Receptor and secreted targets of Wnt-1/beta-catenin signalling in mouse mammary epithelial cells.

BackgroundDeregulation of the Wnt/ beta-catenin signal transduction pathway has been implicated in the pathogenesis of tumours in the mammary gland, colon and other tissues. Mutations in components of this pathway result in beta-catenin stabilization and accumulation, and the aberrant modulation of beta-catenin/TCF target genes. Such alterations in the cellular transcriptional profile are believed to underlie the pathogenesis of these cancers. We have sought to identify novel target genes of this pathway in mouse mammary epithelial cells.MethodsGene expression microarray analysis of mouse mammary epithelial cells inducibly expressing a constitutively active mutant of beta-catenin was used to identify target genes of this pathway.ResultsThe differential expression in response to DeltaNbeta-catenin for five putative target genes, Autotaxin, Extracellular Matrix Protein 1 (Ecm1), CD14, Hypoxia-inducible gene 2 (Hig2) and Receptor Activity Modifying Protein 3 (RAMP3), was independently validated by northern blotting. Each of these genes encodes either a receptor or a secreted protein, modulation of which may underlie the interactions between Wnt/beta-catenin tumour cells and between the tumour and its microenvironment. One of these genes, Hig2, previously shown to be induced by both hypoxia and glucose deprivation in human cervical carcinoma cells, was strongly repressed upon DeltaNbeta-catenin induction. The predicted N-terminus of Hig2 contains a putative signal peptide suggesting it might be secreted. Consistent with this, a Hig2-EGFP fusion protein was able to enter the secretory pathway and was detected in conditioned medium. Mutation of critical residues in the putative signal sequence abolished its secretion. The expression of human HIG2 was examined in a panel of human tumours and was found to be significantly downregulated in kidney tumours compared to normal adjacent tissue.ConclusionsHIG2 represents a novel non-cell autonomous target of the Wnt pathway which is potentially involved in human cancer

Retroviral vectors for establishing tetracycline-regulated gene expression in an otherwise recalcitrant cell line

BACKGROUND: Tetracycline-regulated systems have been used to control the expression of heterologous genes in such diverse organisms as yeast, plants, flies and mice. Adaptation of this prokaryotic regulatory system avoids many of the problems inherent in other inducible systems. There have, however, been many reports of difficulties in establishing functioning stable cell lines due to the cytotoxic effects of expressing high levels of the tetracycline transactivator, tTA, from a strong viral promoter. RESULTS: Here we report the successful incorporation of tetracycline-mediated gene expression in a mouse mammary epithelial cell line, HC11, in which conventional approaches failed. We generated retroviruses in which tTA expression was controlled by one of three promoters: a synthetic tetracycline responsive promoter (TRE), the elongation factor 1-alpha promoter (EF1α) or the phosphoglycerate kinase-1 promoter (PGK), and compared the resulting cell lines to one generated using a cytomegalovirus immediate early gene promoter (CMV). In contrast to cells produced using the CMV and PGK promoters, those produced using the EF1α and TRE promoters expressed high levels of β-galactosidase in a tetracycline-dependent manner. CONCLUSIONS: These novel retroviral vectors performed better than the commercially available system and may have a more general utility in similarly recalcitrant cell lines

In vitro differentiation of human pluripotent stem cells into the B lineage using OP9-MS5 co-culture.

In vitro differentiation of human pluripotent stem cells (hPSCs) offers a genetically tractable system to examine the physiology and pathology of human tissue development and differentiation. We have used this approach to model the earliest stages of human B lineage development and characterize potential target cells for the in utero initiation of childhood B acute lymphoblastic leukemia. Herein, we detail critical aspects of the protocol including reagent validation, controls, and examples of surface markers used for analysis and cell sorting. For complete details on the use and execution of this protocol, please refer to Boiers et al. (2018).Wellcome Trust, Cancer Research UK, Swedish Research Council, Swedish Childhood Cancer Foundatio

DNA ploidy analyses in 218 consecutive Pakistani breast cancer patients: does it add anything?

An analysis was made to evaluate the significance of DNA ploidy in the biology and prognosis of breast carcinoma. This was done by estimating the correlation of DNA ploidy with other established prognostic markers of breast cancer, namely tumor size, tumor grade, lymph node metastasis and S-phase fraction. From 1995 up to year 2000 ploidy analysis was performed on 218 consecutive cases of infiltrating breast carcinoma by flow cytometry using formalin fixed paraffin embedded material. From the laboratory record, data regarding other pathological variables was retrieved. No correlation could be found between DNA ploidy and tumor grade, nor could there be found a correlation with tumor size. For lymph node metastasis there was a significant difference between the proportion of aneuploids and diploids having metastasis in more than 4 lymph nodes. However, no significant difference was found in axillary lymph node positive and negative groups when number of positive lymph nodes was not taken into account. The mean value of S-phase fraction for the aneuploids and the diploids was also insignificantly different. In conclusion DNA ploidy alone did not add much to predict tumor behaviour in terms of known pathologic variables

Single-cell sequencing reveals CD133+CD44--originating evolution and novel stemness related variants in human colorectal cancer

BACKGROUND: Tumor heterogeneity of human colorectal cancer (CRC)-initiating cells (CRCICs) in cancer tissues often represents aggressive features of cancer progression. For high-resolution examination of CRCICs, we performed single-cell whole-exome sequencing (scWES) and bulk cell targeted exome sequencing (TES) of CRCICs to investigate stemness-specific somatic alterations or clonal evolution. METHODS: Single cells of three subpopulations of CRCICs (CD133+CD44+, CD133-CD44+, and CD133+CD44- cells), CRC cells (CRCCs), and control cells from one CRC tissue were sorted for scWES. Then, we set up a mutation panel from scWES data and TES was used to validate mutation distribution and clonal evolution in additional 96 samples (20 patients) those were also sorted into the same three groups of CRCICs and CRCCs. The knock-down experiments were used to analyze stemness-related mutant genes. Neoantigens of these mutant genes and their MHC binding affinity were also analyzed. FINDINGS: Clonal evolution analysis of scWES and TES showed that the CD133+CD44- CRCICs were the likely origin of CRC before evolving into other groups of CRCICs/CRCCs. We revealed that AHNAK2, PLIN4, HLA-B, ALK, CCDC92 and ALMS1 genes were specifically mutated in CRCICs followed by the validation of their functions. Furthermore, four predicted neoantigens of AHNAK2 were identified and validated, which might have applications in immunotherapy for CRC patients. INTERPRETATION: All the integrative analyses above revealed clonal evolution of CRC and new markers for CRCICs and demonstrate the important roles of CRCICs in tumorigenesis and progression of CRCs. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section

A network including TGFβ/Smad4, Gata2 and p57 regulates proliferation of mouse hematopoietic progenitor cells.

Transforming growth factor-β (TGFβ) is a potent inhibitor of hematopoietic stem and progenitor cell proliferation. However, the precise mechanism for this effect is unknown. Here, we have identified the transcription factor Gata2, previously described as an important regulator of hematopoietic stem cell (HSC) function, as an early and direct target gene for TGFβ-induced Smad signaling in hematopoietic progenitor cells. We also report that Gata2 is involved in mediating a significant part of the TGFβ response in primitive hematopoietic cells. Interestingly, the cell cycle regulator and TGFβ signaling effector molecule p57 was found to be upregulated as a secondary response to TGFβ. We observed Gata2 binding upstream of the p57 genomic locus, and importantly loss of Gata2 abolished TGFβ-stimulated induction of p57 as well as the resulting growth arrest of hematopoietic progenitors. Our results connect key molecules involved in HSC self-renewal and reveal a functionally relevant network regulating proliferation of primitive hematopoietic cells

Acute Loss of Cited2 Impairs Nanog Expression and Decreases Self-Renewal of Mouse Embryonic Stem Cells

Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While overexpression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2 is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells.National Portuguese through FCT-Fundacao para a Ciencia e a Tecnologia [PEst-OE/EQB/LA0023/2013, PTDC/SAU-ENB/111702/2009]; Camara Municipal de Oeiras; Merck Sharp & Dhome Foundation-Portugal; CRUK; Leukaemia & Lymphoma Research; Kay Kendall Leukaemia Fund; Wellcome Trust; Medical Research Council; Cancer Research UK [12796, 14633]; Great Ormond Street Hospital Childrens Charity [W1062]; Medical Research Council [MC_U137973817, G1000801g, MC_qA137913

Injury primes mutation-bearing astrocytes for dedifferentiation in later life

Despite their latent neurogenic potential, most normal parenchymal astrocytes fail to dedifferentiate to neural stem cells in response to injury. In contrast, aberrant lineage plasticity is a hallmark of gliomas, and this suggests that tumor suppressors may constrain astrocyte dedifferentiation. Here, we show that p53, one of the most commonly inactivated tumor suppressors in glioma, is a gatekeeper of astrocyte fate. In the context of stab-wound injury, p53 loss destabilized the identity of astrocytes, priming them to dedifferentiate in later life. This resulted from persistent and age-exacerbated neuroinflammation at the injury site and EGFR activation in periwound astrocytes. Mechanistically, dedifferentiation was driven by the synergistic upregulation of mTOR signaling downstream of p53 loss and EGFR, which reinstates stemness programs via increased translation of neurodevelopmental transcription factors. Thus, our findings suggest that first-hit mutations remove the barriers to injury-induced dedifferentiation by sensitizing somatic cells to inflammatory signals, with implications for tumorigenesis

Macrophage-induced blood vessels guide Schwann cell-mediated regeneration of peripheral nerves

The peripheral nervous system has remarkable regenerative capacities in that it can repair a fully cut nerve. This requires Schwann cells to migrate collectively to guide regrowing axons across a 'bridge' of new tissue, which forms to reconnect a severed nerve. Here we show that blood vessels direct the migrating cords of Schwann cells. This multicellular process is initiated by hypoxia, selectively sensed by macrophages within the bridge, which via VEGF-A secretion induce a polarized vasculature that relieves the hypoxia. Schwann cells then use the blood vessels as "tracks" to cross the bridge taking regrowing axons with them. Importantly, disrupting the organization of the newly formed blood vessels in vivo, either by inhibiting the angiogenic signal or by re-orienting them, compromises Schwann cell directionality resulting in defective nerve repair. This study provides important insights into how the choreography of multiple cell-types is required for the regeneration of an adult tissue