A slow-cycling LGR5 tumour population mediates basal cell carcinoma relapse after therapy.

Basal cell carcinoma (BCC) is the most frequent cancer in humans and results from constitutive activation of the Hedgehog pathway1. Several Smoothened inhibitors are used to treat Hedgehog-mediated malignancies, including BCC and medulloblastoma2. Vismodegib, a Smoothened inhibitor, leads to BCC shrinkage in the majority of patients with BCC3, but the mechanism by which it mediates BCC regression is unknown. Here we used two genetically engineered mouse models of BCC4 to investigate the mechanisms by which inhibition of Smoothened mediates tumour regression. We found that vismodegib mediates BCC regression by inhibiting a hair follicle-like fate and promoting the differentiation of tumour cells. However, a small population of tumour cells persists and is responsible for tumour relapse following treatment discontinuation, mimicking the situation found in humans5. In both mouse and human BCC, this persisting, slow-cycling tumour population expresses LGR5 and is characterized by active Wnt signalling. Combining Lgr5 lineage ablation or inhibition of Wnt signalling with vismodegib treatment leads to eradication of BCC. Our results show that vismodegib induces tumour regression by promoting tumour differentiation, and demonstrates that the synergy between Wnt and Smoothened inhibitors is a clinically relevant strategy for overcoming tumour relapse in BCC.

YAP and TAZ are essential for basal and squamous cell carcinoma initiation.

YAP and TAZ are key downstream regulators of the Hippo pathway, regulating cell proliferation and differentiation. YAP and TAZ activation has been reported in different cancer types. However, it remains unclear whether they are required for the initiation of major skin malignancies like basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Here, we analyze the expression of YAP and TAZ in these skin cancers and evaluate cancer initiation in knockout mouse models. We show that YAP and TAZ are nuclear and highly expressed in different BCC types in both human and mice. Further, we find that cells with nuclear YAP and TAZ localize to the invasive front in well-differentiated SCC, whereas nuclear YAP is homogeneously expressed in spindle cell carcinoma undergoing EMT We also show that mouse BCC and SCC are enriched for YAP gene signatures. Finally, we find that the conditional deletion of YAP and TAZ in mouse models of BCC and SCC prevents tumor formation. Thus, YAP and TAZ are key determinants of skin cancer initiation, suggesting that targeting the YAP and TAZ signaling pathway might be beneficial for the treatment of skin cancers.

Deciphering the cells of origin of squamous cell carcinomas.

Squamous cell carcinomas (SCCs) are among the most prevalent human cancers. SCC comprises a wide range of tumours originated from diverse anatomical locations that share common genetic mutations and expression of squamous differentiation markers. SCCs arise from squamous and non-squamous epithelial tissues. Here, we discuss the different studies in which the cell of origin of SCCs has been uncovered by expressing oncogenes and/or deleting tumour suppressor genes in the different cell lineages that compose these epithelia. We present evidence showing that the squamous differentiation phenotype of the tumour depends on the type of mutated oncogene and the cell of origin, which dictate the competence of the cells to initiate SCC formation, as well as on the aggressiveness and invasive properties of these tumours.

Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq.

Mouse heart development arises from Mesp1-expressing cardiovascular progenitors (CPs) that are specified during gastrulation. The molecular processes that control early regional and lineage segregation of CPs have been unclear. We performed single-cell RNA sequencing of wild-type and Mesp1-null CPs in mice. We showed that populations of Mesp1 CPs are molecularly distinct and span the continuum between epiblast and later mesodermal cells, including hematopoietic progenitors. Single-cell transcriptome analysis of Mesp1-deficient CPs showed that Mesp1 is required for the exit from the pluripotent state and the induction of the cardiovascular gene expression program. We identified distinct populations of Mesp1 CPs that correspond to progenitors committed to different cell lineages and regions of the heart, identifying the molecular features associated with early lineage restriction and regional segregation of the heart at the early stage of mouse gastrulation.

Defining the clonal dynamics leading to mouse skin tumour initiation.

The changes in cell dynamics after oncogenic mutation that lead to the development of tumours are currently unknown. Here, using skin epidermis as a model, we assessed the effect of oncogenic hedgehog signalling in distinct cell populations and their capacity to induce basal cell carcinoma, the most frequent cancer in humans. We found that only stem cells, and not progenitors, initiated tumour formation upon oncogenic hedgehog signalling. This difference was due to the hierarchical organization of tumour growth in oncogene-targeted stem cells, characterized by an increase in symmetric self-renewing divisions and a higher p53-dependent resistance to apoptosis, leading to rapid clonal expansion and progression into invasive tumours. Our work reveals that the capacity of oncogene-targeted cells to induce tumour formation is dependent not only on their long-term survival and expansion, but also on the specific clonal dynamics of the cancer cell of origin.

Aberrant epigenome in iPSC-derived dopaminergic neurons from Parkinson's disease patients.

The epigenomic landscape of Parkinson's disease (PD) remains unknown. We performed a genomewide DNA methylation and a transcriptome studies in induced pluripotent stem cell (iPSC)-derived dopaminergic neurons (DAn) generated by cell reprogramming of somatic skin cells from patients with monogenic LRRK2-associated PD (L2PD) or sporadic PD (sPD), and healthy subjects. We observed extensive DNA methylation changes in PD DAn, and of RNA expression, which were common in L2PD and sPD. No significant methylation differences were present in parental skin cells, undifferentiated iPSCs nor iPSC-derived neural cultures not-enriched-in-DAn. These findings suggest the presence of molecular defects in PD somatic cells which manifest only upon differentiation into the DAn cells targeted in PD. The methylation profile from PD DAn, but not from controls, resembled that of neural cultures not-enriched-in-DAn indicating a failure to fully acquire the epigenetic identity own to healthy DAn in PD. The PD-associated hypermethylation was prominent in gene regulatory regions such as enhancers and was related to the RNA and/or protein downregulation of a network of transcription factors relevant to PD (FOXA1, NR3C1, HNF4A, and FOSL2). Using a patient-specific iPSC-based DAn model, our study provides the first evidence that epigenetic deregulation is associated with monogenic and sporadic PD.

Sox9 Controls Self-Renewal of Oncogene Targeted Cells and Links Tumor Initiation and Invasion.

Sox9 is a transcription factor expressed in most solid tumors. However, the molecular mechanisms underlying Sox9 function during tumorigenesis remain unclear. Here, using a genetic mouse model of basal cell carcinoma (BCC), the most frequent cancer in humans, we show that Sox9 is expressed from the earliest step of tumor formation in a Wnt/ß-catenin-dependent manner. Deletion of Sox9 together with the constitutive activation of Hedgehog signaling completely prevents BCC formation and leads to a progressive loss of oncogene-expressing cells. Transcriptional profiling of oncogene-expressing cells with Sox9 deletion, combined with in vivo ChIP sequencing, uncovers a cancer-specific gene network regulated by Sox9 that promotes stemness, extracellular matrix deposition, and cytoskeleton remodeling while repressing epidermal differentiation. Our study identifies the molecular mechanisms regulated by Sox9 that link tumor initiation and invasion.

Induced pluripotent stem cell-based studies of Parkinson's disease: challenges and promises.

A critical step in the development of effective therapeutics to treat Parkinson's disease (PD) is the identification of molecular pathogenic mechanisms underlying this chronically progressive neurodegenerative disease. However, while animal models have provided valuable information about the molecular basis of PD, the lack of faithful cellular and animal models that recapitulate human pathophysiology is delaying the development of new therapeutics. The reprogramming of somatic cells to induced pluripotent stem cells (iPSC) using delivery of defined combinations of transcription factors is a groundbreaking discovery that opens great opportunities for modeling human diseases, including PD, since iPSC can be generated from patients and differentiated into disease-relevant cell types, which would capture the patients' genetic complexity. Furthermore, human iPSC-derived neuronal models offer unprecedented access to early stages of the disease, allowing the investigation of the events that initiate the pathologic process in PD. Recently, human iPSC-derived neurons from patients with familial and sporadic PD have been generated and importantly they recapitulate some PD-related cell phenotypes, including abnormal α-synuclein accumulation in vitro, and alterations in the autophagy machinery. This review highlights the current PD iPSC-based models and discusses the potential future research directions of this field.

Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease.

Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson's disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients' genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type.

Rem2 GTPase controls proliferation and apoptosis of neurons during embryo development.

We have recently found that Rem2 GTPase, highly expressed in human embryonic stem cells (hESC), maintains the cell cycle and controls proper differentiation towards ectoderm, suggesting a role in neuronal development. We describe here the use of the zebrafish (Danio rerio) model to determine the physiological significance of Rem2 during embryogenesis. We show that Rem2 RNA is highly expressed in zebrafish embryos up to 2 hours of development followed by a decrease in expression until 48 hours when afterwards Rem2 is switched on again until 5 days. In situ expression analysis reveals that Rem2 is expressed exclusively in the tectum of the brain and eye of the zebrafish. Rem2 morpholino demonstrates impaired embryo development resulting in loss of neural tissue. We show that the mechanism of action of Rem2 is to control apoptosis and proliferation, peaking at 36 hours of development. Rem2 is down-regulated under general differentiation conditions of hESC and is lower expressed in most differentiated cells; however, it is upregulated with neuronal development. This suggests that Rem2 is critical for neuronal development during embryogenesis by regulating proliferation and apoptosis. We propose a model in which Rem2 GTPase is a key regulator maintaining pluripotency during early stages of embryogenesis and survival of neurons during later embryonic development.

A protocol describing the genetic correction of somatic human cells and subsequent generation of iPS cells.

The generation of patient-specific induced pluripotent stem cells (iPSCs) offers unprecedented opportunities for modeling and treating human disease. In combination with gene therapy, the iPSC technology can be used to generate disease-free progenitor cells of potential interest for autologous cell therapy. We explain a protocol for the reproducible generation of genetically corrected iPSCs starting from the skin biopsies of Fanconi anemia patients using retroviral transduction with OCT4, SOX2 and KLF4. Before reprogramming, the fibroblasts and/or keratinocytes of the patients are genetically corrected with lentiviruses expressing FANCA. The same approach may be used for other diseases susceptible to gene therapy correction. Genetically corrected, characterized lines of patient-specific iPSCs can be obtained in 4-5 months.