Observations that suggest a contribution of altered dermal papilla mitochondrial function to androgenetic alopecia.

Androgenetic alopecia (AGA) is a prevalent hair loss condition in males that develops due to the influence of androgens and genetic predisposition. With the aim of elucidating genes involved in AGA pathogenesis, we modelled AGA with three-dimensional culture of keratinocyte-surrounded dermal papilla (DP) cells. We co-cultured immortalised balding and non-balding human DP cells (DPCs) derived from male AGA patients with epidermal keratinocyte (NHEK) using multi-interfacial polyelectrolyte complexation technique. We observed up-regulated mitochondria-related gene expression in balding compared with non-balding DP aggregates which indicated altered mitochondria metabolism. Further observation of significantly reduced electron transport chain complex activity (complexes I, IV and V), ATP levels and ability to uptake metabolites for ATP generation demonstrated compromised mitochondria function in balding DPC. Balding DP was also found to be under significantly higher oxidative stress than non-balding DP. Our experiments suggest that application of antioxidants lowers oxidative stress levels and improves metabolite uptake in balding DPC. We postulate that the observed up-regulation of mitochondria-related genes in balding DP aggregates resulted from an over-compensatory effort to rescue decreased mitochondrial function in balding DP through the attempted production of new functional mitochondria. In all, our three-dimensional co-culturing revealed mitochondrial dysfunction in balding DPC, suggesting a metabolic component in the aetiology of AGA.

Differential expression of phosphorylated MEK and ERK correlates with aggressive BCC subtypes.

Basal cell carcinoma (BCC) is associated with aberrant Hedgehog (HH) signalling through mutational inactivation of PTCH1; however, there is conflicting data regarding MEK/ERK signalling in BCC and the signalling pathway interactions in these carcinomas. To address this, expression of active phospho (p) MEK and ERK was examined in a panel of 15 non-aggressive and 14 aggressive BCCs. Although not uniformly expressed, both phospho-proteins were detected in the nuclei and/or cytoplasm of normal and tumour-associated epidermal cells however, whereas phospho-MEK (pMEK) was present in all non-aggressive BCCs (14/14), phospho-ERK (pERK) was rarely expressed (2/14). In contrast pERK expression was more prevalent in aggressive tumours (11/14). Interestingly, pMEK was only localized to the tumour mass whereas pERK was expressed in tumours and stroma of aggressive BCCs. Similarly, pERK (but not pMEK) was absent in mouse BCC-like tumours derived from X-ray irradiated Ptch1+/- mice with stromal pERK observed in myofibroblasts of the aggressive variant as well as in the tumour mass. RNA sequencing analysis of tumour epithelium and stroma of aggressive and non-aggressive BCC revealed the upregulation of epidermal growth factor receptor- and ERK-related pathways. Angiogenesis and immune response pathways were also upregulated in the stroma compared with the tumour. PTCH1 suppressed NEB1 immortalized keratinocytes (shPTCH1) display upregulated pERK that can be independent of MEK expression. Furthermore, epidermal growth factor pathway inhibitors affect the HH pathway by suppressing GLI1. These studies reveal differential expression of pERK between human BCC subtypes that maybe active by a pathway independent of MEK.

Tissue stiffening promotes keratinocyte proliferation through activation of epidermal growth factor signaling.

Tissue biomechanics regulate a wide range of cellular functions, but the influences on epidermal homeostasis and repair remain unclear. Here, we examined the role of extracellular matrix stiffness on human keratinocyte behavior using elastomeric substrates with defined mechanical properties. Increased matrix stiffness beyond normal physiologic levels promoted keratinocyte proliferation but did not alter the ability to self-renew or terminally differentiate. Activation of epidermal growth factor (EGF) signaling mediated the proliferative response to matrix stiffness and depended on focal adhesion assembly and cytoskeletal tension. Comparison of normal skin with keloid scar tissue further revealed an upregulation of EGF signaling within the epidermis of stiffened scar tissue. We conclude that matrix stiffness regulates keratinocyte proliferation independently of changes in cell fate and is mediated by EGF signaling. These findings provide mechanistic insights into how keratinocytes sense and respond to their mechanical environment, and suggest that matrix biomechanics may play a role in the pathogenesis keloid scar formation.

An alternative modular 'click-SNAr-click' approach to develop subcellular localised fluorescent probes to image mobile Zn2+ .

Zn2+ is involved in a number of biological processes and its wide-ranging roles at the subcellular level, especially in specific organelles, have not yet been fully established due to a lack of tools to image it effectively. We report a new and efficient modular double 'click' approach towards a range of sub-cellular localised probes for mobile zinc. Through this methodology, endoplasmic reticulum, mitochondria and lysosome localised probes were successfully prepared which show good fluorescence responses to mobile Zn2+in vitro and in cellulo whilst a non-targeting probe was synthesized as a control. The methodology appears to have wide-utility for the generation of sub-cellular localised probes by incorporating specific organelle targeting vectors for mobile Zn2+ imaging.

Culture of the human pilosebaceous unit, hair follicle and sebaceous gland.

Terence Kealey first pioneered the isolation and organ maintenance of human eccrine and sebaceous glands in the early to mid-1980. This led to subsequent methods describing the isolation and culture of human hair follicles, the human pilosebaceous unit as well as the sebaceous duct. The importance of these models in the study of the biology of human skin glands and appendages has been demonstrated in numerous publications and their importance as models for animal replacement, refinement and reduction (3Rs) is increasingly important. In particular, in vitro (ex vivo) hair follicle culture has played a significant part in helping elucidate the role of signalling molecules in regulating hair growth and hair fibre formation and has been especially useful in understanding metabolic aspects of hair growth. However, obtaining sufficient numbers of hair follicles is becoming increasingly difficult as plastic surgery becomes less invasive and smaller skin samples provided. There is therefore an urgent requirement for the next generation of in vitro models using cell lines and tissue engineering, and this has led to the development of immortalised cell lines as well as attempts to model hair follicle embryogenesis in vitro and development of skin on a chip.

Analysis of hedgehog signaling in periocular sebaceous carcinoma.

PURPOSE: Sebaceous carcinoma (SC) is a clinical masquerader of benign conditions resulting in significant eye morbidity, sometimes leading to extensive surgical treatment including exenteration, and even mortality. Little is known about the genetic or molecular basis of SC. This study identifies the involvement of Hedgehog (Hh) signaling in periocular SC. METHODS: Fifteen patients with periocular SC patients were compared to 15 patients with eyelid nodular basal cell carcinoma (nBCC; a known Hh tumor), alongside four normal individuals as a control for physiological Hh expression. Expression of Patched 1 (PTCH1), Smoothened (SMO), and glioma-associated zinc transcription factors (Gli1 and Gli2) were assessed in histological sections using immunohistochemistry and immunofluorescence (IF) techniques. Antibody specificity was verified using Western-blot analysis of a Gli1 over-expressed cancer cell line, LNCaP-Gli1. Semi-quantification compared tumors and control tissue using IF analysis by ImageJ software. RESULTS: Expression of the Hh pathway was observed in SC for all four major components of the pathway. PTCH1, SMO, and Gli2 were more significantly upregulated in SC (P < 0.01) compared to nBCC. Stromal expression of PTCH1 and Gli2 was observed in SC (P < 0.01). In contrast, stromal expression of these proteins in nBCC was similar or down-regulated compared to physiological Hh controls. CONCLUSIONS: The Hh signaling pathway is significantly more upregulated in periocular SC compared to nBCC, a known aberrant Hh pathway tumor. Furthermore, the stroma of the SC demonstrated Hh upregulation, in particular Gli2, compared to nBCC. Targeting of this pathway may be a potential treatment strategy for SC.

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia.

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR-574-3p and miR-720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.

Human hair follicle organ culture: theory, application and perspectives.

For almost a quarter of a century, ex vivo studies of human scalp hair follicles (HFs) have permitted major advances in hair research, spanning diverse fields such as chronobiology, endocrinology, immunology, metabolism, mitochondrial biology, neurobiology, pharmacology, pigmentation and stem cell biology. Despite this, a comprehensive methodological guide to serum-free human HF organ culture (HFOC) that facilitates the selection and analysis of standard HF biological parameters and points out both research opportunities and pitfalls to newcomers to the field is still lacking. The current methods review aims to close an important gap in the literature and attempts to promote standardisation of human HFOC. We provide basic information outlining the establishment of HFOC through to detailed descriptions of the analysis of standard read-out parameters alongside practical examples. The guide closes by pointing out how serum-free HFOC can be utilised optimally to obtain previously inaccessible insights into human HF biology and pathology that are of interest to experimental dermatologists, geneticists, developmental biologists and (neuro-) endocrinologists alike and by highlighting novel applications of the model, including gene silencing and gene expression profiling of defined, laser capture-microdissected HF compartments.

Highly Sensitive Terahertz Metasurface Based on Electromagnetically Induced Transparency-Like Resonance in Detection of Skin Cancer Cells.

Terahertz (THz) metasurfaces based on high Q-factor electromagnetically induced transparency-like (EIT-like) resonances are promising for biological sensing. Despite this potential, they have not often been investigated for practical differentiation between cancerous and healthy cells. The present methodology relies mainly on refractive index sensing, while factors of transmission magnitude and Q-factor offer significant information about the tumors. To address this limitation and improve sensitivity, we fabricated a THz EIT-like metasurface based on asymmetric resonators on an ultra-thin and flexible dielectric substrate. Bright-dark modes coupling at 1.96 THz was experimentally verified, and numerical results and theoretical analysis were presented. An enhanced theoretical sensitivity of 550 GHz/RIU was achieved for a sample with a thickness of 13 µm due to the ultra-thin substrate and novel design. A two-layer skin model was generated whereby keratinocyte cell lines were cultured on a base of collagen. When NEB1-shPTCH (basal cell carcinoma (BCC)) were switched out for NEB1-shCON cell lines (healthy) and when BCC's density was raised from 1 × 105 to 2.5 × 105, a frequency shift of 40 and 20 GHz were observed, respectively. A combined sensing analysis characterizes different cell lines. The findings may open new opportunities for early cancer detection with a fast, less-complicated, and inexpensive method.

Disseminating cells in human oral tumours possess an EMT cancer stem cell marker profile that is predictive of metastasis in image-based machine learning.

Cancer stem cells (CSCs) undergo epithelial-mesenchymal transition (EMT) to drive metastatic dissemination in experimental cancer models. However, tumour cells undergoing EMT have not been observed disseminating into the tissue surrounding human tumour specimens, leaving the relevance to human cancer uncertain. We have previously identified both EpCAM and CD24 as CSC markers that, alongside the mesenchymal marker Vimentin, identify EMT CSCs in human oral cancer cell lines. This afforded the opportunity to investigate whether the combination of these three markers can identify disseminating EMT CSCs in actual human tumours. Examining disseminating tumour cells in over 12,000 imaging fields from 74 human oral tumours, we see a significant enrichment of EpCAM, CD24 and Vimentin co-stained cells disseminating beyond the tumour body in metastatic specimens. Through training an artificial neural network, these predict metastasis with high accuracy (cross-validated accuracy of 87-89%). In this study, we have observed single disseminating EMT CSCs in human oral cancer specimens, and these are highly predictive of metastatic disease.

When oral cancers metastasise ­ that is, when tumour cells invade other parts of the body ­ they typically do so by first colonizing the lymph nodes present in the neck. As this event significantly reduces chances of survival, oral cancer patients often have their neck lymph nodes removed to prevent the spread of the disease. However, this surgery carries risks and leads to longer hospital stays, stressing the need for better ways to predict which oral tumours will metastasise. Evidence from lab-grown cells and mice studies suggest that, in oral cancer, metastasis occurs when some cells in the original tumour go through a process called the epithelial-mesenchymal transition (EMT for short). This transformation allows the cells to detach from the tumour and become invasive. However, it has so far been difficult to observe this process in actual human tumours; this is partly because cells undergoing EMT stop producing the proteins that scientists rely on to distinguish cancer and healthy cells. To address this knowledge gap, Youssef et al. focused on three proteins: two tumour markers, EpCAM and CD24; and Vimentin, which is produced in greater quantities in the invasive mesenchymal state. Previous work had shown that a specific population of oral tumour cells can continue to express all three proteins even when adopting a mesenchymal identity through EMT. Based on this knowledge, Youssef et al. hypothesised that tracking Vimentin, EpCAM and CD24 using fluorescence microscopy would allow them to identify metastasising cells in human samples. An analysis of over 12,000 images from 74 tumours obtained from surgeries revealed that, in the metastatic samples, the cells detaching from primary tumours were more likely to express these three proteins. Finally, Youssef et al. used these images to train a machine learning algorithm. When applied to data from new oral cancer patients, the programme was able to predict whether their tumours were likely to spread with 89% accuracy. If confirmed by further work, and in particular on larger samples, these findings could in the future help clinicians decide which patients with oral cancer would benefit the most from surgery to remove neck lymph nodes.

Magnetism and structure of graphene nanodots with interiors modified by boron, nitrogen, and charge.

The properties (geometry, spin, and charge distribution) of a series of flat hexagonal zigzag edged graphene nanodots (GNDs), with interiors modified by centrally located substituent atoms boron and nitrogen and by positive and negative charge, have been calculated using ab initio density functional theory. The doped series X-GND has the stoichiometry C(6m(2)-1)XH(6m), zigzag size index m = 2, 4, 6, 8, 10 and substituent X = B or N. The undoped parents C(6m(2))H(6m) with m ≤ 8 have spin paired ground states and the parent m = 10 has a spin polarized singlet ground state with edges that alternate α- and ß-spin. The spin on the substituent atom decreases to zero with size index m and magnetization builds on the edges of all the X-GND. This demonstrates translocation of substituent spin and a proximity or directional effect for small m as the edges show different degrees of magnetization. For the largest X-GND (m = 10) the magnetization on edges resembles the calculated triplet S = 1(a) configuration of the parent (four edge spins up and two down) and has a higher apparent symmetry than the C(2v) point group of X-GND. For charged (m = 10) GNDs the edge magnetization has strength comparable to the parent on two parallel edges and weak on the other four in a perimeter pattern that resembles the triplet S = 1(b) configuration of the undoped parent and not the ground configuration of the isoelectronic X-GND molecule. Many of the results can be interpreted by simple Kekulé valence bond structures for an unpaired spin on a network where the substituent site group symmetry is not compatible with the perimeter. A deeper understanding is provided by the properties of the Kohn-Sham orbitals. The calculations of the X-doped GNDs reveal limitations in the use of the hex-radical hypothesis of the parent ground state to systems where foreign atoms lower symmetry and perturb the π- and σ-bond manifolds.

Graphene nanodots with intrinsically magnetic protrusions.

The three step auf bau of a triangular polyaromatic protrusion attached to a larger parent hexagonal shaped graphene nanodot (GND) is described and the dichotomy between intrinsic protrusion localized magnetism and parent extended zigzag edge magnetism is explored using ab initio density functional theory calculations of spin and charge distributions and geometry. Comparison of a three ring with a ten-ring protrusion-GND establishes a pattern for the magnetization of GNDs with larger protrusions and different morphology. The magnetism of the isolated protrusions arises from the mismatch in numbers of sublattice (alternant hydrocarbon) carbon atoms. In the parent, the sublattices are equivalent providing a singlet ground state and the magnetization appears only on long zigzag edges due to exchange interactions operating in a regime of reduced coulombic interactions. We demonstrate that a small protrusion can quench the magnetism of the edge to which it is attached. Concomitantly, the adjacent edges exhibit a small magnetic enhancement, while the remote edges are unperturbed. With size the protrusion can dominate its edge and exert control over the magnetization of other edges. Different multiplicities of the parent moiety were not found. These calculations provide guidance in understanding how the magnetism changes with system shape and in designing nanodots with a specific magnetization.

Steroid synthesis by primary human keratinocytes; implications for skin disease.

Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3ßHSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-(3)H]-pregnenolone through each steroid intermediate to [7-(3)H]-cortisol in cultured PHK. Trilostane (a 3ßHSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data show that PHK are capable of extra-adrenal cortisol synthesis, which could be a fundamental pathway in skin biology with implications in psoriasis and atopic dermatitis.

Triplet states of zigzag edged hexagonal graphene molecules C(6m∗∗2)H(6m) (m = 1, 2, 3, ..., 10) and carbon based magnetism.

The geometry and magnetization (spin distribution) of the series of flat hexagonal zigzag edged molecules C(6m∗∗2)H(6m) (m = 1,2, ..., 10) in their lowest triplet state (S(z) = 1) has been calculated using density functional theory and a connection established from the known benzene (m = 1) triplets to the triplets and singlet ground state of the largest molecules (m = 9, 10). The triplet state potential energy surface has two minima corresponding to distortions from the ground state geometry, such that CC bonds bisected by a C(2)" rotation axis are either longer or shorter. For both geometries, the spin on the carbon atoms forms a pattern that peaks at the middle of an edge and for large index (m) values is the same (apart from sign) as the edge pattern of the hexagonally sectored singlet radical ground state of the largest member C(600)H(60). This similarity suggests that the singlet ground state of the larger (m = 9, 10) zigzag edged hexangulenes is possibly a hex-radical, in some ways analogous to the di- and higher multiradical ground state of the linear acenes C(4m + 2)H(2m + 4) starting around m ≥ 8 and 9. The spin patterns provide guidance in interpreting the multiradical nature of ground and low lying excited states of large hexangulenes and how magnetism evolves with size in molecules with graphene cores.

Magnetism and bonding in graphene nanodots with H modified interior, edge, and apex.

Ab initio density functional theory calculations of hexagonal shaped zigzag edged graphene nanodot molecules, modified by the addition of atomic H to interior and perimeter sites, predict significant changes to the hexagonally sectored spin distribution and chemical bonding of the originals. The redistribution of Kohn-Sham levels at the top of the valence manifold from parent to derivative hint at large changes in the electronic structure. A centrally added H atom creates an occupied level in the middle of the 0.3 eV band gap of the parent molecule and is surrounded by an island of spins. The latter is isolated enough from the perimeter to provide a calibration of the edge spins of the modified parent. Mid-edge addition of a H atom "quenches" the spin on the edge by drawing a p(z)-electron into the C-H bond without reducing the spin on the other edges. Addition of H to an apex carbon atom results in a localized spin freed from the double bond that coexists with the parent spin on the same edge. Saturating the apex double bond by adding two H atoms, returns π-levels shifted in energy and index and parent-like spin patterns on all edges, intact except for small changes on the edges joined at the apex. Taken in unison these results demonstrate how atomic hydrogen and other groups could be used to engineer the magnetism of graphene nanodots.

Watching hair turn grey.

Analysing changes in hair pigmentation may lead to a better understanding of the impacts of 'life events' on human biology and aging.

Human hair follicles operate an internal Cori cycle and modulate their growth via glycogen phosphorylase.

Hair follicles (HFs) are unique, multi-compartment, mini-organs that cycle through phases of active hair growth and pigmentation (anagen), apoptosis-driven regression (catagen) and relative quiescence (telogen). Anagen HFs have high demands for energy and biosynthesis precursors mainly fulfilled by aerobic glycolysis. Histochemistry reports the outer root sheath (ORS) contains high levels of glycogen. To investigate a functional role for glycogen in the HF we quantified glycogen by Periodic-Acid Schiff (PAS) histomorphometry and colorimetric quantitative assay showing ORS of anagen VI HFs contained high levels of glycogen that decreased in catagen. qPCR and immunofluorescence microscopy showed the ORS expressed all enzymes for glycogen synthesis and metabolism. Using human ORS keratinocytes (ORS-KC) and ex vivo human HF organ culture we showed active glycogen metabolism by nutrient starvation and use of a specific glycogen phosphorylase (PYGL) inhibitor. Glycogen in ORS-KC was significantly increased by incubation with lactate demonstrating a functional Cori cycle. Inhibition of PYGL significantly stimulated the ex vivo growth of HFs and delayed onset of catagen. This study defines translationally relevant and therapeutically targetable new features of HF metabolism showing that human scalp HFs operate an internal Cori cycle, synthesize glycogen in the presence of lactate and modulate their growth via PYGL activity.

Early growth response 2 (EGR2) is a novel regulator of the senescence programme.

Senescence, a state of stable growth arrest, plays an important role in ageing and age-related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programmes, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up-regulated during senescence, and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to both the ARF and p16 promoters. Loss of EGR2 down-regulates p16 levels and increases the pool of p16- p21- 'reversed' cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a direct transcriptional activator of the p16/pRB and ARF/p53/p21 pathways in senescence and a novel marker of senescence.

Upregulation of FOXM1 induces genomic instability in human epidermal keratinocytes.

BACKGROUND: The human cell cycle transcription factor FOXM1 is known to play a key role in regulating timely mitotic progression and accurate chromosomal segregation during cell division. Deregulation of FOXM1 has been linked to a majority of human cancers. We previously showed that FOXM1 was upregulated in basal cell carcinoma and recently reported that upregulation of FOXM1 precedes malignancy in a number of solid human cancer types including oral, oesophagus, lung, breast, kidney, bladder and uterus. This indicates that upregulation of FOXM1 may be an early molecular signal required for aberrant cell cycle and cancer initiation. RESULTS: The present study investigated the putative early mechanism of UVB and FOXM1 in skin cancer initiation. We have demonstrated that UVB dose-dependently increased FOXM1 protein levels through protein stabilisation and accumulation rather than de novo mRNA expression in human epidermal keratinocytes. FOXM1 upregulation in primary human keratinocytes triggered pro-apoptotic/DNA-damage checkpoint response genes such as p21, p38 MAPK, p53 and PARP, however, without causing significant cell cycle arrest or cell death. Using a high-resolution Affymetrix genome-wide single nucleotide polymorphism (SNP) mapping technique, we provided the evidence that FOXM1 upregulation in epidermal keratinocytes is sufficient to induce genomic instability, in the form of loss of heterozygosity (LOH) and copy number variations (CNV). FOXM1-induced genomic instability was significantly enhanced and accumulated with increasing cell passage and this instability was increased even further upon exposure to UVB resulting in whole chromosomal gain (7p21.3-7q36.3) and segmental LOH (6q25.1-6q25.3). CONCLUSION: We hypothesise that prolonged and repeated UVB exposure selects for skin cells bearing stable FOXM1 protein causes aberrant cell cycle checkpoint thereby allowing ectopic cell cycle entry and subsequent genomic instability. The aberrant upregulation of FOXM1 serves as a 'first hit' where cells acquire genomic instability which in turn predisposes cells to a 'second hit' whereby DNA-damage checkpoint response (eg. p53 or p16) is abolished to allow damaged cells to proliferate and accumulate genetic aberrations/mutations required for cancer initiation.