Project description:Current interventions for endometriosis mainly involve hormone therapies but have limited efficacy and unacceptable side effects, due to the lack of selectivity to distinguish between endometriosis and endometrial tissues. Elucidating the molecular mechanism underlying rapid growth of endometrial-like stromal cells, one of the main components of endometriotic lesions, will pave a path for more effective treatment of endometriosis. In the current study, we utilized transcriptome sequencing to compare the transcriptional profiles of endometrial-like stromal cells from endometriosis and endometrial tissues and demonstrated that Homeobox C4 (HOXC4) is preferentially expressed in endometriotic lesions. HOXC4 is indispensable for the proliferation of stromal cells from endometriosis, but not those from endometrial tissues. Mechanistically, HOXC4 acts as a transcription factor to promote the expression of Slit Guidance Ligand 2 (SLIT2) and thereby, increases the p38 MAPK activity via the SLIT2 receptor Roundabout Guidance Receptor 1 (ROBO1). Considering the essential role of the p38 MAPK activity in facilitating the development of ectopic endometrium, our findings strongly support the idea of HOXC4, as well as the SLIT2-ROBO1 axis, being as potential therapeutic targets for endometriosis.
Project description:Roundabout (ROBO) 1 and 2 are transmembrane receptors that bind secreted SLIT ligands through their extracellular domains (ECD), and signal through their cytoplasmic domains to modulate the cytoskeleton and regulate cell migration, adhesion, and proliferation. SLIT-ROBO signaling was discovered as a guidance cue for axons in the developing nervous system, but ROBO receptors are expressed by many additional cell types and more broadly regulate organ morphogenesis, as well as cancer and pathological ocular neovascularization. As pharmacological tools to prevent SLIT-ROBOR signaling are lacking, herein we developed human monoclonal antibodies (mAb ) against the ROBO1 and 2 ECD. One antibody that inhibited in vitro SLIT2 signaling through ROBO1/2 reduced ocular neovascularization in two pathological models in vivo. Single cell RNA sequencing revealed that antibody treatment affected several cell types relevant to angiogenesis, including endothelial cells, pericytes, and a heterogeneous population of myeloid cells. mAb treatment improved Blood-Retina Barrier integrity and prevented pathological pericyte activation. SLIT-ROBO signaling inhibition prevented pathological activation of myeloid cells, which was phenocopied by both myeloid cell specific ablation of Robo1 and 2 and by knockout of the downstream effector Pi3kγ. Anti-ROBO1/2 blocking antibodies may thus provide a new promising strategy to combat inflammation in blinding eye diseases.
Project description:Id1 and its closely related family member Id3 are expressed by a diversity of stem and progenitor cells. We show that Id1/3 are required for the self-renewal and proliferation of triple negative breast cancer (TNBC) cells both in vitro and in vivo. Furthermore, we identified that Id1/3 negatively regulates the tumour suppressor gene Robo1. Depletion of Robo1 could rescue the proliferative defect induced by Id1/3 knockdown. To understand the mechanisms by which Robo1 rescues cell proliferation in Id1/3 depleted cells, we performed RNA-Sequencing on 4T1 cells with Dox-inducible Id1/3 KD and/or Robo1 depletion using siRNA. We conclude that following Id1/3 knockdown, Robo1 is induced and exerts anti-proliferative effects via suppression of a Myc transcriptional program.
Project description:The goal of the study was to compare gene expression of Robo1+/+ and Robo1-/- luminal progenitors. Total RNAs were then extracted from FACS purified luminal progenitor cells, harvested from Robo1+/+ or Robo1-/- mice (n=3 per genotype, two animals per n) using TRIreagent LS (Sigma, T3934). Poly(A)+ RNA sequencing libraries were made from each sample using the TruSeq RNA library preparation kit v.1 (Illumina). Illumina RNA PolyA library preparation guide. A total of 6 libraries were created by PCR amplification with Illumina barcoding primers using kit recommended conditions and quantified using a Bioanalyzer DNA 1000 kit (Agilent).
