Inhibition of UV-Induced Stress Signaling and Inflammatory Responses in SKH-1 Mouse Skin by Topical Small-Molecule PD-L1 Blockade.

The immune checkpoint ligand PD-L1 has emerged as a molecular target for skin cancer therapy and might also hold promise for preventive intervention targeting solar UV light-induced skin damage. In this study, we have explored the role of PD-L1 in acute keratinocytic photodamage testing the effects of small-molecule pharmacological inhibition. Epidermal PD-L1 upregulation in response to chronic photodamage was established using immunohistochemical and proteomic analyses of a human skin cohort, consistent with earlier observations that PD-L1 is upregulated in cutaneous squamous cell carcinoma. Topical application of the small-molecule PD-L1 inhibitor BMS-202 significantly attenuated UV-induced activator protein-1 transcriptional activity in SKH-1 bioluminescent reporter mouse skin, also confirmed in human HaCaT reporter keratinocytes. RT-qPCR analysis revealed that BMS-202 antagonized UV induction of inflammatory gene expression. Likewise, UV-induced cleavage of procaspase-3, a hallmark of acute skin photodamage, was attenuated by topical BMS-202. NanoString nCounter transcriptomic analysis confirmed downregulation of cutaneous innate immunity- and inflammation-related responses, together with upregulation of immune response pathway gene expression. Further mechanistic analysis confirmed that BMS-202 antagonizes UV-induced PD-L1 expression both at the mRNA and protein levels in SKH-1 epidermis. These data suggest that topical pharmacological PD-L1 antagonism using BMS-202 shows promise for skin protection against photodamage.

Exposure to chlorinated drinking water alters the murine fecal microbiota.

An abundant body of scientific studies and regulatory guidelines substantiates antimicrobial efficacy of freshwater chlorination ensuring drinking water safety in large populations worldwide. In contrast to the purposeful use of chlorination ensuring antimicrobial safety of drinking water, only a limited body of research has addressed the molecular impact of chlorinated drinking water exposure on the gut microbiota. Here, for the first time, we have examined the differential effects of drinking water regimens stratified by chlorination agent [inorganic (HOCl) versus chloramine (TCIC)] on the C57BL/6J murine fecal microbiota. To this end, we exposed C57BL/6J mice to chlorinated drinking water regimens followed by fecal bacterial microbiota analysis at the end of the three-week feeding period employing 16S rRNA sequencing. α-diversity was strongly reduced when comparing chlorinated versus control drinking water groups and community dissimilarities (ß-diversity) were significant between groups even when comparing HOCl and TCIC. We detected significant differences in fecal bacterial composition as a function of drinking water chlorination observable at the phylum and genus levels. Differential abundance analysis of select amplicon sequence variants (ASVs) revealed changes as a function of chlorination exposure [up: Lactobacillus ASV1; Akkermansia muciniphila ASV7; Clostridium ss1 ASV10; down: Ileibacterium valens ASV5; Desulfovibrio ASV11; Lachnospiraceae UCG-006 ASV15]. Given the established complexity of murine and human gastrointestinal microbiota and their role in health and disease, the translational relevance of the chlorination-induced changes documented by us for the first time in the fecal murine microbiota remains to be explored.

Systemic deuteration of SCID mice using the water-isotopologue deuterium oxide (D2 O) inhibits tumor growth in an orthotopic bioluminescent model of human pancreatic ductal adenocarcinoma.

Since its initial discovery as a natural isotopologue of dihydrogen oxide (1 H2 O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water (2 H2 O [D2 O, also referred to as "heavy water"]). Using a panel of cultured human pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2 O-induced phenotypic antiproliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential array analysis revealed early modulation of stress response gene expression in both BxPC-3 and PANC-1 PDAC cells elicited by D2 O (90%; ≤6 h; upregulated: HMOX1, NOS2, CYP2E1, CRYAB, DDIT3, NFKBIA, PTGS1, SOD2, PTGS2; downregulated: RUNX1, MYC, HSPA8, HSPA1A) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (≤6 h) onset of D2 O-induced MAP-kinase signaling (p-JNK, p-p38) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP). Next, we tested the chemotherapeutic efficacy of D2 O-based drinking water supplementation in an orthotopic PDAC model employing firefly luciferase-expressing BxPC-3-FLuc cells in SCID mice. First, feasibility and time course of systemic deuteration (30% D2 O in drinking water; 21 days) were established using time-resolved whole-body proton magnetic resonance imaging and isotope-ratio mass spectrometry-based plasma (D/H)-analysis. D2 O-supplementation suppressed tumor growth by almost 80% with downregulated expression of PCNA, MYC, RUNX1, and HSP70 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration significantly reduces orthotopic tumor burden in a murine PDAC xenograft model.

The Drinking Water and Swimming Pool Disinfectant Trichloroisocyanuric Acid Causes Chlorination Stress Enhancing Solar UV-Induced Inflammatory Gene Expression in AP-1 Transgenic SKH-1 Luciferase Reporter Mouse Skin.

Freshwater sanitation and disinfection using a variety of chemical entities as chlorination agents is an essential public health intervention ensuring water safety for populations at a global scale. Recently, we have published our observation that the small molecule oxidant, innate immune factor and chlorination agent HOCl antagonize inflammation and photocarcinogenesis in murine skin exposed topically to environmentally relevant concentrations of HOCl. Chlorinated isocyanuric acid derivatives (including the chloramines trichloroisocyanuric acid [TCIC] and dichloroisocyanuric acid [DCIC]) are used worldwide as alternate chlorination agents serving as HOCl precursor and stabilizer compounds ensuring sustained release in aqueous environments including public water systems, recreational pools and residential hot tubs. Here, for the first time, we have examined the cutaneous TCIC-induced transcriptional stress response (in both an organotypic epidermal model and in AP-1 luciferase reporter SKH-1 mouse skin), also examining molecular consequences of subsequent treatment with solar ultraviolet (UV) radiation. Taken together, our findings indicate that cutaneous delivery of TCIC significantly enhances UV-induced inflammation (as profiled at the gene expression level), suggesting a heretofore unrecognized potential to exacerbate UV-induced functional and structural cutaneous changes. These observations deserve further molecular investigations in the context of TCIC-based freshwater disinfection with health implications for populations worldwide.

The Glycolysis-derived α-Dicarbonyl Metabolite Methylglyoxal is a UVA-photosensitizer Causing the Photooxidative Elimination of HaCaT Keratinocytes with Induction of Oxidative and Proteotoxic Stress Response Gene Expression†.

Cellular oxidative stress contributes to solar ultraviolet (UV) radiation-induced skin photoaging and photocarcinogenesis. Light-driven electron and energy transfer reactions involving non-DNA chromophores are a major source of reactive oxygen species (ROS) in skin, and the molecular identity of numerous endogenous chromophores acting as UV-photosensitizers has been explored. Methylglyoxal (MG), a glycolytic byproduct bearing a UV-active α-dicarbonyl-chromophore, is generated under metabolic conditions of increased glycolytic flux, associated with posttranslational protein adduction in human tissue. Here, we undertook a photophysical and photochemical characterization of MG substantiating its fluorescence properties (Stokes shift), phosphorescence lifetime, and quantum yield of singlet oxygen (1 O2 ) formation. Strikingly, upon UV-excitation (290 nm), a clear emission (around 490 nm) was observed (phosphorescence-lifetime: 224.2 milliseconds). At micromolar concentrations, MG acts as a UVA-photosensitizer targeting human HaCaT-keratinocytes inducing photooxidative stress and caspase-dependent cell death substantiated by zVADfmk-rescue and Alexa-488 caspase-3 flow cytometry. Transcriptomic analysis indicated that MG (photoexcited by noncytotoxic doses of UVA) elicits expression changes not observable upon isolated MG- or UVA-treatment, with upregulation of the proteotoxic (CRYAB, HSPA6) and oxidative (HMOX1) stress response. Given the metabolic origin of MG and its role in human pathology, future investigations should address the potential involvement of MG-photosensitizer activity in human skin photodamage.

Hypochlorous Acid: From Innate Immune Factor and Environmental Toxicant to Chemopreventive Agent Targeting Solar UV-Induced Skin Cancer.

A multitude of extrinsic environmental factors (referred to in their entirety as the 'skin exposome') impact structure and function of skin and its corresponding cellular components. The complex (i.e. additive, antagonistic, or synergistic) interactions between multiple extrinsic (exposome) and intrinsic (biological) factors are important determinants of skin health outcomes. Here, we review the role of hypochlorous acid (HOCl) as an emerging component of the skin exposome serving molecular functions as an innate immune factor, environmental toxicant, and topical chemopreventive agent targeting solar UV-induced skin cancer. HOCl [and its corresponding anion (OCl-; hypochlorite)], a weak halogen-based acid and powerful oxidant, serves two seemingly unrelated molecular roles: (i) as an innate immune factor [acting as a myeloperoxidase (MPO)-derived microbicidal factor] and (ii) as a chemical disinfectant used in freshwater processing on a global scale, both in the context of drinking water safety and recreational freshwater use. Physicochemical properties (including redox potential and photon absorptivity) determine chemical reactivity of HOCl towards select biochemical targets [i.e. proteins (e.g. IKK, GRP78, HSA, Keap1/NRF2), lipids, and nucleic acids], essential to its role in innate immunity, antimicrobial disinfection, and therapeutic anti-inflammatory use. Recent studies have explored the interaction between solar UV and HOCl-related environmental co-exposures identifying a heretofore unrecognized photo-chemopreventive activity of topical HOCl and chlorination stress that blocks tumorigenic inflammatory progression in UV-induced high-risk SKH-1 mouse skin, a finding with potential implications for the prevention of human nonmelanoma skin photocarcinogenesis.

Design, Physicochemical Characterization, and In Vitro Permeation of Innovative Resatorvid Topical Formulations for Targeted Skin Drug Delivery.

Nonmelanoma skin cancers (NMSCs) are the most common malignancies worldwide and affect more than 5 million people in the United States every year. NMSC is directly linked to the excessive exposure of the skin to solar ultraviolet (UV) rays. The toll-like receptor 4 (TLR4) antagonist, resatorvid (TAK-242), is a novel prototype chemo preventive agent that suppresses the production of inflammation mediators induced by UV exposure. This study aimed to design and develop TAK-242 into topical formulations using FDA-approved excipients, including DermaBaseTM, PENcreamTM, polyethylene glycol (PEG)-400, propylene glycol (PG), carbomer gel, hyaluronic acid (HA) gel, and Pluronic® F-127 poloxamer triblock copolymer gel for the prevention of skin cancer. The physicochemical properties of raw TAK-242, which influence the compatibility and solubility in the selected base materials, were confirmed using X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), hot-stage microscopy (HSM), Raman spectroscopy, and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopic analysis. The permeation behavior of TAK-242 from the prepared formulations was determined using Strat-M® transdermal diffusion membranes, and 3D cultured primary human-derived epidermal keratinocytes (EpiDermTM). Despite TAK-242's high molecular weight and hydrophobicity, it can permeate through reconstructed human epidermis from all formulations. The findings, reported for the first time in this study, emphasize the capabilities of the topical application of TAK-242 via these multiple innovative topical drug delivery formulation platforms.

Mefloquine induces ER stress and apoptosis in BRAFi-resistant A375-BRAFV600E /NRASQ61K malignant melanoma cells targeting intracranial tumors in a bioluminescent murine model.

Molecularly targeted therapeutics have revolutionized the treatment of BRAFV600E -driven malignant melanoma, but the rapid development of resistance to BRAF kinase inhibitors (BRAFi) presents a significant obstacle. The use of clinical antimalarials for the investigational treatment of malignant melanoma has shown only moderate promise, attributed mostly to inhibition of lysosomal-autophagic adaptations of cancer cells, but identification of specific antimalarials displaying single-agent antimelanoma activity has remained elusive. Here, we have screened a focused library of clinically used artemisinin-combination therapeutic (ACT) antimalarials for the apoptotic elimination of cultured malignant melanoma cell lines, also examining feasibility of overcoming BRAFi-resistance comparing isogenic melanoma cells that differ only by NRAS mutational status (BRAFi-sensitive A375-BRAFV600E /NRASQ61 vs. BRAFi-resistant A375-BRAFV600E /NRASQ61K ). Among ACT antimalarials tested, mefloquine (MQ) was the only apoptogenic agent causing melanoma cell death at low micromolar concentrations. Comparative gene expression-array analysis (A375-BRAFV600E /NRASQ61 vs. A375-BRAFV600E /NRASQ61K ) revealed that MQ is a dual inducer of endoplasmic reticulum (ER) and redox stress responses that precede MQ-induced loss of viability. ER-trackerTM DPX fluorescence imaging and electron microscopy indicated ER swelling, accompanied by rapid induction of ER stress signaling (phospho-eIF2α, XBP-1s, ATF4). Fluo-4 AM-fluorescence indicated the occurrence of cytosolic calcium overload observable within seconds of MQ exposure. In a bioluminescent murine model employing intracranial injection of A375-Luc2 (BRAFV600E /NRASQ61K ) cells, an oral MQ regimen efficiently antagonized brain tumor growth. Taken together, these data suggest that the clinical antimalarial MQ may be a valid candidate for drug repurposing aiming at chemotherapeutic elimination of malignant melanoma cells, even if metastasized to the brain and BRAFi-resistant.

Vemurafenib Drives Epithelial-to-Mesenchymal Transition Gene Expression in BRAF Inhibitor‒Resistant BRAFV600E/NRASQ61K Melanoma Enhancing Tumor Growth and Metastasis in a Bioluminescent Murine Model.

BRAF inhibitor (BRAFi) resistance compromises long-term survivorship of patients with malignant melanoma, and mutant NRAS is a major mediator of BRAFi resistance. In this study, employing phenotypic and transcriptomic analysis of isogenic melanoma cells that differ only by NRAS mutational status (BRAFi-sensitive A375-BRAFV600E/NRASQ61 vs. BRAFi-resistant A375-BRAFV600E/NRASQ61K), we show that BRAFi (vemurafenib) treatment selectively targets BRAFV600E/NRASQ61K cells upregulating epithelial-to-mesenchymal transition (EMT) gene expression, paradoxically promoting invasiveness and metastasis in vitro and in vivo. First, NanoString nCounter transcriptomic analysis identified the upregulation of specific gene expression networks (EMT and EMT to metastasis) as a function of NRASQ61K status. Strikingly, BRAFi treatment further exacerbated the upregulation of genes promoting EMT in BRAFV600E/NRASQ61K cells (with opposing downregulation of EMT-driver genes in the BRAFV600E/NRASQ61 genotype) as detected by EMT-focused RT2 Profiler qPCR array analysis. In BRAFV600E/NRASQ61K cells, BRAFi treatment enhanced proliferation and invasiveness, together with activation of phosphorylated protein kinase B (Ser473), with opposing phenotypic effects observable in BRAFV600E/NRASQ61 cells displaying downregulation of phosphorylated protein kinase B and phosphorylated extracellular signal-regulated kinase 1/2. In a SCID mouse bioluminescent melanoma metastasis model, BRAFi treatment enhanced lung tumor burden imposed by BRAFV600E/NRASQ61K cells while blocking BRAFV600E/NRASQ61 metastasis. These preclinical data document the BRAFi-driven enhancement of tumorigenesis and metastasis in BRAFi-resistant human BRAFV600E/NRASQ61K melanoma, a finding with potential clinical implications for patients with NRAS-driven BRAFi-resistant tumors receiving BRAFi treatment.

Solar simulated ultraviolet radiation inactivates HCoV-NL63 and SARS-CoV-2 coronaviruses at environmentally relevant doses.

The germicidal properties of short wavelength ultraviolet C (UVC) light are well established and used to inactivate many viruses and other microbes. However, much less is known about germicidal effects of terrestrial solar UV light, confined exclusively to wavelengths in the UVA and UVB regions. Here, we have explored the sensitivity of the human coronaviruses HCoV-NL63 and SARS-CoV-2 to solar-simulated full spectrum ultraviolet light (sUV) delivered at environmentally relevant doses. First, HCoV-NL63 coronavirus inactivation by sUV-exposure was confirmed employing (i) viral plaque assays, (ii) RT-qPCR detection of viral genome replication, and (iii) infection-induced stress response gene expression array analysis. Next, a detailed dose-response relationship of SARS-CoV-2 coronavirus inactivation by sUV was elucidated, suggesting a half maximal suppression of viral infectivity at low sUV doses. Likewise, extended sUV exposure of SARS-CoV-2 blocked cellular infection as revealed by plaque assay and stress response gene expression array analysis. Moreover, comparative (HCoV-NL63 versus SARS-CoV-2) single gene expression analysis by RT-qPCR confirmed that sUV exposure blocks coronavirus-induced redox, inflammatory, and proteotoxic stress responses. Based on our findings, we estimate that solar ground level full spectrum UV light impairs coronavirus infectivity at environmentally relevant doses. Given the urgency and global scale of the unfolding SARS-CoV-2 pandemic, these prototype data suggest feasibility of solar UV-induced viral inactivation, an observation deserving further molecular exploration in more relevant exposure models.

Glyoxalase 1 Expression as a Novel Diagnostic Marker of High-Grade Prostatic Intraepithelial Neoplasia in Prostate Cancer.

Glyoxalase 1 (GLO1) is an enzyme involved in the detoxification of methylglyoxal (MG), a reactive oncometabolite formed in the context of energy metabolism as a result of high glycolytic flux. Prior clinical evidence has documented GLO1 upregulation in various tumor types including prostate cancer (PCa). However, GLO1 expression has not been explored in the context of PCa progression with a focus on high-grade prostatic intraepithelial neoplasia (HGPIN), a frequent precursor to invasive cancer. Here, we have evaluated GLO1 expression by immunohistochemistry in archival tumor samples from 187 PCa patients (stage 2 and 3). Immunohistochemical analysis revealed GLO1 upregulation during tumor progression, observable in HGPIN and PCa versus normal prostatic tissue. GLO1 upregulation was identified as a novel hallmark of HGPIN lesions, displaying the highest staining intensity in all clinical patient specimens. GLO1 expression correlated with intermediate-high risk Gleason grade but not with patient age, biochemical recurrence, or pathological stage. Our data identify upregulated GLO1 expression as a molecular hallmark of HGPIN lesions detectable by immunohistochemical analysis. Since current pathological assessment of HGPIN status solely depends on morphological features, GLO1 may serve as a novel diagnostic marker that identifies this precancerous lesion.

Solar simulated ultraviolet radiation inactivates HCoV-NL63 and SARS-CoV-2 coronaviruses at environmentally relevant doses.

The germicidal properties of short wavelength ultraviolet C (UVC) light are well established and used to inactivate many viruses and other microbes. However, much less is known about germicidal effects of terrestrial solar UV light, confined exclusively to wavelengths in the UVA and UVB regions. Here, we have explored the sensitivity of the human coronaviruses HCoV-NL63 and SARS-CoV-2 to solar-simulated full spectrum ultraviolet light (sUV) delivered at environmentally relevant doses. First, HCoV-NL63 coronavirus inactivation by sUV-exposure was confirmed employing (i) viral plaque assays, (ii) RT-qPCR detection of viral genome replication, and (iii) infection-induced stress response gene expression array analysis. Next, a detailed dose-response relationship of SARS-CoV-2 coronavirus inactivation by sUV was elucidated, suggesting a half maximal suppression of viral infectivity at low sUV doses. Likewise, extended sUV exposure of SARS-CoV-2 blocked cellular infection as revealed by plaque assay and stress response gene expression array analysis. Moreover, comparative (HCoV-NL63 versus SARS-CoV-2) single gene expression analysis by RT-qPCR confirmed that sUV exposure blocks coronavirus-induced redox, inflammatory, and proteotoxic stress responses. Based on our findings, we estimate that solar ground level full spectrum UV light impairs coronavirus infectivity at environmentally relevant doses. Given the urgency and global scale of the unfolding SARS-CoV-2 pandemic, these prototype data suggest feasibility of solar UV-induced viral inactivation, an observation deserving further molecular exploration in more relevant exposure models.

Topical hypochlorous acid (HOCl) blocks inflammatory gene expression and tumorigenic progression in UV-exposed SKH-1 high risk mouse skin.

Hypochlorous acid (HOCl) is the active oxidizing principle underlying drinking water disinfection, also delivered by numerous skin disinfectants and released by standard swimming pool chemicals used on a global scale, a topic of particular relevance in the context of the ongoing COVID-19 pandemic. However, the cutaneous consequences of human exposure to HOCl remain largely unknown, posing a major public health concern. Here, for the first time, we have profiled the HOCl-induced stress response in reconstructed human epidermis and SKH-1 hairless mouse skin. In addition, we have investigated the molecular consequences of solar simulated ultraviolet (UV) radiation and HOCl combinations, a procedure mimicking co-exposure experienced for example by recreational swimmers exposed to both HOCl (pool disinfectant) and UV (solar radiation). First, gene expression elicited by acute topical HOCl exposure was profiled in organotypic human reconstructed epidermis. Next, co-exposure studies (combining topical HOCl and UV) performed in SKH-1 hairless mouse skin revealed that the HOCl-induced cutaneous stress response blocks redox and inflammatory gene expression elicited by subsequent acute UV exposure (Nos2, Ptgs2, Hmox1, Srxn1), a finding consistent with emerging clinical evidence in support of a therapeutic role of topical HOCl formulations for the suppression of inflammatory skin conditions (e.g. atopic dermatitis, psoriasis). Likewise, in AP-1 transgenic SKH-1 luciferase-reporter mice, topical HOCl suppressed UV-induced inflammatory signaling assessed by bioluminescent imaging and gene expression analysis. In the SKH-1 high-risk mouse model of UV-induced human keratinocytic skin cancer, topical HOCl blocked tumorigenic progression and inflammatory gene expression (Ptgs2, Il19, Tlr4), confirmed by immunohistochemical analysis including 3-chloro-tyrosine-epitopes. These data illuminate the molecular consequences of HOCl-exposure in cutaneous organotypic and murine models assessing inflammatory gene expression and modulation of UV-induced carcinogenesis. If translatable to human skin these observations provide novel insights on molecular consequences of chlorination stress relevant to environmental exposure and therapeutic intervention.

Deuterium Oxide (D2O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma.

There are two stable isotopes of hydrogen, protium (1H) and deuterium (2H; D). Cellular stress response dysregulation in cancer represents both a major pathological driving force and a promising therapeutic target, but the molecular consequences and potential therapeutic impact of deuterium (2H)-stress on cancer cells remain largely unexplored. We have examined the anti-proliferative and apoptogenic effects of deuterium oxide (D2O; 'heavy water') together with stress response gene expression profiling in panels of malignant melanoma (A375V600E, A375NRAS, G361, LOX-IMVI), and pancreatic ductal adenocarcinoma (PANC-1, Capan-2, or MIA PaCa-2) cells with inclusion of human diploid Hs27 skin fibroblasts. Moreover, we have examined the efficacy of D2O-based pharmacological intervention in murine models of human melanoma tumor growth and metastasis. D2O-induction of apoptosis was substantiated by AV-PI flow cytometry, immunodetection of PARP-1, and pro-caspase 3 cleavage, and rescue by pan-caspase inhibition. Differential array analysis revealed early modulation of stress response gene expression in both A375 melanoma and PANC-1 adenocarcinoma cells elicited by D2O (90%; ≤6 h) (upregulated: CDKN1A, DDIT3, EGR1, GADD45A, HMOX1, NFKBIA, or SOD2 (up to 9-fold; p < 0.01)) confirmed by independent RT-qPCR analysis. Immunoblot analysis revealed rapid onset of D2O-induced stress response phospho-protein activation (p-ERK, p-JNK, p-eIF2α, or p-H2AX) or attenuation (p-AKT). Feasibility of D2O-based chemotherapeutic intervention (drinking water (30% w/w)) was demonstrated in a severe combined immunodeficiency (SCID) mouse melanoma metastasis model using luciferase-expressing A375-Luc2 cells. Lung tumor burden (visualized by bioluminescence imaging) was attenuated by D2O, and inhibition of invasiveness was also confirmed in an in vitro Matrigel transwell invasion assay. D2O supplementation also suppressed tumor growth in a murine xenograft model of human melanoma, and median survival was significantly increased without causing adverse effects. These data demonstrate for the first time that systemic D2O administration impairs growth and metastasis of malignant melanoma through the pharmacological induction of deuterium (2H)-stress.

Translational Advances in Cancer Prevention Agent Development (TACPAD) Virtual Workshop on Immunomodulatory Agents: Report.

The National Cancer Institute (NCI) Division of Cancer Prevention (DCP) convened the "Translational Advances in Cancer Prevention Agent Development (TACPAD) Workshop on Immunomodulatory Agents" as a virtual 2-day workshop on September 13 to 14, 2021. The main goals of this workshop were to foster the exchange of ideas and potentially new collaborative interactions among leading cancer immunoprevention researchers from basic and clinical research and highlight new and emerging trends in immunoprevention. The workshop included an overview of the mechanistic classes of immunomodulatory agents and three sessions covering the gamut from preclinical to clinical studies. The workshop convened individuals working in immunology and cancer prevention to discuss trends in discovery and development of immunomodulatory agents individually and in combination with other chemopreventive agents or vaccines.

The Endogenous Tryptophan-derived Photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) is a Nanomolar Photosensitizer that Can be Harnessed for the Photodynamic Elimination of Skin Cancer Cells in Vitro and in Vivo.

UV-chromophores contained in human skin may act as endogenous sensitizers of photooxidative stress and can be employed therapeutically for the photodynamic elimination of malignant cells. Here, we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan-derived photoproduct and endogenous aryl hydrocarbon receptor agonist, displays activity as a nanomolar sensitizer of photooxidative stress, causing the photodynamic elimination of human melanoma and nonmelanoma skin cancer cells in vitro and in vivo. FICZ is an efficient UVA/Visible photosensitizer having absorbance maximum at 390 nm (ε = 9180 L mol-1  cm-1 ), and fluorescence and singlet oxygen quantum yields of 0.15 and 0.5, respectively, in methanol. In a panel of cultured human squamous cell carcinoma and melanoma skin cancer cells (SCC-25, HaCaT-ras II-4, A375, G361, LOX), photodynamic induction of cell death was elicited by the combined action of solar simulated UVA (6.6 J cm-2 ) and FICZ (≥10 nm), preceded by the induction of oxidative stress as substantiated by MitoSOX Red fluorescence microscopy, comet detection of Fpg-sensitive oxidative genomic lesions and upregulated stress response gene expression (HMOX1, HSPA1A, HSPA6). In SKH1 "high-risk" mouse skin, an experimental FICZ/UVA photodynamic treatment regimen blocked the progression of UV-induced tumorigenesis suggesting feasibility of harnessing FICZ for the photooxidative elimination of malignant cells in vivo.

Genomic GLO1 deletion modulates TXNIP expression, glucose metabolism, and redox homeostasis while accelerating human A375 malignant melanoma tumor growth.

Glyoxalase 1 (encoded by GLO1) is a glutathione-dependent enzyme detoxifying the glycolytic byproduct methylglyoxal (MG), an oncometabolite involved in metabolic reprogramming. Recently, we have demonstrated that GLO1 is overexpressed in human malignant melanoma cells and patient tumors and substantiated a novel role of GLO1 as a molecular determinant of invasion and metastasis in melanoma. Here, employing NanoString™ gene expression profiling (nCounter™ 'PanCancer Progression Panel'), we report that CRISPR/Cas 9-based GLO1 deletion from human A375 malignant melanoma cells alters glucose metabolism and redox homeostasis, observable together with acceleration of tumorigenesis. Nanostring™ analysis identified TXNIP (encoding thioredoxin-interacting protein), a master regulator of cellular energy metabolism and redox homeostasis, displaying the most pronounced expression change in response to GLO1 elimination, confirmed by RT-qPCR and immunoblot analysis. TXNIP was also upregulated in CRISPR/Cas9-engineered DU145 prostate carcinoma cells lacking GLO1, and treatment with MG or a pharmacological GLO1 inhibitor (TLSC702) mimicked GLO1_KO status, suggesting that GLO1 controls TXNIP expression through regulation of MG. GLO1_KO status was characterized by (i) altered oxidative stress response gene expression, (ii) attenuation of glucose uptake and metabolism with downregulation of gene expression (GLUT1, GFAT1, GFAT2, LDHA) and depletion of related key metabolites (glucose-6-phosphate, UDP-N-acetylglucosamine), and (iii) immune checkpoint modulation (PDL1). While confirming our earlier finding that GLO1 deletion limits invasion and metastasis with modulation of EMT-related genes (e.g. TGFBI, MMP9, ANGPTL4, TLR4, SERPINF1), we observed that GLO1_KO melanoma cells displayed a shortened population doubling time, cell cycle alteration with increased M-phase population, and enhanced anchorage-independent growth, a phenotype supported by expression analysis (CXCL8, CD24, IL1A, CDKN1A). Concordantly, an accelerated growth rate of GLO1_KO tumors, accompanied by TXNIP overexpression and metabolic reprogramming, was observable in a SCID mouse melanoma xenograft model, demonstrating that A375 melanoma tumor growth and metastasis can be dysregulated in opposing ways as a consequence of GLO1 elimination.

Activation of NRF2 by topical apocarotenoid treatment mitigates radiation-induced dermatitis.

Radiation therapy is a frontline treatment option for cancer patients; however, the effects of radiotherapy on non-tumor tissue (e.g. radiation-induced dermatitis) often worsen patient quality of life. Previous studies have implicated the importance of redox balance in preventing dermatitis, specifically in reference to modulation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling pathway. Due to the cytoprotective functions of transcriptional target genes of NRF2, we investigated how modulation of NRF2 expression could affect DNA damage, oxidative stress, and cell viability in response to radiotherapy. Specifically, it was noted that NRF2 knockdown sensitized human skin keratinocytes to ionizing radiation; likewise, genetic ablation of NRF2 in vivo increased radiosensitivity of murine epidermis. Oppositely, pharmacological induction of NRF2 via the apocarotenoid bixin lowered markers of DNA damage and oxidative stress, while preserving viability in irradiated keratinocytes. Mechanistic studies indicated that topical pretreatment using bixin as an NRF2 activator antagonized initial DNA damage by raising cellular glutathione levels. Additionally, topical application of bixin prevented radiation-induced dermatitis, epidermal thickening, and oxidative stress in the skin of SKH1 mice. Overall, these data indicate that NRF2 is critical for mitigating the harmful skin toxicities associated with ionizing radiation, and that topical upregulation of NRF2 via bixin could prevent radiation-induced dermatitis.

The sunless tanning agent dihydroxyacetone induces stress response gene expression and signaling in cultured human keratinocytes and reconstructed epidermis.

Sunless (chemical) tanning is widely regarded as a safe alternative to solar UV-induced skin tanning known to be associated with epidermal genotoxic stress, but the cutaneous biology impacted by chemical tanning remains largely unexplored. Chemical tanning is based on the formation of melanin-mimetic cutaneous pigments ('melanoidins') from spontaneous amino-carbonyl ('glycation') reactions between epidermal amino acid/protein components and reactive sugars including the glycolytic ketose dihydroxyacetone (DHA). Here, we have examined the cutaneous effects of acute DHA-exposure on cultured human HaCaT keratinocytes and epidermal reconstructs, profiled by gene expression array analysis and immunodetection. In keratinocytes, DHA-exposure performed at low millimolar concentrations did not impair viability while causing a pronounced cellular stress response as obvious from rapid activation of phospho-protein signal transduction [p-p38, p-Hsp27(S15/S78), p-eIF2α] and gene expression changes (HSPA6, HMOX1, CRYAB, CCL3), not observable upon exposure to the non-ketose, tanning-inactive DHA-control glycerol. Formation of advanced glycation end products (AGEs) from posttranslational protein-adduction was confirmed by quantitative mass spectrometric detection of N-ε-(carboxyethyl)-l-lysine (CEL) and N7-carboxyethyl-l-arginine, and skin cells with CRISPR-Cas9-based elimination of the carbonyl stress response gene GLO1 (encoding glyoxalase 1) displayed hypersensitivity to DHA-cytotoxicity. In human epidermal reconstructs a topical use-relevant DHA-dose regimen elicited a comparable stress response as revealed by gene expression array (HSPA1A, HSPA6, HSPD1, IL6, DDIT3, EGR1) and immunohistochemical analysis (CEL, HO-1, p-Hsp27-S78). In DHA-treated SKH-1 hairless mouse skin IHC-detection revealed epidermal occurrence of CEL- and p-Hsp27-epitopes. For comparison, stress response gene expression array analysis was performed in epidermis exposed to a supra-erythemal dose of solar simulated UV (2 MEDs), identifying genes equally or differentially sensitive to either one of these cutaneous stimuli [DHA ('sunless tanning') versus solar UV ('sun-induced tanning')]. Given the worldwide use of chemical tanners in consumer products, these prototype data documenting a DHA-induced specific cutaneous stress response deserve further molecular exploration in living human skin.

Genetic Target Modulation Employing CRISPR/Cas9 Identifies Glyoxalase 1 as a Novel Molecular Determinant of Invasion and Metastasis in A375 Human Malignant Melanoma Cells In Vitro and In Vivo.

Metabolic reprogramming is a molecular hallmark of cancer. Recently, we have reported the overexpression of glyoxalase 1 (encoded by GLO1), a glutathione-dependent enzyme involved in detoxification of the reactive glycolytic byproduct methylglyoxal, in human malignant melanoma cell culture models and clinical samples. However, the specific role of GLO1 in melanomagenesis remains largely unexplored. Here, using genetic target modulation, we report the identification of GLO1 as a novel molecular determinant of invasion and metastasis in malignant melanoma. First, A375 human malignant melanoma cells with GLO1 deletion (A375-GLO1_KO) were engineered using CRISPR/Cas9, and genetic rescue clones were generated by stable transfection of KO clones employing a CMV-driven GLO1 construct (A375-GLO1_R). After confirming GLO1 target modulation at the mRNA and protein levels (RT-qPCR, immunodetection, enzymatic activity), phenotypic characterization indicated that deletion of GLO1 does not impact proliferative capacity while causing significant sensitization to methylglyoxal-, chemotherapy-, and starvation-induced cytotoxic stress. Employing differential gene expression array analysis (A375-GLO1_KO versus A375-GLO1_WT), pronounced modulation of epithelial--mesenchymal transition (EMT)-related genes [upregulated: CDH1, OCLN, IL1RN, PDGFRB, SNAI3; (downregulated): BMP1, CDH2, CTNNB1, FN1, FTH1, FZD7, MELTF, MMP2, MMP9, MYC, PTGS2, SNAI2, TFRC, TWIST1, VIM, WNT5A, ZEB1, and ZEB2 (up to tenfold; p < 0.05)] was observed-all of which are consistent with EMT suppression as a result of GLO1 deletion. Importantly, these expression changes were largely reversed upon genetic rescue employing A375-GLO1_R cells. Differential expression of MMP9 as a function of GLO1 status was further substantiated by enzymatic activity and ELISA analysis; phenotypic assessment revealed the pronounced attenuation of morphological potential, transwell migration, and matrigel 3D-invasion capacity displayed by A375-GLO1_KO cells, reversed again in genetic rescue clones. Strikingly, in a SCID mouse metastasis model, lung tumor burden imposed by A375-GLO1_KO cells was strongly attenuated as compared to A375-GLO1_WT cells. Taken together, these prototype data provide evidence in support of a novel function of GLO1 in melanoma cell invasiveness and metastasis, and ongoing investigations explore the function and therapeutic potential of GLO1 as a novel melanoma target.