Stephen J. Goldie

Sox2 modulates the function of two distinct cell lineages in mouse skin.

In postnatal skin the transcription factor Sox2 is expressed in the dermal papilla (DP) of guard/awl/auchene hair follicles and by mechanosensory Merkel cells in the touch domes of guard hairs. To investigate the consequences of Sox2 ablation in skin we deleted Sox2 in DP cells via Blimp1Cre and in Merkel cells via K14Cre. Loss of Sox2 from the DP did not inhibit hair follicle morphogenesis or establishment of the dermis and hypodermis. However, Sox2 expression in the DP was necessary for postnatal maintenance of awl/auchene hair follicles. Deletion of Sox2 via K14Cre resulted in a decreased number of Merkel cells but had no effect on other epithelial compartments or on the dermis. The reduced number of Merkel cells did not affect the number or patterning of guard hairs, nerve density or the interaction of nerve cells with the touch domes. We conclude that Sox2 is a marker of two distinct lineages in the skin and regulates the number of differentiated cells in the case of the Merkel cell lineage and hair follicle type in the case of the DP.

FRMD4A Upregulation in Human Squamous Cell Carcinoma Promotes Tumor Growth and Metastasis and Is Associated with Poor Prognosis

New therapeutic strategies are needed to improve treatment of head and neck squamous cell carcinoma (HNSCC), an aggressive tumor with poor survival rates. FRMD4A is a human epidermal stem cell marker implicated previously in epithelial polarity that is upregulated in SCC cells. Here, we report that FRMD4A upregulation occurs in primary human HNSCCs where high expression levels correlate with increased risks of relapse. FRMD4A silencing decreased growth and metastasis of human SCC xenografts in skin and tongue, reduced SCC proliferation and intercellular adhesion, and stimulated caspase-3 activity and expression of terminal differentiation markers. Notably, FRMD4A attenuation caused nuclear accumulation of YAP, suggesting a potential role for FRMD4A in Hippo signaling. Treatment with the HSP90 inhibitor 17-DMAG or ligation of CD44 with hyaluronan caused nuclear depletion of FRMD4A, nuclear accumulation of YAP and reduced SCC growth and metastasis. Together, our findings suggest FRMD4A as a novel candidate therapeutic target in HNSCC based on the key role in metastatic growth we have identified.

Mimicking normal tissue architecture and perturbation in cancer with engineered micro-epidermis

Correct tissue architecture is essential for normal physiology, yet there have been few attempts to recreate tissues using micro-patterning. We have used polymer brush micro-engineering to generate a stratified micro-epidermis with fewer than 10 human keratinocytes. Epidermal stem cells are captured on 100 μm diameter circular collagen-coated disks. Within 24 h they assemble a stratified micro-tissue, in which differentiated cells have a central suprabasal location. For rings with a non-adhesive centre of up to 40 μm diameter, cell–cell and cell–matrix adhesive interactions together result in correct micro-epidermis assembly. Assembly requires actin polymerization, adherens junctions and desmosomes, but not myosin II-mediated contractility nor coordinated cell movement. Squamous cell carcinoma cells on micro-patterned rings exhibit disturbed architecture that correlates with the characteristics of the original tumours. The micro-epidermis we have generated provides a new platform for screening drugs that modulate tissue assembly, quantifying tissue stratification and investigating the properties of tumour cells.

Rewiring of an Epithelial Differentiation Factor, miR-203, to Inhibit Human Squamous Cell Carcinoma Metastasis

Summary Metastatic colonization of distant organs underpins the majority of human-cancer-related deaths, including deaths from head and neck squamous cell carcinoma (HNSCC). We report that miR-203, a miRNA that triggers differentiation in multilayered epithelia, inhibits multiple postextravasation events during HNSCC lung metastasis. Inducible reactivation of miR-203 in already established lung metastases reduces the overall metastatic burden. Using an integrated approach, we reveal that miR-203 inhibits metastasis independently of its effects on differentiation. In vivo genetic reconstitution experiments show that miR-203 inhibits lung metastasis by suppressing the prometastatic activities of three factors involved in cytoskeletal dynamics (LASP1), extracellular matrix remodeling (SPARC), and cell metabolism (NUAK1). Expression of miR-203 and its downstream effectors correlates with HNSCC overall survival outcomes, indicating the therapeutic potential of targeting this signaling axis.

Upregulation of CD26 expression in epithelial cells and stromal cells during wound-induced skin tumour formation.

We have previously described InvEE transgenic mice in which non-dividing, differentiating epidermal cells express oncogenically activated MAPK kinase 1 (MEK1). Skin wounding triggers tumour formation in InvEE mice via a mechanism that involves epidermal release of IL-1α and attraction of a pro-tumorigenic inflammatory infiltrate. To look for potential effects on the underlying connective tissue, we screened InvEE and wild-type epidermis for differential expression of cytokines and immune modulators. We identified a single protein, CD26 (dipeptidyl peptidase-4). CD26 serum levels were not increased in InvEE mice. In contrast, CD26 was upregulated in keratinocytes expressing mutant MEK1 and in the epithelial compartment of InvEE tumours, where it accumulated at cell–cell borders. CD26 expression was increased in dermal fibroblasts following skin wounding but was downregulated in tumour stroma. CD26 activity was stimulated by calcium-induced intercellular adhesion in keratinocytes, suggesting that the upregulation of CD26 in InvEE epidermis is due to expansion of the differentiated cell layers. IL-1α treatment of dermal fibroblasts stimulated CD26 activity, and therefore epidermal IL-1α release may contribute to the upregulation of CD26 expression in wounded dermis. Pharmacological blockade of CD26, via Sitagliptin, reduced growth of InvEE tumours, while combined inhibition of IL-1α and CD26 delayed tumour onset and reduced tumour incidence. Our results demonstrate that inappropriate activation of MEK1 in the epidermis leads to changes in dermal fibroblasts that, like the skin inflammatory infiltrate, contribute to tumour formation.

Integrative genomic and functional analysis of human oral squamous cell carcinoma cell lines reveals synergistic effects of FAT1 and CASP8 inactivation

Oral squamous cell carcinoma (OSCC) is genetically highly heterogeneous, which contributes to the challenges of treatment. To create an in vitro model that accurately reflects this heterogeneity, we generated a panel of HPV-negative OSCC cell lines. By whole exome sequencing of the lines and matched patient blood samples, we demonstrate that the mutational spectrum of the lines is representative of primary OSCC in The Cancer Genome Atlas. We show that loss of function mutations in FAT1 (an atypical cadherin) and CASP8 (Caspase 8) frequently occur in the same tumour. OSCC cells with inactivating FAT1 mutations exhibited reduced intercellular adhesion. Knockdown of FAT1 and CASP8 individually or in combination in OSCC cells led to increased cell migration and clonal growth, resistance to Staurosporine-induced apoptosis and, in some cases, increased terminal differentiation. The OSCC lines thus represent a valuable resource for elucidating the impact of different mutations on tumour behaviour.

Irrigation of cutaneous squamous cell carcinoma wounds to prevent local recurrence

Surgical excision of non-melanoma skin cancers is the mainstay of treatment, and a common procedure performed by clinicians from various backgrounds and training levels. Despite efforts to excise squamous cell carcinomas (SCCs) with a margin of normal tissue, some tumours are incompletely excised or cells are “seeded” into the wound allowing local recurrence. Following surgical oncology procedures many operators irrigate wounds or body cavities with water rather than saline. The logic being that water will induce an osmotic shift of fluid into the cells, causing them to lyse. There is little evidence to support this practice. Work with human material was carried out in compliance with the UK Human Tissue Act (2004) and approved by the National Research Ethics Service (08/H0306/30). SCC13 is cell line established from a facial cutaneous SCC, from a 56-year old female. Cells were labelled with a lentivirus yellow fluorescent protein (YFP)/luciferase reporter and grown in triplicate (100 cells per dish) to form colony forming assays (CFAs). Cells were also added on top of de-epithelialised dermis (DEDs) (100 cells per DED), as an ex-vivo model, to simulate the operative wound environment. After 30 min, media was removed and plates irrigated with either FAD standard keratinocyte culture medium, sterile Milli-Q water, 0.9% saline or a 10% betadine solution. After 5 min all irrigation fluids were rinsed and replaced with standard FAD culture medium. Following 2 weeks in standard culture conditions CFAs were fixed with 4% PFA and stained with 0.1% Toluidine Blue (Figure 1). DEDs were imaged using a UV lamp to detect the YFP label (Figure 2), then treated with Dluciferin to activate the luciferase reporter. Luciferase activity was imaged and quantified using the Xenogen In-

Worrying number of cases of immolation while wearing sheep costumes

burns that had been sustained from direct contact with ice cubes and salt as part of a ‘‘Salt and Ice Challenge’’. The Salt and Ice Challenge is a new craze popularized by social media networking sites including ‘‘You Tube’’ and ‘‘Facebook’’ [1–3]. The subject applies salt to their skin and then presses an ice cube onto the salt. The salt melts the ice in an endothermic reaction and ultimately further lowers the temperature at the skin surface. The aim of the challenge is to see how long the subject can withstand the pain of inducing a burn. In our case the patient was under the influence of alcohol and peer pressure when he carried out his 3 attempts. At the time he was unaware of the significance of his actions and the potential harm he was causing himself. He explained that some of his friends had carried out the challenge previously and were keen to see how long he would tolerate the burning. Typing ‘‘Salt and Ice Challenge’’ into Internet search engines, returns many alarming reports of burns sustained while attempting this challenge. The behaviour appears to have originated in USA and many cases are reported in the lay press, the most significant reported injury was sustained by a twelve year old boy in Pittsburg, Pennsylvania, who sustained an approximately 2% total body surface area partial thickness burns in the shape of a crucifix on his back [4–6]. There is, understandably, great concern amongst parental organizations in the USA, however, to the best of our knowledge there have not been any cases reported in the scientific literature [7].