Aleata A. Triplett

Impaired Alveologenesis and Maintenance of Secretory Mammary Epithelial Cells in Jak2 Conditional Knockout Mice

ABSTRACT Jak2 is a hormone-receptor-coupled kinase that mediates the tyrosine phosphorylation and activation of signal transducers and activators of transcription (Stat). The biological relevance of Jak2-Stat signaling in hormone-responsive adult tissues is difficult to investigate since Jak2 deficiency leads to embryonic lethality. We generated Jak2 conditional knockout mice to study essential functions of Jak2 during mammary gland development. The mouse mammary tumor virus-Cre-mediated excision of the first coding exon resulted in a Jak2 null mutation that uncouples signaling from the prolactin receptor (PRL-R) to its downstream mediator Stat5 in the presence of normal and supraphysiological levels of PRL. Jak2-deficient females were unable to lactate as a result of impaired alveologenesis. Unlike Stat5a knockouts, multiple gestation cycles could not reverse the Jak2-deficient phenotype, suggesting that neither other components of the PRL-R signaling cascade nor other growth factors and their signal transducers were able to compensate for the loss of Jak2 function to activate Stat5 in vivo. A comparative analysis of Jak2-deficient mammary glands with transplants from Stat5a/b knockouts revealed that Jak2 deficiency also impairs the pregnancy-induced branching morphogenesis. Jak2 conditional mutants therefore resemble PRL-R knockouts more closely, which suggested that Jak2 deficiency might affect additional PRL-R downstream mediators other than Stat5a and Stat5b. To address whether Jak2 is required for the maintenance of PRL-responsive, differentiating alveolar cells, we utilized a transgenic strain that expresses Cre recombinase under regulatory elements of the whey acidic protein gene (Wap). The Wap-Cre-mediated excision of Jak2 resulted in a negative selection of differentiated alveolar cells, suggesting that Jak2 is required not only for the proliferation and differentiation of alveolar cells but also for their maintenance during lactation.

Tsg101 Is Essential for Cell Growth, Proliferation, and Cell Survival of Embryonic and Adult Tissues

ABSTRACT Tumor susceptibility gene 101 (Tsg101) was identified in a random mutagenesis screen for potential tumor suppressors in NIH 3T3 cells. Altered transcripts of this gene have been detected in sporadic breast cancers and many other human malignancies. However, the involvement of this gene in neoplastic transformation and tumorigenesis is still elusive. Using gene targeting, we generated genetically engineered mice with a floxed allele of Tsg101. We investigated essential functions of this gene in vivo and examined whether the loss of function of Tsg101 results in tumorigenesis. Conventional knockout mice were generated through Cre-mediated excision of the first coding exon in the germ line of mouse mammary tumor virus (MMTV)-Cre transgenic mice. The complete ablation of Tsg101 in the developing embryo resulted in death around implantation. In contrast, mammary gland-specific knockout mice developed normally but were unable to nurse their young as a result of impaired mammogenesis during late pregnancy. Neither heterozygous null mutants nor somatic knockout mice developed mammary tumors after a latency of 2 years. The Cre-mediated deletion of Tsg101 in primary cells demonstrated that this gene is essential for the growth, proliferation, and survival of mammary epithelial cells. In summary, our results suggest that Tsg101 is required for normal cell function of embryonic and adult tissues but that this gene is not a tumor suppressor for sporadic forms of breast cancer.

Parity-induced mammary epithelial cells facilitate tumorigenesis in MMTV-neu transgenic mice.

Using a Cre-lox-based genetic labeling technique, we have recently discovered a parity-induced mammary epithelial subtype that is abundant in nonlactating and nonpregnant, parous females. These mammary epithelial cells serve as alveolar progenitors in subsequent pregnancies, and transplantation studies revealed that they possess features of multipotent progenitors such as self-renewal and the capability to contribute to ductal and alveolar morphogenesis. Here, we report that these cells are the cellular targets for transformation in MMTV-neu transgenic mice that exhibit accelerated mammary tumorigenesis in multiparous animals. The selective ablation of this epithelial subtype reduces the onset of tumorigenesis in multiparous MMTV-neu transgenics. There is, however, experimental evidence to suggest that parity-induced mammary epithelial cells may not be the only cellular targets in other MMTV-promoter-based transgenic strains. In particular, the heterogeneous MMTV-wnt1 lesions predominantly express the ductal differentiation marker Nkcc1 that is absent in MMTV-neu-derived tumors. Our observations support the idea that tumors originate from distinctly different epithelial subtypes in selected MMTV-promoter-driven cancer models and that diverse oncogenes might exert discrete effects on particular mammary epithelial subtypes.

DNA Methylation in Folbp1 Knockout Mice Supplemented with Folic Acid during Gestation

Periconceptional folic acid supplementation has been shown to prevent up to 70% of neural tube and other birth defects in humans; however, the mechanism is still unknown. In this study, we tested whether defective intracellular folate transport, as achieved by inactivation of the murine folate-binding protein 1 (Folbp1), affects global DNA methylation in the liver and brain from gestational day (GD) 15 embryos. Complete Folbp1 inactivation is embryolethal but can be reversed by maternal folinic acid (FA) supplementation, and thus we also tested the effect of FA supplementation on DNA methylation in Folbp1 fetuses. Overall, the extent of global DNA methylation seems to be similar across all genotypes in unsupplemented control Folbp1 mice; however, explicit conclusions regarding Folbp1(-/-) fetuses were not possible because only a single living unsupplemented fetus was viable at GD 15. FA supplementation induced global DNA hypomethylation across all genotypes. FA-induced hypomethylation is most likely due to its ability to inhibit the enzyme glycine hydroxymethyltransferase, thereby inhibiting the homocysteine remethylation cycle necessary to regenerate S-adenosylmethionine, the methyl donor for DNA methyltransferases. Our hypothesis was that due to defective folate transport in Folbp1(-/-) embryos and fetuses, DNA would be hypomethylated, thereby altering the temporal expression of critical genes necessary for normal embryonic development. However, these results suggest that an extended examination of changes in DNA methylation prior to GD 15 is required to unequivocally prove or disprove the hypothesis.

Cell Cycle Arrest and Cell Death Are Controlled by p53-dependent and p53-independent Mechanisms in Tsg101-deficient Cells

Our previous studies have shown that cells conditionally deficient in Tsg101 arrested at the G1/S cell cycle checkpoint and died. We created a series of Tsg101 conditional knock-out cell lines that lack p53, p21Cip1, or p19Arf to determine the involvement of the Mdm2-p53 circuit as a regulator for G1/S progression and cell death. In this new report we show that the cell cycle arrest in Tsg101-deficient cells is p53-dependent, but a null mutation of the p53 gene is unable to maintain cell survival. The deletion of the Cdkn1a gene in Tsg101 conditional knock-out cells resulted in G1/S progression, suggesting that the p53-dependent G1 arrest in the Tsg101 knock-out is mediated by p21Cip1. The Cre-mediated excision of Tsg101 in immortalized fibroblasts that lack p19Arf seemed not to alter the ability of Mdm2 to sequester p53, and the p21-mediated G1 arrest was not restored. Based on these findings, we propose that the p21-dependent cell cycle arrest in Tsg101-deficient cells is an indirect consequence of cellular stress and not caused by a direct effect of Tsg101 on Mdm2 function as previously suggested. Finally, the deletion of Tsg101 from primary tumor cells that express mutant p53 and that lack p21Cip1 expression results in cell death, suggesting that additional transforming mutations during tumorigenesis do not affect the important role of Tsg101 for cell survival.

Stat5 Promotes Survival of Mammary Epithelial Cells through Transcriptional Activation of a Distinct Promoter in Akt1

ABSTRACT The signal transducer and activator of transcription 5 (Stat5) plays a pivotal role in the proliferation, secretory differentiation, and survival of mammary epithelial cells. However, there is little information about Stat5 target genes that facilitate these biological processes. We provide here experimental evidence that the prolactin-mediated phosphorylation of Stat5 regulates the transcriptional activation of the Akt1 gene. Stat5 binds to consensus sequences within the Akt1 locus in a growth factor-dependent manner to initiate transcription of a unique Akt1 mRNA from a distinct promoter, which is only active in the mammary gland. Elevating the levels of active Akt1 restores the expression of cyclin D1 and proliferation of Jak2-deficient mammary epithelial cells, which provides evidence that Akt1 acts downstream of Jak/Stat signaling. The ligand-inducible expression of Stat5 in transgenic females mediates a sustained upregulation of Akt1 in mammary epithelial cells during the onset of postlactational involution. Stat5-expressing mammary glands exhibit a delay in involution despite induction of proapoptotic signaling events. Collectively, the results of the present study elucidate an underlying mechanism by which active Stat5 mediates evasion from apoptosis and self-sufficiency in growth signals.

Developmental Outcome of Levetiracetam, Its Major Metabolite in Humans, 2-Pyrrolidinone N-Butyric Acid, and Its Enantiomer (R)-α-ethyl-oxo-pyrrolidine Acetamide in a Mouse Model of Teratogenicity

Summary:  Purpose: The purpose of this study was to test the teratogenic potential of the antiepileptic drug (AED) levetiracetam (LEV), its major metabolite in humans, 2‐pyrrolidone‐N‐butyric acid (PBA), and enantiomer, (R)‐α‐ethyl‐oxo‐pyrrolidine acetamide (REV), in a well‐established mouse model.

Janus kinase 2 is required for the initiation but not maintenance of prolactin-induced mammary cancer

The prolactin receptor (PRLR), its associated Janus kinase 2 (Jak2) and the signal transducer and activator of transcription 5 (Stat5) are essential for normal mammary gland development. Owing to the upregulation of the PRLR and the local synthesis of its ligand in neoplastic cells, it has been proposed that PRL can act as a local growth factor in human breast cancers. This notion is supported by experimental evidence in transgenic mice, which showed that the mammary-specific expression of PRL contributes to carcinogenesis in vivo. To assess the importance of Jak2/Stat5 signaling during mammary cancer initiation and progression, we generated a PRL-induced mammary cancer model that allows the functional ablation of the Jak2 gene in the mammary epithelium before and after neoplastic transformation. Collectively, the results of this study show that the functional ablation of Jak2 protects against the onset of PRL-induced mammary tumorigenesis, suggesting that targeting this kinase is a relevant strategy for mammary cancer prevention. Surprisingly, Jak2 deficiency did not affect the growth and survival of PRL-induced mammary cancer cells in culture and in vivo. Consequently, Jak2 cannot be a sole therapeutic target to treat the established disease. PRL-induced mammary cancers exhibited an upregulation of ErbB2 and other ErbB receptor tyrosine kinases that may supersede the functionality of PRLR signaling through Jak2.

Dormant Cancer Cells Contribute to Residual Disease in a Model of Reversible Pancreatic Cancer

The initiation and progression of pancreatic ductal adenocarcinoma (PDAC) is governed by a series of genetic and epigenetic changes, but it is still unknown whether these alterations are required for the maintenance of primary and metastatic PDAC. We show here that the c-Myc oncogene is upregulated throughout the entire process of neoplastic progression in human PDAC and in genetically engineered mice that express mutant Kras. To experimentally address whether c-Myc is essential for the growth and survival of cancer cells, we developed a novel mouse model that allows a temporally and spatially controlled expression of this oncogene in pancreatic progenitors and derived lineages of the exocrine pancreas. Unlike previous reports, upregulation of c-Myc was sufficient to induce the formation of adenocarcinomas after a short latency without additional genetic manipulation of cell survival pathways. Deficiency in Cdkn2a increased the rate of metastasis but had no effect on tumor latency or c-Myc-mediated cancer maintenance. Despite a macroscopically complete regression of primary, metastatic, and transplantable tumors following the ablation of c-Myc, some cancer cells remained dormant. A significant number of these residual neoplastic cells expressed cancer stem cell markers, and re-expression of exogenous c-Myc in these cells led to rapid cancer recurrence. Collectively, the results of this study suggest that c-Myc plays a significant role in the progression and maintenance of PDAC, but besides targeting this oncogene or its downstream effectors, additional therapeutic strategies are necessary to eradicate residual cancer cells to prevent disease recurrence.

Targeting Janus Kinase 2 in Her2/neu-Expressing Mammary Cancer: Implications for Cancer Prevention and Therapy

The Janus kinase 2 (Jak2) is essential for normal mammary gland development, but this tyrosine kinase and its main effector, signal transducer and activator of transcription 5, are also active in a significant subset of human breast cancers. We have recently reported that Jak2 controls the expression and nuclear accumulation of cyclin D1. Because this particular D-type cyclin has been suggested to be a key mediator for ErbB2-associated mammary tumorigenesis, we deleted Jak2 from ErbB2-expressing mammary epithelial cells prior to tumor onset and in neoplastic cells to address whether this tyrosine kinase plays a role in the initiation as well as progression of mammary cancer. Similar to cyclin D1-deficient mice, the functional ablation of Jak2 protects against the onset of mammary tumorigenesis. In contrast, the deletion of Jak2 from neoplastic cells or the acute, ligand-inducible down-regulation of this tyrosine kinase in an orthotopic transplant model did not affect the growth and survival of cancer cells. The constitutive activation of ErbB2 signaling, which is an initial event in the formation of mammary cancer, was able to override the functional role of Jak2 in regulating the expression of Akt1 and cyclin D1. This might be a compensatory mechanism that explains why Jak2 is a relevant target for preventing the initiation but not the progression of ErbB2-associated mammary cancer.