Erika Ginsburg

CD44posCD49fhiCD133/2hi Defines Xenograft-Initiating Cells in Estrogen Receptor–Negative Breast Cancer

Defining the populations of tumor-initating cells that are present in tumors is a first step in developing therapeutics to target these cells. We show here that both CD44(pos)CD24(neg) and CD44(pos)CD24(pos) cell populations in estrogen receptor (ER) alpha-negative breast tumors are tumorigenic in murine xenograft models. We also describe a third population of xenograft-initiating cells (XIC) enriched in CD44(pos)CD49f(hi)CD133/2(hi) cells that display heightened tumorigenicity, self-renewal in vivo, and the capacity to give rise to functional and molecular heterogeneity. Consistent with their capacity for self-renewal, these cells express elevated levels of Sox2, Bmi-1, and/or Nanog and their CpG islands are hypermethylated relative to nontumorigenic cells. These differences in methylome regulation may be responsible for the dramatic functional differences between the two populations. The identification of CD44(pos)CD49f(hi)CD133/2(hi) XIC in ER-negative tumors may lead to expanded understanding of these tumors and ultimately the development of therapeutics designed to specifically target the cells.

Dynamic regulation of CD24 and the invasive, CD44posCD24neg phenotype in breast cancer cell lines.

IntroductionThe invasive, mesenchymal phenotype of CD44posCD24neg breast cancer cells has made them a promising target for eliminating the metastatic capacity of primary tumors. It has been previously demonstrated that CD44neg/lowCD24pos breast cancer cells lack the ability to give rise to their invasive CD44posCD24neg counterpart. Here we demonstrate that noninvasive, epithelial-like CD44posCD24pos cells readily give rise to invasive, mesenchymal CD44posCD24neg progeny in vivo and in vitro. This interconversion was found to be dependent upon Activin/Nodal signaling.MethodsBreast cancer cell lines were sorted into CD44posCD24pos and CD44posCD24neg populations to evaluate their progeny for the expression of CD44, CD24, and markers of a mesenchymal phenotype. The populations, separated by fluorescence activated cell sorting (FACS) were injected into immunocompromised mice to evaluate their tumorigenicity and invasiveness of the resulting xenografts.ResultsCD24 expression was dynamically regulated in vitro in all evaluated breast cancer cell lines. Furthermore, a single noninvasive, epithelial-like CD44posCD24pos cell had the ability to give rise to invasive, mesenchymal CD44posCD24neg progeny. Importantly, this interconversion occurred in vivo as CD44posCD24pos cells gave rise to xenografts with locally invasive borders as seen in xenografts initiated with CD44posCD24neg cells. Lastly, the ability of CD44posCD24pos cells to give rise to mesenchymal progeny, and vice versa, was blocked upon ablation of Activin/Nodal signaling.ConclusionsOur data demonstrate that the invasive, mesenchymal CD44posCD24neg phenotype is under dynamic control in breast cancer cell lines both in vitro and in vivo. Furthermore, our observations suggest that therapies targeting CD44posCD24neg tumor cells may have limited success in preventing primary tumor metastasis unless Activin/Nodal signaling is arrested.

Transcriptional and spatiotemporal regulation of prolactin receptor mRNA and cooperativity with progesterone receptor function during ductal branch growth in the mammary gland

Ductal branching within the mammary gland is stimulated by prolactin (PRL) and progesterone (P) acting through their receptors (PRLR and PR). Analysis of mammary gland PRLR expression revealed increasing expression of the long form (L‐PRLR) and two of the three short forms (S1‐ and S3‐PRLR) during puberty that became maximal late in pubescence and early gestation, then declined during gestation. By contrast, S2‐PRLR mRNA levels remained constant. Examination of stromal PRLR revealed the consistent expression of L‐PRLR mRNA. By contrast, S1‐PRLR was present only in the mammary fat pad of neonates, whereas high neonatal expression of S2‐PRLR became undetectable during puberty. Stromal expression of S3‐PRLR decreased to low levels during puberty and was undetectable during lactation and involution. Exogenous PRL stimulated DNA synthesis in both epithelial and adjacent stromal cells in vivo. Distribution of PRLR mRNA in mammary epithelium was homogeneous before puberty and heterogeneous during puberty, gestation, and early lactation. A mutual role for PRLR and PR was suggested wherein PR mRNA increased beyond 6 weeks to maximal levels during puberty and gestation then became undetectable during lactation. In situ hybridization revealed that PR mRNA distribution is homogeneous in the ductal epithelium before 6 weeks and heterogenous during puberty and gestation and that PRLR and PR are similarly distributed in the ductal epithelium. Neither hormone stimulated DNA synthesis in mammary glands of ovariectomized females while their effects interacted markedly. These results demonstrate differential PRLR transcription by epithelial and stromal cells and a similar distribution of PRLR and PR that may facilitate the interaction between P and PRL during ductal branching in the mammary gland.

In vitro stimulation by paraquat of reactive oxygen-mediated lipid peroxidation in rat lung microsomes

Abstract Paraquat significantly stimulated lipid peroxidation in rat lung microsomes without the addition of exogenous iron. The ability of paraquat to stimulate this lipid peroxidation was dependent on the presence of adequate reducing equivalents (NADPH), aerobic conditions, and the duration of incubation, viz. optimal in vitro conditions. Even greater paraquat-mediated lipid peroxidation was observed if incubations were conducted under O2 or if vitamin E-deficient microsomes were utilized, factors which have previously been reported to increase the in vivo pulnonary toxicity of paraquat. Superoxide dismutase (3 μg/ml) significantly inhibited paraquat-stimulated lipid peroxidation in rat lung microsomes (73%), demonstrating a pivotal role for superoxide in this process. Thus, the redox cycling of paraquat and accompanying reactive oxygen generation was capable of mediating lipid peroxidation not only in mouse lung and rat liver microsomes but also in rat lung microsomes.

Studies on the in vitro interaction of mitomycin C, nitrofurantoin and paraquat with pulmonary microsomes: Stimulation of reactive oxygen-dependent lipid peroxidation☆

In vitro experiments were performed to evaluate the capacity of the redox cycling compounds mitomycin C (MC), nitrofurantoin (NF) and paraquat (PQ) to stimulate pulmonary microsomal lipid peroxidation. It was observed that the interaction of MC, NF or PQ with rat or mouse lung microsomes in the presence of an NADPH-generating system and an O2 atmosphere resulted in significant lipid peroxidation. All three compounds demonstrated similar concentration dependency, similar time courses and the ability to generate lipid peroxidation-associated chemiluminescence. The stimulation of lipid peroxidation by MC, NF or PQ was inhibited significantly by superoxide dismutase, glutathione, ascorbic acid, catalase and EDTA, agents which either scavenge reactive oxygen and/or prevent the generation of secondary reactive oxygen metabolites. In addition, the ability of MC or NF, but not PQ, to stimulate lipid peroxidation was reduced significantly following preincubation with microsomes and NADPH under N2 (15-20 min) prior to incubation under O2. During this period under N2. MC and NF underwent reductive metabolism of their quinone and nitro moieties respectively. Thus, it appears that under aerobic conditions the pulmonary microsomal-mediated redox cycling of MC, NF and PQ is accompanied by the stimulation of reactive oxygen-dependent lipid peroxidation.

Msx-1 and Msx-2 in mammary gland development.

Homeobox genes do not generally function alone to determine cell fate and morphogenesis. Rather it is the distinct combination of various members of the homeobox family of genes and their spatiotemporal patterns of expression that determine cell identity and function. Functional redundancy often makes it difficult to clearly discern the role of any one given homeobox gene. The roles that Msx1 and Msx2 play in branching morphogenesis of the mammary gland are only now becoming more evident. Many signaling pathways and transcription factors are implicated in how these homeobox genes correctly determine the morphological development of the gland. Overexpression of Msx1 and Msx2 may also be involved in tumorigenesis. Additional studies are needed to elucidate the roles of these genes in both breast development and cancer.

S179D Prolactin Increases Vitamin D Receptor and p21 through Up-regulation of Short 1b Prolactin Receptor in Human Prostate Cancer Cells

In this study, we further investigated the mechanisms by which pseudophosphorylated prolactin (S179D PRL) inhibits the growth of human prostate cancer cells. When treated with S179D PRL for 3 days, LnCAP cells responded by increasing expression of the vitamin D receptor (VDR) and the cell cycle regulatory molecule, p21, whereas PC3 and DU145 cells did not. After 5 days of treatment, both PC3 and DU145 cells responded. Untreated LnCAP cells express the short 1b form (SF1b) of the human prolactin receptor, but DU145 and PC3 cells express only low amounts of this receptor until elevated by treatment with S179D PRL. DU145 and PC3 cells become sensitive to the negative effects of S179D PRL on cell number after induction of the SF1b. Transfection of either SF1b or SF1a into PC3 or DU145 cells made them sensitive to S179D PRL in the 3-day time frame, a finding that was not duplicated by transfection with the long form of the receptor. Treatment of LnCAP cells with S179D PRL increased long-term activation of extracellular signal-regulated kinase 1/2 (ERK1/2). This did not occur in PC3 and DU145 cells until transfection with SF1a/SF1b. Blockade of ERK signaling eliminated S179D PRL-stimulated expression of the VDR and p21 in LnCAP cells and transfected PC3 and DU145 cells. We conclude that initiation of alternative splicing to produce SF1b, and subsequent altered signaling, contribute to the growth inhibitory mechanisms of S179D PRL. This is the first indication of a role for short prolactin receptors in the regulation of cell proliferation.

Bronchiolar epithelial damage and impairment of pulmonary microsomal monooxygenase activity in mice by naphthalene

Abstract Injection of a single dose of naphthalene into C57BL/6J mice (225 mg/kg, ip) produced a significant (30–70%) and prolonged (8–15 days) impairment in pulmonary microsomal monooxygenase activities without altering these activities in liver microsomes. The time course of naphthalene-induced morphologic damage to bronchiolar epithelium paralleled compromises in pulmonary monooxygenase activity. No concomitant alterations in hepatic morphology were observed. Five microsomal enzymes were studied: benzphetamine N -demethylase, aryl hydrocarbon hydroxylase, NADPH cytochrome c reductase, 7-ethoxyresorufin O -deethylase (a cytochrome P -448-dependent enzyme), and styrene epoxide hydrolase (a cytochrome P -450-independent enzyme). In general, the time course of the inhibition of these pulmonary enzymes was similar but the magnitude of the inhibition varied somewhat. Maximum inhibition of enzyme activity occurred about 3 days after naphthalene administration; 7-ethoxyresorufin O -deethylase activity was reduced to about 30% of control values whereas benzphetamine N -demethylase declined to about 70% of control. The remaining enzymes clustered midway between these extremes at about 50% of control values. Inhibited activities remained at relatively constant levels between Days 3 and 8 and by Day 15, there was a clear trend returning toward controls. Despite this trend, three of the six pulmonary enzyme activities examined remained significantly below control levels 15 days after a single dose of the hydrocarbon. Histologically, the pulmonary nonciliated bronchiolar epithelial (Clara) cell was the primary target of naphthalene toxicity. At early time points and at low magnifications, it appeared as if the entire bronchiolar epithelium was undergoing necrosis and sloughing into the lumen. However, higher magnifications revealed residual ciliated epithelium. The distribution of Clara cell damage appeared to vary considerably. One could find bronchioles that appeared completely denuded of epithelium and others in the same section whose Clara cells had been spared or, alternatively, were in the process of regeneration. The results are discussed in relation to recent work which has shown selective covalent binding of naphthalene to pulmonary Clara cells.

Local regulation of human breast xenograft models

Breast cancer studies implant human cancer cells under the renal capsule, subcutaneously, or orthotopically and often use estrogen supplementation and immune suppressants (etoposide) in xenograft mouse models. However, cell behavior is significantly impacted by signals from the local microenvironment. Therefore, we investigated how the combinatorial effect of the location of injection and procedural differences affected xenograft characteristics. Patient‐derived breast cancer cells were injected into mouse abdominal or thoracic mammary glands ± estrogen and/or etoposide pretreatment. Abdominal xenografts had increased tumor incidence and volume, and decreased latency (P < 0.001) compared to thoracic tumors. No statistically significant difference in tumor volume was found in abdominal xenografts treated ± estrogen or etoposide; however, etoposide suppressed tumor volume in thoracic xenografts (P < 0.02). The combination of estrogen and etoposide significantly decreased tumor incidence in both sites. In addition, mice treated ± estradiol were injected orthotopically or subcutaneously with well‐characterized breast cancer cell lines (MCF7, ZR75‐1, MDA MB‐231, or MCF10Ca1h). Orthotopic injection increased tumor volume; growth varied with estrogen supplementation. Location also altered methylation status of several breast cancer‐related gene promoters. Lastly, vascularization of orthotopic tumors was significantly enhanced compared to subcutaneous tumors. These data suggest that optimal xenograft success occurs with orthotopic abdominal injections and illustrate molecular details of the compelling influence of the local microenvironment on in vivo models. J. Cell. Physiol. 224: 795–806, 2010. Published 2010 Wiley‐Liss, Inc.

The effects of adriamycin in vitro and in vivo on hepatic microsomal drug-metabolizing enzymes: role of microsomal lipid peroxidation.

The quinone-containing anticancer drug adriamycin augments the reduction of dioxygen to reactive oxygen species and thereby stimulates (sixfold) NADPH-dependent microsomal lipid peroxidation. In vitro the extensive adriamycin-promoted peroxidation depleted (85%) rat liver microsomal cytochrome P-450, severely inhibited cytochrome P-450-dependent monooxygenation (70%), and glucose-6-phosphatase activity (80%), and activated (450%) UDP-glucuronyltransferase activity. When lipid peroxidation was blocked by EDTA, adriamycin selectively decreased cytochrome P-450 and aminopyrine N-demethylase activity; NADPH-cytochrome c reductase, UDP-glucuronyltransferase, and glucose-6-phosphatase activities were unchanged. Washing and resedimenting peroxidized microsomes to remove adriamycin and soluble lipid peroxidation products failed to restore enzyme activities to control values. Adriamycin administered subacutely (5 mg/kg × three doses) to rats significantly descreased hepatic microsomal cytochrome P-450 content and reduced aminopyrine N-demethylase and NADPH-cytochrome c reductase activities compared to saline-treated controls. Microsomal lipid peroxidation was increased following the above adriamycin treatment. Thus, these data suggested that adriamycin was capable of impairing hepatic drug metabolism in vitro by stimulating membrane lipid peroxidation in a manner similar to carbon tetrachloride; the mechanism by which adriamycin treatment in vivo decreased the activity of the drug monooxygenase system remains unclear.