Keratinocyte sonic hedgehog up-regulation 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 NRAS, 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

Role of Patched1 in Epidermal Homeostasis

Abstract – The Role of Patched1 in Epidermal Homeostasis Hedgehog (Hh) signalling is a critical pathway involved in the development of many, if not all, organ systems. However the abnormal activation of Hh signalling in fully developed adult organs leads to cancer. Mutation of the Hh signal receptor, Patched1 (Ptc1), causes Naevoid Basal Cell Carcinoma Syndrome, which presents with developmental defects and cancer predisposition. The activation of Hh signalling is seen in a wide range of non-inherited cancer types also, including Medulloblastoma and Basal Cell Carcinoma (BCC) of the skin. BCC is the most common form of human cancer and over 90% of cases are linked to abnormally high Hh signalling. Hh signalling is known to regulate hair follicle morphogenesis during development and more recently has been linked to modulation of the embryonic epidermal stem cell compartment. However both the mechanisms behind this process and the mechanism behind its induction of BCC are still uncharacterised. The aim of this project was to determine the role of Ptc1 in the skin, particularly the adult stem cell compartment, and the role of Hh signalling in BCC formation. The deletion of Ptc1 specifically in the adult epidermis was enabled by the creation of a K14-Cre Recombinase induced Ptc1 Conditional (K14-Cre:Ptc1C/C) transgenic mouse line. Proliferation was increased throughout the epithelia and BCC-like lesions developed within 4 weeks of Ptc1 deletion. This indicates that Hh signalling plays a critical role in repressing cell turnover in the interfollicular epithelium (IFE) and bulge region in the adult despite being previously reported not to play a role in this area. Ptc1 deletion in the epithelia was also found to promote the IFE lineage over hair follicles and expand the expression of many proposed stem cell markers, including K15, Sox9 and p63. K14-Cre:Ptc1C/C transgenic mice also exhibited a severe growth defect, linked to low levels of Igf1 hormone in the serum. Igf1 binding protein alteration in the skin was determined to be the most likely cause and prompted the investigation of Igf axis signalling in Ptc1 deleted epidermis. Insulin-like growth factor binding protein 2 was found to localise to the bulge or stem cell region of the hair follicle, and was increased in K14-Cre:Ptc1C/C epidermis. Igfbp2 was coincident with a loss of PI3K/Akt signal translation. The majority of human BCC samples also expressed Igfbp2 at much higher levels than surrounding normal tissue indicating these results are relevant to the human BCC condition also. Interestingly Hh activation was also shown to increase p38 MAPK throughout the epidermis indicating it is a universal target of Hh signalling in the skin. In summary we have found that Hh signal activation in the epidermis promotes the bulge/stem cell and interfollicular lineages of the skin at the expense of hair follicles. Finally the modulation of PI3K/Akt signalling by Igfbp2 in the bulge is perhaps mediating the effect of Hh signalling via the promotion of the bulge lineage leading to the development of BCC

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 β-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

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 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 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

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

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. Cancer Prev Res; 3(10); 1222-34. (c) 2010 AACR

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

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

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

Murine basal cell carcinoma leads to tumor mediated alterations in endocrine lgf1 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