Timothy L. Scott

UV Radiation and the Skin

UV radiation (UV) is classified as a “complete carcinogen” because it is both a mutagen and a non-specific damaging agent and has properties of both a tumor initiator and a tumor promoter. In environmental abundance, UV is the most important modifiable risk factor for skin cancer and many other environmentally-influenced skin disorders. However, UV also benefits human health by mediating natural synthesis of vitamin D and endorphins in the skin, therefore UV has complex and mixed effects on human health. Nonetheless, excessive exposure to UV carries profound health risks, including atrophy, pigmentary changes, wrinkling and malignancy. UV is epidemiologically and molecularly linked to the three most common types of skin cancer, basal cell carcinoma, squamous cell carcinoma and malignant melanoma, which together affect more than a million Americans annually. Genetic factors also influence risk of UV-mediated skin disease. Polymorphisms of the melanocortin 1 receptor (MC1R) gene, in particular, correlate with fairness of skin, UV sensitivity, and enhanced cancer risk. We are interested in developing UV-protective approaches based on a detailed understanding of molecular events that occur after UV exposure, focusing particularly on epidermal melanization and the role of the MC1R in genome maintenance.

Repair of Oxidative DNA Damage and Cancer: Recent Progress in DNA Base Excision Repair

SIGNIFICANCE Reactive oxygen species (ROS) are generated by exogenous and environmental genotoxins, but also arise from mitochondria as byproducts of respiration in the body. ROS generate DNA damage of which pathological consequence, including cancer is well established. Research efforts are intense to understand the mechanism of DNA base excision repair, the primary mechanism to protect cells from genotoxicity caused by ROS. RECENT ADVANCES In addition to the notion that oxidative DNA damage causes transformation of cells, recent studies have revealed how the mitochondrial deficiencies and ROS generation alter cell growth during the cancer transformation. CRITICAL ISSUES The emphasis of this review is to highlight the importance of the cellular response to oxidative DNA damage during carcinogenesis. Oxidative DNA damage, including 7,8-dihydro-8-oxoguanine, play an important role during the cellular transformation. It is also becoming apparent that the unusual activity and subcellular distribution of apurinic/apyrimidinic endonuclease 1, an essential DNA repair factor/redox sensor, affect cancer malignancy by increasing cellular resistance to oxidative stress and by positively influencing cell proliferation. FUTURE DIRECTIONS Technological advancement in cancer cell biology and genetics has enabled us to monitor the detailed DNA repair activities in the microenvironment. Precise understanding of the intracellular activities of DNA repair proteins for oxidative DNA damage should provide help in understanding how mitochondria, ROS, DNA damage, and repair influence cancer transformation.

Prolonged treatment of fair-skinned mice with topical forskolin causes persistent tanning and UV protection.

We previously reported that topical application of forskolin to the skin of fair‐skinned MC1R‐defective mice with epidermal melanocytes resulted in accumulation of eumelanin in the epidermis and was highly protective against UV‐mediated cutaneous injury. In this report, we describe the long‐term effects of chronic topical forskolin treatment in this animal model. Forskolin‐induced eumelanin production persisted through 3 months of daily applications, and forskolin‐induced eumelanin remained protective against UV damage as assessed by minimal erythematous dose (MED). No obvious toxic changes were noted in the skin or overall health of animals exposed to prolonged forskolin therapy. Body weights were maintained throughout the course of topical forskolin application. Topical application of forskolin was associated with an increase in the number of melanocytes in the epidermis and thickening of the epidermis due, at least in part, to an accumulation of nucleated keratinocytes. Together, these data suggest in this animal model, short‐term topical regular application of forskolin promotes eumelanin induction and that over time, topical forskolin treatment is associated with persistent melanization, epidermal cell accumulation, and skin thickening.

Pigment-independent cAMP-mediated epidermal thickening protects against cutaneous UV injury by keratinocyte proliferation

The epidermis increases pigmentation and epidermal thickness in response to ultraviolet exposure to protect against UV‐associated carcinogenesis; however, the contribution of epidermal thickness has been debated. In a humanized skin mouse model that maintains interfollicular epidermal melanocytes, we found that forskolin, a small molecule that directly activates adenylyl cyclase and promotes cAMP generation, up‐regulated epidermal eumelanin accumulation in fair‐skinned melanocortin‐1‐receptor (Mc1r)‐defective animals. Forskolin‐induced pigmentation was associated with a reproducible expansion of epidermal thickness irrespective of melanization or the presence of epidermal melanocytes. Rather, forskolin‐enhanced epidermal thickening was mediated through increased keratinocyte proliferation, indirectly through secreted factor(s) from cutaneous fibroblasts. We identified keratinocyte growth factor (Kgf) as a forskolin‐induced fibroblast‐derived cytokine that promoted keratinocyte proliferation, as forskolin induced Kgf expression both in the skin and in primary fibroblasts. Lastly, we found that even in the absence of pigmentation, forskolin‐induced epidermal thickening significantly diminished the amount of UV‐A and UV‐B that passed through whole skin and reduced the amount of UV‐B‐associated epidermal sunburn cells. These findings suggest the possibility of pharmacologic‐induced epidermal thickening as a novel UV‐protective therapeutic intervention, particularly for individuals with defects in pigmentation and adaptive melanization.

Noninvasive Liquid Diet Delivery of Stable Isotopes into Mouse Models for Deep Metabolic Network Tracing

Delivering isotopic tracers for metabolic studies in rodents without overt stress is challenging. Current methods achieve low label enrichment in proteins and lipids. Here, we report noninvasive introduction of 13C6-glucose via a stress-free, ad libitum liquid diet. Using NMR and ion chromatography-mass spectrometry, we quantify extensive 13C enrichment in products of glycolysis, the Krebs cycle, the pentose phosphate pathway, nucleobases, UDP-sugars, glycogen, lipids, and proteins in mouse tissues during 12 to 48 h of 13C6-glucose feeding. Applying this approach to patient-derived lung tumor xenografts (PDTX), we show that the liver supplies glucose-derived Gln via the blood to the PDTX to fuel Glu and glutathione synthesis while gluconeogenesis occurs in the PDTX. Comparison of PDTX with ex vivo tumor cultures and arsenic-transformed lung cells versus xenografts reveals differential glucose metabolism that could reflect distinct tumor microenvironment. We further found differences in glucose metabolism between the primary PDTX and distant lymph node metastases.Isotope tracer administration for probing metabolism in vivo is important to assess metabolic functions in a relevant physiological setting. Here, the authors report a non-invasive method of administering 13C6- glucose to mouse models via liquid diet feeding to achieve deep metabolic network coverage.

Inorganic arsenic inhibits the nucleotide excision repair pathway and reduces the expression of XPC

Chronic exposure to arsenic, most often through contaminated drinking water, has been linked to several types of cancer in humans, including skin and lung cancer. However, the mechanisms underlying its role in causing cancer are not well understood. There is evidence that exposure to arsenic can enhance the carcinogenicity of UV light in inducing skin cancers and may enhance the carcinogenicity of tobacco smoke in inducing lung cancers. The nucleotide excision repair (NER) pathway removes different types of DNA damage including those produced by UV light and components of tobacco smoke. The aim of the present study was to investigate the effect of sodium arsenite on the NER pathway in human lung fibroblasts (IMR-90 cells) and primary mouse keratinocytes. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts (6-4 PP) and cyclobutane pyrimidine dimers (CPDs). We find a concentration-dependent inhibition of the removal of 6-4 PPs and CPDs in both cell types treated with arsenite. Treatment of both cell types with arsenite resulted in a significant reduction in the abundance of XPC, a protein that is critical for DNA damage recognition in NER. The abundance of RNA expressed from several key NER genes was also significantly reduced by treatment of IMR-90 cells with arsenite. Finally, treatment of IMR-90 cells with MG-132 abrogated the reduction in XPC protein, suggesting an involvement of the proteasome in the reduction of XPC protein produced by treatment of cells with arsenic. The inhibition of NER by arsenic may reflect one mechanism underlying the role of arsenic exposure in enhancing cigarette smoke-induced lung carcinogenesis and UV light-induced skin cancer, and it may provide some insights into the emergence of arsenic trioxide as a chemotherapeutic agent.

Purification and growth of melanocortin 1 receptor (Mc1r)-defective primary murine melanocytes is dependent on stem cell factor (SFC) from keratinocyte-conditioned media

The melanocortin 1 receptor (MC1R) is a transmembrane Gs-coupled surface protein found on melanocytes that binds melanocyte-stimulating hormone and mediates activation of adenylyl cyclase and generation of the second messenger cyclic AMP (cAMP). MC1R regulates growth and differentiation of melanocytes and protects against carcinogenesis. Persons with loss-of-function polymorphisms of MC1R tend to be UV-sensitive (fair-skinned and with a poor tanning response) and are at high risk for melanoma. Mechanistic studies of the role of MC1R in melanocytic UV responses, however, have been hindered in part because Mc1r-defective primary murine melanocytes have been difficult to culture in vitro. Until now, effective growth of murine melanocytes has depended on cAMP stimulation with adenylyl cyclase-activating or phosphodiesterase-inhibiting agents. However, rescuing cAMP in the setting of defective MC1R signaling would be expected to confound experiments directly testing MC1R function on melanocytic UV responses. In this paper, we report a novel method of culturing primary murine melanocytes in the absence of pharmacologic cAMP stimulation by incorporating conditioned supernatants containing stem cell factor derived from primary keratinocytes. Importantly, this method seems to permit similar pigment expression by cultured melanocytes as that found in the skin of their parental murine strains. This novel approach will allow mechanistic investigation into MC1R’s role in the protection against UV-mediated carcinogenesis and determination of the role of melanin pigment subtypes on UV-mediated melanocyte responses.

Polyubiquitination of apurinic/apyrimidinic endonuclease 1 by Parkin.

Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential protein crucial for repair of oxidized DNA damage not only in genomic DNA but also in mitochondrial DNA. Parkin, a tumor suppressor and Parkinsons disease (PD) associated gene, is an E3 ubiquitin ligase crucial for mitophagy. Although DNA damage is known to induce mitochondrial stress, Parkins role in regulating DNA repair proteins has not been elucidated. In this study, we examined the possibility of Parkin‐dependent ubiquitination of APE1. Ectopically expressed APE1 was degraded by Parkin and PINK1 via polyubiquitination in mouse embryonic fibroblast cells. PD‐causing mutations in Parkin and PINK1 abrogated APE1 ubiquitination. Interaction of APE1 with Parkin was observed by co‐immunoprecipitation, proximity ligation assay, and co‐localization in the cytoplasm. N‐terminal deletion of 41 amino acid residues in APE1 significantly reduced the Parkin‐dependent APE1 degradation. These results suggested that Parkin directly ubiquitinated N‐terminal Lys residues in APE1 in the cytoplasm. Modulation of Parkin and PINK1 activities under mitochondrial or oxidative stress caused moderate but statistically significant decrease of endogenous APE1 in human cell lines including SH‐SY5Y, HEK293, and A549 cells. Analyses of glioblastoma tissues showed an inverse relation between the expression levels of APE1 and Parkin. These results suggest that degradation of endogenous APE1 by Parkin occur when cells are stressed to activate Parkin, and imply a role of Parkin in maintaining the quality of APE1, and loss of Parkin may contribute to elevated APE1 levels in glioblastoma.

Preclinical evaluation of novel fatty acid synthase inhibitors in primary colorectal cancer cells and a patient-derived xenograft model of colorectal cancer

Fatty Acid Synthase (FASN), a key enzyme of de novo lipogenesis, is upregulated in many cancers including colorectal cancer (CRC); increased FASN expression is associated with poor prognosis. Potent FASN inhibitors (TVBs) developed by 3-V Biosciences demonstrate anti-tumor activity in vitro and in vivo and a favorable tolerability profile in a Phase I clinical trial. However, CRC characteristics associated with responsiveness to FASN inhibition are not fully understood. We evaluated the effect of TVB-3664 on tumor growth in nine CRC patient-derived xenografts (PDXs) and investigated molecular and metabolic changes associated with CRC responsiveness to FASN inhibition. CRC cells and PDXs showed a wide range of sensitivity to FASN inhibition. TVB-3664 treatment showed significant response (reduced tumor volume) in 30% of cases. Anti-tumor effect of TVB-3664 was associated with a significant decrease in a pool of adenine nucleotides and alterations in lipid composition including a significant reduction in fatty acids and phospholipids and an increase in lactosylceramide and sphingomyelin in PDXs sensitive to FASN inhibition. Moreover, Akt, Erk1/2 and AMPK were major oncogenic pathways altered by TVBs. In summary, we demonstrated that novel TVB inhibitors show anti-tumor activity in CRC and this activity is associated with a decrease in activation of Akt and Erk1/2 oncogenic pathways and significant alteration of lipid composition of tumors. Further understanding of genetic and metabolic characteristics of tumors susceptible to FASN inhibition may enable patient selection and personalized medicine approaches in CRC.

Collagen prolyl 4-hydroxylase 1 is essential for HIF-1α stabilization and TNBC chemoresistance

Collagen prolyl 4-hydroxylase (P4H) expression and collagen hydroxylation in cancer cells are necessary for breast cancer progression. Here, we show that P4H alpha 1 subunit (P4HA1) protein expression is induced in triple-negative breast cancer (TNBC) and HER2 positive breast cancer. By modulating alpha ketoglutarate (α-KG) and succinate levels P4HA1 expression reduces proline hydroxylation on hypoxia-inducible factor (HIF) 1α, enhancing its stability in cancer cells. Activation of the P4HA/HIF-1 axis enhances cancer cell stemness, accompanied by decreased oxidative phosphorylation and reactive oxygen species (ROS) levels. Inhibition of P4HA1 sensitizes TNBC to the chemotherapeutic agent docetaxel and doxorubicin in xenografts and patient-derived models. We also show that increased P4HA1 expression correlates with short relapse-free survival in TNBC patients who received chemotherapy. These results suggest that P4HA1 promotes chemoresistance by modulating HIF-1-dependent cancer cell stemness. Targeting collagen P4H is a promising strategy to inhibit tumor progression and sensitize TNBC to chemotherapeutic agents.Hyperactivation of HIF-1α is crucial in progression of triple-negative breast cancer, but how HIF-1α stability is maintained in a hypoxia-independent manner is unclear. Here, the authors show collagen prolyl-4-hydroylase 1 stabilises HIF-1α and is involved in chemoresistance in TNBC.