Unraveling the Functional Heterogeneity of Human Skin at Single-Cell Resolution.

The skin consists of several cell populations, including epithelial, immune, and stromal cells. Recently, there has been a significant increase in single-cell RNA-sequencing studies, contributing to the development of a consensus Human Skin Cell Atlas. The aim is to understand skin biology better and identify potential therapeutic targets. The present review utilized previously published single-cell RNA-sequencing datasets to explore human skin's cellular and functional heterogeneity. Additionally, it summarizes the functional significance of newly identified cell subpopulations in processes such as wound healing and aging.

YBX1 mediates translation of oncogenic transcripts to control cell competition in AML.

Persistence of malignant clones is a major determinant of adverse outcome in patients with hematologic malignancies. Despite the fact that the majority of patients with acute myeloid leukemia (AML) achieve complete remission after chemotherapy, a large proportion of them relapse as a result of residual malignant cells. These persistent clones have a competitive advantage and can re-establish disease. Therefore, targeting strategies that specifically diminish cell competition of malignant cells while leaving normal cells unaffected are clearly warranted. Recently, our group identified YBX1 as a mediator of disease persistence in JAK2-mutated myeloproliferative neoplasms. The role of YBX1 in AML, however, remained so far elusive. Here, inactivation of YBX1 confirms its role as an essential driver of leukemia development and maintenance. We identify its ability to amplify the translation of oncogenic transcripts, including MYC, by recruitment to polysomal chains. Genetic inactivation of YBX1 disrupts this regulatory circuit and displaces oncogenic drivers from polysomes, with subsequent depletion of protein levels. As a consequence, leukemia cells show reduced proliferation and are out-competed in vitro and in vivo, while normal cells remain largely unaffected. Collectively, these data establish YBX1 as a specific dependency and therapeutic target in AML that is essential for oncogenic protein expression.

Hyaluronic acid conjugates for topical treatment of skin cancer lesions.

Skin cancer is one of the most common types of cancer in the United States and worldwide. Topical products are effective for treating cancerous skin lesions when surgery is not feasible. However, current topical products induce severe irritation, light-sensitivity, burning, scaling, and inflammation. Using hyaluronic acid (HA), we engineered clinically translatable polymer-drug conjugates of doxorubicin and camptothecin termed, DOxorubicin and Camptothecin Tailored at Optimal Ratios (DOCTOR) for topical treatment of skin cancers. When compared to the clinical standard, Efudex, DOCTOR exhibited high cancer-cell killing specificity with superior safety to healthy skin cells. In vivo studies confirmed its efficacy in treating cancerous lesions without irritation or systemic absorption. When tested on patient-derived primary cells and live-skin explants, DOCTOR killed the cancer with a selectivity as high as 21-fold over healthy skin tissue from the same donor. Collectively, DOCTOR provides a safe and potent option for treating skin cancer in the clinic.

Novel approaches for managing aged skin and nonmelanoma skin cancer.

The process of aging influences every bodily organ and tissue, and those with rapid epithelial cell turnover, are particularly affected. The most visible of these, however, is the skin (including the epidermis), the largest human organ that provides a barrier to external insults, structure to the body and its movements, facilitates thermoregulation, harbors immune cells, and incorporates sensory neurons (including mechanoreceptors, nociceptors, and thermoreceptors). Skin aging has traditionally been categorized into intrinsic and extrinsic, with the latter nearly exclusively restricted to "photoaging," (i.e., aging due to exposure to solar or artificial ultraviolet radiation). However, both intrinsic and extrinsic aging share similar causes, including oxidative damage, telomere shortening, and mitochondrial senescence. Also, like other malignancies, the risk of malignant and nonmalignant lesions increases with age. Herein, we review the most recent findings in skin aging and nonmelanoma skin cancer, including addition to traditional and developing therapies.

The RNA-binding protein YBX1 regulates epidermal progenitors at a posttranscriptional level.

The integrity of stratified epithelia depends on the ability of progenitor cells to maintain a balance between proliferation and differentiation. While much is known about the transcriptional pathways underlying progenitor cells' behavior in the epidermis, the role of posttranscriptional regulation by mRNA binding proteins-a rate-limiting step in sculpting the proteome-remains poorly understood. Here we report that the RNA binding protein YBX1 (Y-box binding protein-1) is a critical effector of progenitors' function in the epidermis. YBX1 expression is restricted to the cycling keratinocyte progenitors in vivo and its genetic ablation leads to defects in the architecture of the skin. We further demonstrate that YBX1 negatively controls epidermal progenitor senescence by regulating the translation of a senescence-associated subset of cytokine mRNAs via their 3' untranslated regions. Our study establishes YBX1 as a posttranscriptional effector required for maintenance of epidermal homeostasis.

Sustained Akt Activity Is Required to Maintain Cell Viability in Seborrheic Keratosis, a Benign Epithelial Tumor.

Seborrheic keratoses (SKs) are common benign skin tumors that share many morphological features with their malignant counterpart, squamous cell carcinoma. SKs frequently have acquired oncogenic mutations in the receptor tyrosine kinase/phosphatidylinositol 3-kinase/Akt signaling cascade. We developed a reliable culture system to study SKs in vitro and screened these cells using a library of selective kinase inhibitors to evaluate effects on cell survival. These benign tumors are sensitive to inhibition by ATP-competitive Akt inhibitors, including A-443654 and GSK690693. RNA interference-mediated Akt suppression mimicked the effects of enzyme inhibition in cultured cells. Akt inhibition suppressed phosphorylation of downstream targets of Akt kinase that are critical for cell survival, including MDM2 and FOXO3a, and induced apoptosis. Cell death was also dependent on p53, mutations in which, although common in cutaneous squamous cell carcinoma, have not been identified in SKs. Intact explants of SKs were also sensitive to Akt inhibition. In addition to the obvious therapeutic implications of these findings, identifying the signaling characteristics that differentiate benign and malignant tumors may inform our understanding of the malignant state.

Small GTPase RhoE/Rnd3 is a critical regulator of Notch1 signaling.

Aberrations of Notch signaling have been implicated in a variety of human cancers. Oncogenic mutations in NOTCH1 are common in human T-cell leukemia and lymphomas. However, loss-of-function somatic mutations in NOTCH1 arising in solid tumors imply a tumor suppressor function, which highlights the need to understand Notch signaling more completely. Here, we describe the small GTPase RhoE/Rnd3 as a downstream mediator of Notch signaling in squamous cell carcinomas (SCC) that arise in skin epithelia. RhoE is a transcriptional target of activated Notch1, which is attenuated broadly in SCC cells. RhoE depletion suppresses Notch1-mediated signaling in vitro, rendering primary keratinocytes resistant to Notch1-mediated differentiation and thereby favoring a proliferative cell fate. Mechanistic investigations indicated that RhoE controls a key step in Notch1 signaling by mediating nuclear translocation of the activated portion of Notch1 (N1IC) through interaction with importins. Our results define RhoE as a Notch1 target that is essential for recruitment of N1IC to the promoters of Notch1 target genes, establishing a regulatory feedback loop in Notch1 signaling. This molecular circuitry may inform distinct cell fate decisions to Notch1 in epithelial tissues, where carcinomas such as SCC arise.

Decreased DNA repair capacity of UV-irradiated cells following interferon treatment.

The aim of this study was to examine the effect of interferons (IFNs) on the recovery of UV-damaged cells by means of measuring cell viability rates. The influence of the recombinant human interferons IFN-alpha, IFN-beta and IFN-gamma on the repair capacity of the UV-irradiated human cell lines WISH and HeLa was studied. The ability of cells to repair UV-induced damage was determined by the comet assay and both short- and long-term survival assays in proliferating cell cultures. We found that INFs negatively regulated DNA repair in cells damaged by UV light. One day after treatment, in both cell lines tested, IFN-alpha had a stronger inhibitory effect than IFN-gamma. Combined treatment with different IFNs exhibited a stronger inhibitory effect on cell recovery than treatment with each of them. The protein kinase inhibitor wortmanin further aggravated the effect of IFNs on cell survival.

The kinase p38 alpha serves cell type-specific inflammatory functions in skin injury and coordinates pro- and anti-inflammatory gene expression.

The mitogen-activated protein kinase p38 mediates cellular responses to injurious stress and immune signaling. Among the many p38 isoforms, p38 alpha is the most widely expressed in adult tissues and can be targeted by various pharmacological inhibitors. Here we investigated how p38 alpha activation is linked to cell type-specific outputs in mouse models of cutaneous inflammation. We found that both myeloid and epithelial p38 elicit inflammatory responses, yet p38 alpha signaling in each cell type served distinct inflammatory functions and varied depending on the mode of skin irritation. In addition, myeloid p38 alpha limited acute inflammation via activation of anti-inflammatory gene expression dependent on mitogen- and stress-activated kinases. Our results suggest a dual function for p38 alpha in the regulation of inflammation and show mixed potential for its inhibition as a therapeutic strategy.

Influence of interferons on the repair of UV-damaged DNA.

The capacity for nucleotide excision repair of a normal (WISH) and three tumour (MCF-7, HeLa, Namalva) cell lines treated with human recombinant interferons (hrIFN-alpha and hrIFN-gamma) was compared by the host cell reactivation assay. The cells were transfected with in vitro UV-damaged plasmid DNA (pEGFP-N1). The repair capacity was determined by measuring the fluorescence intensity of the expressed marker protein in total cell lysates. The correlation between the interferon-induced NO content and the suppressive effect of interferons on DNA repair was shown. The decrease of repair activity and NO induction by hrIFN-alpha were greatest in WISH, followed by MCF-7, Namalva and HeLa cells, whereas hrIFN-gamma was the best NO inducer and inhibitor for the repair of Namalva, followed by WISH, MCF-7 and HeLa cells. Our data clearly show that the two types of interferon have a strong inhibitory effect on the repair of UV-damaged DNA and this effect is cell type-dependent.

Spontaneous transfection of mammalian cells with plasmid DNA.

A simple method for spontaneous transfection into mammalian cells (both adherent and suspension in culture) with plasmid DNA is described. This method does not require any specific DNA carrier or technical device and can be applied for obtaining both transient and stably transfected cells. The efficiency of spontaneous transfection is slightly lower in comparison with that of the conventional calcium phosphate and lipofectin transfection methods and does not depend on the type of cell culture used.

Human interferon gamma: significance of the C-terminal flexible domain for its biological activity.

The significance of the C-terminal part of human interferon gamma (hIFNgamma) for its biological activity was studied by 3(')-end gene mutagenesis. A series of nine derivative genes obtained by systemic deletion of three codons was constructed and expressed in Escherichia coli LE392. It was shown that the yield of recombinant protein gradually decreased and the solubility gradually increased with truncation of the C terminus. To avoid artifacts related to the imperfect folding of the proteins during purification, the biological activity of the hIFNgamma proteins was measured in clear cell lysates containing the soluble fractions only. The deletion of the C terminus had a two-step effect on both hIFNgamma antiviral and antiproliferative activities. Whereas the removal of the last 3, 6, and 9 C-terminal amino acids led to a gradual increase (up to 10 times) in biological activity of hIFNgamma, the deletion of more than 9 amino acids had an opposite effect. The truncation of the whole unstructured C-terminal domain resulted in a 10-fold decrease (but not in a complete loss) in biological activity of hIFNgamma. The latter was sequestered upon deletion of 24 amino acids, 3 of which belonged to the alpha-helical domain F.

Fluorescence studies on denaturation and stability of recombinant human interferon-gamma.

Unfolding/folding transitions of recombinant human interferon-gamma (hIFNgamma) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy. At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 M and deltaG0 = 13.4 kJ/mol) than urea (C* = 2.8 M and deltaG0 = 11.7 kJ/mol). The close deltaG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNgamma is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNgamma remains native in the pH range of 4.8-9.5. Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified. Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNgamma, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNgamma.