A novel proneural function of Asense is integrated with the sequential actions of Delta-Notch, L'sc and Su(H) to promote the neuroepithelial to neuroblast transition.

In order for neural progenitors (NPs) to generate distinct populations of neurons at the right time and place during CNS development, they must switch from undergoing purely proliferative, self-renewing divisions to neurogenic, asymmetric divisions in a tightly regulated manner. In the developing Drosophila optic lobe, neuroepithelial (NE) cells of the outer proliferation center (OPC) are progressively transformed into neurogenic NPs called neuroblasts (NBs) in a medial to lateral proneural wave. The cells undergoing this transition express Lethal of Scute (L'sc), a proneural transcription factor (TF) of the Acheate Scute Complex (AS-C). Here we show that there is also a peak of expression of Asense (Ase), another AS-C TF, in the cells neighboring those with transient L'sc expression. These peak of Ase cells help to identify a new transitional stage as they have lost NE markers and L'sc, they receive a strong Notch signal and barely exhibit NB markers. This expression of Ase is necessary and sufficient to promote the NE to NB transition in a more robust and rapid manner than that of l'sc gain of function or Notch loss of function. Thus, to our knowledge, these data provide the first direct evidence of a proneural role for Ase in CNS neurogenesis. Strikingly, we found that strong Delta-Notch signaling at the lateral border of the NE triggers l'sc expression, which in turn induces ase expression in the adjacent cells through the activation of Delta-Notch signaling. These results reveal two novel non-conventional actions of Notch signaling in driving the expression of proneural factors, in contrast to the repression that Notch signaling exerts on them during classical lateral inhibition. Finally, Suppressor of Hairless (Su(H)), which seems to be upregulated late in the transitioning cells and in NBs, represses l'sc and ase, ensuring their expression is transient. Thus, our data identify a key proneural role of Ase that is integrated with the sequential activities of Delta-Notch signaling, L'sc, and Su(H), driving the progressive transformation of NE cells into NBs.

Minibrain drives the Dacapo-dependent cell cycle exit of neurons in the Drosophila brain by promoting asense and prospero expression.

A key aim of neurodevelopmental research is to understand how precursor cells decide to stop dividing and commence their terminal differentiation at the correct time and place. Here, we show that minibrain (mnb), the Drosophila ortholog of the Down syndrome candidate gene DYRK1A, is transiently expressed in newborn neuronal precursors known as ganglion cells (GCs). Mnb promotes the cell cycle exit of GCs through a dual mechanism that regulates the expression of the cyclin-dependent kinase inhibitor Dacapo, the homolog of vertebrate p27(Kip1) (Cdkn1b). Mnb upregulates the expression of the proneural transcription factor (TF) Asense, which promotes Dacapo expression. Mnb also induces the expression of Prospero, a homeodomain TF that in turn inhibits the expression of Deadpan, a pan-neural TF that represses dacapo In addition to its effects on Asense and Prospero, Mnb also promotes the expression of the neuronal-specific RNA regulator Elav, strongly suggesting that Mnb facilitates neuronal differentiation. These actions of Mnb ensure the precise timing of neuronal birth, coupling the mechanisms that regulate neurogenesis, cell cycle control and terminal differentiation of neurons.

Association of Notch pathway down-regulation with Triple Negative/Basal-like breast carcinomas and high tumor-infiltrating FOXP3+ Tregs.

T regulatory cells (Tregs) are a lineage of lymphocytes involved in immune response suppression that are characterized by the expression of the forkhead box P3 (FOXP3) transcription factor. Notch pathway regulates FOXP3 transcription in Tregs, but its role in breast cancer is unknown. We aimed at studying whether Notch pathway regulates FOXP3 expression and Tregs content in breast cancer, and its association with luminal breast carcinomas. We analyzed by quantitative Real-Time PCR the mRNA levels of FOXP3, Notch pathway genes (Notch1, Notch2, Notch4 and Jagged1) and STAT3 in a series of 152 breast carcinomas including hormone receptor-positive and -negative phenotypes (luminal and Triple Negative/Basal-like). We also studied the protein expression of Notch1, STAT3 and FOXP3 by immunohistochemistry. High FOXP3 mRNA levels correlated with larger tumor size (p=0.010), histological grade 3 (p=0.008) and positive lymph-node status (p=0.031). Also, low levels of Notch pathway genes mRNA correlated with poor prognostic factors such as larger tumor size, positive lymph-node status, tumor phenotype and infiltrating tumor Tregs. A survival analysis for the patients showed that large tumor size, histological grade 3, vascular invasion, infiltrating Tregs and low Notch1 mRNA expression were significantly associated with a decreased patients' overall survival (p≤0.05). On a multivariate analysis, high Tregs content (HR=3.00, 95% CI 1.04-8.90, p=0.042) and low Notch1 mRNA levels (HR=3.33, 95% CI 1.02-10.86, p=0.046) were independent markers for overall survival. Our results support that the Notch pathway up-regulation promotes luminal breast carcinomas, whereas down-regulation correlates with the expression of FOXP3, favors tumor Tregs infiltration and associates with Triple Negative/Basal-like tumors.

Increased insulin-like growth factor-1 receptor mRNA expression predicts poor survival in immunophenotypes of early breast carcinoma.

The biology of breast carcinoma shows a great variation, reflected by the recent classification of phenotypes based on DNA microarrays or immunohistochemistry. The aim of this study was to determine the prevalence of insulin-like growth factor-1 receptor (IGF1R) in breast carcinoma subtypes and the impact on the outcome. We studied 197 consecutive breast carcinoma patients in stage I-II treated conservatively. Phenotypes were assessed on the basis of the expressions of ER/PR, HER2, Ki67, p53, Bcl2, CK5/6 and EGFR. Moreover, IGF1R expression (α-subunit and ß-phosphorylated/active form) was evaluated by immunohistochemistry, IGF1R mRNA levels by quantitative RT-PCR and IGF1R mutations by direct DNA sequencing. Overall, 40% (78/197) of tumors were luminal A, 24% (48/197) luminal B, 19% (37/197) HER2-positive and 17% (34/197) basal/triple-negative. Luminal A tumors were predominantly of low grade, without necrosis, presenting in older patients as a ≤2-cm unilateral mass (all P ≤ 0.046). α-IGF1R overexpression was observed more frequently in luminal A (49%) cases, followed by luminal B (20%), HER2-positive area under the curve (22%) and basal/triple-negative cases (9%) (P = 0.01) with similar results for mRNA levels (53, 24, 13 and 10%, respectively) (P = 0.038), but without differences for mutations (P = NS). High IGF1R mRNA correlated with poor patient survival among subtypes (P = 0.004) (Kaplan-Meier; log-rank test). For overall survival, only histological grade and IGF1R mRNA emerged as significant predictors (P ≤ 0.034; Cox regression). Increased IGF1R mRNA implies poorer patient prognosis among the different subtypes, and that may be associated with the lack of responsiveness to tamoxifen in cases with a positive hormone receptor status. Our results highlight the biological and clinical relevance of IGF1R in early breast carcinoma subtypes, and provide knowledge to assist in treatment decision.

Methylation analysis of MLH1 improves the selection of patients for genetic testing in Lynch syndrome.

Inactivation of MLH1 due to promoter hypermethylation strongly suggests a sporadic origin, providing exclusion criteria for Lynch syndrome. The aim of this study is to compare the utility of methylation analysis of MLH1 and BRAF V600E mutations for the selection of patients with MLH1 negative colorectal cancer for genetic testing. MLH1 methylation status was evaluated by MethyLight and methylation-specific MLPA (MS-MLPA) in tumor DNA from 73 colorectal cancer patients with loss of MLH1 protein expression. These tumors were analyzed for BRAF V600E mutations, and genetic testing for germline MLH1 mutations was performed in all corresponding patients. Ten patients had germline mutations in MLH1 and none of their tumors showed significant MLH1 methylation or BRAF V600E mutation. MLH1 genetic testing excluded patients by MethyLight in 47 patients (64%), by MS-MLPA in 49 (67%), and BRAF V600E mutation in only 25 patients (34%) (chi(2) P = 0.00001). Specificity was 75% for MethyLight, 78% for MS-MLPA and 40% for BRAF V600E mutation. The use of MethyLight or MS-MLPA instead of BRAF mutation resulted in a cost reduction of 41% and 45%, respectively, per every MLH1 mutation detected. Taken together, methylation analysis of MLH1 shows better performance characteristics than BRAF V600E mutation in the selection of patients for genetic testing of MLH1, especially when using MS-MLPA.

The position and function of the Notch-mediated eye growth organizer: the roles of JAK/STAT and four-jointed.

In many animal systems, the local activation of patterning signals in spatially confined regions (organizers) is crucial for promoting the growth of developing organs. Nevertheless, how organizers are set up and how their activity influences global organ growth remains poorly understood. In the Drosophila eye, local Notch activation establishes a conserved dorsal-ventral organizer that promotes growth. The dorsal selector Iroquois complex defines the position of the organizer at the mid-first instar, and through its ligand, unpaired, the Janus kinase (JAK)/signal transducers and activator of transcription (STAT) pathway is thought to mediate global growth downstream of the organizer. However, here we show that the unpaired/JAK/STAT pathway is actually a fundamental element in the spatial control of the organizer, upstream from Notch activation. Furthermore, we identify four-jointed, a target of the Fat and Hippo tumour-suppressor pathways, as a mediator of the growth controlled by the organizer. These findings redefine the process of organizer formation and function, and they identify four-jointed as a regulatory node, integrating multiple growth-control pathways.

Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing.

Cancer is both a genetic and an epigenetic disease. Inactivation of tumour-suppressor genes by epigenetic changes is frequently observed in human cancers, particularly as a result of the modifications of histones and DNA methylation. It is therefore important to understand how these damaging changes might come about. By studying tumorigenesis in the Drosophila eye, here we identify two Polycomb group epigenetic silencers, Pipsqueak and Lola, that participate in this process. When coupled with overexpression of Delta, deregulation of the expression of Pipsqueak and Lola induces the formation of metastatic tumours. This phenotype depends on the histone-modifying enzymes Rpd3 (a histone deacetylase), Su(var)3-9 and E(z), as well as on the chromodomain protein Polycomb. Expression of the gene Retinoblastoma-family protein (Rbf) is downregulated in these tumours and, indeed, this downregulation is associated with DNA hypermethylation. Together, these results establish a mechanism that links the Notch-Delta pathway, epigenetic silencing pathways and cell-cycle control in the process of tumorigenesis.

Growth and specification of the eye are controlled independently by Eyegone and Eyeless in Drosophila melanogaster.

Control of growth determines the size and shape of organs. Localized signals known as 'organizers' and members of the Pax family of proto-oncogenes are both elements in this control. Pax proteins have a conserved DNA-binding paired domain, which is presumed to be essential for their oncogenic activity. We present evidence that the organizing signal Notch does not promote growth in eyes of D. melanogaster through either Eyeless (Ey) or Twin of eyeless (Toy), the two Pax6 transcription factors. Instead, it acts through Eyegone (Eyg), which has a truncated paired domain, consisting of only the C-terminal subregion. In humans and mice, the sole PAX6 gene produces the isoform PAX6(5a) by alternative splicing; like Eyegone, this isoform binds DNA though the C terminus of the paired domain. Overexpression of human PAX6(5a) induces strong overgrowth in vivo, whereas the canonical PAX6 variant hardly effects growth. These results show that growth and eye specification are subject to independent control and explain hyperplasia in a new way.