MMTV RNA packaging requires an extended long-range interaction for productive Gag binding to packaging signals.

The packaging of genomic RNA (gRNA) into retroviral particles relies on the specific recognition by the Gag precursor of packaging signals (Psi), which maintain a complex secondary structure through long-range interactions (LRIs). However, it remains unclear whether the binding of Gag to Psi alone is enough to promote RNA packaging and what role LRIs play in this process. Using mouse mammary tumor virus (MMTV), we investigated the effects of mutations in 4 proposed LRIs on gRNA structure and function. Our findings revealed the presence of an unsuspected extended LRI, and hSHAPE revealed that maintaining a wild-type-like Psi structure is crucial for efficient packaging. Surprisingly, filter-binding assays demonstrated that most mutants, regardless of their packaging capability, exhibited significant binding to Pr77Gag, suggesting that Gag binding to Psi is insufficient for efficient packaging. Footprinting experiments indicated that efficient RNA packaging is promoted when Pr77Gag binds to 2 specific sites within Psi, whereas binding elsewhere in Psi does not lead to efficient packaging. Taken together, our results suggest that the 3D structure of the Psi/Pr77Gag complex regulates the assembly of viral particles around gRNA, enabling effective discrimination against other viral and cellular RNAs that may also bind Gag efficiently.

B-to-A transition in target DNA during retroviral integration.

Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of tDNA recognition during integration, we have solved the 3.5 Å resolution cryo-EM structure of the mouse mammary tumor virus (MMTV) strand transfer complex (STC) intasome. The tDNA adopts an A-like conformation in the region encompassing the sites of vDNA joining, which exposes the sugar-phosphate backbone for IN-mediated strand transfer. Examination of existing retroviral STC structures revealed conservation of A-form tDNA in the analogous regions of these complexes. Furthermore, analyses of sequence preferences in genomic integration sites selectively targeted by six different retroviruses highlighted consistent propensity for A-philic sequences at the sites of vDNA joining. Our structure additionally revealed several novel MMTV IN-DNA interactions, as well as contacts seen in prior STC structures, including conserved Pro125 and Tyr149 residues interacting with tDNA. In infected cells, Pro125 substitutions impacted the global pattern of MMTV integration without significantly altering local base sequence preferences at vDNA insertion sites. Collectively, these data advance our understanding of retroviral intasome structure and function, as well as factors that influence patterns of vDNA integration in genomic DNA.

A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag.

Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem-loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.

Unconventional p97/VCP-Mediated Endoplasmic Reticulum-to-Endosome Trafficking of a Retroviral Protein.

Mouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the endoplasmic reticulum (ER) membrane into a functional N-terminal signal peptide (SP) and the C terminus (Rem-CT). Rem-CT lacks a membrane-spanning domain and a known ER retention signal, and yet it was not detectably secreted into cell supernatants. Inhibition of intracellular trafficking by the drug brefeldin A (BFA), which interferes with the ER-to-Golgi secretory pathway, resulted in dramatically reduced intracellular Rem-CT levels that were not rescued by proteasomal or lysosomal inhibitors. A Rem mutant lacking glycosylation was cleaved into SP and Rem-CT but was insensitive to BFA, suggesting that unglycosylated Rem-CT does not reach this BFA-dependent compartment. Treatment with endoglycosidase H indicated that Rem-CT does not traffic through the Golgi apparatus. Analysis of wild-type Rem-CT and its glycosylation mutant by confocal microscopy revealed that both were primarily localized to the ER lumen. A small fraction of wild-type Rem-CT, but not the unglycosylated mutant, was colocalized with Rab5-positive (Rab5+) early endosomes. The expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (containing a mutation of threonine to asparagine at position 31 [T31N]) mimicked the effects of BFA by reducing Rem-CT levels and increased Rem-CT association with early and late endosomes. Inhibition of the AAA ATPase p97/VCP rescued Rem-CT in the presence of BFA or DN Arf1 and prevented localization to Rab5+ endosomes. Thus, Rem-CT uses an unconventional p97-mediated scheme for trafficking to early endosomes. IMPORTANCE Mouse mammary tumor virus is a complex retrovirus that encodes a regulatory/accessory protein, Rem. Rem is a precursor protein that is processed at the endoplasmic reticulum (ER) membrane by signal peptidase. The N-terminal SP uses the p97/VCP ATPase to elude ER-associated degradation to traffic to the nucleus and serve a human immunodeficiency virus Rev-like function. In contrast, the function of the C-terminal glycosylated cleavage product (Rem-CT) is unknown. Since localization is critical for protein function, we used mutants, inhibitors, and confocal microscopy to localize Rem-CT. Surprisingly, Rem-CT, which lacks a transmembrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation and the p97 ATPase for early endosome trafficking without passage through the Golgi apparatus. Thus, Rem-CT uses a novel intracellular trafficking pathway, potentially impacting host antiviral immunity.

Effects of two types of energy restriction on methylation levels of adiponectin receptor 1 and leptin receptor overlapping transcript in a mouse mammary tumour virus-transforming growth factor-α breast cancer mouse model.

The role of adiponectin and leptin signalling pathways has been suggested to play important roles in the protective effects of energy restriction (ER) on mammary tumour (MT) development. To study the effects of ER on the methylation levels in adiponectin receptor 1 (AdipoR1) and leptin receptor overlapping transcript (Leprot) genes using the pyrosequencing method in mammary fat pad tissue, mouse mammary tumour virus-transforming growth factor-α (MMTV-TGF-α) female mice were randomly assigned to ad libitum (AL), chronic ER (CER, 15 % ER) or intermittent ER (3 weeks AL and 1 week 60 % ER in cyclic periods) groups at 10 weeks of age until 82 weeks of age. The methylation levels of AdipoR1 in the CER group were higher than those in the AL group at week 49/50 (P < 0·05), while the levels of methylation for AdipoR1 and Leprot genes were similar among the other groups. Also, the methylation levels at CpG2 and CpG3 regions of the promoter region of the AdipoR1 gene in the CER group were three times higher (P < 0·05), while CpG1 island of Leprot methylation was significantly lower compared with the other groups (P < 0·05). Adiponectin and leptin gene expression levels were consistent with the methylation levels. We also observed a change with ageing in methylation levels of these genes. These results indicate that different types of ER modify methylation levels of AdipoR1 and Leprot in different ways and CER had a more significant effect on methylation levels of both genes. Epigenetic regulation of these genes may play important roles in the preventive effects of ER against MT development and ageing processes.

An essential role of CBL and CBL-B ubiquitin ligases in mammary stem cell maintenance.

The ubiquitin ligases CBL and CBL-B are negative regulators of tyrosine kinase signaling with established roles in the immune system. However, their physiological roles in epithelial tissues are unknown. Here, we used MMTV-Cre-mediated Cbl gene deletion on a Cbl-b null background, as well as a tamoxifen-inducible mammary stem cell (MaSC)-specific Cbl and Cbl-b double knockout (Cbl/Cbl-b DKO) using Lgr5-EGFP-IRES-CreERT2, to demonstrate a mammary epithelial cell-autonomous requirement of CBL and CBL-B in the maintenance of MaSCs. Using a newly engineered tamoxifen-inducible Cbl and Cbl-b deletion model with a dual fluorescent reporter (Cblflox/flox; Cbl-bflox/flox; Rosa26-CreERT; mT/mG), we show that Cbl/Cbl-b DKO in mammary organoids leads to hyperactivation of AKT-mTOR signaling with depletion of MaSCs. Chemical inhibition of AKT or mTOR rescued MaSCs from Cbl/Cbl-b DKO-induced depletion. Our studies reveal a novel, cell-autonomous requirement of CBL and CBL-B in epithelial stem cell maintenance during organ development and remodeling through modulation of mTOR signaling.

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA.

Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5' untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

Virions at the gates: receptors and the host-virus arms race.

All viruses need to bind to specific receptor molecules on the surface of target cells to initiate infection. Virus-receptor binding is highly specific, and this specificity determines both the species and the cell type that can be infected by a given virus. In some well-studied cases, the virus-binding region on the receptor has been found to be unrelated to the receptor's normal cellular function. Resistance to virus infection can thus evolve by selection of mutations that alter amino acids in the binding region with minimal effect on normal function. This sort of positive selection can be used to infer the history of the host-virus "arms race" during their coevolution. In a new study, Demogines et al. use a combination of phylogenetic, structural, and virological analysis to infer the history and significance of positive selection on the transferrin receptor TfR1, a housekeeping protein required for iron uptake and the cell surface receptor for at least three different types of virus. The authors show that only two parts of the rodent TfR1 molecule have been subject to positive selection and that these correspond to the binding sites for two of these viruses-the mouse mammary tumor virus (a retrovirus) and Machupo virus (an arenavirus). They confirmed this result by introducing the inferred binding site mutations into the wild-type protein and testing for receptor function. Related arenaviruses are beginning to spread in human populations in South America as the cause of often fatal hemorrhagic fevers, and, although Demogines et al. could find no evidence of TfR1 mutations in this region that might have been selected as a consequence of human infection, the authors identified one such mutation in Asian populations that affects infection with these viruses.

The chemokine receptor CCR6 facilitates the onset of mammary neoplasia in the MMTV-PyMT mouse model via recruitment of tumor-promoting macrophages.

BACKGROUND: The expression of the chemokine receptor CCR6 has been previously correlated with higher grades and stages of breast cancer and decreased relapse-free survival. Also, its cognate chemokine ligand CCL20 has been reported to induce proliferation of cultured human breast epithelial cells. METHODS: To establish if CCR6 plays a functional role in mammary tumorigenesis, a bigenic MMTV-PyMT CCR6-null mouse was generated and mammary tumor development was assessed. Levels of tumor-infiltrating immune cells within tumor-bearing mammary glands from MMTV-PyMT Ccr6 (WT) and Ccr6 (-/-) mice were also analyzed. RESULTS: Deletion of CCR6 delayed tumor onset, significantly reduced the extent of initial hyperplastic outgrowth, and decreased tumor incidence in PyMT transgenic mice. CCR6 was then shown to promote the recruitment of pro-tumorigenic macrophages to the tumor site, facilitating the onset of neoplasia. CONCLUSIONS: This study delineated for the first time a role for CCR6 in the development of breast cancer, and demonstrated a critical function for this receptor in maintaining the pro-tumorigenic cancer microenvironment.

Mammary epithelial-specific disruption of c-Src impairs cell cycle progression and tumorigenesis.

The tyrosine kinase c-Src is activated in a large proportion of breast cancers, in which it is thought to play a key role in promoting the malignant phenotype. c-Src activity is also elevated in transgenic mouse models of breast cancer, including the widely used polyomavirus middle-T antigen (PyVmT) model, which provides an opportunity to study the importance of c-Src in mammary tumorigenesis. However, germline c-Src deletion in mammary epithelial and stromal compartments complicates the interpretation of in vivo tumorigenesis studies as a result of severe defects in mammary gland development. We have therefore engineered a mouse strain in which deletion of c-Src can be targeted to the mammary epithelium. We demonstrate that mammary epithelial disruption of c-Src impairs proliferation and tumor progression driven by PyVmT in vivo. Whereas related kinases substitute for c-Src in PyVmT signaling, c-Src ablation impairs cell cycle progression with decreased cyclin expression and elevated expression of cyclin-dependent kinase inhibitors. Our data indicate that c-Src has essential and unique functions in proliferation and tumor progression in this mouse model that may also be important in certain contexts in some human breast cancers.

Disruption of bone morphogenetic protein receptor 2 (BMPR2) in mammary tumors promotes metastases through cell autonomous and paracrine mediators.

Bone morphogenetic proteins (BMPs) are members of the TGF-ß superfamily of signaling molecules. BMPs can elicit a wide range of effects in many cell types and have previously been shown to induce growth inhibition in carcinoma cells as well as normal epithelia. Recently, it has been demonstrated that BMP4 and BMP7 are overexpressed in human breast cancers and may have tumor suppressive and promoting effects. We sought to determine whether disruption of the BMP receptor 2 (BMPR2) would alter mammary tumor progression in mice that express the Polyoma middle T antigen. Mice expressing Polyoma middle T antigen under the mouse mammary tumor virus promoter were combined with mice that have doxycycline-inducible expression of a dominant-negative (DN) BMPR2. We did not observe any differences in tumor latency. However, mice expressing the BMPR2-DN had a fivefold increase in lung metastases. We characterized several cell autonomous changes and found that BMPR2-DN-expressing tumor cells had higher rates of proliferation. We also identified unique changes in inflammatory cells and secreted chemokines/cytokines that accompanied BMPR2-DN-expressing tumors. By immunohistochemistry, it was found that BMPR2-DN primary tumors and metastases had an altered reactive stroma, indicating specific changes in the tumor microenvironment. Among the changes we discovered were increased myeloid derived suppressor cells and the chemokine CCL9. BMP was shown to directly regulate CCL9 expression. We conclude that BMPR2 has tumor-suppressive function in mammary epithelia and microenvironment and that disruption can accelerate mammary carcinoma metastases.

APOBEC3 inhibition of mouse mammary tumor virus infection: the role of cytidine deamination versus inhibition of reverse transcription.

The apolipoprotein B editing complex 3 (APOBEC3) family of proteins is a group of intrinsic antiviral factors active against a number of retroviral pathogens, including HIV in humans and mouse mammary tumor virus (MMTV) in mice. APOBEC3 restricts its viral targets through cytidine deamination of viral DNA during reverse transcription or via deaminase-independent means. Here, we used virions from the mammary tissue of MMTV-infected inbred wild-type mice with different allelic APOBEC3 variants (APOBEC3(BALB) and APOBEC3(BL/6)) and knockout mice to determine whether cytidine deamination was important for APOBEC3's anti-MMTV activity. First, using anti-murine APOBEC3 antiserum, we showed that both APOBEC3 allelic variants are packaged into the cores of milk-borne virions produced in vivo. Next, using an in vitro deamination assay, we determined that virion-packaged APOBEC3 retains its deamination activity and that allelic differences in APOBEC3 affect the sequence specificity. In spite of this in vitro activity, cytidine deamination by virion-packaged APOBEC3 of MMTV early reverse transcription DNA occurred only at low levels. Instead, the major means by which in vivo virion-packaged APOBEC3 restricted virus was through inhibition of early reverse transcription in both cell-free virions and in vitro infection assays. Moreover, the different wild-type alleles varied in their ability to inhibit this step. Our data suggest that while APOBEC3-mediated cytidine deamination of MMTV may occur, it is not the major means by which APOBEC3 restricts MMTV infection in vivo. This may reflect the long-term coexistence of MMTV and APOBEC3 in mice.

Expression of a phosphorylated substrate domain of p130Cas promotes PyMT-induced c-Src-dependent murine breast cancer progression.

Elevated expression of p130Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. p130Cas is a downstream target of the tyrosine kinase c-Src. Signaling mediated by p130Cas through its phosphorylated substrate domain (SD) and interaction with effector molecules directly promotes tumor progression. We previously developed a constitutively phosphorylated p130Cas SD molecule, Src*/SD (formerly referred to as Src*/CasSD), which acts as decoy molecule and attenuates the transformed phenotype in v-crk-transformed murine fibroblasts and human breast cancer cells. To test the function of this molecule in vivo, we established mouse mammary tumor virus (MMTV)-long terminal repeat-Src*/SD transgenic mice in which mammary gland development and tumor formation were analyzed. Transgenic expression of the Src*/SD molecule under the MMTV-long terminal repeat promoter did not interfere with normal mammary gland development or induce tumors in mice observed for up to 11 months. To evaluate the effects of the Src*/SD molecule on tumor development in vivo, we utilized the MMTV-polyoma middle T-antigen (PyMT) murine breast cancer model that depends on c-Src. PyMT mice crossed with Src*/SD mice displayed accelerated tumor formation. The earlier onset of tumors can be explained by the interaction of the Src* domain with PyMT and targeting the fused phosphorylated SD to the membrane. At membrane compartments, it might integrate membrane-associated active signaling complexes leading to increased proliferation measured by phospho-Histone H3 staining. Although these results were unexpected, they emphasize the importance of preventing the membrane association of Src*/SD when employed as decoy molecule.

Bisected, complex N-glycans and galectins in mouse mammary tumor progression and human breast cancer.

Bisected, complex N-glycans on glycoproteins are generated by the glycosyltransferase MGAT3 and cause reduced cell surface binding of galectins. Previously, we showed that MGAT3 reduces growth factor signaling and retards mammary tumor progression driven by the Polyoma middle T antigen (PyMT) expressed in mammary epithelium under the mouse mammary tumor virus (MMTV) promoter. However, the penetrance of the tumor phenotype became variable in mixed FVB/N and C57BL/6 female mice and we therefore investigated a congenic C57BL/6 Mgat3(-/-)/MMTV-PyMT model. In the absence of MGAT3, C57BL/6 Mgat3(-/-)/MMTV-PyMT females exhibited accelerated tumor appearance and increased tumor burden, glucose uptake in tumors and lung metastasis. Nevertheless, activation of extracellular signal-regulated kinase (ERK)1/2 or protein kinase B (AKT) was reduced in ∼20-week C57BL/6 MMTV-PyMT tumors lacking MGAT3. Activation of focal adhesion kinase (FAK), protein tyrosine kinase Src, and p38 mitogen-activated protein kinase were similar to that of controls. All the eight mouse galectin genes were expressed in mammary tumors and tumor epithelial cells (TECs), but galectin-2 and -12 were not detected by western analysis in tumors, and galectin-7 was not detected in 60% of the TEC lines. From microarray data reported for human breast cancers, at least 10 galectin and 7 N-glycan N-acetylglucosaminyl (GlcNAc)-transferase (MGAT) genes are expressed in tumor tissue, and expression often varies significantly between different breast cancer subtypes. Thus, in summary, while MGAT3 and bisected complex N-glycans retard mouse mammary tumor progression, genetic background may modify this effect; identification of key galectins that promote mammary tumor progression in mice is not straightforward because all the eight galectin genes are expressed; and high levels of MGAT3, galectin-4, -8, -10, -13 and -14 transcripts correlate with better relapse-free survival in human breast cancer.

Requirements for mouse mammary tumor virus Rem signal peptide processing and function.

Mouse mammary tumor virus (MMTV) encodes a Rev-like protein, Rem, which is involved in the nuclear export and expression of viral RNA. Previous data have shown that all Rev-like functions are localized to the 98-amino-acid signal peptide (SP) at the N terminus of MMTV Rem or envelope proteins. MMTV-SP uses endoplasmic reticulum-associated degradation (ERAD) for protein trafficking. Rem cleavage by signal peptidase in the ER is necessary for MMTV-SP function in a reporter assay, but many requirements for trafficking are not known. To allow detection and localization of both MMTV-SP and the C-terminal cleavage product, we prepared plasmids expressing green fluorescent protein (GFP) tags. N-terminal Rem tagging led to protein accumulation relative to untagged Rem and allowed signal peptidase cleavage but reduced its specific activity. C-terminal tagging also led to Rem accumulation yet dramatically reduced cleavage, GFP fluorescence, and activity relative to N-terminally tagged Rem (GFPRem). Substitutions of an invariant leucine at position 71 between the known RNA-binding and nuclear export sequences interfered with GFPRem accumulation and activity but not cleavage. Similarly, deletion of 100 or 150 C-terminal amino acids from GFPRem dramatically reduced both Rem and MMTV-SP levels and function. Removal of the entire C terminus (203 amino acids) restored both protein levels and activity of MMTV-SP. Only C-terminal GFP tagging, and not other modifications, appeared to trap Rem in the ER membrane. Thus, Rem conformation in both the ER lumen and cytoplasm determines cleavage, retrotranslocation, and MMTV-SP function. These mutants further characterize intermediates in Rem trafficking and have implications for all proteins affected by ERAD.

Retroviral Rem protein requires processing by signal peptidase and retrotranslocation for nuclear function.

Mouse mammary tumor virus (MMTV) is a complex murine retrovirus that encodes an HIV Rev-like export protein, Rem, from a doubly spliced version of envelope (Env) mRNA. Previously, the N-terminal 98-amino acid sequence of Rem, which is identical to Env signal peptide (SP), and full-length Rem were shown to be functional in a reporter assay that measures a postexport function. Here we show that MMTV-infected cells or cells transfected with rem or env cDNAs express SP, which is the active component in the reporter assay. Uncleaved Rem was partially glycosylated, but mutations in both glycosylation sites within the C terminus prevented Rem function. Mutations that reduced Rem or Env cleavage by signal peptidase greatly reduced SP levels and functional activity in the reporter assay and allowed accumulation of the uncleaved protein. Fluorescence microscopy revealed that GFP-tagged cleavage-site mutants are unstable and lack fluorescence compared with wild-type Rem, suggesting improper folding. Proteasome inhibitors allowed accumulation of uncleaved Rem relative to SP and increased reporter activity, consistent with SP retrotranslocation and proteasome escape before nuclear entry. Expression of a dominant-negative p97 ATPase did not alter levels of unprocessed Rem and SP but decreased reporter activity, suggesting p97-facilitated retrotranslocation of SP. Our results provide an example of a SP that is processed by signal peptidase and retrotranslocated to allow nuclear localization and function.

Determination of mTOR signal pathway in MMTV-TGFα mice ovary at different ages.

Transforming growth factor alpha (TGFα), a member of the epidermal growth factor (EGF) family, regulates cell proliferation, differentiation, and development, and involves follicular development and viability. In ovaries, TGFα is shown localized in granulosa cells (GCs) of primary follicles, theca cells (TCs) of pre-antral, antral and pre-ovulatory follicles. TGFα overexpression in mouse mammary tumor virus (MMTV-TGFα) transgenic mice causes mammary tumor after 50 weeks. However, follicular development and preservation of the ovarian follicle reserve-mediating follicle stimulating hormone (FSH) response are unknown. Mammalian target of rapamycin (mTOR) is a key regulator for cell proliferation, growth, differentiation, and apoptosis, and important for ovarian folliculogenesis and oocyte maturation. The study aim determines TGFα overexpression during folliculogenesis via mTOR signaling pathway in ovaries from 10-, 18-, 50-, and 82-week-old MMTV-TGFα mice. Histological analysis was performed, along with western blot for mTOR, p-mTOR, P70S6K, PCNA, and Caspase-3, and quantitative RNA (qRT-PCR) for mTOR and P70S6K. Developing follicles number decreased and atretic follicles number increased with aging in MMTV-TGFα mice ovary. Ovaries at 18 and 82 weeks had decreased PCNA and increased Caspase-3 protein expression levels as compared to 10-week ovaries. Protein expression levels of mTOR and p-mTOR decreased gradually from ovaries at 10-18 weeks, increased at 50 weeks and decreased again at 82 weeks. These results indicate that TGFα may be one regulator of healthy follicular development and affect mTOR signaling pathway during ovarian aging. Thus, over-expression of TGFα might lead to reduced ovarian reserve and premature ovarian insufficiency.

Selenoproteins reduce susceptibility to DMBA-induced mammary carcinogenesis.

Selenium is an essential micronutrient in the diet of humans and other mammals. Based largely on animal studies and epidemiological evidence, selenium is purported to be a promising cancer chemopreventive agent. However, the biological mechanisms by which chemopreventive activity takes place are poorly understood. It remains unclear whether selenium acts in its elemental form, through incorporation into organic compounds, through selenoproteins or any combination of these. The purpose of this study was to determine whether selenoproteins mitigate the risk of developing chemically induced mammary cancer. Selenoprotein expression was ablated in mouse mammary epithelial cells through genetic deletion of the selenocysteine (Sec) tRNA gene (Trsp), whose product, designated selenocysteine tRNA, is required for selenoprotein translation. Trsp floxed and mouse mammary tumor virus (MMTV)-cre mice were crossed to achieve tissue-specific excision of Trsp in targeted mammary glands. Eight- to twelve-week-old second generation Trsp(fl/+);wt, Trsp(fl/+);MMTV-cre, Trsp(fl/fl);wt and Trsp(fl/fl);MMTV-cre female mice were administered standard doses of the carcinogen, 7,12-dimethylbenzylbenz[a]antracene. Our results revealed that heterozygous, Trsp(fl/+);MMTV-cre mice showed no difference in tumor incidence, tumor rate and survival compared with the Trsp(fl/+);wt mice. However, 54.8% of homozygous Trsp(fl/f)(l);MMTV-cre mice developed mammary tumors and exhibited significantly shorter survival than the corresponding Trsp(fl/fl);wt mice, where only 36.4% developed tumors. Loss of the homozygous Trsp alleles was associated with the reduction of selenoprotein expression. The results suggest that mice with reduced selenoprotein expression have increased susceptibility to developing carcinogen-induced mammary tumors and that a major protective mechanism against carcinogen-induced mammary cancer requires the expression of these selenoproteins.

Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells.

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co-expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth-promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial-mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co-transfectants exhibited invasive properties in three-dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf-1, a specific antagonist of the Wnt/ß-catenin pathway, or short hairpin RNA targeting ß-catenin expression demonstrated that the invasive activity was not mediated by ß-catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context-dependent. While Rspo2 and Wnt1 act synergistically in the ß-catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures.

A role for KAI1 in promotion of cell proliferation and mammary gland hyperplasia by the gp78 ubiquitin ligase.

Expression of gp78, an E3 ubiquitin ligase in endoplasmic reticulum-associated degradation, is associated with tumor malignancy. To study gp78 overexpression in mammary gland development and tumorigenicity, we generated murine mammary tumor virus (MMTV) long terminal repeat-driven gp78 transgenic mice. Embryos carrying the gp78 transgene cassette were implanted in FVB surrogate mothers, and two founders with high copy integration showed elevated gp78 expression at both transcript and protein levels at the virgin stage and at 12 days gestation. Transgenic mammary glands showed increased ductal branching, dense alveolar lobule formation, and secondary terminal end bud development. Bromodeoxyuridine staining showed increased proliferation in hyperplastic ductal regions at the virgin stage and at 12 days gestation compared with wild type mice. Reduced expression of the metastasis suppressor KAI1, a gp78 endoplasmic reticulum-associated degradation substrate, demonstrates that gp78 ubiquitin ligase activity is increased in MMTV-gp78 mammary gland. Similarly, metastatic MDA-435 cells exhibit increased gp78 expression, decreased KAI1 expression, and elevated proliferation compared with nonmetastatic MCF7 cells whose proliferation was enhanced upon knockdown of KAI1. Importantly, stable gp78 knockdown HEK293 cells showed increased KAI1 expression and reduced proliferation that was rescued upon KAI1 knockdown, demonstrating that gp78 regulation of cell proliferation is mediated by KAI1. Mammary tumorigenesis was not observed in repeatedly pregnant MMTV-long terminal repeat-gp78 transgenic mice over a period of 18 months post-birth. Elevated gp78 ubiquitin ligase activity is therefore not sufficient for mammary tumorigenesis. However, the hyperplastic phenotype observed in mammary glands of MMTV-gp78 transgenic mice identifies a novel role for gp78 expression in enhancing mammary epithelial cell proliferation and nontumorigenic ductal outgrowth.