using Moloney murine leukemia virus reverse transcriptase (Life Technologies) and random primers. The primers for mouse Net1A were 5⬘-GTGGCGCATGATGAGATCG-3⬘ (forward) and 5⬘-CATCTAAGACTCGGATCGTCCTT-3⬘ (reverse); the primers for mouse Net1 were 5⬘-CGGCGAACGAGAGATGCTC-3⬘ (forward) and 5⬘-CTCCTTCAAATCAAGGCTGCTA-3⬘ (reverse); the primers for mouse glyceraldehyde-3-phosphate dehydrogenase were 5⬘-AGGTCGGTGTGAACGGATTTG-3⬘ (forward) and 5⬘-TGTAGACCATGTAGTTGAGGTCA-3⬘ (reverse). To quantify the expression of both Net1 and Net1A mRNAs, Net1-specific quantitative PCR primers were designed toward a region encoded by exons 9 and 10 that is located in the loxPflanked deletion region. The primers were 5⬘-ACATTCTCGTGAACTGGTTA-3⬘ (forward) and 5⬘-GCTGGAGGAAGTCTTGGA-3⬘ (reverse). To quantify the expression of ER␣, progesterone receptor (PR), and their target genes, ER␣-specific quantitative PCR primers were 5⬘-CCTCCCGCCTTCTACAGGT-3⬘ (forward) and 5⬘-CACACGGCACAGTAGCGAG-3⬘ (reverse); PR-A/B primers were 5⬘-CGCAGGTTCTCCACACG TC-3⬘ (forward) and 5⬘-GATCGGTATAGGCGAGACTACA GAC-3⬘(reverse);PR-Bprimerswere5⬘-CACAGTATGGCTTTGA TTCCTTACCTC-3⬘ (forward) and 5⬘-TGCCCTCTTAAAGAA GACCTTGC-3⬘ (reverse) (25); amphiregulin primers were 5⬘GCCATTATGCAGCTGCTTTGGAGC-3⬘ (forward) and 5⬘TGTTTTTCTTGGGCTTAATCACCT-3⬘ (reverse) (26); receptor activator of nuclear factor-␬B ligand (RANKL) primers were 5⬘TTAGCATTCAGGTGTCCAACC-3⬘ (forward) and 5⬘-CGTGG GCCATGTCTCTTAGTA-3⬘ (reverse) (27) and calcitonin-related polypeptide-␣ (Calca), primers were 5⬘-GAAGAAGAAGTTCGCCTGCT-3⬘ (forward) and 5⬘-GATTCCCACACCGCTTAGAT-3⬘ (28). Relative mRNA abundance was determined by real-time PCR (Roche LightCycler 480) with SYBR green master mix (Sigma) and normalized to Gapdh. Amplification conditions included a preincubation at 95°C for 3 minutes followed by amplification of the target DNA for 45 cycles (95°C for 45 sec, 57°C for 45 sec, and 72°C for 45 sec) and fluorescence collection at 60°C.

Statistical analysis Unpaired, two-tailed Student t tests were used for all statistical tests. Values of P ⬍ .05 were considered significant, as indicated in figure legends.

Results Generation of Net1 knockout mice Because the tissue distribution of Net1 expression has not been assessed in the mouse, we assayed for the expres-


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sion of Net1 isoforms by real-time PCR and Western blotting. Previously Net1 was shown by Northern analysis to be highly expressed in human placenta, with lower amounts in the brain, lungs, liver, and muscle. However, this study did not distinguish between Net1 isoforms (29). Using real-time PCR to test for Net1 isoform expression in tissues from adult C57BL/6 mice, we observed that the longer Net1 isoform had a distinct distribution from Net1A, with the highest levels in the testis and lower levels in many other tissues. The Net1A isoform was expressed at higher levels than Net1 in many tissues, with the notable exception of the testis. Both Net1 and Net1A were poorly expressed in the brain, heart, and skeletal muscle (Figure 1, A and B). Interestingly, Western blotting of whole tissue extracts showed a distribution of NET1 proteins that did not exactly match that observed for the mRNA (Figure 1C). This may reflect posttranslational regulation of NET1 stability, which has been observed in cultured cell lines (30, 31). Importantly, Western blotting results indicated a high expression of NET1 in estrogenresponsive tissues such as the mammary gland, uterus, and ovaries (Figure 1C). To generate a targeted deletion of the Net1 gene in mice, a Net1 targeting vector with loxP sites flanking exons 4 –10 of the Net1 gene was obtained from the Knockout Mouse Project consortium. This region encodes the catalytic DH domain and a portion of PH domain, both of which are common to Net1 and Net1A (Figure 1D). Exons 1–3 of Net1 and exon 1 of Net1A, which are not targeted and encode polypeptides of 85 and 31 amino acids, respectively, are not likely to produce stable proteins when expressed in vivo because their expression is undetectable when transfected into cultured cells (Frost, J. A., unpublished observations). Thus, this deletion strategy would be expected to completely eliminate expression of Net1 isoforms. The targeting vector was electroporated into C57BL/6 ES cells, and a correctly recombined ES cell clone was microinjected into BALB/c blastocysts to generate chimeric mice. Net1 null mice were subsequently produced by breeding with ActinFlpe transgenic mice to remove the neomycin and LacZ cassettes and then to Gdf9-Cre transgenic mice to generate a whole-body deletion of the loxP-flanked region. PCR genotyping confirmed the deletion of Net1 (Figure 1E), and the loss of Net1 and Net1A mRNA expression in the spleen and mammary gland was confirmed by real-time PCR (Figure 1F). Loss of NET1 protein expression in the mammary gland was confirmed by Western blotting (Figure 1G). Characterization of mammary gland development in Net1-deficient mice Net1-deficient mice were born in the expected Mendelian ratio and were able to nurse their young, indicating


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continues until adulthood at 12 weeks (3). During this time the ductal epithelial cells invade the mammary adipose tissue through a process of ductal extension and branching, ultimately filling the entire fat pad. Thus, we examined mammary gland development in wild-type and Net1 knockout mice at 6, 8, 10, and 12 weeks of age. Mice were not assessed for the stage of estrus, and six mice per genotype were analyzed. This analysis showed that Net1 deficiency slows mammary gland development (Figure 2). At 6 weeks of age, the mammary glands in Net1-deficient mice exhibited significantly less fat pad filling with reduced ductal branching and diminished fat pad invasion (Figure 2). By 12 weeks of age, the degree of fat pad invasion and filling in Net1 knockout mice was indistinguishable from wild-type mice. However, there was still a significant defect in the degree of ductal branching (Figure 2, B–D). These data indicate that the loss of NET1 expression compromises mammary gland development, with the most profound effect FIGURE 1. Creation of Net1 deficient mice. A and B, Analysis of Net1 and Net1A mRNA expression in C57BL/6 adult mouse tissues by real-time PCR. Values are normalized to Gapdh on ductal branching. expression and are the average of three organs per tissue. Errors are SEM. C, Analysis of NET1 Mammary gland branching morprotein expression in C57BL/6 adult mouse tissues by Western blotting. Shown are representative phogenesis requires collective epitheblots from two mice per organ. Bl, bladder; C Br, brain;, colon; Ep, epididymis; H, heart; K, lial cell migration and invasion of the kidney; Li, liver; Lu, lung; MG, mammary gland; O, ovary; Sp, spleen; St, stomach; SV, seminal vesicle; U, uterus. D, Exon structure of the mouse Net1 gene and sites of inserted loxP sites. fat pad (8) and collective epithelial cell Alternate Net1 and Net1A transcription start sites are shown. E, PCR genotyping of Net1⫹/⫹, motility requires Rac1 and RhoA ac⫹/⫺ ⫺/⫺ Net1 , and Net1 mice. Shown is a representative example. F, Real-time PCR analysis of tivation (33, 34). Importantly, we Net1 and Net1A mRNA in mammary gland and spleen from Net1⫹/⫹, Net1⫹/⫺, and Net1⫺/⫺ mice. PCR primers are designed to a region common to both Net1 isoforms. Results are have shown previously that the normalized to Gapdh expression. Shown is a representative analysis from three independent Net1A isoform contributes to human experiments. Errors are SEM. G, Western blotting of NET1 and vinculin expression in mammary breast cancer cell adhesion and motilglands from Net1⫹/⫹ and Net1⫺/⫺ mice. Shown is a representative experiment from three independent experiments. ity, in part through the regulation of RhoA-dependent myosin light-chain that Net1 is not essential for the formation of a functional phosphorylation (13, 22). Thus, we examined whether mymammary gland. However, many genes that contribute to osin light-chain phosphorylation was compromised in Net1 mammary gland development exhibit initial developmen- knockout mice. In wild-type mice, we observed robust staintal defects that are overcome later in adulthood or during ing for myosin light-chain phosphorylated on its activating pregnancy (32). To determine whether Net1 contributes site, serine 19, both within the duct and in the surrounding to mammary gland development in pubertal mice, we adipose tissue. However, in Net1-deficient mice, myosin collected the inguinal mammary glands from mice at dif- light-chain phosphorylation was significantly attenuated ferent ages and assessed ductal tree formation by whole- (Figure 3A). Phosphorylation of the myosin light-chain mount analysis. Mammary gland development in phosphatase regulatory subunit MYPT1, which is regulated C57BL/6 mice normally begins at 3 weeks of age and by RhoA, also showed a significant decrease in the Net1-


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gether, these data indicate that NET1 is required for optimal phosphorylation of RhoA targets required for actomyosin contraction in the mammary gland. Because cell motility is necessary for mammary gland development, the reduced phosphorylation of MLC2 and MYPT1 may contribute to the impaired fat pad invasion and branching observed in Net1-deficient mice. Net1 deletion inhibits mammary epithelial cell proliferation, ER␣ expression, and transcription of ER␣ target genes Ductal epithelial cells invade the mammary fat pad through a structure called the TEB, and proliferation of epithelial cells within the TEB is an essential aspect of the elongation and branching mechanisms (35). We have previously obFIGURE 2. Whole-mount analysis of developing mammary glands in wild-type and Net1served that Net1 is required for mideficient mice. A, Whole mounts of inguinal mammary glands from 6-week-old Net1⫹/⫹ and totic progression (24), and others ⫺/⫺ Net1 mice. Shown are representative images (n ⫽ 6 mice/genotype). Scale bar, 1000 ␮m. B, have found that Net1 contributes to Whole mounts of inguinal mammary glands from 12-week-old mice. Shown are representative images (n ⫽ 6 mice/genotype). Scale bar, 1000 ␮m. C, Quantification of percent fat pad filling MCF7 breast cancer cell proliferaby the ductal tree at 6 and 12 weeks in Net1⫹/⫹ and Net1⫺/⫺ mice. Averages are from six mice tion (23). Thus, we examined per genotype per age. Errors are SEM. **, P ⬍ .01. D, Quantification of number of branch points whether deletion of Net1 affected per duct length. Averages are from six mice for each age. Errors are SEM. **, P ⬍ .01. E, Quantification of duct invasion past the lymph node. Averages are from six mice for each age. proliferation within TEBs and maErrors are SEM. **, P ⬍ .01. ture ducts by testing for expression of the proliferative marker Ki67. We deficient mammary glands (Figure 3B). Interestingly, phosobserved that wild-type mice exhibited a high percentphorylation of EZRIN, which is also a RhoA target, was not age of proliferating cells within TEB structures at 6 affected in Net1-deficient mice. This is consistent with the weeks of age. However, there was a dramatic reduction observed role of Net1 in controlling only a subset of RhoA in the number of Ki67-positive cells in the TEBs of signaling events human breast cancer cells (14). Taken toNet1-deficient mice (Figure 4, A and C). In ducts that had completed development at 6 weeks, there was little difference between wild-type and Net1-deficient mice, indicating that NET1 was not required for maintenance of ductal integrity (Figure 4, B and D). By 12 weeks of age, proliferation had largely ceased in the mammary epithelial cell population of wildFIGURE 3. Deletion of Net1 attenuates phosphorylation of the Rho kinase substrates MLC2 and type mice because by this time the MYPT1. A, Immunohistochemistry for phosphorylated myosin light-chain 2 (pMLC2) in Net1⫹/⫹ and Net1⫺/⫺ inguinal mammary glands. Samples were counterstained with hematoxylin. Shown mammary ductal tree was largely are representative fields. Mammary glands from three mice per genotype were analyzed. Scale complete. However, in Net1-defibars, 50 ␮m. B, Phosphorylation of the regulatory subunit of myosin light-chain phosphatase cient animals, there was still a sig(pMYPT1) and EZRIN (pEZRIN) in Net1⫹/⫹ and Net1⫺/⫺ mouse mammary glands. Representative Western blots are shown. Mammary glands from three mice per genotype were analyzed. nificant degree of proliferation in


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deficient mammary glands. We observed that loss of NET1 expression resulted in a striking reduction in the number of ductal epithelial cells expressing ER␣ protein in 6-week-old mice (Figure 5A). Attenuated ER␣ expression was maintained at 12 weeks (Figure 5B). Using real-time PCR, we observed a trend toward reduced ER␣ mRNA expression at 6 weeks of age and a statistically significant reduction at 12 weeks, indicating that the loss of ER␣ protein was at least partially due to reduced ER␣ transcription (Figure 5, C and D). Consistent with this finding, we observed reduced mRNA expression of the ER␣ target gene Cyclin D1- in Net1-deficient mammary glands at both 6 and 12 weeks of age (Figure 5, C and D). There was also a trend toward reduced expression of the ER␣ target gene Progesterone receptor (isoforms A and B) (PR-A/B) at both 6 and 12 weeks that did not reach statistical significance (Figure 5, C–E). Similarly, there was a trend toward a reduced expression of the PR target genes Amphiregulin (Areg), Calcitonin related polypeptide alpha (Calca), and RANKL in mammary glands from 12-week animals, but this also did not FIGURE 4. Net1 deletion impairs mammary epithelial cell proliferation. A, reach statistical significance (Figure Immunohistochemistry for the proliferation marker Ki67 in mammary gland TEBs from 6-week5E). These data indicate that ER␣ exold Net1⫹/⫹ and Net1⫺/⫺ mice. Tissues were counterstained with hematoxylin. Shown are pression is impaired in Net1-deficient representative fields. Three mice per genotype were analyzed. Scale bars, 50 ␮m. B, mice. Moreover, this adversely imImmunohistochemistry for Ki67 in mammary gland ducts from 6-week-old Net1⫹/⫹ and Net1⫺/⫺ mice. Tissues were counterstained with hematoxylin. Shown are representative fields. Three mice pacts the expression of ER␣ target per genotype were analyzed. Scale bars, 50 ␮m. C and D, Quantification of Ki67-positive genes required for proliferation, such epithelial cells in TEBs and mature ducts in mammary glands from 6-week-old (n ⫽ 4) and 12as Cyclin D1 and may also reduce the week-old (n ⫽ 3) mice. Shown is the average percent of Ki67-positive cells. Errors are SEM. ***, P ⬍ .001. expression of genes required for ductal branching, such as PR and its the remaining TEBs as well as in the mature ducts, downstream transcriptional targets. indicating that development of the ductal tree was ongoing (Figure 4, C and D). Importantly, there was no Net1 deletion causes mammary gland difference between wild-type and Net1-deficient mice disorganization and elevated collagen deposition in staining for the apoptotic marker cleaved caspase 3, Mammary gland ducts are composed of a single outer indicating that Net1 deletion did not alter the rate of layer of myoepithelial cells surrounding an inner layer of apoptosis in this tissue (not shown). luminal epithelial cells. To assess whether Net1 deletion A major regulator of mammary epithelial cell prolifera- affected ductal organization, we costained mammary tion during mammary gland development is the ER␣ (36). gland sections for the myoepithelial marker CYTOKERTo determine whether Net1 deletion affected ER␣ function, ATIN 5 and luminal epithelial cell marker CYTOKERAwe tested for altered ER␣ expression and signaling in Net1- TIN 8 (37). Wild-type mammary gland ducts exhibited a


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FIGURE 5. Net1 deletion inhibits mammary gland ER␣ expression and function. A, Immunohistochemistry for ER␣ expression in mammary glands from 6-week-old Net1⫹/⫹ and Net1⫺/⫺ mice. Shown are representative fields. Three mice per genotype were analyzed. Scale bars, 50 ␮m. B, Western blot of ER␣ expression in 12-week-old Net1⫹/⫹ and Net1⫺/⫺ mice. Expression of vinculin was used as a loading control. C, Quantification of ER␣, progesterone receptor isoforms A and B (PR-A/B), and cyclin D1 mRNA expression by real-time PCR in mammary glands from 6-week-old mice. Shown is the average from six mice per genotype. Errors are SEM. **, P ⬍ .01. D, Quantification of ER␣, PR-A/B, and cyclin D1 mRNA expression in mammary glands from 12-week-old mice. Shown is the average from four mice per genotype. Errors are SEM. *, P ⬍ .05. E, Quantification of progesterone receptor isoform B (PR-B), amphiregulin (Areg), calcitonin-related polypeptide alpha (Calca), and RANK ligand (RANKL) mRNA expression in mammary glands in 12-week-old mice. Shown is the average expression from four mice per genotype.

normal, one- to two-layer-thick luminal epithelium surrounded by a single, continuous myoepithelial cell layer. However, loss of Net1 led to a disorganization of the myoepithelial and luminal epithelial cells that became progressively worse with age. By 12 weeks of age, focal regions of disorganization were observed with both the myoepithelial and luminal epithelial cells stacked in multiple layers (Figure 6, A and B). Moreover, in these regions of disorganization, there was a noticeable intermingling of the myoepithelial and luminal epithelial cells. The constituents and abundance of the extracellular matrix can profoundly affect ductal organization within the mammary gland (38). Moreover, increased deposition of collagen has been shown to promote mammary gland tumorigenesis in mice and correlates with tumor progression in humans (39 – 42). Thus, we tested whether collagen deposition was altered in Net1-deficient mice using Masson’s trichrome stain. We observed that there was significantly more collagen deposition surrounding mammary gland ducts in Net1-deficient mice (Figure 6, C and D). This increase in collagen deposition was maintained throughout mammary gland development (Figure 6D). Notably, there was also a significant increase in cellularity surrounding mammary ducts in Net1 knockout mice (Figure 6C). This is significant as stromal fibroblasts are major regulators of collagen deposition. Disorganization of a

structured epithelium is often caused by an epithelial-tomesenchymal transition (EMT) (43, 44). To ascertain whether this had occurred in Net1-deficient mice, we stained cells for the epithelial marker E-cadherin and the mesenchymal marker vimentin. However, we did not observe significant changes in E-cadherin or in vimentin expression in the ductal epithelial cells, indicating that a canonical EMT had not taken place (Figure 6, E and F). Defects in ductal branching and organization are cell autonomous Estrogen and progesterone secreted by the ovaries, as well as GH secreted by the pituitary gland, function as master regulators of mammary gland development during puberty, whereas growth factors produced locally by the mammary epithelium and stroma fine-tune the developmental process (3, 32, 45). Because we had deleted Net1 from the entire animal, it was possible that the phenotypes we observed were due to effects on tissues apart from the mammary gland. To understand the extent to which phenotypes observed in Net1-deficient mice were cell autonomous, we performed MEC transplants into immunocompromised mice with normal endocrine function. Mammary epithelial cells were isolated from adult, age-matched wild-type and Net1 knockout mice and injected into the cleared inguinal fat pads of immunocom-


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FIGURE 6. Net1 deletion results in disorganization of mature mammary gland ducts and elevated periductal collagen deposition. A and B, Fluorescence microscopy of paraffin-embedded mammary gland sections from 6- or 12-week-old mice. Sections were stained for the myoepithelial marker cytokeratin 5 (green), the luminal epithelial marker cytokeratin 8 (red), and DNA (blue). Shown are representative images from at least three mice per genotype per age. Scale bars, 50 ␮m. C, Masson’s trichrome staining of mammary glands from 12week-old mice. Collagen is stained in blue. Shown are representative images. Three mice per genotype were analyzed. Scale bars, 50 ␮m. D, Quantification of collagen staining area surrounding ducts in Net1⫹/⫹ and Net1⫺/⫺ mice. Shown are the averages from three mice per genotype per age. Errors are SEM. ***, P ⬍ .001. E, E-cadherin (red) and cytokeratin 8 (CK8, green) staining of mammary glands from Net1⫹/⫹ and Net1⫺/⫺ mice. Shown are representative images. Three mice per genotype were analyzed. Scale bars, 50 ␮m. F, vimentin (green) and cytokeratin 5 (CK5, red) staining of mammary glands from Net1⫹/⫹ and Net1⫺/⫺ mice. Shown are representative images. Three mice per genotype were analyzed. Scale bars, 50 ␮m.

promised SCID/Beige mice. Six or eight weeks later, the mammary glands were harvested and analyzed. In wildtype MEC transplants, expansion of the ductal tree was nearly complete at 6 weeks and had terminated by 8 weeks. In the Net1-deficient MEC transplants, fat pad filling was nearly the same as in wild-type transplants (Figure 7, A and B), indicating that the delayed fat pad filling observed in Net1 knockout mice was due to factors extrinsic to the MECs. On the other hand, branching morphogenesis was significantly reduced in the Net1 deficient transplants at both time points tested (Figure 7A, C), indicating that this was a cell autonomous effect.


FIGURE 7. Net1-deficient mammary epithelial cell transplants exhibit reduced ductal branching. A, MECs were harvested from 4-week-old Net1⫹/⫹ and Net1⫺/⫺ mice and transplanted into the cleared inguinal mammary fat pads of 4-week-old SCID Beige immunocompromised mice. Six or 8 weeks later, the reconstituted mammary glands were assessed by whole-mount analysis. Shown are representative mammary glands 6 weeks after MEC transplant. Scale bars, 1000 ␮m. B and C, Quantification of percentage ductal fat pad filling and branch points per duct length in Net1⫹/⫹ and Net1⫺/⫺ mice. Six mice per genotype were analyzed in 6-week transplants; three per genotype were analyzed in 8-week transplants. Errors are SEM. *, P ⬍ .05.

When we tested for Ki67 and ER␣ expression, we found that mammary glands derived from Net1-deficient MECs exhibited dramatically fewer proliferating cells (Figure 8, A and B) and also expressed less ER␣ protein (Figure 8C). Moreover, similar to the phenotype in Net1 knockout mice, MLC phosphorylation was also significantly reduced in the Net1-deficient MEC transplants (Figure 8D). The reduction in ER␣ expression and ductal epithelial cell proliferation in the Net1-deficient transplants was somewhat surprising because these events normally contribute to the rate of fat pad filling. This may indicate that additional, stromal cell input to ductal elongation was compromised in Net1-deficient mice. Unfortunately, we were unable to determine whether Net1 is expressed in the mammary gland stroma because an antibody suitable for immunohistochemical detection of NET1 in mouse tissues was unavailable. We then examined whether ductal organization was compromised in Net1-deficient transplants. Intriguingly, we again observed a striking disorganization of the luminal epithelial and myoepithelial layers that was comparable with that observed in Net1 knockout mice (Figure 9A). Similarly, Net1-deficient MEC transplants also exhibited increased collagen deposition surrounding the ducts (Figure 9, B and C). Taken together, these data indicate that most the phenotypes observed in Net1-defi-


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tonomous because they are recapitulated in a mammary epithelial cell transplant model with normal hormonal and pituitary function. Net1 is an important regulator of RhoA signaling in breast cancer cells, in which it controls focal adhesion formation and myosin lightchain phosphorylation during cell adhesion and motility (13, 22). Significantly, we found that Net1 deletion results in reduced phosphorylation of MLC2 and the regulatory subunit of myosin phosphatase MYPT1. Thus, these data indicate that Net1 contributes to RhoA-dependent actomyosin contraction in vivo as well as in isolated cells. Because RhoA signals to many intracellular effectors (46), it is intriguing that inhibition of Net1 expression in breast cancer cells as well as in vivo FIGURE 8. Net1-deficient MEC transplants exhibit reduced proliferation, ER␣ expression, and has such a specific effect. This imMLC2 phosphorylation. A, Immunohistochemistry for Ki67 expression in 6-week mammary gland plies that particular RhoA guanidine transplants from Net1⫹/⫹ and Net1⫺/⫺ MECs. Shown are representative images. Three mammary glands per genotype were analyzed. Scale bars, 50 ␮m. B, Quantification of Ki67 staining in nucleotide exchange factors (GEFs) ductal epithelial cells from 6-week MEC transplants. Shown is the average from three transplants may be dedicated to specific RhoA per genotype. Errors are SEM. ***, P ⬍ .001. C, Immunohistochemistry for ER␣ expression in ⫹/⫹ ⫺/⫺ signaling pathways. Because actomymammary glands from Net1 - and Net1 -deficient MEC transplants at 6 weeks. Shown are representative images from three transplants per genotype. Scale bars, 50 ␮m. D, osin contraction is an essential aspect Immunohistochemistry for pMLC2 in mammary glands from Net1⫹/⫹- and Net1⫺/⫺-deficient of cell motility, loss of contractility in MEC transplants at 6 weeks. Shown are representative images from three transplants per Net1-deleted mammary epithelial genotype. Scale bars, 50 ␮m. cells may contribute to the delay in mammary gland development. cient mice, with the notable exception of reduced fat pad Previous studies addressing the role of RhoA in mamfilling, are recapitulated in the Net1 deficient MEC mary gland development have focused on the RhoA inactransplants. tivating genes p190A- and p190B-RhoGAP (10 –12, 47). Deletion of one allele of p190B-RhoGAP, which would be expected to increase RhoA signaling, delayed mamDiscussion mary gland development and was associated with rePrevious studies have demonstrated that Net1 is required duced proliferation within TEBs and decreased expresfor breast cancer cell motility, extracellular matrix invasion of the insulin receptor substrates insulin receptor sion, and proliferation (13, 22, 24). Moreover, Net1 is substrate-1 and -2 (11). Alternatively, overexpression of overexpressed in breast cancer and may contribute to de- p190B-RhoGAP in the mammary epithelium, which creased metastasis-free survival (19, 23). However, the should decrease RhoA signaling, also produced a delay in role of Net1 in normal mammary gland development and mammary gland development. However, this defect was function has not been described. To understand the con- characterized by decreased ductal elongation, increased tribution of Net1 to mammary gland development, we ductal branching, and disorganization of the myoepithehave generated a conditional Net1 knockout mouse lial and luminal epithelial layers. It was also associated model. Using this model, we show that the Net1 deletion with increased collagen deposition in the stroma (12). slows mammary gland development by inhibiting ductal Surprisingly, the mammary gland phenotypes of Net1extension and branching and leads to disorganization of deficient mice, which have reduced RhoA signaling, reepithelial architecture and increased periductal collagen semble aspects of both models. This is difficult to recondeposition. Moreover, most these phenotypes are cell au- cile unless one considers that some of the Net1 or p190-


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FIGURE 9. Ductal disorganization and excess collagen deposition in Net1-deficient MEC mammary gland transplants. A, Immunofluorescence for cytokeratin 5 (green), cytokeratin 8 (red), and DNA (blue) in Net1⫹/⫹ and Net1⫺/⫺-deficient MEC transplants. Shown are representative images from 6-week-old transplants. Three mice per genotype were analyzed. Scale bars, 50 ␮m. B, Masson’s trichrome staining of Net1⫹/⫹ and Net1⫺/⫺ mammary gland MEC transplants. Shown are representative images from 6-week-old transplants. Three mice per genotype were analyzed. Scale bars, 50 ␮m. C, Quantification of collagen staining area from wild-type and Net1deficient MEC transplants at 6 weeks. Shown is the collagen average staining area for three mice per genotype. Errors are SEM. ***, P ⬍ .001.

RhoGAP knockout phenotypes may due to mechanisms independent of effects on RhoA activity, as suggested by cell culture and biochemical studies. For example, we have recently shown that Net1 contributes to mitotic progression in a RhoA-independent manner (24). Moreover, the p190-RhoGAP proteins function as GTPase activating proteins (GAPs) for Rac1 and Cdc42 in addition to RhoA (48, 49). Thus, it is possible that the phenotypes of Net1 or p190A/B-RhoGAP transgenic mice are complicated by effects on signaling pathways other than those regulated by RhoA. The finding that Net1 deletion compromised ER␣ expression and signaling was unexpected, because RhoA has not been shown previously to control ER␣ expression or activity. Nevertheless, this effect is likely to contribute to the observed delay in mammary gland development because estrogen stimulates cell proliferation within TEBs, and ER␣ expression is essential for mammary gland development (1, 3, 36, 50). The apparent disconnect between reduced Cyclin D1 expression in Net1-deficient mammary glands and the continued proliferation of mammary epithelial cells at 12 weeks may reflect the different assays used to assess these readouts because Cyclin D1 expression was assessed in the mammary gland as a whole, whereas Ki67 staining was assessed in individual epithelial cells. It likely also reflects the complexity of ER␣ function during mammary gland development,


which drives mammary epithelial cell proliferation by eliciting the secretion of multiple mitogens from surrounding stromal cells (3). Thus, it is possible that the reduction in Cyclin D1 expression results from altered paracrine signaling in stromal as well as epithelial cells that we have not measured. An intriguing feature of mammary gland development in Net1-deficient mice is the progressive disorganization of the myoepithelial and luminal epithelial cells within mature ducts. This phenotype is reminiscent of the hyperplastic ducts observed in mammary glands overexpressing p190B-RhoGAP (12). The disorganization in Net1deficient animals is unlikely to be due to a classic EMT because we did not observe alterations in E-cadherin or vimentin expression in the ducts. Nor did we find evidence of Mothers against DPP homolog 2 (Smad2) phosphorylation (not shown), suggesting that TGF␤ signaling was not elevated. However, EMT is a general term for a wide range of phenotypes (43, 51), and there is some evidence to suggest that Net1 proteins may repress the ability of cells to switch to a mesenchymal-like state. For instance, Net1A is degraded in gastrulating chick embryo epithelial cells, which undergo an EMT-like transition. Moreover, forced expression of Net1A blocks this process, suggesting that Net1A may repress EMT in vivo (52). Furthermore, we have observed that Net1A knockdown in human breast cancer cells switches their morphology to a more mesenchymal phenotype during extracellular matrix invasion characterized by the activation of ␤1-integrin and high levels of matrix metalloproteinase 14 (MT1-MMP) expression (13). These data would support a role for Net1A in repressing EMT-like transitions, and it is possible that an atypical EMT process has occurred in the areas of ductal disorganization in Net1deficient mice. The elevated collagen deposition in in Net1-deficient mammary glands may also contribute to ductal disorganization because increased matrix density has been shown to cause disorganization and aberrant proliferation of breast epithelial cells in vitro. For example, increased collagen density causes a disorganization of mammary acini in vitro by stimulating integrin activation (53). Increased collagen density also promotes cell proliferation and motility (54, 55), which in the Net1-deficient mouse may result in local overgrowth and disorganization of the luminal and myoepithelial cells. Altered collagen deposition may also affect branching morphogenesis in the developing mammary gland because migrating mammary epithelial cells invade the fat pad by following collagen tracks and arrange these tracks through RhoA-dependent Rho kinase activation (56).


doi: 10.1210/me.2014-1128

In summary, our data indicate that NET1 is an important regulator of mammary gland development in the mouse. Net1 deletion delays mammary gland development and inhibits branching morphogenesis, with a specific loss of ER␣ expression and activity. It also results in disorganization of the luminal and myoepithelial cells in early adulthood. It will be interesting in the future to assess whether this disorganization increases with age or after mammary gland remodeling that accompanies pregnancy and involution. In addition, because Net1 has been shown to control breast cancer cell motility and proliferation in vitro, it will be important in future work to assess its contribution to breast tumorigenesis and metastasis.


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Acknowledgments We thank Jeff Rosen (Baylor College of Medicine) and members of his laboratory for their time and extremely generous help. We also thank Eun Hyeon Song for the kind help in the mouse genotyping and colony maintenance. In addition, we thank the members of the Dessauer, Clark, Denicourt, and Frost laboratories for their helpful suggestions during the laboratory meetings.

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Address all correspondence and requests for reprints to: Jeffrey A. Frost, PhD, Associate Professor, University of Texas Health Science Center at Houston, Department of Integrative Biology and Pharmacology, 6431 Fannin Street, Houston, TX 77030. E-mail: [email protected]. This work was supported by Grant CA116356 from the National Cancer Institute and Cancer Prevention and Research Institute of Texas Grant RP100502 (both to J.A.F.). Disclosure Summary: The authors have nothing to declare.

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The long noncoding RNA Neat1 is required for mammary gland development and lactation.

securin is required for the branching morphogenesis of the mammary gland and suppresses mammary tumorigenesis.

Epithelial Xbp1 is required for cellular proliferation and differentiation during mammary gland development.

Mammary gland specific knockdown of the physiological surge in Cx26 during lactation retains normal mammary gland development and function.

Microbial colonization is required for normal neurobehavioral development in zebrafish.

Oligopeptidase A is required for normal phage P22 development.

Progesterone action in normal mouse mammary gland.

The RhoGEF GEF-H1 is required for oncogenic RAS signaling via KSR-1.

PTEN is required to maintain luminal epithelial homeostasis and integrity in the adult mammary gland.

Actin filament-associated protein 1 is required for cSrc activity and secretory activation in the lactating mammary gland.

MicroRNAs in the development and neoplasia of the mammary gland.

SIRT6 is required for normal retinal function.

Steroid metabolism in normal mammary gland and in the dimethylbenzanthracene-induced mammary tumor of rats.

Alix is required during development for normal growth of the mouse brain.

The brain is required for normal muscle and nerve patterning during early Xenopus development.

Cerebral Cortex Expression of Gli3 Is Required for Normal Development of the Lateral Olfactory Tract.

Coordinated activity of Spry1 and Spry2 is required for normal development of the external genitalia.

Stromal matrix metalloproteinase-11 is involved in the mammary gland postnatal development.

EGF receptor regulation in normal mouse mammary gland.

CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation.

Prolactin stimulation of alpha-lactalbumin in normal primate mammary gland.

The mammary gland.

Peroxisomal membrane channel Pxmp2 in the mammary fat pad is essential for stromal lipid homeostasis and for development of mammary gland epithelium in mice.

The Deleted in Liver Cancer 1 (Dlc1) tumor suppressor is haploinsufficient for mammary gland development and epithelial cell polarity.