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Pediatric liver transplant (LT) recipients are often transplanted at a young age, precluding them from receiving live virus vaccinations (LVV) such as varicella (VZV) vaccine and measles, mumps and rubella. This places them at profound risk for vaccine preventable illness. We sought to detail safety of vaccination. This was a retrospective cohort study of pediatric LT recipients at two children's hospitals. Among 204 LT recipients included in the study, 97 received at least one LVV after LT. Six patients who did not receive LVV after transplant had evidence of vaccine-preventable infection following vaccination (one disseminated VZV disease, five VZV-related rash), while one patient who received LVV after transplant developed a diffuse VZV-related rash. Rejection rates were the same between those that did and did not receive a live virus vaccine post-transplant. There were no serious adverse events caused by vaccination post-transplant. LVV following LT was safe at our two institutions, although there exist limitations in our study due to its retrospective study design. Larger scale studies should be performed to evaluate the effectiveness of LVV in relation to immunosuppression.

Background: Fecal shedding of SARS-CoV-2 has raised concerns about transmission through fecal microbiota transplantation (FMT) procedures. While many tests have been authorized for diagnosis of COVID-19 using respiratory samples, no fully validated stool tests for detection of SARS-CoV-2 are currently available. We sought to adapt and validate an available test specifically for detection of SARS-CoV-2 in human stool. Methods: Stool samples were spiked with inactivated SAR-CoV-2 virus for development and validation of the assay. A modified version of the CDC rRT-PCR SARS-CoV-2 test was used for detection of virus. Analytical sensitivity, assay reproducibility, and sample stability under a variety of storage conditions were assessed. We also performed the assay on stool samples collected from known COVID positive individuals. Findings: The lower limit of detection (LoD) of the assay was found to be 3000 viral RNA copies per gram of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Samples were relatively stable in all buffers tested at both 4 o C and ambient temperature, with the exception of storage in STAR buffer at ambient temperature. Assay sensitivity was slightly diminished in low-copy-number samples after a single freeze-thaw cycle at -80°C. Thirty contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, we detected SARS-CoV-2 RNA in the stool of known COVID-19 positive individuals using this method. Interpretation: This is a sensitive, reproducible, and validated assay for detection of SARS-CoV-2 RNA in human stool with potential uses in FMT donor screening, sewage monitoring, and further research into the impact of fecal shedding on the epidemiology of this pandemic. Funding: National Institute for Allergy and Infectious Diseases, NIH. Center for Biologics Evaluation and Research, FDA. Declaration of Interests: None to declare.

Marine-derived n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been shown to inhibit mammary carcinogenesis. However, evidence regarding plant-based α-linolenic acid (ALA), the major n-3 PUFA in the Western diet, remains equivocal. The objective of this study was to examine the effect of lifelong exposure to plant- or marine-derived n-3 PUFAs on pubertal mammary gland and tumor development in MMTV-neu(ndl)-YD5 mice. It is hypothesized that lifelong exposure to n-3 PUFA reduces terminal end buds during puberty leading to delayed tumor onset, volume and multiplicity. It is further hypothesized that plant-derived n-3 PUFAs will exert dose-dependent effects. Harems of MMTV-FVB males were bred with wild-type females and fed either a (1) 10% safflower (10% SF, n-6 PUFA, control), (2) 10% flaxseed (10% FS), (3) 7% safflower plus 3% flaxseed (3% FS) or (4) 7% safflower plus 3% menhaden (3% FO) diet. Female offspring were maintained on parental diets. Compared to SF, 10% FS and 3% FO reduced (P

Obesity is associated with inflammation and has been shown to increase breast cancer severity. The objective of this study was to examine the effect of fish oil (FO) supplementation in obesity-associated mammary tumorigenesis in the MMTV-neu(ndl)-YD5 mouse model of human epidermal growth factor receptor-2 positive BC. Female mice were fed one of three diets for 16 weeks: i) high fat diet [HF, % kacl: 41.2% lard, 18.7% corn oil (CO)], ii) an isocaloric HF plus menhaden FO diet (HF+FO, % kcal: 41.2 lard, 13.4% CO, 5.3% FO), iii) low fat diet (LF, % kcal: 4.7% lard, 6% CO). HF mice had increased body weight, visceral adipose weight and serum hormone concentrations (increased leptin and resistin; decreased adiponectin) versus LF, which was attenuated in the HF+FO group versus HF (P.05). Compared to HF, tumor onset was delayed in HF+FO and LF mice (P0.05). Compared to HF, HF+FO reduced mammary tumor multiplicity (-27%), tumor weight (-46%) and total tumor volume (-50%) (P0.05). Additionally, HF+FO reduced mammary tumor multiplicity (-33%), tumor weight (-39%) and total tumor volume (-60%) versus LF. HF+FO improved mammary tumor apoptosis status with increased expression of pro-apoptotic Bad and decreased expression of anti-apoptotic Bcl-xLmediators versus HF (P0.05). Additionally, HF+FO decreased tumor protein expression of activated Akt, NFκB p65 and STAT3, versus HF (P0.05). Tumor mRNA expression of inflammatory mediators TNFα, IL-6 and leptin were reduced in HF+FO, whereas IL-10 expression was increased compared to HF (P0.05). Collectively these results demonstrate the efficacy of FO supplementation for improving obesity-associated breast cancer outcomes.

Summary There is increasing interest in therapeutically exploiting metabolic differences between normal and cancer cells. We show that kinase inhibitors (KIs) and biguanides synergistically and selectively target a variety of cancer cells. Synthesis of non-essential amino acids (NEAAs) aspartate, asparagine, and serine, as well as glutamine metabolism, are major determinants of the efficacy of KI/biguanide combinations. The mTORC1/4E-BP axis regulates aspartate, asparagine, and serine synthesis by modulating mRNA translation, while ablation of 4E-BP1/2 substantially decreases sensitivity of breast cancer and melanoma cells to KI/biguanide combinations. Efficacy of the KI/biguanide combinations is also determined by HIF-1α-dependent perturbations in glutamine metabolism, which were observed in VHL-deficient renal cancer cells. This suggests that cancer cells display metabolic plasticity by engaging non-redundant adaptive mechanisms, which allows them to survive therapeutic insults that target cancer metabolism.

The Software as a Service (SaaS) industry is under attack by the likes of companies like Salesforce.com. Salesforce.com, founded in 1999, has taken advantage of a disruptive business model innovation to gain significant market penetration for the Customer Relationship Management (CRM) arena. The company went public in 2004 and up until December 2011 has acquired numerous other technology companies, which individually introduced disruptive innovation solutions, to enhance Salesforce.com’s ability to offer cloud based technology and solutions to its customers.This article will analyze, Salesforce.com, one of the most successful examples of a company that has been able to transition from a traditional management model to a radical management model. The factors affecting the transformation influenced every aspect of the organization from strategy development through execution.

Mesenchymal dysplasia (mes) mice expressing a C-terminally truncated version of the Hedgehog (Hh)-ligand receptor, Patched-1 (Ptch1), exhibit a limited spectrum of developmental defects including blocked ductal morphogenesis of the mammary gland during puberty. Given that the Hh-ligands can stimulate signalling cascades distinct from the canonical pathway involving Smo and the Gli-family proteins and that Ptch1 binds to factors harbouring SH3-domains, we determined whether the mes mammary gland defect could be rescued by activating non-canonical signalling pathways downstream of Ptch1. We demonstrate here that expression of constitutively active c-src (c-src(Act)) in mammary epithelial cells overcomes the block to mammary epithelial morphogenesis in mes mice. Specifically, MMTV-directed expression of c-src(Act) rescued blocked ductal morphogenesis in mes mice, albeit only after animals were more than 15 weeks of age. The overall morphology resembled wild type mice expressing c-src(Act) although 40% of mes/MMTV-c-src(Act) mice exhibited terminal end buds at 24 weeks of age. C-src(Act) restored the proliferative capacity of mes epithelial cells, self-renewal capacity of mammary progenitor cells and increased the expression of Esr1, Ptch1 and Gli1. These data reveal the cooperative interactions between signalling cascades involving c-src and Ptch1 and suggest that Hh-signalling may be permissive for c-src/Esr1-dependent mammary gland morphogenesis.

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.

Metastasis remains the leading cause of cancer morbidity and mortality. ErbB2, a metastasis-promoting oncoprotein, is overexpressed in 50–60% of noninvasive ductal carcinoma in situ (DCIS). However, only 25% of invasive breast cancer (IBC) overexpress ErbB2, indicating that ErbB2 alone is not sufficient to drive metastasis and additional risk factors are necessary for the progression of ErbB2-overexpressing DCIS to IBC. A recent study published in Cancer Cell identified 14-3-3ξ as a risk factor aiding the transition of ErbB2-overexpressing DCIS into IBC. Furthermore, the study elucidated molecular mechanisms by which ErbB2 and 14-3-3ξ co-overexpression drives metastasis. Namely, ErbB2 promotes cell motility and migration via the activation of Src, while 14-3-3ξ induces epithelial-mesenchymal transition by activating TGFβ pathway to reduce cell adhesion. On the other hand, two studies recently published in British Journal of Cancer and Oncogene provide mechanistic insight into how ErbB signalling is transduced via Src, focal adhesion kinase and Ste20-like kinase to regulate focal adhesion turnover and modulate cell motility and migration. Taken together, these studies reveal that metastasis engages a variety of players that must show team spirit to win the game of spreading.

Overexpression and/or amplification of the ErbB-2 oncogene as well as inactivation of the PTEN tumor suppressor are two important genetic events in human breast carcinogenesis. To address the biological impact of conditional inactivation of PTEN on ErbB-2-induced mammary tumorigenesis, we generated a novel transgenic mouse model that utilizes the murine mammary tumor virus (MMTV) promoter to directly couple expression of activated ErbB-2 and Cre recombinase to the same mammary epithelial cell (MMTV-NIC). Disruption of PTEN in the mammary epithelium of the MMTV-NIC model system dramatically accelerated the formation of multifocal and highly metastatic mammary tumors, which exhibited homogenous pathology. PTEN-deficient/NIC-induced tumorigenesis was associated with an increase in angiogenesis. Moreover, inactivation of PTEN in the MMTV-NIC mouse model resulted in hyperactivation of the phosphatidylinositol 3′-kinase/Akt signaling pathway. However, like the parental strain, tumors obtained from PTEN-deficient/NIC mice displayed histopathological and molecular features of the luminal subtype of primary human breast cancer. Taken together, our findings provide important implications in understanding the molecular determinants of mammary tumorigenesis driven by PTEN deficiency and ErbB-2 activation and could provide a valuable tool for testing the efficacy of therapeutic strategies that target these critical signaling pathways.

SummaryLysophosphatidic acid (LPA) acts through high-affinity G protein-coupled receptors to mediate a plethora of physiological and pathological activities associated with tumorigenesis. LPA receptors and autotaxin (ATX/LysoPLD), the primary enzyme producing LPA, are aberrantly expressed in multiple cancer lineages. However, the role of ATX and LPA receptors in the initiation and progression of breast cancer has not been evaluated. We demonstrate that expression of ATX or each edg family LPA receptor in mammary epithelium of transgenic mice is sufficient to induce a high frequency of late-onset, estrogen receptor (ER)-positive, invasive, and metastatic mammary cancer. Thus, ATX and LPA receptors can contribute to the initiation and progression of breast cancer.

Individual protein tyrosine kinases and phosphatases target multiple substrates; this may generate conflicting signals, possibly within a single pathway. Protein-tyrosine phosphatase ϵ (PTPϵ) performs two potentially opposing roles: in Neu-induced mammary tumors, PTPϵ activates Src downstream of Neu, whereas in other systems PTPϵ can indirectly down-regulate MAP kinase signaling. We now show that the latter effect is mediated at least in part via the adaptor protein Shc. PTPϵ binds and dephosphorylates Shc in vivo, reducing the association of Shc with Grb2 and inhibiting downstream ERK activation. PTPϵ binds Shc in a phosphotyrosine-independent manner mediated by the Shc PTB domain and aided by a sequence of 10 N-terminal residues in PTPϵ. Surprisingly, PTPϵ dephosphorylates Shc in a kinase-dependent manner; PTPϵ targets Shc in the presence of Src but not in the presence of Neu. Using a series of point mutants of Shc and Neu, we show that Neu protects Shc from dephosphorylation by binding the PTB domain of Shc, most likely competing against PTPϵ for binding the same domain. In agreement, PTPϵ dephosphorylates Shc in mouse embryo fibroblasts but not in Neu-induced mammary tumor cells. We conclude that in the context of Neu-induced mammary tumor cells, Neu prevents PTPϵ from targeting Shc and from reducing its promitogenic signal while phosphorylating PTPϵ and directing it to activate Src in support of mitogenesis. In so doing, Neu contributes to the coherence of the promitogenic role of PTPϵ in this system.

Stat5 and Stat3, two members of the Stat (signal transducer and activator of transcription) family, are known to play critical roles in mammopoiesis/lactogenesis and involution, respectively, in the mammary gland. Phosphotyrosine phosphatase Shp2 has been shown to dephosphorylate and thus inactivate both Stat5 and Stat3 in vitro. Paradoxically, cell culture studies also suggest a positive role of Shp2 in promoting prolactin-stimulated Stat5 activation. We have shown here that selective deletion of Shp2 in mouse mammary glands suppresses Stat5 activity during pregnancy and lactation, resulting in significant impairment of lobulo-alveolar outgrowth and lactation. In contrast, Stat3 activity was slightly up-regulated shortly before/at involution, leading to normal epithelial cell apoptosis/involution in Shp2-deficient mammary gland. Thus, Shp2 acts to promote Stat5 activation by the JAK2·prolactin receptor complex, while negatively modulating Stat3 activity before the onset of involution. This is the first demonstration that Shp2 manipulates Stat5 and Stat3 activities reciprocally in mammary epithelial cells, providing novel insight into the complex mechanisms for regulation of various Stat family members by a cytoplasmic tyrosine phosphatase.

Amplification of the ErbB2 locus, which encodes a receptor tyrosine kinase, is common in aggressive breast tumors and correlates with poor prognosis. The mechanisms underlying ErbB2-mediated breast carcinoma progression remain incompletely defined. To examine the role of the signaling and cell-adhesion receptor beta 4 integrin during ErbB2-mediated tumorigenesis, we introduced a targeted deletion of the beta 4 signaling domain into a mouse model of ErbB2-induced mammary carcinoma. Loss of beta 4 signaling suppresses mammary tumor onset and invasive growth. Ex vivo studies indicate that beta 4 forms a complex with ErbB2 and enhances activation of the transcription factors STAT3 and c-Jun. STAT3 contributes to disruption of epithelial adhesion and polarity, while c-Jun is required for hyperproliferation. Finally, deletion of the beta 4 signaling domain enhances the efficacy of ErbB2-targeted therapy. These results indicate that beta 4 integrin promotes tumor progression by amplifying ErbB2 signaling and identify beta 4 as a potential target for molecular therapy of breast cancer.

The angiogenic molecule, vascular endothelial growth factor (VEGF), is a critical regulator of normal and pathologic angiogenesis. ErbB2, an epidermal growth factor receptor family member whose overexpression in mammary tumors is correlated with poor patient prognosis, has been implicated as a positive modulator of VEGF expression. Mammary tumor cells overexpressing ErbB2 (NAFA cells) and a normal mouse mammary cell line (HC11) transfected with ErbB2 expression vectors were used to study the effects of ErbB2 overexpression on VEGF regulation. We found that ErbB2 overexpression led to an increase in endogenous VEGF mRNA as well as ErbB3 protein levels in HC11 cells. Additionally, we determined that ErbB2 overexpression-mediated upregulation of VEGF involves at least two distinct promoter elements, one previously identified as the hypoxia responsive element and the other the core promoter region (-161 to -51bp), which is specifically controlled via two adjacent SP1 binding sites (-80 to -60bp).

Interfering with Ets transcription factor function reverses multiple aspects of the transformed phenotype of mouse or human tumor cells. However, the unknown number of individual Ets factors expressed in any cellular context and the similar DNA binding specificities of Ets family members complicates the identification of those that mediate transformation. By utilizing quantitative PCR assays for 25 mouse Ets factors, we analyzed the expression of essentially the entire Ets family in normal mammary tissue, mammary-related cell lines, and mammary tumors. In normal mammary tissue, 24 Ets factors were expressed. Even clonal derived cell lines expressed 14-20 Ets members. The most abundant Ets factor mRNAs measured in normal mammary tissue were Elk4, Elf1, and Ets2. Subtractive analysis of mammary tissue identified which Ets factors were predominantly expressed in the myeloid/lymphoid or epithelial cell compartments. Comparison of Ets factor expression in normal mammary tissue and mammary tumors identified significantly elevated expression of Pse/PDEF, Ese2/Elf5, Ese3/Ehf, TEL/Etv6, and Elf2/NERF in mammary tumors and confirmed previously reported alterations in expression of Ese1/Elf3 and the PEA3 subfamily. Expression of 13 Ets target genes, implicated in various aspects of tumor progression, was also analyzed. Altered expression of particular Ets target genes was significantly correlated with particular Ets factors (e.g. maspin and Ese2), suggesting specific in vivo regulatory roles. Together, this comprehensive analysis revealed unexpectedly diverse Ets family gene expression, characterized novel Ets factor changes in mammary tumors, and implicated specific Ets factors in the regulation of multiple genes involved in mammary tumor progression.

Animal models are powerful tools to analyze the mechanism of the induction of human breast cancer. Here we report a detailed analysis of mammary tumor progression in one mouse model of breast cancer caused by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium, and its comparison to human breast tumors. In PyMT mice, four distinctly identifiable stages of tumor progression from premalignant to malignant stages occur in a single primary tumor focus and this malignant transition is followed by a high frequency of distant metastasis. These stages are comparable to human breast diseases classified as benign or in situ proliferative lesions to invasive carcinomas. In addition to the morphological similarities with human breast cancer, the expression of biomarkers in PyMT-induced tumors is also consistent with those associated with poor outcome in humans. These include a loss of estrogen and progesterone receptors as well as integrin-beta1 expression and the persistent expression of ErbB2/Neu and cyclinD1 in PyMT-induced tumors as they progress to the malignant stage. An increased leukocytic infiltration was also closely associated with the malignant transition. This study demonstrates that the PyMT mouse model is an excellent one to understand the biology of tumor progression in humans.

The cyclin D1 gene is overexpressed in breast tumors and encodes a regulatory subunit of cyclin-dependent kinases that phosphorylate the retinoblastoma protein. pp60c-src activity is frequently increased in breast tumors; however, the mechanisms governing pp60c-src regulation of the cell cycle in breast epithelium are poorly understood. In these studies, pp60v-src induced cyclin D1 protein levels and promoter activity (48-fold) in MCF7 cells. Cyclin D1-associated kinase activity and protein levels were increased in mammary tumors from murine mammary tumor virus-pp60c-src527F transgenic mice. Optimal induction of cyclin D1 by pp60v-srcinvolved the extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase members of the mitogen-activated protein kinase family. Cyclin D1 promoter activation by pp60v-srcinvolved a cAMP response element-binding protein (CREB)/activating transcription factor 2 (ATF-2) binding site. Dominant negative mutants of CREB and ATF-2 but not c-Jun inhibited pp60v-srcinduction of cyclin D1. pp60v-src induction of CREB was blocked by the p38 inhibitor SB203580 or by mutation of CREB at Ser133. pp60v-src induction of ATF-2 was abolished by the c-Jun N-terminal kinase inhibitor JNK-interacting protein-1 or by mutation of ATF-2 at Thr69 and Thr71. CREB and ATF-2, which bind to a common pp60v-src response element, are transcriptionally activated by distinct mitogen-activated protein kinases. Induction of cyclin D1 activity by pp60v-src may contribute to breast tumorigenesis through phosphorylation and inactivation of the retinoblastoma protein.

Neu (c-erbB2) is a proto-oncogene product that encodes an epidermal growth factor-like receptor tyrosine kinase. Amplification of wild-type c-Neu and mutational activation of Neu (Neu T) have been implicated in oncogenic transformation of cultured fibroblasts and mammary tumorigenesis in vivo. Here, we examine the relationship between Neu tyrosine kinase activity and caveolin-1 protein expression in vitro and in vivo. Recent studies have suggested that caveolins may function as negative regulators of signal transduction. Our current results show that mutational activation of c-Neu down-regulates caveolin-1 protein expression, but not caveolin-2, in cultured NIH 3T3 and Rat 1 cells. Conversely, recombinant overexpression of caveolin-1 blocks Neu-mediated signal transduction in vivo. These results suggest a reciprocal relationship between c-Neu tyrosine kinase activity and caveolin-1 protein expression. We next analyzed a variety of caveolin-1 deletion mutants to map this caveolin-1-dependent inhibitory activity to a given region of the caveolin-1 molecule. Results from this mutational analysis show that this functional in vivo inhibitory activity is contained within caveolin-1 residues 32-95. In accordance with these in vivo studies, a 20-amino acid peptide derived from this region (the caveolin-1 scaffolding domain) was sufficient to inhibit Neu autophosphorylation in an in vitro kinase assay. To further confirm or refute the relevance of our findings in vivo, we next examined the expression levels of caveolin-1 in mammary tumors derived from c-Neu transgenic mice. Our results indicate that dramatic reduction of caveolin-1 expression occurs in mammary tumors derived from c-Neu-expressing transgenic mice and other transgenic mice expressing downstream effectors of Neu-mediated signal transduction, such as Src and Ras. Taken together, our data suggest that a novel form of reciprocal negative regulation exists between c-Neu and caveolin-1.

Mammary hyperplasias, dysplasias and tumors have been described in many strains of transgenic mice. Many of the transgenes produce characteristic disturbances of growth, development and neoplasia. The disturbances can now be classified into groups. A classification of transgenic tumors and an atlas illustrating some characteristic examples of common morphological changes in transgenic mammary tissues is provided.