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1.  Herpes simplex virus serotype and entry receptor availability alter CNS disease in a mouse model of neonatal HSV 
Pediatric research  2014;76(6):528-534.
Outcomes of neonates with herpes simplex virus (HSV) encephalitis are worse after infection with HSV-2 when compared with HSV-1. The proteins herpes virus entry mediator (HVEM) and nectin-1 mediate HSV entry into susceptible cells. Prior studies have shown receptor-dependent differences in pathogenesis that depend on route of inoculation and host developmental age.
We investigated serotype-related differences in HSV disease and their relationship to entry receptor availability in a mouse model of encephalitis.
Mortality was attenuated in seven-day-old wild-type (WT) mice inoculated with HSV-1(F) when compared with HSV-2(333). No serotype-specific differences were seen after inoculation of adult mice. HSV-1 pathogenesis was also attenuated relative to HSV-2 in newborn but not adult mice lacking HVEM or nectin-1. HSV-2 requires nectin-1 for encephalitis in adult but not newborn mice; in contrast, nectin-1 was important for HSV-1 pathogenesis in both age groups. Early viral replication was independent of age, viral serotype, or mouse genotype, suggesting host responses influence outcomes. In this regard, significantly greater amounts of inflammatory mediators were detected in brain homogenates from WT newborns 2 days after infection compared with adults and receptor-knockout newborns.
Dysregulation of inflammatory responses induced by infection may influence the severity of HSV encephalitis.
PMCID: PMC4233006  PMID: 25198371
2.  c-Src drives intestinal regeneration and transformation 
The EMBO Journal  2014;33(13):1474-1491.
The non-receptor tyrosine kinase c-Src, hereafter referred to as Src, is overexpressed or activated in multiple human malignancies. There has been much speculation about the functional role of Src in colorectal cancer (CRC), with Src amplification and potential activating mutations in up to 20%of the human tumours, although this has never been addressed due to multiple redundant family members. Here, we have used the adult Drosophila and mouse intestinal epithelium as paradigms to define a role for Src during tissue homeostasis, damage-induced regeneration and hyperplasia. Through genetic gain and loss of function experiments, we demonstrate that Src is necessary and sufficient to drive intestinal stem cell (ISC) proliferation during tissue self-renewal, regeneration and tumourigenesis. Surprisingly, Src plays a non-redundant role in the mouse intestine, which cannot be substituted by the other family kinases Fyn and Yes. Mechanistically, we show that Src drives ISC proliferation through upregulation of EGFR and activation of Ras/MAPK and Stat3 signalling. Therefore, we demonstrate a novel essential role for Src in intestinal stem/progenitor cell proliferation and tumourigenesis initiation in vivo.
PMCID: PMC4194090  PMID: 24788409
Apc; intestinal stem cells; regeneration; Src; tumourigenesis
3.  Akt1 and Akt2 play distinct roles in the initiation and metastatic phases of mammary tumor progression 
Cancer research  2009;69(12):5057-5064.
The PI3K/Akt survival pathway is often dysregulated in cancer. Our previous studies have demonstrated that coexpression of activated Akt1 with activated ErbB2 or polyoma virus middle T antigen uncoupled from the PI3K pathway (PyVmT Y315/322F) accelerates mammary tumor development but cannot rescue the metastatic phenotype associated with these models. Here we report the generation of transgenic mice expressing activated Akt2 in the mammary epithelium. Like the MMTV-Akt1 strain, mammary-specific expression of Akt2 delayed mammary gland involution. However, in contrast to Akt1, coexpression of Akt2 with activated ErbB2 or PyVmT Y315/322F in the mammary glands of transgenic mice did not impact the latency of tumor development. Strikingly Akt2 coexpresssion markedly increased the incidence of pulmonary metastases in both tumor models demonstrating a unique role in tumor progression. Together these observations argue that these highly conserved kinases have distinct biological and biochemical outputs that play opposing roles in mammary tumor induction and metastasis.
PMCID: PMC4151524  PMID: 19491266
Akt; ErbB2; PyVmT; mammary tumorigenesis; metastasis
4.  ERBB2 Deficiency Alters an E2F-1-Dependent Adaptive Stress Response and Leads to Cardiac Dysfunction 
Molecular and Cellular Biology  2014;34(23):4232-4243.
The tyrosine kinase receptor ERBB2 is required for normal development of the heart and is a potent oncogene in breast epithelium. Trastuzumab, a monoclonal antibody targeting ERBB2, improves the survival of breast cancer patients, but cardiac dysfunction is a major side effect of the drug. The molecular mechanisms underlying how ERBB2 regulates cardiac function and why trastuzumab is cardiotoxic remain poorly understood. We show here that ERBB2 hypomorphic mice develop cardiac dysfunction that mimics the side effects observed in patients treated with trastuzumab. We demonstrate that this phenotype is related to the critical role played by ERBB2 in cardiac homeostasis and physiological hypertrophy. Importantly, genetic and therapeutic reduction of ERBB2 activity in mice, as well as ablation of ERBB2 signaling by trastuzumab or siRNAs in human cardiomyocytes, led to the identification of an impaired E2F-1-dependent genetic program critical for the cardiac adaptive stress response. These findings demonstrate the existence of a previously unknown mechanistic link between ERBB2 and E2F-1 transcriptional activity in heart physiology and trastuzumab-induced cardiac dysfunction.
PMCID: PMC4248744  PMID: 25246633
5.  Biomarker-guided sequential targeted therapies to overcome therapy resistance in rapidly evolving highly aggressive mammary tumors 
Cell Research  2014;24(5):542-559.
Combinatorial targeted therapies are more effective in treating cancer by blocking by-pass mechanisms or inducing synthetic lethality. However, their clinical application is hampered by resistance and toxicity. To meet this important challenge, we developed and tested a novel concept of biomarker-guided sequential applications of various targeted therapies using ErbB2-overexpressing/PTEN-low, highly aggressive breast cancer as our model. Strikingly, sustained activation of ErbB2 and downstream pathways drives trastuzumab resistance in both PTEN-low/trastuzumab-resistant breast cancers from patients and mammary tumors with intratumoral heterogeneity from genetically-engineered mice. Although lapatinib initially inhibited trastuzumab-resistant mouse tumors, tumors by-passed the inhibition by activating the PI3K/mTOR signaling network as shown by the quantitative protein arrays. Interestingly, activation of the mTOR pathway was also observed in neoadjuvant lapatinib-treated patients manifesting lapatinib resistance. Trastuzumab + lapatinib resistance was effectively overcome by sequential application of a PI3K/mTOR dual kinase inhibitor (BEZ235) with no significant toxicity. However, our p-RTK array analysis demonstrated that BEZ235 treatment led to increased ErbB2 expression and phosphorylation in genetically-engineered mouse tumors and in 3-D, but not 2-D, culture, leading to BEZ235 resistance. Mechanistically, we identified ErbB2 protein stabilization and activation as a novel mechanism of BEZ235 resistance, which was reversed by subsequent treatment with lapatinib + BEZ235 combination. Remarkably, this sequential application of targeted therapies guided by biomarker changes in the tumors rapidly evolving resistance doubled the life-span of mice bearing exceedingly aggressive tumors. This fundamentally novel approach of using targeted therapies in a sequential order can effectively target and reprogram the signaling networks in cancers evolving resistance during treatment.
PMCID: PMC4011340  PMID: 24675532
sequential therapy; tumor evolution; targeted therapy; trastuzumab resistance; BEZ235; ErbB2 stabilization
6.  Early Viral Suppression Improves Neurocognitive Outcomes in HIV-infected Children 
AIDS (London, England)  2015;29(3):295-304.
To estimate the association of age of viral suppression and central nervous system penetration effectiveness (CPE) score with neurocognitive functioning among school-age children with perinatally-acquired HIV infection (PHIV+).
We analyzed data from two U.S.-based multisite prospective cohort studies.
Multivariable general linear regression models were used to evaluate associations of age at viral suppression and CPE scores [of initial ART regimen and weighted average] with WISC-III or WISC-IV neurocognitive assessments [full scale IQ (FSIQ); performance IQ/ perceptual reasoning index (PIQ/PRI); and verbal IQ/ verbal comprehension index (VIQ/VCI)], adjusted for demographic and clinical covariates. Sensitivity analyses were stratified by birth cohort (before vs after 1996).
396 PHIV+ children were included. Estimated differences in mean FSIQ (comparing virally suppressed vs. unsuppressed children) by each age cutoff were 3.7, 2.2, 3.2, 4.4, and 3.9 points at ages 1, 2, 3, 4, and 5, respectively. For PIQ/PRI, estimated mean differences were 3.7, 2.4, 2.2, 4.6, and 4.5 at ages 1 through 5 respectively. In both cases, these differences were significant only at the age 4 and 5 thresholds. After stratifying by birth cohort the association between age at suppression and cognitive function persisted only among those born after 1996. Age at viral suppression was not associated with VIQ/VCI; CPE score was not associated with FSIQ, verbal comprehension or perceptual reasoning indices.
Virologic suppression during infancy or early childhood is associated with improved neurocognitive outcomes in school-aged PHIV+ children. In contrast, CPE scores showed no association with neurocognitive outcomes.
PMCID: PMC4332557  PMID: 25686678
Paediatrics; HIV; cognition; antiretroviral therapy; viral load; central nervous system
7.  Loss of periostin/OSF-2 in ErbB2/Neu-driven tumors results in androgen receptor-positive molecular apocrine-like tumors with reduced Notch1 activity 
Periostin (Postn) is a secreted cell adhesion protein that activates signaling pathways to promote cancer cell survival, angiogenesis, invasion, and metastasis. Interestingly, Postn is frequently overexpressed in numerous human cancers, including breast, lung, colon, pancreatic, and ovarian cancer.
Using transgenic mice expressing the Neu oncogene in the mammary epithelium crossed into Postn-deficient animals, we have assessed the effect of Postn gene deletion on Neu-driven mammary tumorigenesis.
Although Postn is exclusively expressed in the stromal fibroblasts of the mammary gland, Postn deletion does not affect mammary gland outgrowth during development or pregnancy. Furthermore, we find that loss of Postn in the mammary epithelium does not alter breast tumor initiation or growth in mouse mammary tumor virus (MMTV)-Neu expressing mice but results in an apocrine-like tumor phenotype. Surprisingly, we find that tumors derived from Postn-null animals express low levels of Notch protein and Hey1 mRNA but increased expression of androgen receptor (AR) and AR target genes. We show that tumor cells derived from wild-type animals do not proliferate when transplanted in a Postn-null environment but that this growth defect is rescued by the overexpression of active Notch or the AR target gene prolactin-induced protein (PIP/GCDFP-15).
Together our data suggest that loss of Postn in an ErbB2/Neu/HER2 overexpression model results in apocrine-like tumors that activate an AR-dependent pathway. This may have important implications for the treatment of breast cancers involving the therapeutic targeting of periostin or Notch signaling.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0513-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4355979  PMID: 25592291
8.  Differential Reliance on Autophagy for Protection from HSV Encephalitis between Newborns and Adults 
PLoS Pathogens  2015;11(1):e1004580.
Newborns are more susceptible to severe disease from infection than adults, with maturation of immune responses implicated as a major factor. The type I interferon response delays mortality and limits viral replication in adult mice in a model of herpes simplex virus (HSV) encephalitis. We found that intact type I interferon signaling did not control HSV disease in the neonatal brain. However, the multifunctional HSV protein γ34.5 involved in countering type I interferon responses was important for virulence in the brain in both age groups. To investigate this observation further, we studied a specific function of γ34.5 which contributes to HSV pathogenesis in the adult brain, inhibition of the cellular process of autophagy. Surprisingly, we found that the beclin binding domain of γ34.5 responsible for inhibiting autophagy was dispensable for HSV disease in the neonatal brain, as infection of newborns with the deletion mutant decreased time to mortality compared to the rescue virus. Additionally, a functional beclin binding domain in HSV γ34.5 did not effectively inhibit autophagy in the neonate, unlike in the adult. Type I IFN responses promote autophagy in adult, a finding we confirmed in the adult brain after HSV infection; however, in the newborn brain we observed that autophagy was activated through a type I IFN-independent mechanism. Furthermore, autophagy in the wild-type neonatal mouse was associated with increased apoptosis in infected regions of the brain. Observations in the mouse model were consistent with those in a human case of neonatal HSV encephalitis. Our findings reveal age-dependent differences in autophagy for protection from HSV encephalitis, indicating developmental differences in induction and regulation of this innate defense mechanism after HSV infection in the neonatal brain.
Author Summary
Disease after infection with a pathogen results from an intersection between the infectious agent and the host. Newborns are particularly susceptible to infectious illness compared to adults, and HSV infection commonly results in devastating encephalitis. We studied the interaction of HSV with the type I interferon pathway and found that a specific activity of the viral protein γ34.5, which counters host autophagy to promote encephalitis in adults, was not required to cause disease in newborns. Furthermore, autophagy was not inhibited by HSV in the neonate and was not activated by type I interferon signaling, unlike in the adult. Activated autophagy was associated with increased apoptosis, which may contribute to the increased pathology in newborns. Our findings reveal development-specific differences in the pathogenesis of HSV encephalitis, including a distinct role for autophagy in the neonatal brain.
PMCID: PMC4287605  PMID: 25569138
9.  14-3-3ζ Orchestrates Mammary Tumor Onset and Progression via miR221-Mediated Cell Proliferation 
Cancer research  2013;74(1):363-373.
14-3-3ζ is overexpressed in over 40% of breast cancers but its pathophysiological relevance to tumorigenesis has not been established. Here we show that 14-3-3ζ overexpression is sufficient to induce tumorigenesis in a transgenic mouse model of breast cancer. MMTV-LTR promoter driven HA-14-3-3ζ transgenic mice (MMTV-HA-14-3-3ζ) developed mammary tumors whereas control mice did not. Whey acidic protein promoter driven HA-14-3-3ζ transgenic mice (WAP-HA-14-3-3ζ) developed hyperplastic lesions and showed increased susceptibility to carcinogen-induced tumorigenesis. When crossed with MMTV-neu transgenic mice, 14-3-3ζ.neu transgenic mice exhibited accelerated mammary tumorigenesis and metastasis compared to MMTV-neu mice. Mechanistically, 14-3-3ζ overexpression enhanced MAPK/c-Jun signaling leading to increased miR-221 transcription, which inhibited p27 CDKI translation, and consequently, promoted cell proliferation. Importantly, this 14-3-3ζ/miR-221/p27/proliferation axis is also functioning in patients' breast tumors and associates with high grade cancers. Taken together, our findings show that 14-3-3ζ overexpression has a causal role in mammary tumorigenesis and progression, acting through miR-221 in cooperation with known oncogenic events to drive neoplastic cell proliferation.
PMCID: PMC3947305  PMID: 24197133
Breast cancer; 14-3-3ζ; microRNA; transgenic mice
10.  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.
PMCID: PMC3536411  PMID: 23097453
11.  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.
PMCID: PMC3656553  PMID: 23687576
12.  Receptor Tyrosine Kinase Signaling Favors a Protumorigenic State in Breast Cancer Cells by Inhibiting the Adaptive Immune Response 
Cancer research  2010;70(20):7776-7787.
Using transgenic mouse models of breast cancer that ablate Src homology and collagen A (ShcA) expression or oncogene-coupled ShcA signaling, we previously showed that this adaptor is critical for mammary tumor onset and progression. We now provide the first evidence that ShcA regulates mammary tumorigenesis, in part, through its ability to regulate the adaptive immune response. Inactivation of ShcA signaling within tumor cells results in extensive CD4+ T-cell infiltration and induction of a humoral immune response in mammary tumors. This is associated with a robust CTL response in preneoplastic lesions that are deficient in ShcA signaling. Moreover, mammary tumor progression of ShcA-deficient hyperplasias is accelerated in a T cell–deficient background. We also uncover a clinically relevant correlation between high ShcA expression and low CTL infiltration in human breast cancers. Finally, we define a novel ShcA-regulated immune signature that functions as an independent prognostic marker of survival in human epidermal growth factor receptor 2+ and basal breast cancers. We reveal a novel role for tumor cell–derived ShcA in the establishment and maintenance of an immunosuppressive state.
PMCID: PMC3660232  PMID: 20924104
13.  Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer 
Cancer cell  2013;24(2):213-228.
Accelerated glucose metabolism is a common feature of cancer cells. Hexokinases catalyze the first committed step of glucose metabolism. Hexokinase 2 (HK2) is expressed at high level in cancer cells, but only in a limited number of normal adult tissues. Using Hk2 conditional knockout mice, we showed that HK2 is required for tumor initiation and maintenance in mouse models of KRas-driven lung cancer, and ErbB2-driven breast cancer, despite continued HK1 expression. Similarly HK2 ablation inhibits the neoplastic phenotype of human lung and breast cancer cells in vitro and in vivo. Systemic Hk2 deletion is therapeutic in mice bearing lung tumors without adverse physiological consequences. Hk2 deletion in lung cancer cells suppressed glucose-derived ribonucleotides and impaired glutamine-derived carbon utilization in anaplerosis.
PMCID: PMC3753022  PMID: 23911236
14.  Deletion of Cd151 reduces mammary tumorigenesis in the MMTV/PyMT mouse model 
BMC Cancer  2014;14:509.
Tetraspanins are transmembrane proteins that serve as scaffolds for multiprotein complexes containing, for example, integrins, growth factor receptors and matrix metalloproteases, and modify their functions in cell adhesion, migration and transmembrane signaling. CD151 is part of the tetraspanin family and it forms tight complexes with β1 and β4 integrins, both of which have been shown to be required for tumorigenesis and/or metastasis in transgenic mouse models of breast cancer. High levels of the tetraspanin CD151 have been linked to poor patient outcome in several human cancers including breast cancer. In addition, CD151 has been implicated as a promoter of tumor angiogenesis and metastasis in various model systems.
Here we investigated the effect of Cd151 deletion on mammary tumorigenesis by crossing Cd151-deficient mice with a spontaneously metastasising transgenic model of breast cancer induced by the polyoma middle T antigen (PyMT) driven by the murine mammary tumor virus promoter (MMTV).
Cd151 deletion did not affect the normal development and differentiation of the mammary gland. While there was a trend towards delayed tumor onset in Cd151−/− PyMT mice compared to Cd151+/+ PyMT littermate controls, this result was only approaching significance (Log-rank test P-value =0.0536). Interestingly, Cd151 deletion resulted in significantly reduced numbers and size of primary tumors but did not appear to affect the number or size of metastases in the MMTV/PyMT mice. Intriguingly, no differences in the expression of markers of cell proliferation, apoptosis and blood vessel density was observed in the primary tumors.
The findings from this study provide additional evidence that CD151 acts to enhance tumor formation initiated by a range of oncogenes and strongly support its relevance as a potential therapeutic target to delay breast cancer progression.
PMCID: PMC4226978  PMID: 25012362
Tetraspanin; CD151; Breast; Cancer; Metastasis
15.  Mammary tumour development is dose-dependently inhibited by n-3 polyunsaturated fatty acids in the MMTV-neu(ndl)-YD5 transgenic mouse model 
Breast cancer is attributable to modifiable risk factors including the intake of dietary n-3 polyunsaturated fatty acids (PUFA). A key piece of evidence, yet to be addressed, that would demonstrate a causal relationship between n-3 PUFA and breast cancer, is a dose-dependent effect of n-3 PUFA on tumour outcomes. Thus, the objective of the present study was to determine whether n-3 PUFA reduces mammary gland tumor outcomes in a dose-dependent manner in female MMTV-neu(ndl)-YD5 transgenic mice, an aggressive model of human breast cancer.
Harems were provided one of three experimental diets comprised of 0, 3 or 9% (w/w) menhaden fish oil containing n-3 PUFA. Female offspring were weaned onto the same parental diet and maintained on their respective diet for 20 weeks. Tumour onset, size and multiplicity were measured throughout the study. Fatty acid composition of mammary gland and tumours were determined by gas–liquid chromatography.
Tumour size was significantly (p < 0.05) reduced in a dose-dependent manner. n-3 PUFA were also incorporated in a dose-dependent manner; differential incorporation was observed for eicosapentaenoic and docosapentaenoic acids into mammary gland tissue, while docosahexaenoic acid was preferentially incorporated into tumours.
Overall, the present study provides fundamental knowledge about the dose-dependent effect of n-3 PUFA on tumour outcomes in a pre-clinical model and also sheds light on the differential role of individual n-3 PUFA on tumour outcomes.
PMCID: PMC4064525  PMID: 24916956
n-3 PUFA; Eicosapentaenoic acid; Docosahexaenoic acid; Mammary gland; MMTV-neu mice; Breast cancer; Tumour; Tumour multiplicity; Tumour volume; Phospholipids
16.  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.
PMCID: PMC3359786  PMID: 22666282
17.  RUNX2 correlates with subtype-specific breast cancer in a human tissue microarray, and ectopic expression of Runx2 perturbs differentiation in the mouse mammary gland 
Disease Models & Mechanisms  2014;7(5):525-534.
RUNX2, a master regulator of osteogenesis, is oncogenic in the lymphoid lineage; however, little is known about its role in epithelial cancers. Upregulation of RUNX2 in cell lines correlates with increased invasiveness and the capacity to form osteolytic disease in models of breast and prostate cancer. However, most studies have analysed the effects of this gene in a limited number of cell lines and its role in primary breast cancer has not been resolved. Using a human tumour tissue microarray, we show that high RUNX2 expression is significantly associated with oestrogen receptor (ER)/progesterone receptor (PR)/HER2-negative breast cancers and that patients with high RUNX2 expression have a poorer survival rate than those with negative or low expression. We confirm RUNX2 as a gene that has a potentially important functional role in triple-negative breast cancer. To investigate the role of this gene in breast cancer, we made a transgenic model in which Runx2 is specifically expressed in murine mammary epithelium under the control of the mouse mammary tumour virus (MMTV) promoter. We show that ectopic Runx2 perturbs normal development in pubertal and lactating animals, delaying ductal elongation and inhibiting lobular alveolar differentiation. We also show that the Runx2 transgene elicits age-related, pre-neoplastic changes in the mammary epithelium of older transgenic animals, suggesting that elevated RUNX2 expression renders such tissue more susceptible to oncogenic changes and providing further evidence that this gene might have an important, context-dependent role in breast cancer.
PMCID: PMC4007404  PMID: 24626992
RUNX2; Breast cancer; Transgenic model; Mammary development
18.  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 
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).
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.
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.
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.
PMCID: PMC3978996  PMID: 24457046
19.  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.
PMCID: PMC2880222  PMID: 20424120
Akt; breast cancer; metastasis; transgenic mouse; invasion
20.  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.
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 analysed 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. After DNA vaccination, the splenocyte T-cell response to overlapping peptides from UL46 and UL47 was more than 500 gamma interferon spot-forming units per 106 responder cells. 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.
PMCID: PMC2675279  PMID: 19264627
21.  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.
PMCID: PMC3556997  PMID: 22773664
22.  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.
PMCID: PMC3752779  PMID: 21954288
translational control; PERK; eIF2α phosphorylation; Akt/PKB; endoplasmic reticulum stress; oxidative stress; chemotherapeutic drugs
23.  LKB1 is a central regulator of tumor initiation and pro-growth metabolism in ErbB2-mediated breast cancer 
Cancer & Metabolism  2013;1:18.
Germline and somatic mutations in STK11, the gene encoding the serine/threonine kinase LKB1, are strongly associated with tumorigenesis. While loss of LKB1 expression has been linked to breast cancer, the mechanistic role of LKB1 in regulating breast cancer development, metastasis, and tumor metabolism has remained unclear.
We have generated and analyzed transgenic mice expressing ErbB2 in the mammary epithelium of LKB1 wild-type or LKB1-deficient mice. We have also utilized ErbB2-expressing breast cancer cells in which LKB1 levels have been reduced using shRNA approaches. These transgenic and xenograft models were characterized for the effects of LKB1 loss on tumor initiation, growth, metastasis and tumor cell metabolism.
We demonstrate that loss of LKB1 promotes tumor initiation and induces a characteristic shift to aerobic glycolysis (‘Warburg effect’) in a model of ErbB2-mediated breast cancer. LKB1-deficient breast cancer cells display enhanced early tumor growth coupled with increased cell migratory and invasive properties in vitro. We show that ErbB2-positive tumors deficient for LKB1 display a pro-growth molecular and phenotypic signature characterized by elevated Akt/mTOR signaling, increased glycolytic metabolism, as well as increased bioenergetic markers both in vitro and in vivo. We also demonstrate that mTOR contributes to the metabolic reprogramming of LKB1-deficient breast cancer, and is required to drive glycolytic metabolism in these tumors; however, LKB1-deficient breast cancer cells display reduced metabolic flexibility and increased apoptosis in response to metabolic perturbations.
Together, our data suggest that LKB1 functions as a tumor suppressor in breast cancer. Loss of LKB1 collaborates with activated ErbB2 signaling to drive breast tumorigenesis and pro-growth metabolism in the resulting tumors.
PMCID: PMC4178213  PMID: 24280377
Breast cancer; ErbB2; LKB1; Metabolism
24.  Phosphatase and Tensin Homologue Deleted on Chromosome 10 Deficiency Accelerates Tumor Induction in a Mouse Model of ErbB-2 Mammary Tumorigenesis 
Cancer research  2008;68(7):2122-2131.
Loss of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and amplification or elevated expression of ErbB-2 are both involved in human breast cancer. To directly test the importance of these genetic events in mammary tumorigenesis, we have assessed whether mammary-specific disruption of PTEN could cooperate with activation of ErbB-2. Transgenic mice expressing ErbB-2 under the transcriptional control of its endogenous promoter (ErbB-2KI) were interbred with mice carrying conditional PTEN alleles and an MMTV/Cre transgene. Loss of one or both PTEN alleles resulted in a dramatic acceleration of mammary tumor onset and an increased occurrence of lung metastases in the ErbB-2KI strain. Tumor progression in PTEN-deficient/ErbB-2KI strains was associated with elevated ErbB-2 protein levels, which were not due to ErbB-2 amplification or to a dramatic increase in ErbB-2 transcripts. Moreover, the PTEN-deficient/ErbB-2KI–derived mouse mammary tumors display striking morphologic heterogeneity in comparison with the homogeneous pathology of the ErbB-2KI parental strain. Therefore, inactivation of PTEN would not only have a dramatic effect on ErbB-2–induced mammary tumorigenesis but would also lead to the formation of mammary tumors that, in part, display pathologic and molecular features associated with the basal-like subtype of primary human breast cancer.
PMCID: PMC2752841  PMID: 18381417
25.  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.
PMCID: PMC3693553  PMID: 22461506

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