Sean E. Egan

High-level Coexpression of JAG1 and NOTCH1 Is Observed in Human Breast Cancer and Is Associated with Poor Overall Survival

Aberrant activation of Notch receptors has been shown to cause mammary tumors in mice. We therefore used in situ hybridization to analyze expression of Notch ligands and receptors in human breast cancer. High levels of JAG1 and NOTCH1 were noted in a subset of tumors with poor prognosis pathologic features (P < 0.05). We therefore used tissue microarrays to analyze the expression of these genes in a collection of breast cancers from patients representing a wide spectrum of clinical stages, and from whom associated follow-up survival data was available (n = 184). Patients with tumors expressing high levels of JAG1 or NOTCH1 had a significantly poorer overall survival compared with patients expressing low levels of these genes [5-year survival rate of 42% versus 65% and median survival of 50 versus 83 months, respectively, for JAG1(Hi vs. Lo) (P = 0.01); 49% versus 64% and 53 versus 91 months, respectively, for NOTCH1(Hi vs. Lo) (P = 0.02)]. Moreover, a synergistic effect of high-level JAG1 and high-level NOTCH1 coexpression on overall survival was observed (5-year survival rate of 32% and median survival of 40 months; P = 0.003). These data (a) identify novel prognostic markers for breast cancer, (b) suggest a mechanism whereby Notch is activated in aggressive breast tumors, and (c) may identify a signaling pathway activated in poor prognosis breast cancer which can be therapeutically targeted.

The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15

Clathrin‐mediated endocytosis is a multistep process which requires interaction between a number of conserved proteins. We have cloned two mammalian genes which code for a number of endocytic adaptor proteins. Two of these proteins, termed Ese1 and Ese2, contain two N‐terminal EH domains, a central coiled‐coil domain and five C‐terminal SH3 domains. Ese1 is constitutively associated with Eps15 proteins to form a complex with at least 14 protein–protein interaction surfaces. Yeast two‐hybrid assays have revealed that Ese1 EH and SH3 domains bind epsin family proteins and dynamin, respectively. Overexpression of Ese1 is sufficient to block clathrin‐mediated endocytosis in cultured cells, presumably through disruption of higher order protein complexes, which are assembled on the endogenous Ese1–Eps15 scaffold. The Ese1–Eps15 scaffold therefore links dynamin, epsin and other endocytic pathway components.

Subversion of the T/B Lineage Decision in the Thymus by Lunatic Fringe-Mediated Inhibition of Notch-1

Notch-1 signaling is essential for lymphoid progenitors to undergo T cell commitment, but the mechanism has not been defined. Here we show that thymocytes ectopically expressing Lunatic Fringe, a modifier of Notch-1 signaling, induce lymphoid progenitors to develop into B cells in the thymus. This cell fate switch resulted from Lunatic Fringe-mediated inhibition of Notch-1 function, as revealed by experiments utilizing lymphoid progenitors in which Notch-1 activity was genetically manipulated. These data identify Lunatic Fringe as a potent regulator of Notch-1 during the T/B lineage decision and show that an important function of Notch-1 in T cell commitment is to suppress B cell development in the thymus.

High-level JAG1 mRNA and protein predict poor outcome in breast cancer

Notch receptors regulate cell fate determination, stem cell self-renewal, proliferation and apoptosis. We previously reported that elevated mRNA expression of the Notch ligand JAG1 identifies breast cancer patients with a poor prognosis. Here we show through immunohistochemical analysis of the same breast cancer cases (N=127) that patients with tumors expressing high levels of JAG1 protein had a worse outcome than those with tumors expressing low levels (10-year survival 26 vs 48%, and median survival 63 vs 108 months, respectively; P=0.03). We also describe the novel application of the Allred score to quantify JAG1 mRNA and protein expression levels. Using the Allred score, patients with tumors expressing high levels of JAG1 mRNA had a worse outcome than those with tumors expressing low levels (10-year survival 16 vs 47%, and median survival 43 months vs 100 months, respectively; P<0.001). Interestingly, when tumors were classified as either high or low for JAG1 mRNA or protein expression, there was only 65% agreement (κ=0.08) between the two methods of expression analysis. When JAG1 mRNA and protein data were combined, patients with tumors expressing low levels of both had a 10-year survival of 53% and median survival of 131 months. In comparison, patients with tumors expressing either high levels of JAG1 protein, mRNA or both had reduced 10-year survival and median survival (31%, 19%, 11% and 77, 43, 23 months respectively; P<0.0001). There was marginal evidence of an interaction effect (P=0.055), which indicated that the prognostic value of JAG1 protein was limited to the JAG1 mRNA-low subgroup. These data show that the Allred score can be used to rapidly quantify JAG1 mRNA and protein levels in breast cancer to identify patients who have a significant survival disadvantage and who may benefit from therapies (such as γ-secretase inhibitors) that target signaling through the Notch pathway.

Notch signaling in mammary development and oncogenesis.

With the discovery of an activated Notch oncogene as a causative agent in mouse mammary tumor virus induced breast cancer in mice, the potential role for Notch signaling in normal and pathological mammary development was revealed. Subsequently, Notch receptors have been found to regulate normal development in many organ systems. In addition, inappropriate Notch signaling has been implicated in cancer of several tissues in humans and animal model systems. Here we review important features of the Notch system, and how it may regulate development and cancer in the mammary gland. A large body of literature from studies in Drosophila and C. elegans has not only revealed molecular details of how the Notch proteins signal to control biology, but shown that Notch receptor activation helps to define how other signaling pathways are interpreted. In many ways the Notch system is used to define the context in which other pathways function to control proliferation, differentiation, cell survival, branching morphogenesis, asymmetric cell division, and angiogenesis—all processes which are critical for normal development and function of the mammary gland.

Lunatic and Manic Fringe Cooperatively Enhance Marginal Zone B Cell Precursor Competition for Delta-like 1 in Splenic Endothelial Niches

Notch2 activation induced by Delta-like-1 (DL1) drives development of splenic marginal zone (MZ) B cells, an innate-like lineage that protects against sepsis. DL1 interacts with Notch2 weakly, but it is not known whether enhancement of DL1-induced Notch2 activation by Fringe glycosyltransferases is important for MZ B cell development. Furthermore, DL1-expressing cells that promote MZ B cell development have not been identified. We show that Lunatic Fringe (Lfng) and Manic Fringe (Mfng) cooperatively enhanced the DL1-Notch2 interaction to promote MZ B cell development. We also identified radio-resistant red pulp endothelial cells in the splenic MZ that express high amounts of DL1 and promoted MZ B generation. Finally, MZ B cell precursor competition for DL1 homeostatically regulated entry into the MZ B cell pool. Our study has revealed that the Fringe-Notch2 interaction has important functions in vivo and provides insights into mechanisms regulating MZ B cell development.

RB1 and p53 at the crossroad of EMT and triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease that includes Basal-like and Claudin-low tumors. The Claudin-low tumors are enriched for features associated with epithelial-to-mesenchymal transition (EMT) and possibly for tumor initiating cells. Primary TNBCs respond relatively well to conventional chemotherapy; however, metastatic disease is virtually incurable. Thus, there is a great interest in identifying specific therapeutic targets for TNBC. The tumor suppressor RB1 is frequently lost in Basal-like breast cancer. To test for a causative role of RB1 gene loss in BC and for its effect on specific subtypes, we deleted mouse Rb in mammary stem/bipotent progenitor cells. This led to diverse mammary tumors including TNBC, with a subset of the latter containing p53 mutations and exhibiting features of Basal-like BC or EMT. Combined mutation of Rb and p53 in mammary stem/bipotent progenitors induced EMT type tumors. Here, we review our findings and those of others, which connect Rb and p53 to EMT in TNBC. Furthermore, we discuss how by understanding this circuit and its vulnerabilities, we may identify novel therapy for TNBC.

BHLHB2 Controls Bdnf Promoter 4 Activity and Neuronal Excitability

Brain-derived neurotrophic factor (BDNF), via activation of TrkB receptors, mediates vital physiological functions in the brain, ranging from neuronal survival to synaptic plasticity, and has been implicated in the pathophysiology of neurodegenerative disorders. Although transcriptional regulation of the BDNF gene (Bdnf) has been extensively studied, much remains to be understood. We discovered a sequence within Bdnf promoter 4 that binds the basic helix-loop-helix protein BHLHB2 and is a target for BHLHB2-mediated transcriptional repression. NMDA receptor activation de-repressed promoter 4-mediated transcription and correlated with reduced occupancy of the promoter by BHLHB2 in cultured hippocampal neurons. Bhlhb2 gene −/− mice showed increased hippocampal exon 4-specific Bdnf mRNA levels compared with +/+ littermates under basal and activity-dependent conditions. Bhlhb2 knock-out mice also showed increased status epilepticus susceptibility, suggesting that BHLHB2 alters neuronal excitability. Together, these results support a role for BHLHB2 as a new modulator of Bdnf transcription and neuronal excitability.

The miR-17-92 cluster expands multipotent hematopoietic progenitors whereas imbalanced expression of its individual oncogenic miRNAs promotes leukemia in mice

The miR-17-92 cluster and its 6 encoded miRNAs are frequently amplified and aberrantly expressed in various malignancies. This study demonstrates that retroviral-mediated miR-17-92 overexpression promotes expansion of multipotent hematopoietic progenitors in mice. Cell lines derived from these miR-17-92-overexpressing mice are capable of myeloid and lymphoid lineage differentiation, and recapitulate the normal lymphoid phenotype when transplanted to nonobese diabetic/severe combined immunodeficiency mice. However, overexpression of individual miRNAs from this locus, miR-19a or miR-92a, results in B-cell hyperplasia and erythroleukemia, respectively. Coexpression of another member of this cluster miR-17, with miR-92a, abrogates miR-92a-induced erythroleukemogenesis. Accordingly, we identified several novel miR-92a and miR-17 target genes regulating erythroid survival and proliferation, including p53. Expression of this critical target results in marked growth inhibition of miR-92a erythroleukemic cells. In both murine and human leukemias, p53 inactivation contributed to the selective overexpression of oncogenic miR-92a and miR-19a, and down-regulation of tumor-suppressive miR-17. This miR-17-92 expression signature was also detected in p53- B-cell chronic lymphocytic leukemia patients displaying an aggressive clinical phenotype. These results revealed that imbalanced miR-17-92 expression, also mediated by p53, directly transforms the hematopoietic compartment. Thus examination of such miRNA expression signatures should aid in the diagnosis and treatment of cancers displaying miR-17-92 gene amplification.

Transcriptomic classification of genetically engineered mouse models of breast cancer identifies human subtype counterparts

BackgroundHuman breast cancer is a heterogeneous disease consisting of multiple molecular subtypes. Genetically engineered mouse models are a useful resource for studying mammary cancers in vivo under genetically controlled and immune competent conditions. Identifying murine models with conserved human tumor features will facilitate etiology determinations, highlight the effects of mutations on pathway activation, and should improve preclinical drug testing.ResultsTranscriptomic profiles of 27 murine models of mammary carcinoma and normal mammary tissue were determined using gene expression microarrays. Hierarchical clustering analysis identified 17 distinct murine subtypes. Cross-species analyses using three independent human breast cancer datasets identified eight murine classes that resemble specific human breast cancer subtypes. Multiple models were associated with human basal-like tumors including TgC3(1)-Tag, TgWAP-Myc and Trp53-/-. Interestingly, the TgWAPCre-Etv6 model mimicked the HER2-enriched subtype, a group of human tumors without a murine counterpart in previous comparative studies. Gene signature analysis identified hundreds of commonly expressed pathway signatures between linked mouse and human subtypes, highlighting potentially common genetic drivers of tumorigenesis.ConclusionsThis study of murine models of breast carcinoma encompasses the largest comprehensive genomic dataset to date to identify human-to-mouse disease subtype counterparts. Our approach illustrates the value of comparisons between species to identify murine models that faithfully mimic the human condition and indicates that multiple genetically engineered mouse models are needed to represent the diversity of human breast cancers. The reported trans-species associations should guide model selection during preclinical study design to ensure appropriate representatives of human disease subtypes are used.