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1.  Inducible and coupled expression of the polyomavirus middle T antigen and Cre recombinase in transgenic mice: an in vivo model for synthetic viability in mammary tumour progression 
Introduction
Effective in vivo models of breast cancer are crucial for studying the development and progression of the disease in humans. We sought to engineer a novel mouse model of polyomavirus middle T antigen (PyV mT)-mediated mammary tumourigenesis in which inducible expression of this well-characterized viral oncoprotein is coupled to Cre recombinase (TetO-PyV mT-IRES-Cre recombinase or MIC).
Methods
MIC mice were crossed to the mouse mammary tumour virus (MMTV)-reverse tetracycline transactivator (rtTA) strain to generate cohorts of virgin females carrying one or both transgenes. Experimental (rtTA/MIC) and control (rtTA or MIC) animals were administered 2 mg/mL doxycycline beginning as early as eight weeks of age and monitored for mammary tumour formation, in parallel with un-induced controls of the same genotypes.
Results
Of the rtTA/MIC virgin females studied, 90% developed mammary tumour with complete penetrance to all glands in response to doxycycline and a T50 of seven days post-induction, while induced or un-induced controls remained tumour-free after one year of induction. Histological analyses of rtTA/MIC mammary glands and tumour revealed that lesions followed the canonical stepwise progression of PyV mT tumourigenesis, from hyperplasia to mammary intraepithelial neoplasia/adenoma, carcinoma, and invasive carcinoma that metastasizes to the lung; at each of these stages expression of PyV mT and Cre recombinase transgenes was confirmed. Withdrawal of doxycycline from rtTA/MIC mice with end-stage mammary tumours led to rapid regression, yet animals eventually developed PyV mT-expressing and -non-expressing recurrent masses with varied tumour histopathologies.
Conclusions
We have successfully created a temporally regulated mouse model of PyV mT-mediated mammary tumourigenesis that can be used to study Cre recombinase-mediated genetic changes simultaneously. While maintaining all of the hallmark features of the well-established constitutive MMTV-PyV mT model, the utility of this strain derives from the linking of PyV mT and Cre recombinase transgenes; mammary epithelial cells are thereby forced to couple PyV mT expression with conditional ablation of a given gene. This transgenic mouse model will be an important research tool for identifying synthetic viable genetic events that enable PyV mT tumours to evolve in the absence of a key signaling pathway.
doi:10.1186/bcr3603
PMCID: PMC3978996  PMID: 24457046
2.  Concomitant Targeting of Tumor Cells and Induction of T-cell Response Synergizes to Effectively Inhibit Trastuzumab-Resistant Breast Cancer 
Cancer research  2012;72(17):4417-4428.
Trastuzumab is an iconic rationally designed targeted therapy for HER2-positive breast cancers. However, the low response rate and development of resistance call for novel approaches for the treatment of patients. Here, we report that concurrent targeting of tumor cells and activation of T cells in the tumor microenvironment results in a synergistic inhibitory effect on tumor growth and overcomes resistance in two distinct PTEN loss–mediated trastuzumab-resistant mammary tumor mouse models. In vivo combination treatment with HER2/Neu antibody and Akt inhibitor triciribine effectively inhibited tumor growth in both models via inhibiting PI3K/AKT and mitogen-activated protein kinase signaling accompanied by increased T-cell infiltration in the tumor microenvironment. We showed that both CD8+ and CD4+ T cells were essential to the optimal antitumor effect of this combination treatment in an IFN- γ–dependent manner. Importantly, the antitumor activities of HER2/Neu antibody and triciribine combination treatment were further improved when coinhibitory receptor cytotoxic T-lymphocyte–associated antigen 4 was blocked to enhance the T-cell response. Our data indicate that multitargeted combinatorial therapies targeting tumor cells and concomitantly enhancing T-cell response in the tumor microenvironment could cooperate to exert maximal therapeutic activity, suggesting a promising clinical strategy for treating trastuzumab-resistant breast cancers and other advanced malignancies.
doi:10.1158/0008-5472.CAN-12-1339-T
PMCID: PMC3556997  PMID: 22773664
3.  Akt determines cell fate through the negative regulation of the PERK-eIF2α phosphorylation pathway 
Science signaling  2011;4(192):ra62.
Metazoans respond to various forms of environmental stress by inducing the phosphorylation of the α subunit of the translation initiation factor eIF2 at serine 51 (eIF2αP), a modification that leads to a global inhibition of mRNA translation. Herein, we demonstrate that eIF2αP is induced by pharmacological inhibition of the phosphoinositide-3-kinase (PI3K)-Akt pathway as well as by genetic or small interfering (si)RNA-mediated ablation of Akt. Increased eIF2αP is an evolutionary conserved process that involves the endoplasmic reticulum (ER)-resident protein kinase PERK, which is negatively regulated by Akt-dependent phosphorylation at threonine 799. PERK activity and eIF2αP are downregulated by activated Akt in mouse mammary gland tumors as well as in cells exposed to ER stress or oxidative stress leading to the induction of cell survival or death respectively. In unstressed cells, the PERK-eIF2αP pathway guards survival and facilitates adaptation to the deleterious effects of PI3K or Akt inactivation. As such, inactivation of the PERK-eIF2αP arm increases the susceptibility of tumor cells to death by pharmacological inhibitors of PI3K or Akt. Thus, in addition to mTOR the PERK-eIF2αP pathway provides a link between Akt signaling and translational control with implications in tumor formation and treatment.
doi:10.1126/scisignal.2001630
PMCID: PMC3752779  PMID: 21954288
translational control; PERK; eIF2α phosphorylation; Akt/PKB; endoplasmic reticulum stress; oxidative stress; chemotherapeutic drugs
4.  Pathogenesis of Neonatal Herpes Simplex 2 Disease in a Mouse Model Is Dependent on Entry Receptor Expression and Route of Inoculation 
Journal of Virology  2013;87(1):474-481.
Herpes simplex virus (HSV) pathogenesis in mice differs based on availability of the principal entry receptors herpesvirus entry mediator (HVEM) and nectin-1 in a manner dependent upon route of inoculation. After intravaginal or intracranial inoculation of adult mice, nectin-1 is a major mediator of neurologic disease, while the absence of either receptor attenuates disease after ocular infection. We tested the importance of receptor availability and route of infection on disease in mouse models of neonatal HSV. We infected 7-day-old mice lacking neither or one principal HSV receptor or both principal HSV receptors with HSV-2 via a peripheral route (intranasal), via a systemic route (intraperitoneal), or by inoculation directly into the central nervous system (intracranial). Mortality, neurologic disease, and visceral dissemination of virus were significantly attenuated in nectin-1 knockout mice compared with HVEM knockout or wild-type mice after intranasal inoculation. Mice lacking both entry receptors (double-knockout mice) showed no evidence of disease after inoculation by any route. Nectin-1 knockout mice had delayed mortality after intraperitoneal inoculation relative to wild-type and HVEM knockout mice, but virus was able to spread to the brain and viscera in all genotypes except double-knockout mice. Unlike in adult mice, HVEM was sufficient to mediate disease in neonatal mice after direct intracranial inoculation, and the absence of HVEM delayed time to mortality relative to that of wild-type mice. Additionally, in wild-type neonatal mice inoculated intracranially, HSV antigen did not primarily colocalize with NeuN-positive neurons. Our results suggest that differences in receptor expression between adults and newborns may partially explain differences in susceptibility to HSV-2.
doi:10.1128/JVI.01849-12
PMCID: PMC3536411  PMID: 23097453
5.  HER3 Is Required for HER2-Induced Preneoplastic Changes to the Breast Epithelium and Tumor Formation 
Cancer research  2012;72(10):2672-2682.
Increasing evidence suggests that HER2-amplified breast cancer cells use HER3/ErbB3 to drive therapeutic resistance to HER2 inhibitors. However, the role of ErbB3 in the earliest events of breast epithelial transformation remains unknown. Using mouse mammary specific models of Cre-mediated ErbB3 ablation, we show that ErbB3 loss prevents the progressive transformation of HER2-overexpressing mammary epithelium. Decreased proliferation and increased apoptosis were seen in MMTV-HER2 and MMTV-Neu mammary glands lacking ErbB3, thus inhibiting premalignant HER2-induced hyperplasia. Using a transgenic model in which HER2 and Cre are expressed from a single polycistronic transcript, we showed that palpable tumor penetrance decreased from 93.3% to 6.7% upon ErbB3 ablation. Penetrance of ductal carcinomas in situ was also decreased. In addition, loss of ErbB3 impaired Akt and p44/42 phosphorylation in preneoplastic HER2-overexpressing mammary glands and in tumors, decreased growth of preexisting HER2-overexpressing tumors, and improved tumor response to the HER2 tyrosine kinase inhibitor lapatinib. These events were rescued by reexpression of ErbB3, but were only partially rescued by ErbB36F, an ErbB3 mutant harboring six tyrosine-to-phenylalanine mutations that block its interaction with phosphatidyl inositol 3-kinase. Taken together, our findings suggest that ErbB3 promotes HER2-induced changes in the breast epithelium before, during, and after tumor formation. These results may have important translational implications for the treatment and prevention of HER2-amplified breast tumors through ErbB3 inhibition.
doi:10.1158/0008-5472.CAN-11-3594
PMCID: PMC3693553  PMID: 22461506
6.  Selenoproteins reduce susceptibility to DMBA-induced mammary carcinogenesis 
Carcinogenesis  2012;33(6):1225-1230.
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 Trspfl/+;wt, Trspfl/+;MMTV-cre, Trsp fl/fl;wt and Trspfl/fl;MMTV-cre female mice were administered standard doses of the carcinogen, 7,12-dimethylbenzylbenz[a]antracene. Our results revealed that heterozygous, Trspfl/+;MMTV-cre mice showed no difference in tumor incidence, tumor rate and survival compared with the Trspfl/+;wt mice. However, 54.8% of homozygous Trspfl/f l;MMTV-cre mice developed mammary tumors and exhibited significantly shorter survival than the corresponding Trspfl/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.
doi:10.1093/carcin/bgs129
PMCID: PMC3514862  PMID: 22436612
7.  Longitudinal Characterization of Herpes Simplex Virus (HSV) Isolates Acquired From Different Sites in an Immune-Compromised Child: A New HSV Thymidine Kinase Mutation Associated With Resistance 
Background. Herpes simplex virus resistance to acyclovir is well described in immune-compromised patients. Management of prolonged infection and recurrences in such patients may be problematic.
Methods. A patient with neuroblastoma developed likely primary herpes gingivostomatitis shortly after starting a course of chemotherapy, with spread to the eye during treatment with acyclovir. Viral isolates were serially obtained from separate sites after treatment was begun and tested for susceptibility to acyclovir and foscarnet by plaque reduction and plating efficiency assays. The thymidine kinase and DNA polymerase genes from each isolate were sequenced.
Results. Initial isolates from a throat swab, an oral lesion, and conjunctiva were resistant to acyclovir within 13 days of treatment. Subsequent isolates while on foscarnet were initially acyclovir-susceptible, but reactivation of an acyclovir-resistant isolate was subsequently documented while on acyclovir suppression. Genotypic analysis identified a previously unreported UL23 mutation in some resistant isolates. None of the amino acid changes identified in UL30 were associated with resistance.
Conclusions. Phenotypic and genotypic antiviral resistance of herpes simplex isolates may vary from different compartments and over time in individual immune-compromised hosts, highlighting the importance of obtaining cultures from all sites. Phenotypic resistance testing should be considered for isolates obtained from at-risk patients not responding to first-line therapy. Empiric combination treatment with multiple antivirals could be considered in some situations.
doi:10.1093/jpids/pis009
PMCID: PMC3656553  PMID: 23687576
8.  Herpes Simplex Virus-2 Glycoprotein Interaction with HVEM Influences Virus-Specific Recall Cellular Responses at the Mucosa 
Infection of susceptible cells by herpes simplex virus (HSV) requires the interaction of the HSV gD glycoprotein with one of two principal entry receptors, herpes virus entry mediator (HVEM) or nectins. HVEM naturally functions in immune signaling, and the gD-HVEM interaction alters innate signaling early after mucosal infection. We investigated whether the gD-HVEM interaction during priming changes lymphocyte recall responses in the murine intravaginal model. Mice were primed with attenuated HSV-2 expressing wild-type gD or mutant gD unable to engage HVEM and challenged 32 days later with virulent HSV-2 expressing wild-type gD. HSV-specific CD8+ T cells were decreased at the genital mucosa during the recall response after priming with virus unable to engage HVEM but did not differ in draining lymph nodes. CD4+ T cells, which are critical for entry of HSV-specific CD8+ T cells into mucosa in acute infection, did not differ between the two groups in either tissue. An inverse association between Foxp3+ CD4+ regulatory T cells and CD8+ infiltration into the mucosa was not statistically significant. CXCR3 surface expression was not significantly different among different lymphocyte subsets. We conclude that engagement of HVEM during the acute phase of HSV infection influences the antiviral CD8+ recall response by an unexplained mechanism.
doi:10.1155/2012/284104
PMCID: PMC3359786  PMID: 22666282
9.  The Immunologic Basis for Severe Neonatal Herpes Disease and Potential Strategies for Therapeutic Intervention 
Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) infect a large proportion of the world's population. Infection is life-long and can cause periodic mucocutaneous symptoms, but it only rarely causes life-threatening disease among immunocompetent children and adults. However, when HSV infection occurs during the neonatal period, viral replication is poorly controlled and a large proportion of infants die or develop disability even with optimal antiviral therapy. Increasingly, specific differences are being elucidated between the immune system of newborns and those of older children and adults, which predispose to severe infections and reflect the transition from fetal to postnatal life. Studies in healthy individuals of different ages, individuals with primary or acquired immunodeficiencies, and animal models have contributed to our understanding of the mechanisms that control HSV infection and how these may be impaired during the neonatal period. This paper outlines our current understanding of innate and adaptive immunity to HSV infection, immunologic differences in early infancy that may account for the manifestations of neonatal HSV infection, and the potential of interventions to augment neonatal immune protection against HSV disease.
doi:10.1155/2013/369172
PMCID: PMC3626239  PMID: 23606868
10.  Distinct biological roles for the Akt family in mammary tumor progression 
Cancer research  2010;70(11):4260-4264.
The phosphatidylinositol 3' kinase/Akt pathway is frequently dysregulated in cancer which can have unfavorable consequences in terms of cell proliferation, survival, metabolism and migration. Increasing evidence suggests that Akt1, Akt2 and Akt3 play unique roles in breast cancer initiation and progression. We have recently shown that in contrast to Akt1 which accelerates mammary tumor induction in transgenic mice, Akt2 promotes metastasis of tumor cells without affecting the latency of tumor development. Despite the distinct phenotypic outputs resulting from Akt1 or Akt2 activation, very little is known regarding the mode by which such unique functions originate from these highly related kinases. Here we discuss potential mechanisms contributing to the differing functional specificity of Akt1 and Akt2 with respect to migration, invasion and metastasis.
doi:10.1158/0008-5472.CAN-10-0266
PMCID: PMC2880222  PMID: 20424120
Akt; breast cancer; metastasis; transgenic mouse; invasion
11.  Oncogenes and Tumor Suppressor Genes 
Breast cancer progression involves multiple genetic events, which can activate dominant-acting oncogenes and disrupt the function of specific tumor suppressor genes. This article describes several key oncogene and tumor suppressor signaling networks that have been implicated in breast cancer progression. Among the tumor suppressors, the article emphasizes BRCA1/2 and p53 tumor suppressors. In addition to these well characterized tumor suppressors, the article highlights the importance of PTEN tumor suppressor in counteracting PI3K signaling from activated oncogenes such as ErbB2. This article discusses the use of mouse models of human breast that recapitulate the key genetic events involved in the initiation and progression of breast cancer. Finally, the therapeutic potential of targeting these key tumor suppressor and oncogene signaling networks is discussed.
Mouse models can recapitulate genetic events that disrupt signaling by ErbB2, p53, and PTEN during breast cancer progression. They also reveal that mutations in Brca1 can produce different tumor subtypes, depending on the context.
doi:10.1101/cshperspect.a003236
PMCID: PMC2944361  PMID: 20719876
12.  β1-integrins signaling and mammary tumor progression in transgenic mouse models: implications for human breast cancer 
Consistent with their essential role in cell adhesion to the extracellular matrix, integrins and their associated signaling pathways have been shown to be involved in cell proliferation, migration, invasion and survival, processes required in both tumorigenesis and metastasis. β1-integrins represent the predominantly expressed integrins in mammary epithelial cells and have been proven crucial for mammary gland development and differentiation. Here we provide an overview of the studies that have used transgenic mouse models of mammary tumorigenesis to establish β1-integrin as a critical mediator of breast cancer progression and thereby as a potential therapeutic target for the development of new anticancer strategies.
doi:10.1186/bcr2905
PMCID: PMC3326542  PMID: 22264244
13.  Focal adhesion kinase contributes to proliferative potential of ErbB2 mammary tumour cells but is dispensable for ErbB2 mammary tumour induction in vivo 
Introduction
Activation of focal adhesion kinase (FAK) is hypothesized to play an important role in the pathogenesis of human breast cancer.
Methods
To directly evaluate the role of FAK in mammary tumour progression, we have used a conditional FAK mouse model and mouse mammary tumour virus (MMTV)-driven Cre recombinase strain to inactivate FAK in the mammary epithelium of a transgenic mouse model of ErbB2 breast cancer.
Results
Although mammary epithelial disruption of FAK in this model resulted in both a delay in onset and a decrease in the number of neoplastic lesions, mammary tumours occurred in 100% of virgin female mice. All of the tumours and derived metastases that developed were proficient for FAK due to the absence of Cre recombinase expression. The hyperplastic epithelia where Cre-mediated recombination of FAK could be detected exhibited a profound proliferative defect. Consistent with these observations, disruption of FAK in established tumour cells resulted in reduced tumour growth that was associated with impaired proliferation. To avoid the selection for FAK-proficient ErbB2 tumour epithelia through escape of Cre-mediated recombination, we next intercrossed the FAK conditional mice with a separate MMTV-driven ErbB2 strain that co-expressed ErbB2 and Cre recombinase on the same transcriptional unit.
Conclusions
While a delay in tumour induction was noted, FAK-deficient tumours arose in 100% of female animals indicating that FAK is dispensable for ErbB2 tumour initiation. In addition, the FAK-null ErbB2 tumours retained their metastatic potential. We further demonstrated that the FAK-related Pyk2 kinase is still expressed in these tumours and is associated with its downstream regulator p130Cas. These observations indicate that Pyk2 can functionally substitute for FAK in ErbB2 mammary tumour progression.
doi:10.1186/bcr3131
PMCID: PMC3496154  PMID: 22373082
14.  Activation of p21(CIP1/WAF1) in Mammary Epithelium Accelerates Mammary Tumorigenesis and Promotes Lung Metastasis 
While p21 is well known to inhibit cyclin-CDK activity in the nucleus and it has also been demonstrated to have oncogenic properties in different types of human cancers. In vitro studies showed that the oncogenic function of p21is closely related to its cytoplasmic localization. However, it is unclear whether cytoplasmic p21 contributes to tumorigenesis in vivo. To address this question, we generated transgenic mice expressing the Akt-phosphorylated form of p21 (p21T145D) in the mammary epithelium. The results showed that Akt-activated p21 was expressed in the cytoplasm of mammary epithelium. Overexpression of Akt-activated p21 accelerated tumor onset and promoted lung metastasis in MMTV/neu mice, providing evidence that p21, especially cytoplasmic phosphorylated p21, has an oncogenic role in promoting mammary tumorigenesis and metastasis.
doi:10.1016/j.bbrc.2010.10.126
PMCID: PMC3001223  PMID: 21040707
p21; PKB/Akt; mammary tumorigenesis; lung metastasis
15.  PTHrP drives breast tumor initiation, progression, and metastasis in mice and is a potential therapy target 
The Journal of Clinical Investigation  2011;121(12):4655-4669.
Parathyroid hormone–related protein (PTHrP) is a secreted factor expressed in almost all normal fetal and adult tissues. It is involved in a wide range of developmental and physiological processes, including serum calcium regulation. PTHrP is also associated with the progression of skeletal metastases, and its dysregulated expression in advanced cancers causes malignancy-associated hypercalcemia. Although PTHrP is frequently expressed by breast tumors and other solid cancers, its effects on tumor progression are unclear. Here, we demonstrate in mice pleiotropic involvement of PTHrP in key steps of breast cancer — it influences the initiation and progression of primary tumors and metastases. Pthrp ablation in the mammary epithelium of the PyMT-MMTV breast cancer mouse model caused a delay in primary tumor initiation, inhibited tumor progression, and reduced metastasis to distal sites. Mechanistically, it reduced expression of molecular markers of cell proliferation (Ki67) and angiogenesis (factor VIII), antiapoptotic factor Bcl-2, cell-cycle progression regulator cyclin D1, and survival factor AKT1. PTHrP also influenced expression of the adhesion factor CXCR4, and coexpression of PTHrP and CXCR4 was crucial for metastatic spread. Importantly, PTHrP-specific neutralizing antibodies slowed the progression and metastasis of human breast cancer xenografts. Our data identify what we believe to be new functions for PTHrP in several key steps of breast cancer and suggest that PTHrP may constitute a novel target for therapeutic intervention.
doi:10.1172/JCI46134
PMCID: PMC3225988  PMID: 22056386
16.  Activation of MDM2 by Akt in Mammary Epithelium Delays Mammary Involution and Accelerates Mammary Tumorigenesis 
Cancer research  2010;70(19):7684-7689.
Amplification or overexpression of MDM2 promotes a variety of human tumors by degrading tumor suppressor proteins such as p53. Phosphorylation of MDM2 on serines 166 and 186 by the survival kinase Akt inhibits p53-mediated apoptosis. However, it is unclear whether this pathway contributes to normal or malignant pathophysiology in vivo. To address these questions, we generated transgenic mice expressing the Akt-phosphorylated form of MDM2 (MDM2DDS166D/S186D) in the mammary epithelium. Activation of MDM2 delayed mammary gland involution and accelerated tumor progression in MMTV/neu transgenic mice by inhibiting apoptosis in a manner associated with decreased p53 expression. Our findings offer in vivo evidence that activation of MDM2 by Akt contributes to mammary development and tumorigenesis.
doi:10.1158/0008-5472.CAN-09-3231
PMCID: PMC2948588  PMID: 20841468
17.  Transforming Growth Factor-β Regulator SnoN Modulates Mammary Gland Branching Morphogenesis, Postlactational Involution, and Mammary Tumorigenesis 
Cancer research  2010;70(10):4204-4213.
SnoN is an important negative regulator of transforming growth factor-β (TGF-β) signaling that was originally identified as a transforming oncogene in chicken embryonic fibroblasts. Both pro-oncogenic and antioncogenic activities of SnoN have been reported, but its function in normal epithelial cells has not been defined. In the mouse mammary gland, SnoN is expressed at relatively low levels, but it is transiently upregulated at late gestation before being downregulated during lactation and early involution. To assess the effects of elevated levels of SnoN, we generated transgenic mice expressing a SnoN fragment under the control of the mouse mammary tumor virus promoter. In this model system, SnoN elevation increased side-branching and lobular-alveolar proliferation in virgin glands, while accelerating involution in postlactation glands. Increased proliferation stimulated by SnoN was insufficient to induce mammary tumorigenesis. In contrast, elevated levels of SnoN cooperated with polyoma middle T antigen to accelerate the formation of aggressive multifocal adenocarcinomas and to increase the formation of pulmonary metastases. Our studies define functions of SnoN in mammary epithelial cell proliferation and involution, and provide the first in vivo evidence of a pro-oncogenic role for SnoN in mammalian tumorigenesis.
doi:10.1158/0008-5472.CAN-10-0135
PMCID: PMC3098116  PMID: 20460516
18.  Immunobiology of herpes simplex virus and cytomegalovirus infections of the fetus and newborn 
Current immunology reviews  2010;6(1):38-55.
Immunologic “immaturity” is often blamed for the increased susceptibility of newborn humans to infection, but the precise mechanisms and details of immunologic development remain somewhat obscure. Herpes simplex virus (HSV) and cytomegalovirus (CMV) are two of the more common severe infectious agents of the fetal and newborn periods. HSV infection in the newborn most commonly occurs after exposure to the virus during delivery, and can lead to a spectrum of clinical disease ranging from isolated skin-eye-mucous membrane infection to severe disseminated multiorgan disease, often including encephalitis. In contrast to HSV, clinically severe CMV infections early in life are usually acquired during the intrauterine period. These infections can result in a range of clinical disease, including hearing loss and neurodevelopmental delay. However, term newborns infected with CMV after delivery are generally asymptomatic, and older children and adults often acquire infection with HSV or CMV with either no or mild clinical symptoms. The reasons for these widely variable clinical presentations are not completely understood, but likely relate to developmental differences in immune responses.
This review summarizes recent human and animal studies of the immunologic response of the fetus and newborn to these two infections, in comparison to the responses of older children and adults. The immunologic defense of the newborn against each virus is considered under the broader categories of (i) the placental barrier to infection, (ii) skin and mucosal barriers (including antimicrobial peptides), (iii) innate responses, (iv) humoral responses, and (v) cellular responses. A specific focus is made on recent studies of innate and cellular immunity to HSV and CMV.
doi:10.2174/157339510790231833
PMCID: PMC2866044  PMID: 20467462
herpes simplex virus; cytomegalovirus; neonatal immunity; fetal immunity
19.  Loss of STAT1 from Mouse Mammary Epithelium Results in an Increased Neu-Induced Tumor Burden1 
Neoplasia (New York, N.Y.)  2010;12(11):899-905.
Type I and type II classes of interferons (IFNs) signal through the JAK/STAT1 pathway and are known to be important in adaptive and innate immune responses and in protection against tumors. Although STAT1 is widely considered a tumor suppressor, it remains unclear, however, if this function occurs in tumor cells (cell autonomous) or if STAT1 acts primarily through immune cells. Here, the question of whether STAT1 has a cell autonomous role in mammary tumor formation was addressed in a mouse model of ERBB2/neu-induced breast cancer in the absence and presence of STAT1. For this purpose, mice that carry floxed Stat1 alleles, which permit cell-specific removal of STAT1, were generated. To induce tumors only in mammary cells lacking STAT1, Stat1 floxed mice were crossed with transgenic mice that express cre recombinase and the neu oncogene under the mouse mammary tumor virus LTR (Stat1fl/fl NIC). Stat1 was effectively deleted in mammary epithelium of virgin Stat1fl/fl NIC females. Time-to-tumor onset was significantly shorter in Stat1fl/fl NIC females than in WT NIC (Wilcoxon rank sum test, P = .02). The median time-to-tumor onset in the Stat1fl/fl NIC mice was 49.4 weeks, whereas it was 62.4 weeks in the WT NIC mice. These results suggest that STAT1 in mammary epithelial cells may play a role in suppressing tumorigenesis. The Stat1 floxed allele described in this study is also a unique resource to determine the cellular targets of IFNs and STAT1 action, which should aid our understanding and appreciation of these pathways.
PMCID: PMC2978912  PMID: 21076615
20.  Shc is required for ErbB2 induced inhibition of apoptosis but is dispensable for cell proliferation and disruption of cell polarity 
Oncogene  2009;29(2):174-187.
Amplification and overexpression of ErbB2 strongly correlates with aggressive breast cancers. A deeper understanding of pathways downstream of ErbB2 signaling that are required for transformation of human mammary epithelial cells will identify novel strategies for therapeutic intervention in breast cancer. Using an inducible activation of ErbB2 autophosphorylation site mutants and the MCF-10A three-dimensional culture system we investigated pathways used by ErbB2 to transform epithelia. We report that ErbB2 induces cell proliferation and loss of 3D organization by redundant mechanisms whereas it disrupts apical basal polarity and inhibits apoptosis using Tyr 1201 and Tyr 1226/7 respectively. Signals downstream of Tyr 1226/7 were also sufficient to confer paclitaxel resistance. The Tyr1226/7 binds Shc, and knockdown of Shc blocked the ability of ErbB2 to inhibit apoptosis and mediate paclitaxel resistance. Tyr1226/7 is known to activate the Ras/Erk pathway, however, paclitaxel resistance did not correlate with activation of Erk or Akt suggesting the presence of a novel mechanism. Thus, our results demonstrate that targeting pathways used by ErbB2 to inhibit cell death is a better option than targeting cell proliferation pathways. Furthermore, we identify a novel function for Shc as a regulator of apoptosis and drug resistance in human mammary epithelial cells transformed by ErbB2.
doi:10.1038/onc.2009.312
PMCID: PMC2948752  PMID: 19826412
21.  14-3-3ζ Cooperates with ErbB2 to Promote Progression of Ductal Carcinoma in Situ to Invasive Breast Cancer by Inducing Epithelial-Mesenchymal Transition 
Cancer cell  2009;16(3):195-207.
Summary
ErbB2, a metastasis-promoting oncoprotein, is overexpressed in ~25% of invasive/metastatic breast cancers, but in 50–60% of non-invasive ductal carcinomas in situ (DCIS). It has been puzzling how a subset of ErbB2-overexpressing DCIS develops into invasive breast cancer (IBC). We found that co-overexpression of 14-3-3ζ in ErbB2-overexpressing DCIS conferred a higher risk of progression to IBC. ErbB2 and 14-3-3ζ overexpression, respectively, increased cell migration and decreased cell adhesion, two prerequisites of tumor cell invasion. 14-3-3ζ overexpression reduced cell adhesion by activating the TGFβ/Smads pathway that led to ZFHX1B/SIP-1 upregulation, E-cadherin loss, and epithelial-mesenchymal transition (EMT). Importantly, patients whose breast tumors overexpressed both ErbB2 and 14-3-3ζ had higher rates of metastatic recurrence and death than those whose tumors overexpressed only one.
doi:10.1016/j.ccr.2009.08.010
PMCID: PMC2754239  PMID: 19732720
22.  Identification of a Stat3-dependent transcription regulatory network involved in metastatic progression 
Cancer research  2009;69(17):6823-6830.
High levels of activated Stat3 are often found in human breast cancers and can correlate with poor patient outcome. We employed an activated-ErbB2 mouse model of breast cancer to investigate the in vivo role of Stat3 in mammary tumor progression and found that Stat3 does not alter mammary tumor initiation but dramatically affects metastatic progression. Four-fold fewer animals exhibited lung metastases in the absence of Stat3 and a 12-fold reduction in the number of lung lesions was observed in the Stat3-null tumors when compared to tumors from the wild type cohort. The decreased malignancy in Stat3-deficient tumors is attributed to a reduction in both angiogenic and inflammatory responses associated with a Stat3-dependent transcriptional cascade involving C/EBPδ.
doi:10.1158/0008-5472.CAN-09-1684
PMCID: PMC2841985  PMID: 19690134
23.  Cooperative signaling between Wnt1 and integrin-linked kinase induces accelerated breast tumor development 
Introduction
Breast cancer is genetically and clinically a heterogeneous disease. However, the exact contribution of different cell types and oncogenic mutations to this heterogeneity are not well understood. Recently, we discovered an interaction between Wnt and integrin-linked kinase (ILK) within the signaling cascade that regulates cell growth and survival. Interestingly, mammary-specific expression of either one of these proteins has been shown to promote mammary tumorigenesis. In light of our recent findings and to investigate the potential interaction between Wnt and ILK proteins during mammary tumor formation and progression, we established a transgenic mouse model that expresses both Wnt and ILK in mammary epithelial cells.
Methods
A novel transgenic mouse model with mammary-specific expression of both Wnt1 and ILK was generated by crossing the two previously characterized mouse models, MMTV-Wnt1 and MMTV-ILK. The resulting MMTV-Wnt/ILK mice were closely monitored for tumor development and growth, as well as for the tumor onset. The molecular phenotypes of both tumors and premalignant mammary glands were investigated by using biochemical and global gene-expression analysis approaches.
Results
A significant acceleration in mammary tumor incidence and growth was observed in the MMTV-Wnt/ILK mice. Pre-neoplastic mammary glands also display lobuloalveolar hyperplasia and an increase in ductal epithelium proliferation. Apart from elevated expression of Wnt/ILK targets, such as β-catenin and cyclin D1, gene-expression profiling identified the surprising activation of the FOXA1 transcription factor. Upregulation of FOXA1, which is also known as the molecular marker of differentiated mammary luminal cells, was consistent with the expansion of the enriched luminal progenitor population or CD29loCD24hiCD61+ cells in MMTV-Wnt/ILK tumors.
Conclusions
These results show cooperation between Wnt1 and ILK transgenes during mammary carcinogenesis, leading to changes in a transcriptional network, which could dictate a specific breast cancer phenotype with enhanced growth dynamics. The MMTV-Wnt/ILK can be used as a model to identify further the genes downstream of the estrogen receptor-β/FOXA1 and to investigate the mechanisms targeting the expansion of the luminal progenitor cells leading to hyperplasia and tumorigenesis.
doi:10.1186/bcr2592
PMCID: PMC2917033  PMID: 20565980
24.  Herpes simplex virus type 2 tegument proteins contain subdominant T-cell epitopes detectable in BALB/c mice after DNA immunization and infection 
The Journal of general virology  2009;90(Pt 5):1153-1163.
Summary
Cytotoxic T-cells are important in controlling herpes simplex virus type 2 (HSV-2) reactivation and peripheral lesion resolution. Humans latently infected with HSV-2 have cytotoxic T-cells directed against epitopes present in tegument proteins. Studies in mice of immunity to HSV have commonly focused on immunodominant responses in HSV envelope glycoproteins. These antigens have not proved to be an effective prophylactic vaccine target for most of the human population. The murine immune response against HSV tegument proteins has not been explored. We analyzed cellular responses in BALB/c mice directed against the tegument proteins encoded by UL46, UL47, and UL49 and against the envelope glycoprotein gD after DNA vaccination or HSV-2 infection. Splenocyte T-cell responses to overlapping peptides from UL46 and UL47 were more than 500 interferon-γ spot forming units per 106 responder cells after DNA vaccination. Peptide truncation studies, responder cell fractionation, and major histocompatibility complex binding studies identified several CD8+ and CD4+ epitopes. Cellular responses to tegument protein epitopes were also detected after HSV-2 infection. Tegument proteins are rational candidates for further HSV-2 vaccine research.
doi:10.1099/vir.0.008771-0
PMCID: PMC2675279  PMID: 19264627
Herpes simplex virus; Rodent; Cytotoxic T-cells; Viral infection; Vaccination
25.  c-Src Associates with ErbB2 through an Interaction between Catalytic Domains and Confers Enhanced Transforming Potential▿ †  
Molecular and Cellular Biology  2009;29(21):5858-5871.
Previous studies have demonstrated that c-Src tyrosine kinase interacts specifically with ErbB2, but not with other members of the epidermal growth factor receptor (EGFR) family. To identify the site of interaction, we recently used a chimeric EGFR/ErbB2 receptor approach to show that c-Src requires the kinase region of ErbB2 for binding. Here, we demonstrate that retention of a conserved amino acid motif surrounding tyrosine 877 (referred to here as EGFRYHAD) is sufficient to confer binding to c-Src. Surprisingly the association of c-Src was not dependent on its SH2 or SH3 domain or on the phosphorylation or kinase activity of the receptor. We further show that the chimeric EGFRs that contain the Y877 motif are transforming in vitro and in vivo following ligand stimulation. Transformation was also partially dependent on sustained activation of Stat3. Finally, we demonstrate that EGFRs with mutations in the catalytic domain, originally identified in lung cancer and conferring increased sensitivity to gefitinib and erlotinib, two EGFR kinase inhibitors, gained the capacity to bind c-Src. Moreover, transformation by these EGFR mutants was inhibited by Src inhibitors regardless of their sensitivities to gefitinib and erlotinib. These observations have important implications for understanding the molecular basis for resistance to EGFR inhibitors and implicate c-Src as a critical signaling molecule in EGFR mutant-induced transformation.
doi:10.1128/MCB.01731-08
PMCID: PMC2772728  PMID: 19704002

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