Do DLX3 and CD271 Protect Human Keratinocytes from Squamous Tumor Development?

Well-regulated epidermal homeostasis depends on the function of different classes of factors, such as transcription regulators and receptors. Alterations in this homeostatic balance may lead to the development of cutaneous squamous tumorigenesis. The homeobox transcription factor DLX3 is determinant for a p53-dependent regulation of epidermal differentiation and modulates skin carcinogenesis. The maintenance of skin homeostasis also involves the action of neurotrophins (NTs) and their receptors, Trk and CD271. While Trk receptor overexpression is a hallmark of cancer, there are conflicting data on CD271 expression and function in cutaneous SCC (cSCC). Previous studies have reported NT receptors expression in head and neck SSC (HNSCC). We show that CD271 is expressed at low levels in primary cSCC cells and the number of CD271+ cells correlates with cell cohesion in SCC spheroids. In normal epidermis, CD271 is expressed in proliferative progenitor cells and DLX3 in terminally differentiated keratinocytes. Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) increase DLX3 expression. In the absence of a functional BDNF receptor TrkB in keratinocytes, we hypothesize that the BDNF-dependent DLX3 response could be mediated via CD271. Altogether, our results support a putative CD271-DLX3 connection in keratinocytes, which might be crucial to preventing squamous skin cancer

CD271 activation prevents low to high-risk progression of cutaneous squamous cell carcinoma and improves therapy outcomes

Background: Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer, showing a rapid increasing incidence worldwide. Although most cSCC can be cured by surgery, a sizeable number of cases are diagnosed at advanced stages, with local invasion and distant metastatic lesions. In the skin, neurotrophins (NTs) and their receptors (CD271 and Trk) form a complex network regulating epidermal homeostasis. Recently, several works suggested a significant implication of NT receptors in cancer. However, CD271 functions in epithelial tumors are controversial and its precise role in cSCC is still to be defined. Methods: Spheroids from cSCC patients with low-risk (In situ or Well-Differentiated cSCC) or high-risk tumors (Moderately/Poorly Differentiated cSCC), were established to explore histological features, proliferation, invasion abilities, and molecular pathways modulated in response to CD271 overexpression or activation in vitro. The effect of CD271 activities on the response to therapeutics was also investigated. The impact on the metastatic process and inflammation was explored in vivo and in vitro, by using zebrafish xenograft and 2D/3D models. Results: Our data proved that CD271 is upregulated in Well-Differentiated tumors as compared to the more aggressive Moderately/Poorly Differentiated cSCC, both in vivo and in vitro. We demonstrated that CD271 activities reduce proliferation and malignancy marker expression in patient-derived cSCC spheroids at each tumor grade, by increasing neoplastic cell differentiation. CD271 overexpression significantly increases cSCC spheroid mass density, while it reduces their weight and diameter, and promotes a major fold-enrichment in differentiation and keratinization genes. Moreover, both CD271 overexpression and activation decrease cSCC cell invasiveness in vitro. A significant inhibition of the metastatic process by CD271 was observed in a newly established zebrafish cSCC model. We found that the recruitment of leucocytes by CD271-overexpressing cells directly correlates with tumor killing and this finding was further highlighted by monocyte infiltration in a THP-1-SCC13 3D model. Finally, CD271 activity synergizes with Trk receptor inhibition, by reducing spheroid viability, and significantly improves the outcome of photodynamic therapy (PTD) or chemotherapy in spheroids and zebrafish. Conclusion: Our study provides evidence that CD271 could prevent the switch between low to high-risk cSCC tumors. Because CD271 contributes to maintaining active differentiative paths and favors the response to therapies, it might be a promising target for future pharmaceutical development

Ectodermal dysplasias: Classification and organization by phenotype, genotype and molecular pathway

An international advisory group met at the National Institutes of Health in Bethesda, Maryland in 2017, to discuss a new classification system for the ectodermal dysplasias (EDs) that would integrate both clinical and molecular information. We propose the following, a working definition of the EDs building on previous classification systems and incorporating current approaches to diagnosis: EDs are genetic conditions affecting the development and/or homeostasis of two or more ectodermal derivatives, including hair, teeth, nails, and certain glands. Genetic variations in genes known to be associated with EDs that affect only one derivative of the ectoderm (attenuated phenotype) will be grouped as non‐syndromic traits of the causative gene (e.g., non‐syndromic hypodontia or missing teeth associated with pathogenic variants of EDA “ectodysplasin”). Information for categorization and cataloging includes the phenotypic features, Online Mendelian Inheritance in Man number, mode of inheritance, genetic alteration, major developmental pathways involved (e.g., EDA, WNT “wingless‐type,” TP63 “tumor protein p63”) or the components of complex molecular structures (e.g., connexins, keratins, cadherins)

BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network

Several homeodomain (HD) proteins are critical for skeletal patterning and respond directly to BMP2 as an early step in bone formation. RUNX2, the earliest transcription factor proven essential for commitment to osteoblastogenesis, is also expressed in response to BMP2. However, there is a gap in our knowledge of the regulatory cascade from BMP2 signaling to the onset of osteogenesis. Here we show that BMP2 induces DLX3, a homeodomain protein that activates Runx2 gene transcription. Small interfering RNA knockdown studies in osteoblasts validate that DLX3 is a potent regulator of Runx2. Furthermore in Runx2 null cells, DLX3 forced expression suffices to induce transcription of Runx2, osteocalcin, and alkaline phosphatase genes, thus defining DLX3 as an osteogenic regulator independent of RUNX2. Our studies further show regulation of the Runx2 gene by several homeodomain proteins: MSX2 and CDP/cut repress whereas DLX3 and DLX5 activate endogenous Runx2 expression and promoter activity in non-osseous cells and osteoblasts. These HD proteins exhibit distinct temporal expression profiles during osteoblast differentiation as well as selective association with Runx2 chromatin that is related to Runx2 transcriptional activity and recruitment of RNA polymerase II. Runx2 promoter mutagenesis shows that multiple HD elements control expression of Runx2 in relation to the stages of osteoblast maturation. Our studies establish mechanisms for commitment to the osteogenic lineage directly through BMP2 induction of HD proteins DLX3 and DLX5 that activate Runx2, thus delineating a transcriptional regulatory pathway mediating osteoblast differentiation. We propose that the three homeodomain proteins MSX2, DLX3, and DLX5 provide a key series of molecular switches that regulate expression of Runx2 throughout bone formation. <br/

3D Chromatin Architecture, BRD4, and Mediator Have Distinct Roles in Regulating Genome-Wide Transcriptional Bursting and Gene Network

Discontinuous transcription is evolutionarily conserved and a fundamental feature of gene regulation; yet, the exact mechanisms underlying transcriptional bursting are unresolved. Analyses of bursting transcriptome-wide have focused on the role of cis-regulatory elements, but other factors that regulate this process remain elusive. We applied mathematical modeling to single-cell RNA sequencing data to infer bursting dynamics transcriptome-wide under multiple conditions to identify possible molecular mechanisms. We found that Mediator complex subunit 26 (MED26) primarily regulates frequency, MYC regulates burst size, while cohesin and Bromodomain-containing protein 4 (BRD4) can modulate both. Despite comparable effects on RNA levels among these perturbations, acute depletion of MED26 had the most profound impact on the entire gene regulatory network, acting downstream of chromatin spatial architecture and without affecting TATA box-binding protein (TBP) recruitment. These results indicate that later steps in the initiation of transcriptional bursts are primary nodes for integrating gene networks in single cells

Abnormal Placental Development and Early Embryonic Lethality in EpCAM-Null Mice

BACKGROUND: EpCAM (CD326) is encoded by the tacstd1 gene and expressed by a variety of normal and malignant epithelial cells and some leukocytes. Results of previous in vitro experiments suggested that EpCAM is an intercellular adhesion molecule. EpCAM has been extensively studied as a potential tumor marker and immunotherapy target, and more recent studies suggest that EpCAM expression may be characteristic of cancer stem cells. METHODOLOGY/PRINCIPAL FINDINGS: To gain insights into EpCAM function in vivo, we generated EpCAM -/- mice utilizing an embryonic stem cell line with a tacstd1 allele that had been disrupted. Gene trapping resulted in a protein comprised of the N-terminus of EpCAM encoded by 2 exons of the tacstd1 gene fused in frame to betageo. EpCAM +/- mice were viable and fertile and exhibited no obvious abnormalities. Examination of EpCAM +/- embryos revealed that betageo was expressed in several epithelial structures including developing ears (otocysts), eyes, branchial arches, gut, apical ectodermal ridges, lungs, pancreas, hair follicles and others. All EpCAM -/- mice died in utero by E12.5, and were small, developmentally delayed, and displayed prominent placental abnormalities. In developing placentas, EpCAM was expressed throughout the labyrinthine layer and by spongiotrophoblasts as well. Placentas of EpCAM -/- embryos were compact, with thin labyrinthine layers lacking prominent vascularity. Parietal trophoblast giant cells were also dramatically reduced in EpCAM -/- placentas. CONCLUSION: EpCAM was required for differentiation or survival of parietal trophoblast giant cells, normal development of the placental labyrinth and establishment of a competent maternal-fetal circulation. The findings in EpCAM-reporter mice suggest involvement of this molecule in development of vital organs including the gut, kidneys, pancreas, lungs, eyes, and limbs

Epidermal stem cells: the cradle of epidermal determination, differentiation and wound healing

The field of epidermal stem cells has dramatically advanced in the last decade, leading to a better understanding of the molecular factors, signalling pathways and cellular events that identify and characterize stem cells, thus revealing their immense potential for therapeutic use. Furthermore, multipotent epidermal stem cells present the major advantage of easy accessibility with the discovery of their specific location within the bulge of the hair follicle. This review focuses on the most recent findings on epidermal stem cells, and their potential role in initial epidermal commitment, differentiation and wound healing processes in the skin