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Supplementary Table 1. Kaplan-Meier survival statistics for control and experimental mice: This table presents results of analysis on tumor assays.

Supplementary Figure Legends 1-5

Supplementary Figures 1-5. Supplementary Fig. 1. Kaplan-Meier overall survival curves and cause of death data for control and experimental female mice. Supplementary Fig. 2. Mammary pathology analysis and estrogen receptor α staining for experimental mice. Supplementary Fig. 3. Activation of Stat3 and Mapk signaling in mouse mammary tumors. Supplementary Fig. 4. Ras pathway activation is associated with relapse and survival of breast cancer patients independent of PI3K and Stat3 pathway activation. Supplementary Fig. 5. PI3K pathway activation is associated with relapse of patients with Basal-like breast cancer.

Supplementary Figure 8 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Tables 1-3 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 4 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 7 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 9 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure Legends from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 2 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 3 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 6 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 5 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Supplementary Figure 1 from Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

Activated lymphocytes promote inflammation and bone destruction in rheumatoid arthritis (RA), making T cells and B cells therapeutic targets. Indeed, pharmacological blockade of CD28 costimulation using CTLA-4Ig (abatacept), approved for amelioration of RA, renders T cells dormant (anergic). CTLA-4Ig also promotes bone accretion in healthy mice; surprisingly, however, this effect is driven exclusively through upregulation of bone formation, rather than anti-inflammatory effects on resorption. In the study presented here, we utilized T cell receptor β gene and Wnt-10b gene knockout mice to investigate the roles of T cells and Wnt-10b in CTLA-4Ig-induced bone anabolism. Ablation of either T cells or Wnt-10b not only abolished CTLA-4Ig-induced bone anabolism but also, paradoxically, suppressed bone formation leading to bone loss. Stalled bone formation was accompanied by bone marrow stromal cell expression of the Wnt pathway inhibitor sclerostin. Our data suggest that an immunoskeletal pivot may promote or suppress bone formation, depending on the net outcome of CTLA-4Ig action directed independently on T cells and osteoblast-linage cells that counter Wnt-10b-induced bone anabolism, by secretion of sclerostin. While CTLA-4Ig action is tipped in favor of bone formation under physiological conditions, pathological immunodeficiency may lead to suppressed bone formation and skeletal damage.

Endothelial cells transduce mechanical forces from blood flow into intracellular signals required for vascular homeostasis. Here we show that endothelial NOTCH1 is responsive to shear stress, and is necessary for the maintenance of junctional integrity, cell elongation, and suppression of proliferation, phenotypes induced by laminar shear stress. NOTCH1 receptor localizes downstream of flow and canonical NOTCH signaling scales with the magnitude of fluid shear stress. Reduction of NOTCH1 destabilizes cellular junctions and triggers endothelial proliferation. NOTCH1 suppression results in changes in expression of genes involved in the regulation of intracellular calcium and proliferation, and preventing the increase of calcium signaling rescues the cell–cell junctional defects. Furthermore, loss of Notch1 in adult endothelium increases hypercholesterolemia-induced atherosclerosis in the descending aorta. We propose that NOTCH1 is atheroprotective and acts as a mechanosensor in adult arteries, where it integrates responses to laminar shear stress and regulates junctional integrity through modulation of calcium signaling., The arterial wall is subjected to mechanical forces that modulate endothelial cell responses. Here, Mack and colleagues identify a novel role for Notch1 as a mechanosensor in adult arteries, where it ensures junctional integrity through modulation of calcium signalling and limits atherosclerosis.

Breast cancer is associated with alterations in a number of growth factor and hormone-regulated signaling pathways. Mouse models of metastatic breast cancer typically feature mutated oncoproteins that activate PI3K, Stat3, and Ras signaling, but the individual and combined roles of these pathways in breast cancer progression are poorly understood. In this study, we examined the relationship between oncogenic pathway activation and breast cancer subtype by analyzing mouse mammary tumor formation in which each pathway was activated singly or pairwise. All three oncogenes showed cooperation during primary tumor formation, but efficient dissemination was only dependent on Ras. In addition, transcriptional profiling demonstrated that Ras induced adenocarcinomas with molecular characteristics related to human basal-like and HER2+ tumors. In contrast, Ras combined with PIK3CAH1047R, an oncogenic mutant linked to ERα+/luminal breast cancer in humans, induced metastatic luminal B-like tumors. Consistent with these data, elevated Ras signaling was associated with basal-like and HER2+ subtype tumors in humans and showed a statistically significant negative association with estrogen receptor (ER) signaling across all breast cancer. Despite this, there are luminal tumors with elevated Ras signaling. Importantly, when considered as a continuous variable, Ras pathway activation was strongly linked to reduced survival of patients with ERα+ disease independent of PI3K or Stat3 activation. Therefore, our studies suggest that Ras activation is a key determinant for dissemination and poor prognosis of ERα+/luminal breast cancer in humans, and hormone therapy supplemented with Ras-targeting agents may be beneficial for treating this aggressive subtype. Cancer Res; 75(22); 4960–72. ©2015 AACR.

with a similar mechanism of injury in each location. Increasing its content or decreasing the size of the compartment can raise compartment pressure. This triggers a vicious cycle whereby increased venous pressure causes an increased capillary pressure and the increase in exudate increases the content of the compartment further. De-creasing tissue perfusion by reducing arteriolar pressure even-tually increases capillary permeability and causes a further in-crease in exudate. Muscle and nerve necrosis follows, causing the earliest clinical signs of compartment syndrome. Figure 1

Estrogen deficiency expands hemopoietic stem and progenitor cells (HSPCs) and mature blood lineages, but the involved mechanism and the affected HSPC populations are mostly unknown. Here we show that ovariectomy (ovx) expands short-term HSPCs (ST-HSPCs) and improves blood cell engraftment and host survival after bone marrow (BM) transplantation through a dual role of the T-cell costimulatory molecule CD40 ligand (CD40L). This surface receptor is required for ovx to stimulate T-cell production of Wnt10b, a Wnt ligand that activates Wnt signaling in HSPCs and stromal cells (SCs). Moreover, CD40L is required for ovx to increase SC production of the hemopoietic cytokines interleukin (IL)-6, IL-7, and granulocyte macrophage-colony-stimulating factor. Attesting to the relevance of CD40L and Wnt10b, ovx fails to expand ST-HSPCs in CD40L-null mice and in animals lacking global or T-cell expression of Wnt10b. In summary, T cells expressed CD40L, and the resulting increased production of Wnt10b and hemopoietic cytokines by T cells and SCs, respectively, plays a pivotal role in the mechanism by which ovx regulates hemopoiesis. The data suggest that antiestrogens may represent pharmacological targets to improve ST-HSPC function through activation of the microenvironment.

Breast cancer is a highly heterogeneous disease, with several different subtypes being characterized by distinct histology, gene expression patterns, and genetic alterations. The tumor suppressor gene retinoblastoma 1 (RB1) is frequently lost in both luminal-B and triple-negative tumor (TNT; i.e., estrogen receptor–, progesterone receptor–, and human epidermal growth factor receptor 2–negative) breast cancer subtypes. However, a causal role for RB1 loss in different subtypes remains undefined. Here we report that deletion of Rb alone or together with its relative p107 in mouse mammary stem/bipotent progenitor cells induced focal acinar hyperplasia with squamous metaplasia. These lesions progressed into histologically diverse, transplantable mammary tumors with features of either luminal-B or TNT subtypes. The TNTs included basal-like tumors as well as tumors that exhibited epithelial-to-mesenchymal transition (EMT). The EMT-type tumors and a subset of the basal-like tumors, but not luminal-B–like tumors, expressed mutant forms of the tumor suppressor p53. Accordingly, targeted deletion of both Rb and p53 in stem/bipotent progenitors led to histologically uniform, aggressive, EMT-type tumors. Reintroduction of Rb into these tumor cells suppressed growth in vitro and tumor formation in vivo. These results establish a causal role for Rb loss in breast cancer in mice and demonstrate that cooperating oncogenic events, such as mutations in p53, dictate tumor subtype after Rb inactivation.

Following genotoxic stress, the histone H2AX becomes phosphorylated at serine 139 by the ATM/ATR family of kinases. The tumor suppressor BRCA1, also phosphorylated by ATM/ATR kinases, is one of several proteins that colocalize with phospho-H2AX (γ-H2AX) at sites of active DNA repair. Both the precise mechanism and the purpose of BRCA1 recruitment to sites of DNA damage are unknown. Here we show that BRCA1 andγ-H2AX form an acid-stable biochemical complex on chromatin after DNA damage. Maximal association of BRCA1 withγ-H2AX correlates with reduced globalγ-H2AX levels on chromatin late in the repair process. Since BRCA1 is known to have E3 ubiquitin ligase activityin vitro, we examined H2AX for evidence of ubiquitination. We found that H2AX is ubiquitinated at lysines 119 and 119in vivoand that blockage of 26S proteasome function stabilizesγ-H2AX levels within cells. When BRCA1 levels were reduced, ubiquitination of H2AX was also reduced, and the cells retained higher levels of phosphorylated H2AX. These results indicate that BRCA1 is recruited into stable complexes withγ-H2AX and that the complex is involved in attenuation of theγ-H2AX repair signal after DNA damage.

Background: Targeted gene transduction in vivo is the ultimate preferred method for gene delivery. We previously developed targeting lentiviral vectors that specifically recognize cell surface molecules with conjugated antibodies and mediate targeted gene transduction both in vitro and in vivo. Although effective in some experimental settings, the conjugation of virus with antibodies is mediated by the interaction between protein A and the Fc region of antibodies, which is not as stable as covalent conjugation. We have now developed a more stable conjugation strategy utilizing the interaction between avidin and biotin. Methods: We inserted the biotin-adaptor-peptide, which was biotinylated by secretory biotin ligase at specific sites, into our targeting envelope proteins, enabling conjugation of the pseudotyped virus with avidin, streptavidin or neutravidin. Results: When conjugated with avidin-antibody fusion proteins or the complex of avidin and biotinylated targeting molecules, the vectors could mediate specific transduction to targeted cells recognized by the targeting molecules. When conjugated with streptavidin-coated magnetic beads, transduction by the vectors was targeted to the locations of magnets. Conclusions: This targeting vector system can be used for broad applications of targeted gene transduction using biotinylated targeting molecules or targeting molecules fused with avidin. Fil: Morizono, Kouki. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Xie, Yiming. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Helguera, Gustavo Fernando. University of California at Los Angeles; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Daniels, Tracy R.. University of California at Los Angeles; Estados Unidos Fil: Lane T. F.. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Penichet, Manuel L.. University of California at Los Angeles; Estados Unidos. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Chen, Irvin S.Y.. University of California at Los Angeles. School of Medicine; Estados Unidos

Systemic hormones and local growth factor-mediated tissue interactions are essential for mammary gland development. Using phenotypic and transplantation analyses of mice carrying the mesenchymal dysplasia (mes) allele of patched 1 (Ptch1mes), we found that Ptch1mes homozygosity led to either complete failure of gland development, failure of post-pubertal ductal elongation, or delayed growth with ductal dysplasia. All ductal phenotypes could be present in the same animal. Whole gland and epithelial fragment transplantation each yielded unique morphological defects indicating both epithelial and stromal functions for Ptch1. However, ductal elongation was rescued in all cases, suggesting an additional systemic function. Epithelial function was confirmed using a conditional null Ptch1 allele via MMTV-Cre-mediated disruption. In Ptch1mes homozygotes, failure of ductal elongation correlated with diminished estrogen and progesterone receptor expression, but could not be rescued by exogenous ovarian hormone treatment. By contrast, pituitary isografts were able to rescue the ductal elongation phenotype. Thus, Ptch1 functions in the mammary epithelium and stroma to regulate ductal morphogenesis, and in the pituitary to regulate ductal elongation and ovarian hormone responsiveness.

Loss of the CDK inhibitor p27KIP1 is widely linked with poor prognosis in human cancer. In Wnt10b-expressing mammary tumors, levels of p27KIP1 were extremely low; conversely, Wnt10b-null mammary cells expressed high levels of this protein, suggesting Wnt-dependent regulation of p27KIP1. Interestingly we found that Wnt-induced turnover of p27KIP1 was independent from classical SCFSKP2-mediated degradation in both mouse and human cells. Instead, turnover required Cullin 4A and Cullin 4B, components of an alternative E3 ubiquitin ligase induced in response to active Wnt signaling. We found that CUL4A was a novel Wnt target gene in both mouse and human cells and that CUL4A physically interacted with p27KIP1 in Wnt-responding cells. We further demonstrated that both Cul4A and Cul4B were required for Wnt-induced p27KIP1 degradation and S-phase progression. CUL4A and CUL4B are therefore components of a conserved Wnt-induced proteasome targeting (WIPT) complex that regulates p27KIP1 levels and cell cycle progression in mammalian cells.

Overexpression of Wnt10b from the osteocalcin promoter in transgenic mice increases postnatal bone mass. Increases in osteoblast perimeter, mineralizing surface, and bone formation rate without detectable changes in pre-osteoblast proliferation, osteoblast apoptosis, or osteoclast number and activity suggest that, in this animal model, Wnt10b primarily increases bone mass by stimulating osteoblastogenesis. Introduction: Wnt signaling regulates many aspects of development including postnatal accrual of bone. Potential mechanisms for how Wnt signaling increases bone mass include regulation of osteoblast and/or osteoclast number and activity. To help differentiate between these possibilities, we studied mice in which Wnt10b is expressed specifically in osteoblast lineage cells or in mice devoid of Wnt10b. Materials and Methods: Transgenic mice, in which mouse Wnt10b is expressed from the human osteocalcin promoter (Oc-Wnt10b), were generated in C57BL/6 mice. Transgene expression was evaluated by RNase protection assay. Quantitative assessment of bone variables was done by radiography, μCT, and static and dynamic histomorphometry. Mechanisms of bone homeostasis were evaluated with assays for BrdU, TUNEL, and TRACP5b activity, as well as serum levels of C-terminal telopeptide of type I collagen (CTX). The endogenous role of Wnt10b in bone was assessed by dynamic histomorphometry in Wnt10b−/− mice. Results: Oc-Wnt10b mice have increased mandibular bone and impaired eruption of incisors during postnatal development. Analyses of femoral distal metaphyses show significantly higher BMD, bone volume fraction, and trabecular number. Increased bone formation is caused by increases in number of osteoblasts per bone surface, rate of mineral apposition, and percent mineralizing surface. Although number of osteoclasts per bone surface is not altered, Oc-Wnt10b mice have increased total osteoclast activity because of higher bone mass. In Wnt10b−/− mice, changes in mineralizing variables and osteoblast perimeter in femoral distal metaphyses were not observed; however, bone formation rate is reduced because of decreased total bone volume and trabecular number. Conclusions: High bone mass in Oc-Wnt10b mice is primarily caused by increased osteoblastogenesis, with a minor contribution from elevated mineralizing activity of osteoblasts.

To introduce temporal control in genetic experiments targeting the endothelium, we established a mouse line expressing tamoxifen-inducible Cre-recombinase (Cre-ERT2) under the regulation of the vascular endothelial cadherin promoter (VECad). Specificity and efficiency of Cre activity was documented by crossing VECad-Cre-ERT2 with the ROSA26R reporter mouse, in which a floxed-stop cassette has been placed upstream of the beta-galactosidase gene. We found that tamoxifen specifically induced widespread recombination in the endothelium of embryonic, neonatal, and adult tissues. Recombination was also documented in tumor-associated vascular beds and in postnatal angiogenesis assays. Furthermore, injection of tamoxifen in adult animals resulted in negligible excision (lower than 0.4%) in the hematopoietic lineage. The VECad-Cre-ERT2 mouse is likely to be a valuable tool to study the function of genes involved in vascular development, homeostasis, and in complex processes involving neoangiogenesis, such as tumor growth.

The BRCA1 tumor suppressor gene encodes an E3-ubiquitin ligase that has been implicated in several distinct biochemical processes. As the cell cycle progresses, BRCA1 proteins interact transiently with nuclear foci containing DNA replication and DNA double-strand repair machinery. A hallmark of these foci is the presence of S139 phosphorylated histone H2AX. BRCA1 was recently shown to associate with facultative heterochromatin at the inactive X chromosome (Xi), where it may play a role in maintaining gene silencing. As the kinetics of this interaction has not been described, we sought to establish whether association of BRCA1 with the Xi also correlated with replication. Here we demonstrate that the interaction of BRCA1 and the Xi is transient, occurring during late S-phase. This interaction is concomitant with the presence of distinct foci of S139 phospho-H2AX and specifically corresponds with late replication of the Xi. BRCA1 and phospho-H2AX appear on the Xi immediately adjacent to CAF-1, a known marker of replication fork activity. Taken together, these data implicate BRCA1 and the H2AX kinase in replication of facultative heterochromatin on the Xi, most likely in a fashion similar to that performed at sites of DNA replication and double-strand break repair observed on somatic chromosomes.

Adult myoblasts retain plasticity in developmental potential and can be induced to undergo myogenic, adipogenic, or osteoblastogenic differentiation in vitro. In this report, we show that the balance between myogenic and adipogenic potential in myoblasts is controlled by Wnt signaling. Furthermore, this balance is altered during aging such that aspects of both differentiation programs are coexpressed in myoblasts due to decreased Wnt10b abundance. Mimicking Wnt signaling in aged myoblasts through inhibition of glycogen synthase kinase or through overexpression of Wnt10b resulted in inhibition of adipogenic gene expression and sustained or enhanced myogenic differentiation. On the other hand, myoblasts isolated from Wnt10b null mice showed increased adipogenic potential, likely contributing to excessive lipid accumulation in actively regenerating myofibers in vivo in Wnt10b-/- mice. Whereas Wnt10b deficiency contributed to increased adipogenic potential in myoblasts, the augmented myogenic differentiation potential observed is likely the result of a compensatory increase in Wnt7b during differentiation of Wnt10b-/- myoblasts. No such compensation was apparent in aged myoblasts and in fact, both Wnt5b and Wnt10b were down-regulated. Thus, alteration in Wnt signaling in myoblasts with age may contribute to impaired muscle regenerative capacity and to increased muscle adiposity, both characteristic of aged muscle.

Wnts comprise a family of secreted signaling proteins that regulate diverse developmental processes. Activation of Wnt signaling by Wnt10b inhibits differentiation of preadipocytes and blocks adipose tissue development; however, the effect of Wnt10b on other mesenchymal lineages has not been defined. To explore the physiological role of Wnt signaling in bone development, we analyzed FABP4-Wnt10b mice, which express the Wnt10b transgene in marrow. Femurs from FABP4-Wnt10b mice have almost four times as much bone in the distal metaphyses and are mechanically stronger. These mice maintain elevated bone mass at least through 23 months of age. In addition, FABP4-Wnt10b mice are protected from the bone loss characteristic of estrogen deficiency. We used pharmacological and genetic approaches to demonstrate that canonical Wnt signaling stimulates osteoblastogenesis and inhibits adipogenesis of bipotential mesenchymal precursors. Wnt10b shifts cell fate toward the osteoblast lineage by induction of the osteoblastogenic transcription factors Runx2, Dlx5, and osterix and suppression of the adipogenic transcription factors C/EBPα and PPARγ. One mechanism whereby Wnt10b promotes osteoblastogenesis is suppression of PPARγ expression. Finally, Wnt10b-/- mice have decreased trabecular bone and serum osteocalcin, confirming that Wnt10b is an endogenous regulator of bone formation.

Numerous bacterial products such as lipopolysaccharide potently induce type I interferons (IFNs); however, the contribution of this innate response to host defense against bacterial infection remains unclear. Although mice deficient in either IFN regulatory factor (IRF)3 or the type I IFN receptor (IFNAR)1 are highly susceptible to viral infection, we show that these mice exhibit a profound resistance to infection caused by the Gram-positive intracellular bacterium Listeria monocytogenes compared with wild-type controls. Furthermore, this enhanced bacterial clearance is accompanied by a block in L. monocytogenes–induced splenic apoptosis in IRF3- and IFNAR1-deficient mice. Thus, our results highlight the disparate roles of type I IFNs during bacterial versus viral infections and stress the importance of proper IFN modulation in host defense.

Overexpression of human cyclooxygenase 2 (COX-2) in the mammary glands of transgenic mice induces tissue-specific tumorigenic transformation. However, the molecular mechanisms involved are not yet defined. Here we show that COX-2 expressed in the epithelial cell compartment regulates angiogenesis in the stromal tissues of the mammary gland. Microvessel density increased before visible tumor growth and exponentially during tumor progression. Inhibition of prostanoid synthesis with indomethacin strongly decreased microvessel density and inhibited tumor progression. Up-regulation of angiogenic regulatory genes in COX-2 transgenic mammary tissue was also potently inhibited by indomethacin treatment, suggesting that prostanoids released from COX-2-expressing mammary epithelial cells induce angiogenesis. G protein-coupled receptors for the major product, prostaglandin E 2 (PGE 2 ) EP 1-4 , are expressed during mammary gland development, and EP 1,2,4 receptors were up-regulated in tumor tissue. PGE 2 stimulated the expression angiogenic regulatory genes in mammary tumor cells isolated from COX-2 transgenic mice. Such cells are tumorigenic in nude mice; however, treatment with Celecoxib, a COX-2-specific inhibitor, reduced tumor growth and microvessel density. These results define COX-2-derived PGE 2 as a potent inducer of angiogenic switch during mammary cancer progression.

The human BRCA1 tumor suppressor interacts with transcriptional machinery, including RNA polymerase II (RNA pol II). We demonstrated that interaction with RNA pol II is a conserved feature of BRCA1 proteins from several species. We found that full-length BRCA1 proteins universally fail to activate transcription in classic GAL4-UAS one-hybrid assays and that the activity associated with the human BRCA1 C terminus was poorly conserved in closely related homologs of the gene. Fractionation studies demonstrated that BRCA1 proteins from all species tested interacted specifically with hyperphosphorylated pol II (IIO), in preference to hypophosphorylated RNA pol II (IIA) expected at promoters. BRCA1-RNA pol II complexes showed evidence of a multiply phosphorylated heptad repeat domain in the catalytic subunit (p220) of RNA pol II, and the complex was highly functional in transcriptional run-off assays. Interestingly, endogenous BRCA1 associated with a large fraction of the processive RNA pol II activity present in undamaged cells, and the interaction was disrupted by DNA-damaging agents. Preferential interaction with processive RNA pol II in undamaged cells places BRCA1 in position to link late events in transcription with repair processes in eukaryotic cells.

The identification of novel endothelial-derived genes is important in the study of angiogenesis, and may have potential uses in cancer diagnosis and treatment. We performed SSH (suppression subtractive hybridization) on control HUVECs (human umbilical vein endothelial cells) versus HUVECs exposed to tumor-conditioned media. We found that a novel cDNA (GenBank Accession No. AF358829) is differentially expressed in endothelial cells on Northern analysis, and named it endothelial-derived gene-1 (EG-1). This gene product is predicted to encode a 178-aa, 19.5-kDa protein, and is localized to chromosome 4. It has some homology to a mouse cDNA (94%) and a Drosophila cDNA (31%). On Northern analysis, endothelial cells express two EG-1 RNA species (1.2 and 2.4 kb). The expression of either transcripts is upregulated by endothelial cells when exposed to tumor conditioned media. This phenomenon is observed only under sparse conditions (50% confluency). Transcripts are present abundantly in highly vascular tissues such as placenta, testis, and liver. Interestingly, both Northern analysis and in situ hybridization studies show that this gene is expressed in other cell types as well, predominantly the epithelial type. Breast cancer, prostate cancer, and colon cancer cells show elevated expression of the higher 2.4-kb RNA form. Our data suggest that EG-1 is associated with a stimulated state in endothelial and epithelial cells, and may have a role in tumor angiogenesis.

We recently reported that the PPM1l gene encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (named PP2Ce) with highly specific activity towards Inositol-requiring protein-1 (IRE1) and regulates the functional outcome of ER stress. In the present report, we found that the PP2Ce protein is highly expressed in lactating epithelium of the mammary gland. Loss of PP2Ce in vivo impairs physiological unfolded protein response (UPR) and induces stress kinase activation, resulting in loss of milk production and induction of epithelial apoptosis in the lactating mammary gland. This study provides the first in vivo evidence that PP2Ce is an essential regulator of normal lactation, possibly involving IRE1 signaling and ER stress regulation in mammary epithelium.

Epigenetic gene silencing by histone modifications and DNA methylation is essential for cancer development. The molecular mechanism that promotes selective epigenetic changes during tumorigenesis is not understood. We report here that the PIAS1 SUMO ligase is involved in the progression of breast tumorigenesis. Elevated PIAS1 expression was observed in breast tumor samples. PIAS1 knockdown in breast cancer cells reduced the subpopulation of tumor-initiating cells, and inhibited breast tumor growth in vivo. PIAS1 acts by delineating histone modifications and DNA methylation to silence the expression of a subset of clinically relevant genes, including breast cancer DNA methylation signature genes such as cyclin D2 and estrogen receptor, and breast tumor suppressor WNT5A. Our studies identify a novel epigenetic mechanism that regulates breast tumorigenesis through selective gene silencing.

Growth of tumors and metastasis are processes known to require neovascularization. To ascertain the participation of the endogenous angiogenic inhibitor thrombospondin-1 (TSP1) in tumor progression, we generated mammary tumor-prone mice that either lack, or specifically overexpress, TSP1 in the mammary gland. Tumor burden and vasculature were significantly increased in TSP1-deficient animals, and capillaries within the tumor appeared distended and sinusoidal. In contrast, TSP1 overexpressors showed delayed tumor growth or lacked frank tumor development (20% of animals); tumor capillaries showed reduced diameter and were less frequent. Interestingly, absence of TSP1 resulted in increased association of vascular endothelial growth factor (VEGF) with its receptor VEGFR2 and higher levels of active matrix metalloproteinase-9 (MMP9), a molecule previously shown to facilitate both angiogenesis and tumor invasion. In vitro , enzymatic activation of proMMP9 was suppressed by TSP1. Together these results argue for a protective role of endogenous inhibitors of angiogenesis in tumor growth and implicate TSP1 in the in vivo regulation of metalloproteinase-9 activation and VEGF signaling.

Vascular endothelial growth factors (VEGFs) are endothelial cell-specific mitogens with potent angiogenic and vascular permeability-inducing properties. VEGF, VEGF-C, and VEGFRs -1, -2, and -3 were found to be expressed in post-pubertal (virgin) rodent mammary glands. VEGF was increased during pregnancy (5-fold) and lactation (15-19-fold). VEGF-C was moderately increased during pregnancy and lactation (2- and 3-fold respectively). VEGF levels were reduced by approximately 75% in cleared mouse mammary glands devoid of epithelial components, demonstrating that although the epithelial component is the major source of VEGF, approximately 25% is derived from stroma. This was confirmed by the findings (a) that VEGF transcripts were expressed predominantly in ductal and alveolar epithelial cells, and (b) that VEGF protein was localized to ductal epithelial cells as well as to the stromal compartment including vascular structures. VEGF was detected in human milk. Finally, transcripts for VEGFRs -2 and -3 were increased 2-3-fold during pregnancy, VEGFRs -1, -2 and -3 were increased 2-4-fold during lactation, and VEGFRs -2 and -3 were decreased by 20-50% during involution. These results point to a causal role for the VEGF ligand-receptor pairs in pregnancy-associated angiogenesis in the mammary gland, and suggest that they may also regulate vascular permeability during lactation.

Wnt-10b is expressed during the formation of the mammary rudiment in mouse embryos and its expression continues through puberty when the mammary ductal pattern is established under control of ovarian steroids. Recently, viral activation of the Wnt-10b locus has linked its overexpression to mammary tumor formation, suggesting a role for Wnt-10b in patterning and growth-regulation of the mammary gland. To test this notion, we created lines of transgenic mice that express elevated levels of Wnt-10b under the control of the MMTV promoter/enhancer. Overexpression of this gene resulted in profound developmental alterations in the mammary gland, including expanded glandular development and the precocious appearance of alveoli in virgin females. Moreover, transgenic male mice also exhibited dramatic mammary development involving highly branched mammary ducts and gynecomastia. Aberrant expression of Wnt-10b in the mammary rudiments of males evidently bypasses the normal requirement for ovarian hormonal control in stimulating mammary ductal growth and the repressive effects of androgens. In addition to these developmental effects, transgenic mice of both sexes were highly susceptible to the development of mammary adenocarcinomas. Such tumors arose in a solitary manner indicating that Wnt-10b is a proto-oncogene which provides a necessary, but insufficient signal for oncogenesis. Relevant to this, there was no evidence of amplified expression of FGF mRNAs in these tumors though the Fgf's are a class of genes often implicated as collaborators in Wnt-mediated tumor formation. Indeed, co-expression of MMTV-Wnt-10b and MMTV-FGF-3/int-2 resulted in sterile offspring with highly disorganized mammary epithelium, demonstrating a potent interaction between their respective developmental pathways. These results suggest that Wnt-10b, or other Wnt genes expressed early in mammary development, play a role in regulating sexual dimorphism and show potent transforming activity when overexpressed.

Unlimited self renewal capacity and differentiation potential make human pluripotent stem cells (PSC) a promising source for the ex vivo manufacture of red blood cells (RBCs) for safe transfusion. Current methods to induce erythropoiesis from PSC suffer from low yields of RBCs, most of which are immature and contain embryonic and fetal rather than adult hemoglobins. We have previously shown that homodimerization of the intracellular component of MPL (ic-MPL) induces erythropoiesis from human cord blood progenitors. The goal of this study was to investigate the potential of ic-MPL dimerization to induce erythropoiesis from human embryonic stem cells (hESCs) and to identify the signaling pathways activated by this strategy. We present here the evidence that ic-MPL dimerization induces erythropoietin (EPO)-independent erythroid differentiation from hESC by inducing the generation of erythroid progenitors and by promoting more efficient erythroid maturation with increased RBC enucleation as well as increased gamma:epsilon globin ratio and production of beta-globin protein. ic-MPL dimerization is significantly more potent than EPO in inducing erythropoiesis, and its effect is additive to EPO. Signaling studies show that dimerization of ic-MPL, unlike stimulation of the wild type MPL receptor, activates AKT in the absence of JAK2/STAT5 signaling. AKT activation upregulates GATA-1 and FOXO3 transcriptional pathways with resulting inhibition of apoptosis, modulation of cell cycle, and enhanced maturation of erythroid cells. These findings open up potential new targets for the generation of therapeutically relevant RBC products from hPSC. Stem Cells 2014;32:1503–1514

Host responses to chemotherapy can induce resistance mechanisms that facilitate tumor regrowth. To determine the contribution of bone marrow–derived cells (BMDCs), we exposed tumor-bearing mice to chemotherapeutic agents and evaluated the influx and contribution of a genetically traceable subpopulation of BMDCs (vascular endothelial–cadherin-Cre-enhanced yellow fluorescent protein [VE-Cad-Cre-EYFP]). Treatment of tumor-bearing mice with different chemotherapeutics resulted in a three- to 10-fold increase in the influx of VE-Cad-Cre-EYFP. This enhanced influx was accompanied by a significant increase in angiogenesis. Expression profile analysis revealed a progressive change in the EYFP population with loss of endothelial markers and an increase in mononuclear markers. In the tumor, 2 specific populations of VE-Cad-Cre-EYFP BMDCs were identified: Gr1+/CD11b+ and Tie2high/platelet endothelial cell adhesion moleculelow cells, both located in perivascular areas. A common signature of the EYFP population that exits the bone marrow is an increase in Notch. Inducible inactivation of Notch in the EYFP+ BMDCs impaired homing of these BMDCs to the tumor. Importantly, Notch deletion reduced therapy-enhanced angiogenesis, and was associated with an increased antitumor effect of the chemotherapy. These findings revealed the functional significance of a specific population of supportive BMDCs in response to chemotherapeutics and uncovered a new potential strategy to enhance anticancer therapy.

We have isolated genomic and cDNA clones of Brca1, a mouse homolog of the recently cloned breast cancer-associated gene, BRCA1. Brca1 encodes an 1812-amino-acid protein with a conserved zinc finger domain and significant homology to the human protein. Brca1 maps to Chromosome 11 within a region of conserved synteny with human chromosome 17, consistent with the mapping of the human gene to 17q21. Brca1 transcripts are expressed in a variety of cultured cells but reveal a specific and dynamic expression pattern during embryonic development. For example, expression is observed first in the otic vesicle of embryonic day 9.5 (E9.5) embryos. This expression diminishes and is replaced by expression in the neuroectoderm at E10.5. By E11-12.5, higher levels are observed in differentiating keratinocytes and in whisker pad primordia. Transcripts also become evident in epithelial cells of the E14-17 kidney. Brca1 expression occurs in differentiating epithelial cells of several adult organs as well, suggesting a general role in the functional maturation of these tissues. Consistent with this, Brca1 transcripts are expressed in both alveolar and ductal epithelial cells of the mammary gland. During pregnancy, there is a large increase in Brca1 mRNA in mammary epithelial cells, an increase that parallels their functional differentiation. Because high rates of breast cancer are associated with loss of BRCA1 in humans, it is possible that this gene provides an important growth regulatory function in mammary epithelial cells. In addition, increased transcription of mammary Brca1 during pregnancy might contribute, in part, to the reduced cancer risk associated with exposure to pregnancy and lactation.

Transgenic mice harboring the int-2/Fgf-3 protooncogene under transcriptional control of the mouse mammary tumor virus (MMTV) promoter/enhancer exhibit a dramatic, benign hyperplasia of the mammary gland. In one int-2 transgenic line (TG.NX), this growth disturbance is evoked by pregnancy and regresses after parturition. Regression of hyperplastic mammary epithelium is less complete after successive pregnancies, and, within 10 months, most TG.NX mice stochastically develop mammary carcinomas that are transplantable in virgin, syngeneic mice. To identify genes that cooperate with int-2 in cell transformation, we infected TG.NX transgenic mice with MMTV. In a cohort of 14 animals, most mammary tumors represented clonal or oligoclonal outgrowths harboring one to five proviral MMTV integrants. Eight of 35 (23%) MMTV+ tumors exhibited proviral insertion at the Wnt-1 locus. No provirus was detected at the int-2, int-3, or Wnt-3 loci. By Southern analysis, two tumors had proviral insertions at the same genomic location, which was mapped to chromosome 15. Cloning of this int locus identified an additional member of the Wnt gene family. The predicted 389-amino acid protein is most closely related to zebrafish Wnt-10a (58% amino acid identity over 362 residues) and, based on homology analysis, was designated Wnt-10b. This newly discovered Wnt family member was expressed in the embryo and mammary gland of virgin but not pregnant mice and represents a candidate collaborating oncogene of int-2/Fgf-3 in the mammary epithelium.

SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin and BM-40) is a metal-binding glycoprotein secreted by a variety of cultured cells and characteristic of tissues undergoing morphogenesis, remodeling, and repair. Recently it has been shown that SPARC inhibits the progression of the endothelial cell cycle in mid-G1, and that a synthetic peptide (amino acids 54-73 of secreted murine SPARC, peptide 2.1) from a cationic, disulfide-bonded region was in part responsible for the growth-suppressing activity [Funk and Sage (1991): Proc Natl Acad Sci USA 88:2648-2652]. Moreover, SPARC was shown to interact directly with bovine aortic endothelial (BAE) cells through a C-terminal EF-hand sequence comprising a high-affinity Ca(2+)-binding site of SPARC and represented by a synthetic peptide (amino acids 254-273) termed 4.2 [Yost and Sage (1993): J Biol Chem 268:25790-25796]. In this study we show that peptide 4.2 is a more potent inhibitor of DNA synthesis that acts cooperatively with peptide 2.1 to diminish the incorporation of [3H]-thymidine by both BAE and bovine capillary endothelial (BCE) cells. At concentrations of 0.019-0.26 mM peptide 4.2, thymidine incorporation by BAE cells was decreased incrementally, relative to control values, from approximately 100 to 10%. Although somewhat less responsive, BCE cells exhibited a dose-responsive decrement in thymidine incorporation, with a maximal inhibition of 55% at 0.39 mM. The inhibitory effect of peptide 4.2 was essentially independent of heparin and basic fibroblast growth factor and was blocked by anti-SPARC peptide 4.2 IgG, but not by antibodies specific for other domains of SPARC. To identify residues that were necessary for inhibition of DNA synthesis, we introduced single amino acid substitutions into synthetic peptide 4.2 and tested their activities and cell-surface binding characteristics on endothelial cells. Two peptides displayed null to diminished effects in the bioassays that were concentration-dependent: peptide 4.2 K, containing an Asp258 --> Lys substitution, and peptide 4.2 AA, in which the two disulfide-bonded Cys (positions 255 and 271) were changed to Ala residues. Peptide 4.2 K, which failed to fulfill the EF-hand consensus formula, exhibited an anomalous fluorescence emission spectrum, in comparison with the wild-type 4.2 sequence, that was indicative of a compromised affinity for Ca2(+).(ABSTRACT TRUNCATED AT 400 WORDS)

Intermittent parathyroid hormone (iPTH) treatment expands hemopoietic stem and progenitor cells (HSPCs), but the involved mechanisms and the affected HSPC populations are mostly unknown. Here we show that T cells are required for iPTH to expand short-term HSPCs (ST-HSPCs) and improve blood cell engraftment and host survival after BM transplantation. Silencing of PTH/PTH-related protein receptor (PPR) in T cells abrogates the effects of iPTH, thus demonstrating a requirement for direct PPR signaling in T cells. Mechanistically, iPTH expands ST-HSPCs by activating Wnt signaling in HSPCs and stromal cells (SCs) through T-cell production of the Wnt ligand Wnt10b. Attesting to the relevance of Wnt10b, iPTH fails to expand ST-HSPCs in mice with Wnt10b−/− T cells. Moreover, iPTH fails to promote engraftment and survival after BM transplantation in Wnt10b null mice. In summary, direct PPR signaling in T cells and the resulting production of Wnt10b play a pivotal role in the mechanism by which iPTH expands ST-HSPCs. The data suggest that T cells may provide pharmacologic targets for HSPC expansion.

SPARC is a transiently expressed extracellular matrix-binding protein that alters cell shape and regulates endothelial cell proliferation in vitro. In this study, we show that SPARC mRNA and protein are synthesized by endothelial cells during angiogenesis in vivo. SPARC and peptides derived from a cationic region of the protein (amino acids 113-130) stimulated the formation of endothelial cords in vitro; moreover, these peptides stimulated angiogenesis in vivo. Mapping of the active domain demonstrated that the sequence KGHK was responsible for most of the angiogenic activity; substitution of the His residue decreased the effect. We found that proteolysis of SPARC provided a source of KGHK, GHK, and longer peptides that contained these sequences. Although the Cu(2+)-GHK complex had been identified as a mitogen/morphogen in normal human plasma, we found KGHK and longer peptides to be potent stimulators of angiogenesis. SPARC113-130 and KGHK were shown to bind Cu2+ with high affinity; however, previous incubation with Cu2+ was not required for the stimulatory activity. Since a peptide from a second cationic region of SPARC (SPARC54-73) also bound Cu2+ but had no effect on angiogenesis, the angiogenic activity appeared to be sequence specific and independent of bound Cu2+. Thus, specific degradation of SPARC, a matrix-associated protein expressed by endothelial cells during vascular remodeling, releases a bioactive peptide or peptides, containing the sequence (K)GHK, that could regulate angiogenesis in vivo.

PIK3CA, which codes for the p110α catalytic subunit of phosphatidylinositol 3-kinase, is one of the most frequently mutated genes in human breast cancer. Here, we describe a mouse model for PIK3CA-induced breast cancer by using the ROSA26 (R26) knock-in system, in which targeted Pik3ca alleles can be activated through transgenic expression of Cre recombinase. We mated Pik3caH1047R and Pik3cawt knock-in lines with MMTV-Cre transgenics, which express Cre in mammary epithelium. Starting at approximately 5 months of age, female R26-Pik3caH1047R;MMTV-Cre mice, but not control R26-Pik3cawt;MMTV-Cre mice, developed mammary tumors, as well as lymphoid and skin malignancies. R26-Pik3caH1047R;MMTV-Cre mammary tumors were typically either adenosquamous carcinoma or adenomyoepithelioma. As p53 is the most commonly mutated gene in breast cancer, we tested for genetic interaction between Pik3caH1047R and p53 loss-of-function mutations in R26-Pik3caH1047R;p53loxP/+;MMTV-Cre mice. This led to decreased survival of double-mutant animals, which developed lymphoma and mammary tumors with rapid kinetics. Mammary tumors that formed in p53loxP/+;MMTV-Cre conditional mutants were either poorly differentiated adenocarcinoma or spindle cell/EMT, whereas R26-Pik3caH1047R;p53loxP/+;MMTV-Cre mammary tumors were mostly adenosquamous carcinoma or spindle cell/EMT indicating that double-mutant mice develop a distinct spectrum of mammary tumors. Thus, an oncogenic variant of PIK3CA implicated in multiple human breast cancer subtypes can induce a very diverse spectrum of mammary tumors in mice. Furthermore, Pik3caH1047R shows cooperation with p53, which altered the specific tumors that formed. Thus, the two most frequently mutated genes in human breast cancer show cooperation in mammary tumor formation. Cancer Res; 71(7); 2706–17. ©2011 AACR.