Regional-specific calibration enables application of computational evidence for clinical classification of 5' cis-regulatory variants in Mendelian disease.

To date, clinical genetic testing for Mendelian disease variants has focused heavily on exonic coding and intronic gene regions. This multi-step study was undertaken to provide an evidence base for selecting and applying computational approaches for use in clinical classification of 5' cis-regulatory region variants. Curated datasets of clinically reported disease-causing 5' cis-regulatory region variants and variants from matched genomic regions in population controls were used to calibrate six bioinformatic tools as predictors of variant pathogenicity. Likelihood ratio estimates were aligned to code weights following ClinGen recommendations for application of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) classification scheme. Considering code assignment across all reference dataset variants, performance was best for CADD (81.2%) and REMM (81.5%). Optimized thresholds provided moderate evidence toward pathogenicity (CADD, REMM) and moderate (CADD) or supporting (REMM) evidence against pathogenicity. Both sensitivity and specificity of prediction were improved when further categorizing variants based on location in an EPDnew-defined promoter region. Combining predictions (CADD, REMM, and location in a promoter region) increased specificity at the expense of sensitivity. Importantly, the optimal CADD thresholds for assigning ACMG/AMP codes PP3 (≥10) and BP4 (≤8) were vastly different from recommendations for protein-coding variants (PP3 ≥25.3; BP4 ≤22.7); CADD <22.7 would incorrectly assign BP4 for >90% of reported disease-causing cis-regulatory region variants. Our results demonstrate the need to consider a tiered approach and tailored score thresholds to optimize bioinformatic impact prediction for clinical classification of 5' cis-regulatory region variants.

Subtype-Specific Analyses Reveal Infiltrative Basal Cell Carcinomas Are Highly Interactive with their Environment.

Little is known regarding the molecular differences between basal cell carcinoma (BCC) subtypes, despite clearly distinct phenotypes and clinical outcomes. In particular, infiltrative BCCs have poorer clinical outcomes in terms of response to therapy and propensity for dissemination. In this project, we aimed to use exome sequencing and RNA sequencing to identify somatic mutations and molecular pathways leading to infiltrative BCCs. Using whole-exome sequencing of 36 BCC samples (eight infiltrative) combined with previously reported exome data (58 samples), we determine that infiltrative BCCs do not contain a distinct somatic variant profile and carry classical UV-induced mutational signatures. RNA sequencing on both datasets revealed key differentially expressed genes, such as POSTN and WISP1, suggesting increased integrin and Wnt signaling. Immunostaining for periostin and WISP1 clearly distinguished infiltrative BCCs, and nuclear ß-catenin staining patterns further validated the resulting increase in Wnt signaling in infiltrative BCCs. Of significant interest, in BCCs with mixed morphology, infiltrative areas expressed WISP1, whereas nodular areas did not, supporting a continuum between subtypes. In conclusion, infiltrative BCCs do not differ in their genomic alteration in terms of initiating mutations. They display a specific type of interaction with the extracellular matrix environment regulating Wnt signaling.

Regional Variation in Epidermal Susceptibility to UV-Induced Carcinogenesis Reflects Proliferative Activity of Epidermal Progenitors.

To better understand the influence of ultraviolet (UV) irradiation on the initial steps of skin carcinogenesis, we examine patches of labeled keratinocytes as a proxy for clones in the interfollicular epidermis (IFE) and measure their size variation upon UVB irradiation. Multicolor lineage tracing reveals that in chronically irradiated skin, patches near hair follicles (HFs) increase in size, whereas those far from follicles do not change. This is explained by proliferation of basal epidermal cells within 60 µm of HF openings. Upon interruption of UVB, patch size near HFs regresses significantly. These anatomical differences in proliferative behavior have significant consequences for the cell of origin of basal cell carcinomas (BCCs). Indeed, a UV-inducible murine BCC model shows that BCC patches are more frequent, larger, and more invasive near HFs. These findings have major implications for the prevention of field cancerization in the epidermis.

Ectopic expression of SOX18 in Basal cell carcinoma negatively regulates tumour progression.

BACKGROUND: Basal Cell Carcinoma is the most common tumour and yet much remains to be determined regarding the molecular mechanisms that leads to its development. Hedgehog signal activation is sufficient for BCC induction, but the molecular mediators of BCC growth are not well understood. SoxF transcription factor Sox18 has been identified in human BCC, but its role in growth of the tumour is as yet unknown. OBJECTIVE: To determine if Sox18 is involved in the regulation of Basal Cell Carcinoma growth. METHODS: We analysed the function of Sox18 by combining a dominant negative Sox18 mouse model, Sox18+/OP with murine BCC RESULTS: We determine that Sox18 is ectopically expressed in the epidermal cells of a murine model of Basal Cell Carcinoma. We then show that dominant negative mutation of Sox18 increases the severity of murine Basal Cell Carcinoma. Finally, decreased Hey1 in Sox18+/OP BCC suggests Sox18 may negatively regulate BCC progression via Notch signaling. CONCLUSIONS: These data suggest that Sox18 is a hedgehog regulated mediator of tumour suppression within Basal Cell Carcinoma epidermis.

Murine dorsal hair type is genetically determined by polymorphisms in candidate genes that influence BMP and WNT signalling.

Mouse dorsal coat hair types, guard, awl, auchene and zigzag, develop in three consecutive waves. To date, it is unclear if these hair types are determined genetically through expression of specific factors or can change based on their mesenchymal environment. We undertook a novel approach to this question by studying individual hair type in 67 Collaborative Cross (CC) mouse lines and found significant variation in the proportion of each type between strains. Variation in the proportion of zigzag, awl and auchene, but not guard hair, was largely due to germline genetic variation. We utilised this variation to map a quantitative trait locus (QTL) on chromosome 12 that appears to influence a decision point switch controlling the propensity for either second (awl and auchene) or third wave (zigzag) hairs to develop. This locus contains two strong candidates, Sostdc1 and Twist1, each of which carry several ENCODE regulatory variants, specific to the causal allele, that can influence gene expression, are expressed in the developing hair follicle, and have been previously reported to be involved in regulating human and murine hair behaviour, but not hair subtype determination. Both of these genes are likely to play a part in hair type determination via regulation of BMP and/or WNT signalling.

The role of cellular reactive oxygen species in cancer chemotherapy.

Most chemotherapeutics elevate intracellular levels of reactive oxygen species (ROS), and many can alter redox-homeostasis of cancer cells. It is widely accepted that the anticancer effect of these chemotherapeutics is due to the induction of oxidative stress and ROS-mediated cell injury in cancer. However, various new therapeutic approaches targeting intracellular ROS levels have yielded mixed results. Since it is impossible to quantitatively detect dynamic ROS levels in tumors during and after chemotherapy in clinical settings, it is of increasing interest to apply mathematical modeling techniques to predict ROS levels for understanding complex tumor biology during chemotherapy. This review outlines the current understanding of the role of ROS in cancer cells during carcinogenesis and during chemotherapy, provides a critical analysis of the methods used for quantitative ROS detection and discusses the application of mathematical modeling in predicting treatment responses. Finally, we provide insights on and perspectives for future development of effective therapeutic ROS-inducing anticancer agents or antioxidants for cancer treatment.

Temporal Regulation of Natural Killer T Cell Interferon Gamma Responses by ß-Catenin-Dependent and -Independent Wnt Signaling.

Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator ß-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. ß-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and ß-catenin activity shape liver NKT cell functions in vivo in response to the glycolipid antigen, α-galactosylceramide (α-GalCer) using a mouse model. Pharmacologic targeting of ß-catenin activity with ICG001, as well as myeloid-specific genetic ablation of Wntless (Wls), to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific Wls deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of ß-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/ß-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of ß-catenin activity. Our analyses in ICG001-treated mice confirmed a role for ß-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and ß-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of ß-catenin to NKT cell functions.

Keratinocyte Sonic Hedgehog Upregulation Drives the Development of Giant Congenital Nevi via Paracrine Endothelin-1 Secretion.

Giant congenital nevi are associated with clinical complications such as neurocutaneous melanosis and melanoma. Virtually nothing is known about why some individuals develop these lesions. We previously identified the sonic hedgehog (Shh) pathway regulator Cdon as a candidate nevus modifier gene. Here we validate this by studying Cdon knockout mice, and go on to establishing the mechanism by which Shh exacerbates nevogenesis. Cdon knockout mice develop blue nevi without the need for somatic melanocyte oncogenic mutation. In a mouse model carrying melanocyte NRASQ61K, we found that strain backgrounds that carry genetic variants that cause increased keratinocyte Shh pathway activity, as measured by Gli1 and Gli2 expression, develop giant congenital nevi. Shh components are also active adjacent to human congenital nevi. Mechanistically, this exacerbation of nevogenesis is driven via the release of the melanocyte mitogen endothelin-1 from keratinocytes. We then suppressed nevus development in mice using Shh and endothelin antagonists. Our work suggests an aspect of nevus development whereby keratinocyte cytokines such as endothelin-1 can exacerbate nevogenesis, and provides potential therapeutic approaches for giant congenital nevi. Furthermore, it highlights the notion that germline genetic variation, in addition to somatic melanocyte mutation, can strongly influence the histopathological features of melanocytic nevi.

Dominant-negative Sox18 function inhibits dermal papilla maturation and differentiation in all murine hair types.

SOX family proteins SOX2 and SOX18 have been reported as being essential in determining hair follicle type; however, the role they play during development remains unclear. Here, we demonstrate that Sox18 regulates the normal differentiation of the dermal papilla of all hair types. In guard (primary) hair dermal condensate (DC) cells, we identified transient Sox18 in addition to SOX2 expression at E14.5, which allowed fate tracing of primary DC cells until birth. Similarly, expression of Sox18 was detected in the DC cells of secondary hairs at E16.5 and in tertiary hair at E18.5. Dominant-negative Sox18 mutation (opposum) did not prevent DC formation in any hair type. However, it affected dermal papilla differentiation, restricting hair formation especially in secondary and tertiary hairs. This Sox18 mutation also prevented neonatal dermal cells or dermal papilla spheres from inducing hair in regeneration assays. Microarray expression studies identified WNT5A and TNC as potential downstream effectors of SOX18 that are important for epidermal WNT signalling. In conclusion, SOX18 acts as a mesenchymal molecular switch necessary for the formation and function of the dermal papilla in all hair types.

Transgenic flash mice for in vivo quantitative monitoring of canonical Wnt signaling to track hair follicle cycle dynamics.

Hair follicles (HFs) upon development enter a lifelong cycle of growth, regression, and resting. These phases have been extensively studied at the cellular and molecular levels for individual HFs. However, HFs group into domains with coordinated cycling strongly influenced by their environment. These macroscopic hair domains have been difficult to study and can be influenced by physiological or pathological conditions, such as pregnancy or skin wounds. To robustly address this issue, we generated a mouse model for quantitative monitoring of ß-catenin activity reflecting HF cycle dynamics macroscopically by using live bioluminescence imaging. These mice allowed live tracking of HF cycles and development, and highlighted hair regenerative patterns known to occur through macro-environmental cues, including initiation events, propagating anagen and border stability, and allowed refinement of a mechanistic mathematical model that integrates epidermal cell population dynamics into an excitable reaction-diffusion model. HF cycling could be studied in situations of pregnancy, wound healing, hair plucking, as well as in response to cyclosporine or Wnt3a stimulation. In conclusion, we developed a model for analysis of HF cycling at the macroscopic level that will allow refined analysis of hair cycle kinetics as well as its propagation dynamics.

Patched 1 and patched 2 redundancy has a key role in regulating epidermal differentiation.

The Patched 1 (Ptch1) receptor has a pivotal role in inhibiting the activity of the Hedgehog (Hh) pathway and is therefore critical in preventing the onset of many human developmental disorders and tumor formation. However, the functional role of the mammalian Ptch2 paralogue remains elusive, particularly the extent to which it contributes to regulating the spatial and temporal activity of Hh signaling. Here we demonstrate in three independent mouse models of epidermal development that in vivo ablation of both Ptch receptors results in a more severe phenotype than loss of Ptch1 alone. Our studies indicate that concomitant loss of Ptch1 and Ptch2 activity inhibits epidermal lineage specification and differentiation. These results reveal that repression of Hh signaling through a dynamic Ptch regulatory network is a crucial event in lineage fate determination in the skin. In general, our findings implicate Ptch receptor redundancy as a key issue in elucidating the cellular origin of Hh-induced tumors.

Differential effects of ultraviolet irradiation in neonatal versus adult mice are not explained by defective macrophage or neutrophil infiltration.

Epidemiological studies suggest that ultraviolet B exposure (UVR) during childhood is the most important environmental risk factor for melanoma. In accordance, neonatal, but not adult, UVR exacerbates melanoma incidence in mouse models. The inability of neonates, as opposed to adults, to mount a proper neutrophil inflammatory response in the skin upon UVR exposure has been one of the driving hypotheses explaining this observation for the past decade. However, this aspect remains controversial. Here, we evaluated the UVR-induced inflammatory response in neonatal versus adult mice. In neonates, a significant neutrophil infiltration could be identified and quantified using three different antibodies by flow cytometry or immunohistochemistry. On day 1 after UVR, neutrophils were increased by 84-fold and on day 4 macrophages increased by 37-fold compared with nonexposed age-matched skin. When compared with adults, neonatal skin harbored a higher proportion of neutrophils in the myeloid compartment without significant differences in absolute counts. This response was reproduced with different kinetics in C57Bl/6 and FVB mice with a more rapid attenuation of neutrophil counts in the latter. Overall, our results suggest that the greatly increased sensitivity to melanomagenesis in neonates does not result from their incompetence in terms of myeloid inflammatory response to UVR.

Analysis of bulge stem cells from the epidermis using flow cytometry.

The epidermis is a multilayered epithelium consisting of multiple different progenitor cell populations, all of which are important to epidermal function. In order to study these populations, several techniques have been developed that enable specific purification of the different progenitor cell populations. The best characterized stem cell population in the epidermis, and likely the most pluripotent, are the quiescent stem cells in the hair follicle bulge. In this chapter, we provide a method for isolating bulge stem cells from skin of adult mice using fluorescence-activated cell sorting of immunofluorescently labeled keratinocytes. We use the cell surface markers CD34 and α6-integrin for the enrichment of bulge stem cells. This method also contains notes on how to adjust the cytometer settings for a reproducible analysis.

Murine basal cell carcinoma leads to tumor-mediated alterations in endocrine Igf1 signaling.

The intrinsic properties underlying cancer development are extensively studied while the effect of a cancer on the host is often overlooked. Activation of the Hedgehog (Hh) signaling pathway underlies a number of types of common human cancers, yet little is known concerning endocrine signaling in such tumors. Here, we investigated endocrine signaling in a murine model of basal cell carcinoma (BCC) of the skin, the most common cancer. BCCs were generated by the activation of Hh signaling resulting from the specific deletion of the Ptch1 gene in the developing epidermis. Subsequently, a severe growth deficiency was observed in the murine BCC model, and we identified a deficiency of circulating IGF1 (Igf1). We demonstrate that Hh pathway activation in murine BCC induces IGF binding proteins, thereby regulating Igf1 sequestration into the skin and skewing Igf endocrine signaling. Significantly, these results show that Hh-induced tumors can have endocrine effects on normal tissues that in turn can greatly impact the host. This study not only identifies that Igf is important in Hh-associated skin tumors but also exemplifies the need to consider endocrine signaling when interpreting complex in vivo tumor models.

Patched1 inhibits epidermal progenitor cell expansion and basal cell carcinoma formation by limiting Igfbp2 activity.

Basal cell carcinoma (BCC) of the skin is the most common form of cancer, with the majority being caused by mutations in the Patched1 (Ptch1) gene, leading to activation of the Hedgehog (Hh) signaling pathway. Hh signaling is implicated in many tumor types; thus, defining the mechanisms by which Ptch1 regulates tissue proliferation is of paramount importance. Here, we show that the key role of Ptch1 in the skin is to limit the size of the epidermal stem/progenitor compartment and allow hair follicle differentiation. Specifically, loss of Ptch1 leads to the promotion of progenitor cell fate by increasing basal cell proliferation and limiting the progression of basal cells into differentiated hair follicle cell types. Our data indicate that BCCs likely result from hair follicle progenitor cells that, due to Hh signal activation, cannot progress through normal hair follicle differentiation. These data confirm the role of Ptch1 as a negative regulator of epidermal progenitor turnover and also show for the first time that Ptch1 plays a role in the differentiation of the hair follicle lineage. In addition, we show that insulin-like growth factor binding protein 2 (Igfbp2) is upregulated in both murine and human BCCs and that blocking Igfbp2 activity reduces the Hh-mediated expansion of epidermal progenitor cells. We propose that Igfbp2 mediates epidermal progenitor cell expansion and therefore represents an epidermal progenitor cell-specific target of Hh signaling that promotes BCC development.

The Hedgehog receptor Patched1 regulates myeloid and lymphoid progenitors by distinct cell-extrinsic mechanisms.

Hedgehog (Hh) ligands bind to the Patched1 (Ptch1) receptor, relieving repression of Smoothened, which leads to activation of the Hh signaling pathway. Using conditional Ptch1 knockout mice, the aim of this study was to determine the effects of activating the Hh signaling pathway in hematopoiesis. Surprisingly, hematopoietic-specific deletion of Ptch1 did not lead to activation of the Hh signaling pathway and, consequently, had no phenotypic effect. In contrast, deletion of Ptch1 in nonhematopoietic cells produced 2 distinct hematopoietic phenotypes. First, activation of Hh signaling in epithelial cells led to apoptosis of lymphoid progenitors associated with markedly elevated levels of circulating thymic stromal lymphopoietin. Second, activation of Hh signaling in the bone marrow cell niche led to increased numbers of lineage-negative c-kit(+) Sca-1(+) bone marrow cells and mobilization of myeloid progenitors associated with a marked loss of osteoblasts. Thus, deletion of Ptch1 leads to hematopoietic effects by distinct cell-extrinsic mechanisms rather than by direct activation of the Hh signaling pathway in hematopoietic cells. These findings have important implications for therapeutics designed to activate the Hh signaling pathway in hematopoietic cells including hematopoietic stem cells.