Yira Bermudez

Functional protein pathway activation mapping of the progression of normal skin to squamous cell carcinoma.

Reverse phase protein microarray analysis was used to identify cell signaling derangements in squamous cell carcinoma (SCC) compared with actinic keratosis (AK) and upper inner arm (UIA). We analyzed two independent tissue sets with isolation and enrichment of epithelial cells by laser capture microdissection. Set 1 served as a pilot and a means to identify protein pathway activation alterations that could be further validated in a second independent set. Set 1 was comprised of 4 AK, 13 SCC, and 20 UIA. Set 2 included 15 AK, 9 SCCs, and 20 UIAs. Activation of 51 signaling proteins, known to be involved in tumorigenesis, were assessed for set 1 and showed that the MEK–ERK [mitogen-activated protein (MAP)/extracellular signal-regulated (ERK; MEK)] pathway was activated in SCC compared with AK and UIA, and that epidermal growth factor receptor (EGFR) and mTOR pathways were aberrantly activated in SCC. Unsupervised two-way hierarchical clustering revealed that AK and UIA shared a common signaling network activation architecture while SCC was dramatically different. Statistical analysis found that prosurvival signaling through phosphorylation of ASK and 4EBP1 as well as increased Bax and Bak expression was higher in AK compared with UIA. We expanded pathway network activation mapping in set 2 to 101 key signaling proteins, which corroborated activation of MEK–ERK, EGFR, and mTOR pathways through discovery of a number of upstream and downstream signaling molecules within these pathways to conclude that SCC is indeed a pathway activation–driven disease. Pathway activation mapping of SCC compared with AK revealed several interconnected networks that could be targeted with drug therapy for potential chemoprevention and therapeutic applications. Cancer Prev Res; 5(3); 403–13. ©2012 AACR.

Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation

Background Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through Gi-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells. Results Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional Gi-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional. Conclusions The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s) of nicotinic acid receptors in human skin homeostasis.

Activation of the PI3K/Akt/mTOR and MAPK Signaling Pathways in Response to Acute Solar-Simulated Light Exposure of Human Skin

The incidence of skin cancer is higher than all other cancers and continues to increase, with an average annual cost over

Malondialdehyde-derived epitopes in human skin result from acute exposure to solar UV and occur in nonmelanoma skin cancer tissue.

8 billion in the United States. As a result, identifying molecular pathway alterations that occur with UV exposure to strategize more effective preventive and therapeutic approaches is essential. To that end, we evaluated phosphorylation of proteins within the PI3K/Akt and MAPK pathways by immunohistochemistry in sun-protected skin after acute doses of physiologically relevant solar-simulated ultraviolet light (SSL) in 24 volunteers. Biopsies were performed at baseline, 5 minutes, 1, 5, and 24 hours after SSL irradiation. Within the PI3K/Akt pathway, we found activation of Akt (serine 473) to be significantly increased at 5 hours while mTOR (serine 2448) was strongly activated early and was sustained over 24 hours after SSL. Downstream, we observed a marked and sustained increase in phospho-S6 (serine 235/S236), whereas phospho-4E-BP1 (threonines 37/46) was increased only at 24 hours. Within the MAPK pathway, SSL-induced expression of phospho-p38 (threonine 180/tyrosine 182) peaked at 1 to 5 hours. ERK 1/2 was observed to be immediate and sustained after SSL irradiation. Phosphorylation of histone H3 (serine 10), a core structural protein of the nucleosome, peaked at 5 hours after SSL irradiation. The expression of both p53 and COX-2 was increased at 5 hours and was maximal at 24 hours after SSL irradiation. Apoptosis was significantly increased at 24 hours as expected and indicative of a sunburn-type response to SSL. Understanding the timing of key protein expression changes in response to SSL will aid in development of mechanistic-based approaches for the prevention and control of skin cancers. Cancer Prev Res; 8(8); 720–8. ©2015 AACR.

Phase IIB Randomized Study of Topical Difluoromethylornithine and Topical Diclofenac on Sun-Damaged Skin of the Forearm

Cutaneous exposure to solar ultraviolet radiation (UVR) is a causative factor in photoaging and photocarcinogenesis. In human skin, oxidative stress is widely considered a key mechanism underlying the detrimental effects of acute and chronic UVR exposure. The lipid peroxidation product malondialdehyde (MDA) accumulates in tissue under conditions of increased oxidative stress, and the occurrence of MDA-derived protein epitopes, including dihydropyridine-lysine (DHP), has recently been substantiated in human skin. Here we demonstrate for the first time that acute exposure to sub-apoptogenic doses of solar simulated UV light (SSL) causes the formation of free MDA and protein-bound MDA-derived epitopes in cultured human HaCaT keratinocytes and healthy human skin. Immunohistochemical staining revealed that acute exposure to SSL is sufficient to cause an almost twenty-fold increase in general MDA- and specific DHP-epitope content in human skin. When compared to dose-matched solar simulated UVA, complete SSL was more efficient generating both free MDA and MDA-derived epitopes. Subsequent tissue microarray (TMA) analysis revealed the prevalence of MDA- and DHP-epitopes in nonmelanoma skin cancer (NMSC). In squamous cell carcinoma tissue, both MDA- and DHP-epitopes were increased more than threefold as compared to adjacent normal tissue. Taken together, these date demonstrate the occurrence of MDA-derived epitopes in both solar UVR-exposed healthy human skin and NMSC TMA tissue; however, the potential utility of these epitopes as novel biomarkers of cutaneous photodamage and a functional role in the process of skin photocarcinogenesis remain to be explored.

Pilot study on the bioactivity of vitamin d in the skin after oral supplementation.

Prevention of nonmelanoma skin cancers remains a health priority due to high costs associated with this disease. Diclofenac and difluoromethylornithine (DFMO) have demonstrated chemopreventive efficacy for cutaneous squamous cell carcinomas. We designed a randomized study of the combination of DFMO and diclofenac in the treatment of sun-damaged skin. Individuals with visible cutaneous sun damage were eligible. Subjects were randomized to one of the three groups: topical DFMO applied twice daily, topical diclofenac applied daily, or DFMO plus diclofenac. The treatment was limited to an area on the left forearm, and the duration of use was 90 days. We hypothesized that combination therapy would have increased efficacy compared with single-agent therapy. The primary outcome was change in karyometric average nuclear abnormality (ANA) in the treated skin. Individuals assessing the biomarkers were blinded regarding the treatment for each subject. A total of 156 subjects were randomized; 144 had baseline and end-of-study biopsies, and 136 subjects completed the study. The ANA unexpectedly increased for all groups, with higher values correlating with clinical cutaneous inflammation. Nearly all of the adverse events were local cutaneous effects. One subject had cutaneous toxicity that required treatment discontinuation. Significantly more adverse events were seen in the groups taking diclofenac. Overall, the study indicated that the addition of topical DFMO to topical diclofenac did not enhance its activity. Both agents caused inflammation on a cellular and clinical level, which may have confounded the measurement of chemopreventive effects. More significant effects may be observed in subjects with greater baseline cutaneous damage. Cancer Prev Res; 9(2); 128–34. ©2015 AACR. See related article by Tsai and Hawk, p. 125

Abstract 3248: Pilot study on the bioactivity of vitamin D in the skin after oral supplementation

Laboratory studies suggest that vitamin D (VD) supplementation inhibits skin carcinogenesis. However, epidemiologic studies report mixed findings in the association between circulating VD levels and skin cancer risk. We conducted a clinical study to determine whether oral cholecalciferol supplementation would exert direct bioactivity in human skin through modulation of the VD receptor (VDR). We enrolled 25 individuals with serum 25-hydroxyvitamin-D levels <30 ng/mL and with skin photodamage to take 50,000 IU of cholecalciferol biweekly for 8 to 9 weeks. Then, we obtained baseline and end-of-study skin biopsies from photodamaged (PD) and photoprotected (PP) skin, and from benign nevi (BN) and tested for mRNA expression of VDR and cytochrome P450-24 (CYP24), and markers of keratinocytic differentiation. High-dose cholecalciferol supplementation significantly elevated circulating levels of 25-hydroxyvitamin-D (P < 0.0001) and 1,25-dihydroxyvitamin-D (P < 0.0001). VDR expression in PD- and PP-skin showed minimum changes after supplementation. CYP24 expression in PD- and PP-skin was increased after supplementation by 186%, P = 0.08, and 134%, P = 0.07, respectively. In BNs from 11 participants, a trend for higher VDR and CYP24 expression was observed (average of 20%, P = 0.08, and 544%, P = 0.09, respectively). Caspase-14 expression at the basal layer in PD skin samples was the only epidermal differentiation marker that was significantly increased (49%, P < 0.0001). High-dose cholecalciferol supplementation raised serum VD metabolite levels concurrently with CYP24 mRNA and caspase-14 levels in the skin. Our findings of significant variability in the range of VDR and CYP24 expression across study samples represent an important consideration in studies evaluating the role of VD as a skin cancer chemopreventive agent. Cancer Prev Res; 8(6); 563–9. ©2015 AACR.

Abstract 3246: Mapping of functional protein pathway modulations in non-sun exposed skin of healthy volunteers using solar simulated light: A new model for pharmacodynamic testing of skin cancer chemopreventive drugs

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Experimental studies suggest that vitamin D (VD) plays an important role in skin carcinogenesis. In humans, epidemiologic studies have reported mixed findings in the association between circulating vitamin D levels and skin cancer risk. We conducted a pilot clinical study to determine whether oral VD supplementation would exert any bioactivity in human skin. Methods: The study accrued twenty-five healthy individuals with serum 25-hydroxyvitamin D levels <30 ng/mL and with moderate to severe photodamage on the forearms. Participants took 50,000 IU of VD3 capsules twice a week for 8-9 weeks. Baseline and end-of study skin biopsies were obtained from photodamaged (PD) and photoprotected (PP) skin, and benign nevi (BN), when available, for assessment of changes in putative biomarkers of VD activity. Biomarkers evaluated include the mRNA expression of vitamin D receptor (VDR) and cytochrome P450 24 (CYP24) in keratinocytes and available BN. In addition, molecular markers of keratinocytic differentiation (caspase 14 and loricrin protein expression), epidermal thickness, serum levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were assessed. Results: High dose VD supplementation significantly elevated the circulating levels of 25-hydroxyvitamin D from 21.6 ± 5.2 to 70.5 ± 18.2 ng/mL (p < 0.0001) and 1,25-dihydroxyvitamin D from 31.1 ± 12.4 to 51.4 ± 13.5 pg/mL (p < 0.0001). VDR expression in PD- and PP-skin showed minimum changes after VD supplementation. CYP24 expression in PD- and PP-skin showed a non-statistically significant increase after VD supplementation (average of 186% increase, p = 0.08, and 134% increase, p=0.07, respectively). Higher VDR and CYP24 expression was observed in BNs collected at post-intervention than that at baseline from eleven participants, but the difference did not reach statistical significance (average of 20% higher, p = 0.08, and 544% higher, p= 0.09, respectively). The large inter-subject variation in VDR and CYP24 expression may have limited the statistical evaluation. The epidermal differentiation markers did not change significantly after VD supplementation with the exception of a 49% increase (p< 0.0001) in caspase 14 expression at the basal layer in PD skin samples. When epidermal thickness was analyzed, the only significant change was identified when the analysis was stratified by the baseline median thickness. Samples with baseline thickness below or equal to the median exhibited a significant increase in thickness at the end of the intervention. Conclusion: The study showed that following effective oral supplementation based on VD serum levels, subtle indicators of increased keratinocytic differentiation and CYP24 activation can be observed. Future studies evaluating the role of VD as a skin cancer chemopreventive agent with biomarker modulation as an endpoint should be considered before larger intervention studies are implemented in at risk populations. Citation Format: Clara Curiel-Lewandrowski, Jean Tang, Janine Einspahr, Yira Bermudez, Chiu-Hsieh Hsu, Melika Rezaee, Alex Lee, Joe Tangrea, Howard Parnes, David Alberts, H-H. Sherry Chow. Pilot study on the bioactivity of vitamin D in the skin after oral supplementation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3248. doi:10.1158/1538-7445.AM2014-3248

Folate Nutrition in Skin Health and Skin Cancer Prevention

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Efficient secondary prevention methods for non-melanoma skin cancers are needed to supplement primary prevention in high-risk individuals. Pharmacologic effects in UV-induced keratinocyte signaling may predict efficacy of new drugs. In order to understand effects of solar light on signal transduction networks in human skin, we exposed buttocks skin of healthy volunteers to solar-simulated light (SSL) at doses 2x-3x of minimum erythemal dose (MED) using a Multiport UV Solar Simulator (Solar Light Co.). Punch biopsies (6 mm) were collected at baseline, 5 min, 1 hr, 5 hr, and 24 hr post-exposure. Biopsies were split with half fixed in formalin and half snap-frozen for Reverse Phase Protein Microarray (RPMA) analysis to identify relevant signaling networks activated by SSL exposure. 24 subjects ≥ 18 y.o. with Fitzpatrick skin type II or III, with no concurrent illness, cancer, or use of photosensitizing drugs were recruited. 12 were exposed to SSL at 2x MED, 6 at 2.5x MED, and 6 at 3x MED. The activation/phosphorylation or total levels of 128 key signaling proteins and drug targets were measured for each sample. Coordinate network-based analysis was performed on specific signaling pathways that included PI3k/Akt/mTOR, Ras/Raf/MEK/Erk, and Fyn/RSK2. Analyte levels were compared at baseline to those at 5 min, 1 hr, 5 hr, and 24 hr after SSL exposure. Unsupervised and supervised statistical analysis was used with Bonferroni multiple comparison adjustment (p < 0.01). Pathway activation maps were constructed using p values to indicate time-dependence of pathway activation. Differences in MED did not significantly affect expression, so all 24 subjects were analyzed independent of SSL dose. Most pathway modulation occurred within the first 5 hr, with cell death and apoptosis-related endpoints maximal at 24 hr. Many kinases were activated within 5 min and activity increased at 1-5 hr before reversing to baseline or lower at 24 hr. Early and sustained activation of p38/SAPK/ ERK pathways started at 5 min, continued through 5 hr and was sustained at 24 hr. Systemic AKT-mTOR pathway activation was observed from 5 min-1 hr, sustained through 5 hr, and decreased at 24 hr. EGFR-HER3 activation followed a similar pattern. COX2 expression increased at 1 hr and was sustained through 24 hr. AMPK was activated early and sustained through 24 hr since LKB1, the dominant AMPK kinase was activated within 5 min-1hr and sustained. Correlation-based network maps were generated and revealed time-dependent SSL induced pathway activation linkages. This work shows that protein pathway activation mapping of phosphorylated proteins in relevant signaling pathways can be used in future studies to determine pharmacodynamic activity of selective topical agents administered in a test area exposed to SSL to determine drug-induced modification of skin carcinogenesis pathways. Citation Format: Steven P. Stratton, Clara Curiel-Lewandrowski, Janine G. Einspahr, Valerie Calvert, Chengcheng Hu, Yira Bermudez, David S. Alberts, George T. Bowden, Emanuel F. Petricoin. Mapping of functional protein pathway modulations in non-sun exposed skin of healthy volunteers using solar simulated light: A new model for pharmacodynamic testing of skin cancer chemopreventive drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3246. doi:10.1158/1538-7445.AM2014-3246

Abstract 4264: PI3K/Akt/mTOR signaling modulation in solar UV-induced skin carcinogenesis.

This chapter will discuss the role of folate nutrition in the unique environment of human skin. The folates are a family of structurally similar, water-soluble, B vitamins, which have been well documented as vital in promoting human health and preventing disease. Optimized folate nutrient levels support many biochemical processes important for the maintenance and function of healthy skin. This importance is underscored by potential links between folate deficiency and psoriasis, vitiligo, exfoliative dermatitis, glossitis, and skin cancers. Human skin is particularly prone to the development of carcinomas. It is established that skin cancer risk correlates with exposure to the complete carcinogen, ultraviolet radiation (UVR) from sunlight. Total avoidance of solar exposure is impractical.