Pigment epithelium derived factor drives melanocyte proliferation and migration in neurofibromatosis café au lait macules.

Background: RASopathies, which include neurofibromatosis type 1 (NF1), are defined by Ras/mitogen-activated protein kinase (Ras/MAPK) pathway activation. They represent a group of clinically related disorders often characterised by multiple Café au Lait Macules (CALMs). Objectives: To determine, using in depth transcriptomic analysis of NF1 melanocytes from CALM and unaffected skin, (1) the gene(s) responsible for melanocyte proliferation and migration, and (2) the activated signalling pathway(s) in NF1 melanoma. Methods: Classical NF1 (n = 2, who develop tumours) and 3bp deletion NF1 (p. Met992del, who do not develop tumours) (n = 3) patients underwent skin biopsies from CALM and unaffected skin. Melanocytes were isolated and propagated, with five replicates from each tissue sample. DNA and RNA were extracted for mutational analysis and transcriptomic profiling with six replicates per sample. Mechanistic determination was undertaken using melanocyte and melanoma cell lines. Results: All CALMs in NF1 were associated with biallelic NF1 loss, resulting in amplification of Ras/MAPK and Wnt pathway signalling. CALMs were also associated with reduced SERPINF1 gene expression (and pigment epithelium-derived factor (PEDF) levels, the reciprocal protein), a known downstream target of the master regulator of melanocyte differentiation microphthalmia-associated transcription factor (MITF), leading to increased melanocyte proliferation, migration and invasion. In classical NF1 and melanoma, but not 3bp deletion NF1, there was also activation of the PI3K/AKT pathway. Pigment epithelium-derived factor was found to reduce cell proliferation and invasion of NF1 melanoma. Conclusions: Melanocyte proliferation and migration leading to CALMs in NF1 arises from biallelic NF1 loss, resulting in RAS/MAPK pathway activation, and reduced expression of the tumour suppressor PEDF. Activation of the PI3K/AKT pathway in classical NF1 and NF1 melanoma may facilitate tumour growth.

HPV8-induced STAT3 activation led keratinocyte stem cell expansion in human actinic keratoses.

Despite epidermal turnover, the skin is host to a complex array of microbes, including viruses, such as HPV, which must infect and manipulate skin keratinocyte stem cells (KSCs) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induced ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses. Together, these results define the "hit-and-run" mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lack melanosome protection and are thus susceptible to sun light-induced malignant transformation.

Targeting CD20-expressing malignant melanoma cells augments BRAF inhibitor killing.

BACKGROUND: Mutant BRAF targeted therapies remain a standard of care for the treatment of metastatic malignant melanoma (MM); however, high initial response rates are tempered by the persistence of residual MM cells that eventually lead to disease recurrence and mortality. As MM recurrence during targeted therapy can present with the simultaneous occurrence of multiple tumour nodules at the original body sites, we hypothesized the presence of an intrinsically resistant MM cell subpopulation. OBJECTIVES: To identify an MM cell subpopulation that is intrinsically resistant to targeted therapy and possibly responsible for MM recurrence. METHODS: Using melanoma cell lines, we defined culture conditions for the reproducible three-dimensional growth of melanospheres to investigate putative cancer stem cell populations. We undertook RNA sequencing and bioinformatic analysis to characterize cell populations between adherent and nonadherent culture, and cells expressing or not expressing CD20. Furthermore, we defined an in vitro assay to evaluate the killing of melanoma cancer stem cells as a therapeutic test using combination therapies targeting driver mutation and CD20. RESULTS: We described the culture conditions that promote MM cells to form melanospheres with a reproducible colony-forming efficiency rate of 0.3-1.3%. RNA sequencing of melanosphere vs. conventional MM cell cultures (n = 6), irrespective of the BRAF mutation status, showed that melanosphere formation was associated with growth and differentiation transcriptional signatures resembling MM tumours. Importantly, melanosphere formation also led to the emergence of a CD20+ MM cell subpopulation, similar to that observed in primary human MM tumours. CD20+ MM cells were resistant to BRAF inhibitor therapy and, consistent with this finding, demonstrated a Forkhead box protein M1 transcriptomic profile (n = 6). Combining BRAF inhibitor and anti-CD20 antibody treatment led to the additional killing of previously resistant CD20+  BRAF mutant MM cells. CONCLUSIONS: In patients with MM that harbour a CD20+ subpopulation, combined therapy with BRAF inhibitor and anti-CD20 antibody could potentially kill residual MM cells and prevent disease recurrence.

CD200 ectodomain shedding into the tumor microenvironment leads to NK cell dysfunction and apoptosis.

The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor-initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells to suppress MAPK pathway signaling that in turn blocked indirect (IFN-γ release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ-regulated gene transcription and led to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL, which resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell-specific immune checkpoint, which is responsible for NK cell-associated poor outcomes in many cancers.

Genome-wide association study in frontal fibrosing alopecia identifies four susceptibility loci including HLA-B*07:02.

Frontal fibrosing alopecia (FFA) is a recently described inflammatory and scarring type of hair loss affecting almost exclusively women. Despite a dramatic recent increase in incidence the aetiopathogenesis of FFA remains unknown. We undertake genome-wide association studies in females from a UK cohort, comprising 844 cases and 3,760 controls, a Spanish cohort of 172 cases and 385 controls, and perform statistical meta-analysis. We observe genome-wide significant association with FFA at four genomic loci: 2p22.2, 6p21.1, 8q24.22 and 15q2.1. Within the 6p21.1 locus, fine-mapping indicates that the association is driven by the HLA-B*07:02 allele. At 2p22.1, we implicate a putative causal missense variant in CYP1B1, encoding the homonymous xenobiotic- and hormone-processing enzyme. Transcriptomic analysis of affected scalp tissue highlights overrepresentation of transcripts encoding components of innate and adaptive immune response pathways. These findings provide insight into disease pathogenesis and characterise FFA as a genetically predisposed immuno-inflammatory disorder driven by HLA-B*07:02.

Identification of Human Cutaneous Squamous Cell Carcinoma Cancer Stem Cells.

Epithelia are under constant threat from environmental carcinogens and none more so than squamous epithelia, which form the outermost linings of our bodies. Hence malignancies of squamous epithelia are collectively the most common cancer type and with the highest mortality, despite a constant cell turnover and only relatively rare long-lived adult tissue stem cells. Genetic analysis from SCC whole genome sequencing reveals commonality in mutated genes, despite various etiological factors. Most SCC types have been shown to exhibit hierarchical growth, in which a high frequency of cancer stem cells is associated with poor prognosis. For human cutaneous SCC (cSCC), we have shown that cancer stem cells express CD133 and that this population can recreate tumor heterogeneity in a novel in vivo model. CD133+ cSCC cells is small subset of tumor cells (~1%) in the outer layer of cSCC that are highly enriched for tumor-initiating capacity (TIC) (~1/400) compared to unsorted cSCC cells (~1/106). Xenografts of CD133+ cSCC recreated the original cSCC tumor histology and organizational hierarchy, while CD133- cells did not. Only CD133+ cells demonstrated the capacity for self-renewal in serial transplantation studies. Hence, cSCC has the potential to be the ideal model in which to study SCC biology.

Identification of Human Cutaneous Basal Cell Carcinoma Cancer Stem Cells.

The cancer stem cell model states that a subset of tumor cells, called "cancer stem cells," can initiate and propagate tumor growth through self-renewal, high proliferative capacity, and their ability to recreate tumor heterogeneity. In basal cell carcinoma (BCC), we have shown that tumor cells that express the cell surface protein CD200 fulfill the cancer stem cell hypothesis. CD200+ CD45- BCC cells represent 0.05-3.96% of all BCC cells and reside in small clusters at the tumor periphery. Using a novel, reproducible in vivo xenograft growth assay, we determined that tumor-initiating cell (TIC) frequencies are approximately 1 per 1.5 million unsorted BCC cells. The CD200+ CD45- BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200+ CD45- cells, representing ~1500-fold enrichment. The methods used to identify and purify CD200+ CD45- BCC cells, as well as characterize gene expression, are described herein.

HPV-Induced Field Cancerisation: Transformation of Adult Tissue Stem Cell Into Cancer Stem Cell.

Field cancerisation was originally described as a basis for multiple head and neck squamous cell carcinoma (HNSCC) and is a pre-malignant phenomenon that is frequently attributable to oncogenic human papillomavirus (HPV) infection. Our work on ß-HPV-induced cutaneous squamous cell carcinomas identified a novel Lrig1+ hair follicle junctional zone keratinocyte stem cell population as the basis for field cancerisation. Herein, we describe the ability for HPV to infect adult tissue stem cells in order to establish persistent infection and induce their proliferation and displacement resulting in field cancerisation. By review of the HPV literature, we reveal how this mechanism is conserved as the basis of field cancerisation across many tissues. New insights have identified the capacity for HPV early region genes to dysregulate adult tissue stem cell self-renewal pathways ensuring that the expanded population preserve its stem cell characteristics beyond the stem cell niche. HPV-infected cells acquire additional transforming mutations that can give rise to intraepithelial neoplasia (IEN), from environmental factors such as sunlight or tobacco induced mutations in skin and oral cavity, respectively. With establishment of IEN, HPV viral replication is sacrificed with loss of the episome, and the tissue is predisposed to multiple cancer stem cell-driven carcinomas.

ß-HPV Infection Correlates with Early Stages of Carcinogenesis in Skin Tumors and Patient-Derived Xenografts from a Kidney Transplant Recipient Cohort.

Many malignancies that occur in high excess in kidney transplant recipients (KTRs) are due to viruses that thrive in the setting of immunosuppression. Keratinocyte carcinoma (KC), the most frequently occurring cancer type in KTR, has been associated with skin infection by human papillomavirus (HPV) from the beta genus. In this report, we extend our previous investigation aimed at identifying the presence of active ß-HPV infection in skin tumors from KTRs through detection of viral protein expression. Using a combination of antibodies raised against the E4 and L1 proteins of the ß-genotypes, we were able to visualize infection in five tumors [one keratoacanthoma (KA), three actinic keratoses (AKs), and one seborrheic keratoses (SKs)] that were all removed from two patients who had been both transplanted twice, had developed multiple KCs, and presented with a long history of immunosuppression (>30 years). These infected tissues displayed intraepidermal hyperplasia and increased expression of the ΔNp63 protein, which extended into the upper epithelial layers. In addition, using a xenograft model system in nude mice displaying a humanized stromal bed in the site of grafting, we successfully engrafted three AKs, two of which were derived from the aforementioned KTRs and displayed ß-HPV infection in the original tumor. Of note, one AK-derived xenograft, along with its ensuing lymph node metastasis, was diagnosed as squamous cell carcinoma (SCC). In the latter, both ß-HPV infection and ΔNp63 expression were no longer detectable. Although the overall success rate of engrafting was very low, the results of this study show for the first time that ß-HPV+ and ΔNp63+ intraepidermal hyperplasia can indeed progress to an aggressive SCC able to metastasize. Consistent with a series of reports attributing a causative role of ß-HPV at early stages of skin carcinogenesis through ΔNp63 induction and increased keratinocytes stemness, here we provide in vivo evidence that these events are also occurring in the affected skin of KTRs. Due to these ß-HPV-driven molecular pathways, the nascent tumor cell is able to acquire a high enough number of carcinogenic insults that its proliferation and survival will eventually become independent of viral gene expression.

Delayed and localized pemphigus vulgaris after breast cancer radiotherapy.

Breast cancer treatment involving ionizing radiation causes characteristic radiation dermatitis in the majority of patients. The DNA damaging effects of radiation can rarely predispose to primary inflammatory dermatoses, such as pemphigus vulgaris. In such cases, the disease presents with all the hallmarks of the primary dermatosis, but the eruption is limited to the field of irradiation and is often amenable to treatment. In contrast, occurrence of generalized pemphigus vulgaris in this setting may mean cancer recurrence. The mechanism by which radiotherapy induces localized disease remains unknown, but there is likely a loss of self-tolerance which maybe coupled to antigen exposure.

HPV8 Field Cancerization in a Transgenic Mouse Model Is due to Lrig1+ Keratinocyte Stem Cell Expansion.

ß-Human papillomaviruses (HPVs) cause near ubiquitous latent skin infection within long-lived hair follicle (HF) keratinocyte stem cells. In patients with epidermodysplasia verruciformis, ß-HPV viral replication is associated with skin keratosis and cutaneous squamous cell carcinoma. To determine the role of HF keratinocyte stem cells in ß-HPV-induced skin carcinogenesis, we utilized a transgenic mouse model in which the keratin 14 promoter drives expression of the entire HPV8 early region (HPV8tg). HPV8tg mice developed thicker skin in comparison with wild-type littermates consistent with a hyperproliferative epidermis. HF keratinocyte proliferation was evident within the Lrig1+ keratinocyte stem cell population (69 vs. 55%, P < 0.01, n = 7), and not in the CD34+, LGR5+, and LGR6+ keratinocyte stem cell populations. This was associated with a 2.8-fold expansion in Lrig1+ keratinocytes and 3.8-fold increased colony-forming efficiency. Consistent with this, we observed nuclear p63 expression throughout this population and the HF infundibulum and adjoining interfollicular epidermis, associated with a switch from p63 transcriptional activation isoforms to ΔNp63 isoforms in HPV8tg skin. Epidermodysplasia verruciformis keratosis and in some cases actinic keratoses demonstrated similar histology associated with ß-HPV reactivation and nuclear p63 expression within the HF infundibulum and perifollicular epidermis. These findings would suggest that ß-HPV field cancerization arises from the HF junctional zone and predispose to squamous cell carcinoma.

Hair restoration approaches for early onset male androgenetic alopecia.

Society places great emphasis on the presence of hair. Some degree of hair loss is accepted as a normal part of the aging process, in line with the observation that more than 50% of men will develop androgenetic alopecia by the age of 50 years. However, it is possible to understand the psychosocial isolation and distress felt by men with a strong familial predisposition to androgenetic alopecia, who tend to display hair loss in their late teens or twenties. There are currently two drugs which have been licensed for the treatment of male androgenetic alopecia: oral finasteride and topical minoxidil solution which are effective to some extent. Furthermore, upon discontinuing treatment, any gain that has been achieved is quickly lost. Added to which there is an entire market of unproven over the counter products: advertised in the electronic media, local hair salons, and various departmental stores. In this review, we highlight the important advances in the management of male androgenetic alopecia with emphasis on approaches that can lead to more successful and long-term hair restoration for young adults. In particular, we discuss the evidence supporting the use of the follicular unit grafting technique in conjunction with medical treatment before and after the procedure. Moreover, some other alterations of this most popular state of the art hair restoration technique have been mentioned briefly. As a result, patients and physicians seem equally satisfied from this procedure for its naturally looking results which are cosmetically more acceptable and esthetically pleasing for longer period of time.

CD200-expressing human basal cell carcinoma cells initiate tumor growth.

Smoothened antagonists directly target the genetic basis of human basal cell carcinoma (BCC), the most common of all cancers. These drugs inhibit BCC growth, but they are not curative. Although BCC cells are monomorphic, immunofluorescence microscopy reveals a complex hierarchical pattern of growth with inward differentiation along hair follicle lineages. Most BCC cells express the transcription factor KLF4 and are committed to terminal differentiation. A small CD200(+) CD45(-) BCC subpopulation that represents 1.63 ± 1.11% of all BCC cells resides in small clusters at the tumor periphery. By using reproducible in vivo xenograft growth assays, we determined that tumor initiating cell frequencies approximate one per 1.5 million unsorted BCC cells. The CD200(+) CD45(-) BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200(+) CD45(-) cells, representing ~1,500-fold enrichment. CD200(-) CD45(-) BCC cells were unable to form tumors. These findings establish a platform to study the effects of Smoothened antagonists on BCC tumor initiating cell and also suggest that currently available anti-CD200 therapy be considered, either as monotherapy or an adjunct to Smoothened antagonists, in the treatment of inoperable BCC.

Human skin cancer stem cells: a tale of mice and men.

Carcinomas, cancers of epithelial tissues, are the commonest malignancies and cause the greatest cancer mortality worldwide. Among these, the incidence of keratinocyte-derived non-melanoma skin cancers (NMSC), by far the greatest, is increasing rapidly. Yet despite access to tumor tissue, acceptance of human NMSC as a model carcinoma has been hindered by the lack of a reliable xenograft model. Instead, we have relied on the murine two-step carcinogenesis protocol as a reproducible squamous cell carcinoma (SCC) model, but this differs from their human counterpart in cause, site, genetic basis and biological behaviour. By xeno-engraftment of primary human SCC, we were recently successful in demonstrating the presence of primary human SCC cancer stem cells or tumor-initiating cells. These findings once more align the study human SCC as the archetypal carcinoma model. In this review, we describe the evidence for the existence of tumor-initiating cells, with emphasis on skin cancer, limiting our discussions to primary human cancer studies where possible.

Identification and characterization of tumor-initiating cells in human primary cutaneous squamous cell carcinoma.

Primary human squamous cell carcinomas (SCCas) are heterogeneous invasive tumors with proliferating outer layers and inner differentiating cell masses. To determine if tumor-initiating cells (TICs) are present in SCCas, we utilized newly developed reliable in vitro and in vivo xenograft assays that propagate human SCCas, and demonstrated that a small subset of SCCa cells (∼1%) expressing Prominin-1 (CD133) in the outer layers of SCCas were highly enriched for TICs (∼1/400) compared with unsorted SCCa cells (TICs ∼1/10(6)). Xenografts of CD133+ SCCas recreated the original SCCa tumor histology and organizational hierarchy, whereas CD133- cells did not, and only CD133+ cells demonstrated the capacity for self-renewal in serial transplantation studies. We present a model of human SCCas in which tumor projections expand with outer leading edges that contain CD133+ TICs. Successful cancer treatment will likely require that the TICs identified in cancers be targeted therapeutically. The demonstration that TICs are present in SCCas and are enriched in a CD133- expressing subpopulation has not been, to our knowledge, previously reported.

A humanized stromal bed is required for engraftment of isolated human primary squamous cell carcinoma cells in immunocompromised mice.

Epithelial cancers are the most common malignancies and the greatest cause of cancer mortality worldwide. The incidence of keratinocyte-derived (non-melanoma) skin cancers is increasing rapidly. Despite access to abundant tumor tissue and ease of observation, acceptance of non-melanoma skin cancers as model carcinomas has been hindered by the lack of a reliable xenograft model. Herein we describe conditions that allow routine xeno-engraftment of primary human squamous cell carcinoma (SCCa) cells. Tumor development required creation of an appropriate stromal bed before xenografting tumor tissue onto the backs of athymic nude mice. We also demonstrate that the stromal bed must be "humanized" if primary human SCCa is to be propagated from cell suspensions. SCCa xenografts recapitulated the histological grade and phenotype of the original tumors with considerable fidelity, even after serial passage, irrespective of the histological grade of the primary human SCCa. This model, which to our knowledge is previously unreported, can be used for drug testing, as well as for studies that are relevant to the biology of primary human SCCa and other epithelial cancers.

Early use of negative pressure therapy in combination with silver dressings in a difficult breast abscess.

Combining silver-based dressings with negative pressure therapy after radical excision of chronically infected breast disease is a novel application of two technologies. One patient with complex, chronic, infected breast disease underwent radical excision of the affected area and was treated early with a combination of silver-based dressings and topical negative pressure therapy. The wound was then assessed sequentially using clinical measurements of wound area and depth, pain severity scores and level of exudation. It is possible to combine accepted techniques with modern dressing technologies that result in a positive outcome. In this case, the combination of a silver-based dressing with negative pressure therapy following radical excision proved safe and was well tolerated by the patient. Full epithelisation of the wound was achieved and there was no recurrence of the infection for the duration of the treatment.