Dysregulated Notch signaling in breast cancer and liver disease

The evolutionarily conserved Notch signaling pathway regulates crucial aspects of development and tissue homeostasis. This thesis contributes research towards understanding a role of non-canonical Notch signaling in the tumor-stroma interaction of breast cancer, provides a bioinformatics-based technology to study these interactions, and proposes a novel mouse model of the liver disease in Alagille syndrome. In Paper I, we report a novel target for non-canonical Notch signaling in breast cancer, the cytokine IL-6. In human breast cancer cell lines, we observe increased IL-6 mRNA and protein levels when Notch signaling is amplified, in turn activating the JAK/STAT pathway in a p53-dependent, but CSL-independent fashion, via IKKα and IKKβ of the NF-κB pathway. These data add a new facet to the existing body of knowledge on hyperactivated Notch signaling in promoting inflammation in breast tumors. In Paper II, we present and validate a new bioinformatics-based approach of species-specific sequencing (S3). Using an intermixed human tumor and mouse stroma cell population from xenografted cells, we demonstrate a way to decode transcriptomes, separated by their species-specific differences, with 99% accuracy. This technique circumvents current problems in mechanically separating mixed tissue, and paves the way to efficiently analyze in vivo cell-cell interactions. In Paper III, we characterize a mouse strain, with a missense mutation in the Jagged1 gene, as a potential model for the rare genetic disorder Alagille syndrome. We show that this model recapitulates pathologies in the liver, heart, lens and kidney observed in Alagille patients, and identify dysregulated biliary morphogenesis caused by this mutation. We also use the S3 technology, developed in Paper II, to investigate signaling specifically in receptor-expressing cells by wild type and mutated Jagged1. In summary, the work presented in this thesis sheds new light on the role of Notch signaling in breast cancer and liver disease, and provides a novel technology to facilitate the detailed study of cell-cell interactions

Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations.

BACKGROUND & AIMS: Alagille syndrome is a genetic disorder characterized by cholestasis, ocular abnormalities, characteristic facial features, heart defects, and vertebral malformations. Most cases are associated with mutations in JAGGED1 (JAG1), which encodes a Notch ligand, although it is not clear how these contribute to disease development. We aimed to develop a mouse model of Alagille syndrome to elucidate these mechanisms. METHODS: Mice with a missense mutation (H268Q) in Jag1 (Jag1+/Ndr mice) were outbred to a C3H/C57bl6 background to generate a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice). Liver tissues were collected at different timepoints during development, analyzed by histology, and liver organoids were cultured and analyzed. We performed transcriptome analysis of Jag1Ndr/Ndr livers and livers from patients with Alagille syndrome, cross-referenced to the Human Protein Atlas, to identify commonly dysregulated pathways and biliary markers. We used species-specific transcriptome separation and ligand-receptor interaction assays to measure Notch signaling and the ability of JAG1Ndr to bind or activate Notch receptors. We studied signaling of JAG1 and JAG1Ndr via NOTCH 1, NOTCH2, and NOTCH3 and resulting gene expression patterns in parental and NOTCH1-expressing C2C12 cell lines. RESULTS: Jag1Ndr/Ndr mice had many features of Alagille syndrome, including eye, heart, and liver defects. Bile duct differentiation, morphogenesis, and function were dysregulated in newborn Jag1Ndr/Ndr mice, with aberrations in cholangiocyte polarity, but these defects improved in adult mice. Jag1Ndr/Ndr liver organoids collapsed in culture, indicating structural instability. Whole-transcriptome sequence analyses of liver tissues from mice and patients with Alagille syndrome identified dysregulated genes encoding proteins enriched at the apical side of cholangiocytes, including CFTR and SLC5A1, as well as reduced expression of IGF1. Exposure of Notch-expressing cells to JAG1Ndr, compared with JAG1, led to hypomorphic Notch signaling, based on transcriptome analysis. JAG1-expressing cells, but not JAG1Ndr-expressing cells, bound soluble Notch1 extracellular domain, quantified by flow cytometry. However, JAG1 and JAG1Ndr cells each bound NOTCH2, and signaling from NOTCH2 signaling was reduced but not completely inhibited, in response to JAG1Ndr compared with JAG1. CONCLUSIONS: In mice, expression of a missense mutant of Jag1 (Jag1Ndr) disrupts bile duct development and recapitulates Alagille syndrome phenotypes in heart, eye, and craniofacial dysmorphology. JAG1Ndr does not bind NOTCH1, but binds NOTCH2, and elicits hypomorphic signaling. This mouse model can be used to study other features of Alagille syndrome and organ development

Decoding breast cancer tissue-stroma interactions using species-specific sequencing.

Decoding transcriptional effects of experimental tissue-tissue or cell-cell interactions is important; for example, to better understand tumor-stroma interactions after transplantation of human cells into mouse (xenografting). Transcriptome analysis of intermixed human and mouse cells has, however, frequently relied on the need to separate the two cell populations prior to transcriptome analysis, which introduces confounding effects on gene expression

Access Issues and Training Needs of Teachers in the Adult and Community Sector

Optimization of S3 parameters. (XLS 65 kb

Additional file 1: Table S1. of Decoding breast cancer tissue–stroma interactions using species-specific sequencing

Fisher’s exact test for count data (R). (XLSX 10 kb

Additional file 7: Table S6. of Decoding breast cancer tissue–stroma interactions using species-specific sequencing

Gene Ontology. (XLSX 270 kb

Additional file 8: Table S7. of Decoding breast cancer tissue–stroma interactions using species-specific sequencing

Alignment Statistics. (PDF 657 kb

Additional file 3: Figure S1. of Decoding breast cancer tissue–stroma interactions using species-specific sequencing

Pipeline of data processing. Figure S2. Comparison with other methods to separate human and mouse reads, both in terms of sensitivity (A) and specificity (B). Figure S3. Comparison of three species (rat, mouse, human) separation of rat (R1–R3) and mouse (M4, M5) RNA-seq samples, similar to Fig. 1g but with absolute number of reads on the y axis. Figure S4. Comparison of the number of genes up- and downregulated in (A) MDA-MB-231 cells co-cultured with 3T3-L1 cells expressing DLL4 or GFP, (B) MDA-MB-231 cells on immobilized Fc-DLL4 or Fc, and (C) 3T3-L1 cells, expressing DLL4 or GFP, co-cultured with MDA-MB-231 cells. Figure S5. Whole-genome gene expression QC: Depth Saturation. Figure S6. Whole-Genome Gene Expression QC: Density Plots after TMM Normalization. Figure S7. Additional principal component analyses (PCA). Figure S8. Scatterplot of genes in Estrogen-related signaling, differentially expressed in MCF7 cells in vitro and MCF7 cells in tumor. Figure S9. Scatterplots of Table S8 comparison groups. Figure S10. Hierarchical Clustering. Figure S11. FINAK_BREAST_CANCER_SDPP_SIGNATURE: Scatterplot of genes in the stroma-derived prognostic predictor of breast cancer disease outcome (Finak et al. 2008 [59]), differentially expressed in (A) Mammary Gland (MG) compared to MCF7 tumor stroma, (B) MG compared to MDA-MB-231 tumor stroma, and MCF7 tumor stroma compared to MDA-MB-231 tumor stroma. (PDF 11548 kb

Additional file 2: Table S2. of Decoding breast cancer tissue–stroma interactions using species-specific sequencing

p-values and FDR from SAMlike_test_on_rpkms.py. (XLSX 14232 kb