Transitional B cells commit to marginal zone B cell fate by Taok3-mediated surface expression of ADAM10.

Notch2 and B cell antigen receptor (BCR) signaling determine whether transitional B cells become marginal zone B (MZB) or follicular B (FoB) cells in the spleen, but it is unknown how these pathways are related. We generated Taok3-/- mice, lacking the serine/threonine kinase Taok3, and found cell-intrinsic defects in the development of MZB but not FoB cells. Type 1 transitional (T1) B cells required Taok3 to rapidly respond to ligation by the Notch ligand Delta-like 1. BCR ligation by endogenous or exogenous ligands induced the surface expression of the metalloproteinase ADAM10 on T1 B cells in a Taok3-dependent manner. T1 B cells expressing surface ADAM10 were committed to becoming MZB cells in vivo, whereas T1 B cells lacking expression of ADAM10 were not. Thus, during positive selection in the spleen, BCR signaling causes immature T1 B cells to become receptive to Notch ligands via Taok3-mediated surface expression of ADAM10.

Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation.

BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. METHODS: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RESULTS: RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. CONCLUSIONS: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

STAT5 activation promotes progression and chemotherapy resistance in early T-cell precursor acute lymphoblastic leukemia.

Interleukin-7 (IL-7) supports the growth and chemoresistance of T-cell acute lymphoblastic leukemia (T-ALL), particularly the early T-cell precursor subtype (ETP-ALL), which frequently has activating mutations of IL-7 signaling. Signal transducer and activator of transcription (STAT5) is an attractive therapeutic target because it is almost universally activated in ETP-ALL, even in the absence of mutations of upstream activators such as the IL-7 receptor (IL-7R), Janus kinase, and Fms-like tyrosine kinase 3 (FLT3). To examine the role of activated STAT5 in ETP-ALL, we have used a Lmo2-transgenic (Lmo2Tg) mouse model in which we can monitor chemoresistant preleukemia stem cells (pre-LSCs) and leukemia stem cells (LSCs) that drive T-ALL development and relapse following chemotherapy. Using IL-7R-deficient Lmo2Tg mice, we show that IL-7 signaling was not required for the formation of pre-LSCs but essential for their expansion and clonal evolution into LSCs to generate T-ALL. Activated STAT5B was sufficient for the development of T-ALL in IL-7R-deficient Lmo2Tg mice, indicating that inhibition of STAT5 is required to block the supportive signals provided by IL-7. To further understand the role of activated STAT5 in LSCs of ETP-ALL, we developed a new transgenic mouse that enables T-cell specific and doxycycline-inducible expression of the constitutively activated STAT5B1∗6 mutant. Expression of STAT5B1∗6 in T cells had no effect alone but promoted expansion and chemoresistance of LSCs in Lmo2Tg mice. Pharmacologic inhibition of STAT5 with pimozide-induced differentiation and loss of LSCs, while enhancing response to chemotherapy. Furthermore, pimozide significantly reduced leukemia burden in vivo and overcame chemoresistance of patient-derived ETP-ALL xenografts. Overall, our results demonstrate that STAT5 is an attractive therapeutic target for eradicating LSCs in ETP-ALL.

Interplay between the EMT transcription factors ZEB1 and ZEB2 regulates hematopoietic stem and progenitor cell differentiation and hematopoietic lineage fidelity.

The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus-based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML.

Dysregulation of Grainyhead-like 3 expression causes widespread developmental defects.

BACKGROUND: The gene encoding the transcription factor, Grainyhead-like 3 (Grhl3), plays critical roles in mammalian development and homeostasis. Grhl3-null embryos exhibit thoraco-lumbo-sacral spina bifida and soft-tissue syndactyly. Additional studies reveal that these embryos also exhibit an epidermal proliferation/differentiation imbalance. This manifests as skin barrier defects resulting in peri-natal lethality and defective wound repair. Despite these extensive analyses of Grhl3 loss-of-function models, the consequences of gain-of-function of this gene have been difficult to achieve. RESULTS: In this study, we generated a novel mouse model that expresses Grhl3 from a transgene integrated in the Rosa26 locus on an endogenous Grhl3-null background. Expression of the transgene rescues both the neurulation and skin barrier defects of the knockout mice, allowing survival into adulthood. Despite this, the mice are not normal, exhibiting a range of phenotypes attributable to dysregulated Grhl3 expression. In mice homozygous for the transgene, we observe a severe Shaker-Waltzer phenotype associated with hearing impairment. Micro-CT scanning of the inner ear revealed profound structural alterations underlying these phenotypes. In addition, these mice exhibit other developmental anomalies including hair loss, digit defects, and epidermal dysmorphogenesis. CONCLUSION: Taken together, these findings indicate that diverse developmental processes display low tolerance to dysregulation of Grhl3.

The EMT modulator SNAI1 contributes to AML pathogenesis via its interaction with LSD1.

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.

GNrep mouse: A reporter mouse for front-rear cell polarity.

Cell migration is essential during development, regeneration, homeostasis, and disease. Depending on the microenvironment, cells use different mechanisms to migrate. Yet, all modes of migration require the establishment of an intracellular front-rear polarity axis for directional movement. Although front-rear polarity can be easily identified in in vitro conditions, its assessment in vivo by live-imaging is challenging due to tissue complexity and lack of reliable markers. Here, we describe a novel and unique double fluorescent reporter mouse line to study front-rear cell polarity in living tissues, called GNrep. This mouse line simultaneously labels Golgi complexes and nuclei allowing the assignment of a nucleus-to-Golgi axis to each cell, which functions as a readout for cell front-rear polarity. As a proof-of-principle, we validated the efficiency of the GNrep line using an endothelial-specific Cre mouse line. We show that the GNrep labels the nucleus and the Golgi apparatus of endothelial cells with very high efficiency and high specificity. Importantly, the features of fluorescent intensity and localization for both mCherry and eGFP fluorescent intensity and localization allow automated segmentation and assignment of polarity vectors in complex tissues, making GNrep a great tool to study cell behavior in large-scale automated analyses. Altogether, the GNrep mouse line, in combination with different Cre recombinase lines, is a novel and unique tool to study of front-rear polarity in mice, both in fixed tissues or in intravital live imaging. This new line will be instrumental to understand cell migration and polarity in development, homeostasis, and disease.

Lysyl oxidase-like 2 represses Notch1 expression in the skin to promote squamous cell carcinoma progression.

Lysyl oxidase-like 2 (LOXL2) is involved in a wide range of physiological and pathological processes, including fibrosis and tumor progression, implicating intracellular and extracellular functions. To explore the specific in vivo role of LOXL2 in physiological and tumor contexts, we generated conditional gain- and loss-of-function mouse models. Germ-line deletion of Loxl2 promotes lethality in half of newborn mice mainly associated to congenital heart defects, while Loxl2 overexpression triggers male sterility due to epididymal dysfunction caused by epithelial disorganization, fibrosis and acute inflammation. Remarkably, when challenged to chemical skin carcinogenesis, Loxl2-overexpressing mice increased tumor burden and malignant progression, while Loxl2-deficient mice exhibit the opposite phenotypes. Loxl2 levels in premalignant tumors negatively correlate with expression of epidermal differentiation markers and components of the Notch1 pathway. We show that LOXL2 is a direct repressor of NOTCH1. Additionally, we identify an exclusive expression pattern between LOXL2 and members of the canonical NOTCH1 pathway in human HNSCC. Our data identify for the first time novel LOXL2 roles in tissue homeostasis and support it as a target for SCC therapy.

Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium.

Snail family members regulate epithelial-to-mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation-induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.

The pulmonary microvasculature entraps induced vascular progenitor cells (iVPCs) systemically delivered after cardiac ischemia-reperfusion injury: Indication for preservation of heart function via paracrine effects beyond engraftment.

OBJECTIVE: Stem cell-based regenerative therapies have been intensively studied with the aim to define an ideal cell type for the treatment of myocardial infarction. We tested systemically delivered, platelet-targeted induced vascular progenitor cells (iVPCs) to study their potential to salvage damaged myocardium after ischemia-reperfusion injury. METHODS: Using a mouse model of ischemia-reperfusion injury, we tested the potential of platelet-targeted iVPCs (1 × 106 targ-iVPCs) compared to non-targ-iVPCs and a saline control. Bioluminescence imaging, echocardiography, and histological analyses were performed. RESULTS: Four weeks after ischemia-reperfusion injury, systemic delivery of targ-iVPCs led to reduced fibrosis and infarct size (PBS: 25.7 ± 3.9 vs targ-iVPC: 18.4 ± 6.6 vs non-targ-iVPC: 25.1 ± 3.7%I/LV, P < 0.05), increased neovascularization, and restored cardiac function (PBS: 44.0 ± 4.2 vs targ-iVPC: 54.3 ± 4.5 vs non-targ-iVPC: 46.4 ± 3.8%EF, P < 0.01). Cell tracking experiments revealed entrapment of intravenously injected iVPCs in the pulmonary microvasculature in both cell-treated groups. CONCLUSIONS: Systemic delivery of iVPCs after cardiac ischemia-reperfusion injury is limited by pulmonary entrapment of the cells. Nevertheless, targ-iVPCs reduced infarct size, fibrosis, increased neovascularization, and most importantly retained cardiac function. These findings contribute to the mechanistic discussion of cell-based therapy and ultimately identify activated platelet-targeted iVPCs as candidates for cell therapy and also describe cell therapy benefits without the necessity of engrafting.

Oncogenic ZEB2 activation drives sensitivity toward KDM1A inhibition in T-cell acute lymphoblastic leukemia.

Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.

The EMT transcription factor Zeb2 controls adult murine hematopoietic differentiation by regulating cytokine signaling.

Epithelial-to-mesenchymal-transition (EMT) is critical for normal embryogenesis and effective postnatal wound healing, but is also associated with cancer metastasis. SNAIL, ZEB, and TWIST families of transcription factors are key modulators of the EMT process, but their precise roles in adult hematopoietic development and homeostasis remain unclear. Here we report that genetic inactivation of Zeb2 results in increased frequency of stem and progenitor subpopulations within the bone marrow (BM) and spleen and that these changes accompany differentiation defects in multiple hematopoietic cell lineages. We found no evidence that Zeb2 is critical for hematopoietic stem cell self-renewal capacity. However, knocking out Zeb2 in the BM promoted a phenotype with several features that resemble human myeloproliferative disorders, such as BM fibrosis, splenomegaly, and extramedullary hematopoiesis. Global gene expression and intracellular signal transduction analysis revealed perturbations in specific cytokine and cytokine receptor-related signaling pathways following Zeb2 loss, especially the JAK-STAT and extracellular signal-regulated kinase pathways. Moreover, we detected some previously unknown mutations within the human Zeb2 gene (ZFX1B locus) from patients with myeloid disease. Collectively, our results demonstrate that Zeb2 controls adult hematopoietic differentiation and lineage fidelity through widespread modulation of dominant signaling pathways that may contribute to blood disorders.

ZEB2 and LMO2 drive immature T-cell lymphoblastic leukemia via distinct oncogenic mechanisms.

ZEB1 and ZEB2 are structurally related E-box binding homeobox transcription factors that induce epithelial to mesenchymal transitions during development and disease. As such, they regulate cancer cell invasion, dissemination and metastasis of solid tumors. In addition, their expression is associated with the gain of cancer stem cell properties and resistance to therapy. Using conditional loss-of-function mice, we previously demonstrated that Zeb2 also plays pivotal roles in hematopoiesis, controlling important cell fate decisions, lineage commitment and fidelity. In addition, upon Zeb2 overexpression, mice spontaneously develop immature T-cell lymphoblastic leukemia. Here we show that pre-leukemic Zeb2-overexpressing thymocytes are characterized by a differentiation delay at beta-selection due to aberrant activation of the interleukin-7 receptor signaling pathway. Notably, and in contrast to Lmo2-overexpressing thymocytes, these pre-leukemic Zeb2-overexpressing T-cell progenitors display no acquired self-renewal properties. Finally, Zeb2 activation in more differentiated T-cell precursor cells can also drive malignant T-cell development, suggesting that the early T-cell differentiation delay is not essential for Zeb2-mediated leukemic transformation. Altogether, our data suggest that Zeb2 and Lmo2 drive malignant transformation of immature T-cell progenitors via distinct molecular mechanisms.

p120 Catenin-Mediated Stabilization of E-Cadherin Is Essential for Primitive Endoderm Specification.

E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their in vitro differentiation was incomplete. In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment.

A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8.

Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot-Marie-Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces impairments in autophagy that may aggravate the phenotype.

Zeb2 Regulates Cell Fate at the Exit from Epiblast State in Mouse Embryonic Stem Cells.

In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell-fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA-sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast-like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial-to-mesenchymal transition (EMT), and DNA-(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1-binding sites correlate with loss-of-methylation in neural-stimulating conditions, however, after the cells initially acquired the correct DNA-methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re-adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA-methylation with irreversible commitment to differentiation. Stem Cells 2017;35:611-625.

Identification of a clonally expanding haematopoietic compartment in bone marrow.

In mammals, postnatal haematopoiesis occurs in the bone marrow (BM) and involves specialized microenvironments controlling haematopoietic stem cell (HSC) behaviour and, in particular, stem cell dormancy and self-renewal. While these processes have been linked to a number of different stromal cell types and signalling pathways, it is currently unclear whether BM has a homogenous architecture devoid of structural and functional partitions. Here, we show with genetic labelling techniques, high-resolution imaging and functional experiments in mice that the periphery of the adult BM cavity harbours previously unrecognized compartments with distinct properties. These units, which we have termed hemospheres, were composed of endothelial, haematopoietic and mesenchymal cells, were enriched in CD150+ CD48- putative HSCs, and enabled rapid haematopoietic cell proliferation and clonal expansion. Inducible gene targeting of the receptor tyrosine kinase VEGFR2 in endothelial cells disrupted hemospheres and, concomitantly, reduced the number of CD150+ CD48- cells. Our results identify a previously unrecognized, vessel-associated BM compartment with a specific localization and properties distinct from the marrow cavity.

The Snail Family in Normal and Malignant Haematopoiesis.

Snail family proteins are key inducers of the epithelial-mesenchymal transition (EMT), a critical process required for normal embryonic development. They have also been strongly implicated in regulating the EMT-like processes required for tumour cell invasion, migration, and metastasis. Whether these proteins also contribute to normal blood cell development, however, remains to be clearly defined. Increasing evidence supports a role for the Snail family in regulating cell survival, migration, and differentiation within the haematopoietic system, as well as potentially an oncogenic role in the malignant transformation of haematopoietic stem cells. This review will provide a broad overview of the Snail family, including key aspects of their involvement in the regulation and development of solid organ cancer, as well as a discussion on our current understanding of Snail family function during normal and malignant haematopoiesis.

A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours.

Angiogenesis is critical during tumour initiation and malignant progression. Different strategies aimed at blocking vascular endothelial growth factor (VEGF) and its receptors have been developed to inhibit angiogenesis in cancer patients. It has become increasingly clear that in addition to its effect on angiogenesis, other mechanisms including a direct effect of VEGF on tumour cells may account for the efficiency of VEGF-blockade therapies. Cancer stem cells (CSCs) have been described in various cancers including squamous tumours of the skin. Here we use a mouse model of skin tumours to investigate the impact of the vascular niche and VEGF signalling on controlling the stemness (the ability to self renew and differentiate) of squamous skin tumours during the early stages of tumour progression. We show that CSCs of skin papillomas are localized in a perivascular niche, in the immediate vicinity of endothelial cells. Furthermore, blocking VEGFR2 caused tumour regression not only by decreasing the microvascular density, but also by reducing CSC pool size and impairing CSC renewal properties. Conditional deletion of Vegfa in tumour epithelial cells caused tumours to regress, whereas VEGF overexpression by tumour epithelial cells accelerated tumour growth. In addition to its well-known effect on angiogenesis, VEGF affected skin tumour growth by promoting cancer stemness and symmetric CSC division, leading to CSC expansion. Moreover, deletion of neuropilin-1 (Nrp1), a VEGF co-receptor expressed in cutaneous CSCs, blocked VEGF's ability to promote cancer stemness and renewal. Our results identify a dual role for tumour-cell-derived VEGF in promoting cancer stemness: by stimulating angiogenesis in a paracrine manner, VEGF creates a perivascular niche for CSCs, and by directly affecting CSCs through Nrp1 in an autocrine loop, VEGF stimulates cancer stemness and renewal. Finally, deletion of Nrp1 in normal epidermis prevents skin tumour initiation. These results may have important implications for the prevention and treatment of skin cancers.

Gata3 antagonizes cancer progression in Pten-deficient prostates.

Loss of the tumor suppressor PTEN is a common occurrence in prostate cancer. This aberration leads to the ectopic activation of the PI3K-Akt pathway, which promotes tumor growth. Here, we show that the transcription factor Gata3 is progressively lost in Pten-deficient mouse prostate tumors as a result of both transcriptional down-regulation and increased proteasomal degradation. To determine the significance of this loss, we used conditional loss- and gain-of-function approaches to manipulate Gata3 expression levels in prostate tumors. Our results show that Gata3 inactivation in Pten-deficient prostates accelerates tumor invasion. Conversely, enforced expression of GATA3 in Pten-deficient tissues markedly delays tumor progression. In Pten-deficient prostatic ducts, enforced GATA3 prevented Akt activation, which correlated with the down-regulation of Pik3cg and Pik3c2a mRNAs, encoding respectively class I and II PI3K subunits. Remarkably, the majority of human prostate tumors similarly show loss of active GATA3 as they progress to the aggressive castrate-resistant stage. In addition, GATA3 expression levels in hormone-sensitive tumors holds predictive value for tumor recurrence. Together, these data establish Gata3 as an important regulator of prostate cancer progression.