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Supplementary Figure S1 from Loss of Inositol Polyphosphate 5-Phosphatase Is an Early Event in Development of Cutaneous Squamous Cell Carcinoma

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

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.

Cutaneous squamous cell carcinoma (SCC) occurs commonly and can metastasize. Identification of specific molecular aberrations and mechanisms underlying the development and progression of cutaneous SCC may lead to better prognostic and therapeutic approaches and more effective chemoprevention strategies. To identify genetic changes associated with early stages of cutaneous SCC development, we analyzed a series of 40 archived skin tissues ranging from normal skin to invasive SCC. Using high-resolution array-based comparative genomic hybridization, we identified deletions of a region on chromosome 10q harboring the INPP5A gene in 24% of examined SCC tumors. Subsequent validation by immunohistochemistry on an independent sample set of 71 SCC tissues showed reduced INPP5A protein levels in 72% of primary SCC tumors. Decrease in INPP5A protein levels seems to be an early event in SCC development, as it also is observed in 9 of 26 (35%) examined actinic keratoses, the earliest stage in SCC development. Importantly, further reduction of INPP5A levels is seen in a subset of SCC patients as the tumor progresses from primary to metastatic stage. The observed frequency and pattern of loss indicate that INPP5A, a negative regulator of inositol signaling, may play a role in development and progression of cutaneous SCC tumors. Cancer Prev Res; 3(10); 1277–83. ©2010 AACR.

Supplementary Figure 1. Box plot graphs of protein expression in sun-protected skin after 2, 2,5, and 3 MED (x-axis) of SSL-irradiation (SSL) with baseline (green bars), 5 minutes (orange bars), 1 hour (blue bars), 5 hours (red bars), and 24 hours (grey bars) post SSL on the y-axis. Supplementary Figure 2. Box plot graphs of protein expression in sun-protected skin after 2, 2,5, and 3 MED (x-axis) of SSL-irradiation (SSL) with baseline (green bars), 5 minutes (orange bars), 1 hour (blue bars), 5 hours (red bars), and 24 hours (grey bars) post SSL on the y-axis. Supplementary Figure 3. Box plot graphs of protein expression in sun-protected skin after 2, 2,5, and 3 MED (x-axis) of SSL-irradiation (SSL) with baseline (green bars), 5 minutes (orange bars), 1 hour (blue bars), 5 hours (red bars), and 24 hours (grey bars) post SSL on the y-axis.

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

Solar ultraviolet (sUV) irradiation is a major environmental carcinogen that can cause inflammation and skin cancer. The costs and morbidity associated with skin cancer are increasing, and therefore identifying molecules that can help prevent skin carcinogenesis is important. In this study, we identified the p53-related protein kinase (PRPK) as a novel oncogenic protein that is phosphorylated by the T-LAK cell-originated protein kinase (TOPK). Knockdown of TOPK inhibited PRPK phosphorylation and conferred resistance to solar-simulated light (SSL)-induced skin carcinogenesis in mouse models. In the clinic, acute SSL irradiation significantly increased epidermal thickness as well as total protein and phosphorylation levels of TOPK and PRPK in human skin tissues. We identified two PRPK inhibitors, FDA-approved rocuronium bromide (Zemuron®) or betamethasone 17-valerate (Betaderm®) that could attenuate TOPK-dependent PRPK signaling. Importantly, topical application of either rocuronium bromide or betamethasone decreased SSL-induced epidermal hyperplasia, neovascularization, and cutaneous squamous cell carcinoma (cSCC) development in SKH1 (Crl: SKH1-Hrhr) hairless mice by inhibiting PRPK activation, and also reduced expression of the proliferation and oncogenesis markers, COX-2, cyclin D1, and MMP-9. This study is the first to demonstrate that targeting PRPK could be useful against sUV-induced cSCC development.

Cutaneous exposure to solar ultraviolet (UV) radiation is a major causative factor in skin carcinogenesis, and improved molecular strategies for efficacious chemoprevention of non-melanoma skin cancer (NMSC) are urgently needed. Toll-like receptor 4 (TLR4) signaling has been shown to drive skin inflammation, photoimmunosuppression and chemical carcinogenesis. Here we have examined the feasibility of genetic and pharmacological antagonism targeting cutaneous TLR4 for the suppression of UV-induced NF-κB and AP-1 signaling in keratinocytes and mouse skin. Using immunohistochemical and proteomic microarray analysis of human skin, we demonstrate for the first time that a significant increase in expression of TLR4 occurs in keratinocytes during the progression from normal skin to actinic keratosis (AK), also detectible during further progression to squamous cell carcinoma. Next, we demonstrate that siRNA-based genetic TLR4 inhibition blocks UV-induced stress signaling in cultured keratinocytes. Importantly, we observed that resatorvid (TAK-242), a molecularly-targeted clinical TLR4 antagonist, blocks UV-induced NF-κB and MAP kinase/AP-1 activity and cytokine expression (Il-6, Il-8, and Il-10) in cultured keratinocytes and in topically treated murine skin. Taken together, our data reveal that pharmacological TLR4 antagonism can suppress UV-induced cutaneous signaling, and future experiments will explore the potential of TLR4-directed strategies for prevention of NMSC.

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

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

Ultraviolet radiation is an important etiologic factor in skin cancer and a better understanding of how solar stimulated light (SSL) affects signal transduction pathways in human skin which is needed in further understanding activated networks that could be targeted for skin cancer prevention. We utilized Reverse Phase Protein Microarray Analysis (RPPA), a powerful technology that allows for broad-scale and quantitative measurement of the activation/phosphorylation state of hundreds of key signaling proteins and protein pathways in sun-protected skin after an acute dose of two minimal erythema dose (MED) of SSL. RPPA analysis was used to map the altered cell signaling networks resulting from acute doses of solar simulated radiation (SSL). To that end, we exposed sun-protected skin in volunteers to acute doses of two MED of SSL and collected biopsies pre-SSL and post-SSL irradiation. Frozen biopsies were subjected to laser capture microdissection (LCM) and then assessed by RPPA. The activation/phosphorylation or total levels of 128 key signaling proteins and drug targets were selected for statistical analysis. Coordinate network-based analysis was performed on specific signaling pathways that included the PI3k/Akt/mTOR and Ras/Raf/MEK/ERK pathways. Overall, we found early and sustained activation of the PI3K-AKT-mTOR and MAPK pathways. Cell death and apoptosis-related proteins were activated at 5 and 24 h. Ultimately, expression profile patterns of phosphorylated proteins in the epidermal growth factor receptor(EGFR), AKT, mTOR, and other relevant pathways may be used to determine pharmacodynamic activity of new and selective topical chemoprevention agents administered in a test area exposed to SSL to determine drug-induced attenuation or reversal of skin carcinogenesis pathways., Mapping skin cancer-signaling proteins Skin exposure to ultraviolet radiation leads to the activation of proteins involved in carcinogenic pathways. Janine Einspahr and Clara Curiel-Lewandrowski of the Arizona Cancer Center and colleagues in the US exposed normally ultraviolet protected skin of 12 individuals to two times the dose of solar-simulated light needed to induce redness. Skin biopsies were taken before and after exposure and 128 proteins known to be involved in key cancer signaling pathways were examined using ‘reverse phase protein microarray analysis’. They found early and sustained activation of multiple signaling pathways, in addition to activation of cell death and apoptosis-related proteins. The study may serve as a model for investigating the pathways involved in chronic or ultraviolet-induced carcinogenesis, which may ultimately lead to the development of targeted therapies to attenuate or reverse skin cancer pathways.

To develop a quantitative histopathology algorithm to predict which patients with cutaneous squamous cell carcinoma (cSCC) were likely to experience recurrence or metastases.This retrospective study of cSCC lesions compared patients with aggressive disease (n = 40) and those with nonaggressive disease (n = 35). Based on a previous study using nuclear karyometry, we determined that aggressive lesions had a high proportion of a specific nuclear phenotype. The proportion of those nuclei was used to derive an aggressiveness score for each lesion. The mean age of patients was similar in both groups, as were the locations of index lesions.The mean aggressiveness scorefor cases with aggressive lesions was 0.60 ± 0.21 and was 0.28 ± 0.35 for those with nonaggressive lesions. The overall accuracy in properly characterizing lesions was 72%. The area under the receiver operating characteristic curve was 0.80 ± 0.05. In general, the aggressive nuclear phenotype is represented by elevated levels of chromatin clumps and short linear segments of dark chromatin/intense pixels.These data suggest that discriminant functions may be utilized to distinguish between aggressive and nonaggressive lesions at the time of diagnosis.

BACKGROUND: Reduced melanoma risk has been reported with regular use of nonsteroidal anti-inflammatory drugs (NSAIDs). However, the ability of NSAIDs to reach melanocytes in vivo and modulate key biomarkers in preneoplastic lesions such as atypical nevi has not been evaluated. METHODS: This randomized, double-blind, placebo-controlled trial of sulindac was conducted in individuals with atypical nevi (AN) to determine bioavailability of sulindac and metabolites in nevi and effect on apoptosis and vascular endothelial growth factor A (VEGFA) expression in AN. Fifty subjects with AN ≥4 mm in size and 1 benign nevus (BN) were randomized to sulindac (150 mg twice a day) or placebo for 8 weeks. Two AN were randomized for baseline excision, and 2 AN and BN were excised after intervention. RESULTS: Postintervention sulindac, sulindac sulfone, and sulindac sulfide concentrations were 0.31 ± 0.36, 1.56 ± 1.35, and 2.25 ± 2.24 μg/mL in plasma, and 0.51 ± 1.05, 1.38 ± 2.86, and 0.12 ± 0.12 μg/g in BN, respectively. Sulindac intervention did not significantly change VEGFA expression but did increase expression of the apoptotic marker cleaved caspase-3 in AN (increase of 3 ± 33 in sulindac vs decrease of 25 ± 45 in the placebo arm, P = .0056), although significance was attenuated (P = .1103) after adjusting for baseline expression. CONCLUSIONS: Eight weeks of sulindac intervention resulted in high concentrations of sulindac sulfone, a proapoptotic metabolite, in BN but did not effectively modulate VEGFA and cleaved caspase-3 expression. Study limitations included limited exposure time to sulindac and the need to optimize a panel of biomarkers for NSAID intervention studies. Cancer 2012. © 2012 American Cancer Society.

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.

Difluoromethylornithine (DFMO) and nonsteroidal anti-inflammatory drugs have been shown to have a synergistic protective effect against colon cancer. As single agents, both DFMO and diclofenac have demonstrated efficacy in the treatment of actinic keratoses, the cutaneous precursors to cutaneous squamous cell carcinomas. We designed a study investigating the effects of these two medications, alone and in combination, on sun-damaged skin of the forearm. The primary objective is to determine whether a three-month course of combination therapy increases the efficacy over either agent used alone in the treatment of moderately sun-damaged skin. The primary endpoint is the change in karyometric values from skin biopsies before and after treatment. A total of 156 subjects were enrolled in the study and randomized to A: topical DFMO applied BID × 90 days; B: topical diclofenac applied QD × 90 days; or C: topical DFMO applied BID and topical diclofenac applied QD × 90 days. Planned sample size was 138 evaluable subjects to provide 80% power to detect the difference in response rate as measured by karyometric average nuclear abnormality (ANA) between the single-agent DFMO arm and the combination treatment arm at the 2.5% significance level. The results for efficacy and safety will be presented at the AACR Frontiers in Cancer Prevention meeting. Secondary biomarker and clinical endpoints will also be summarized. These findings will provide insight into the potential use of the combination of these medications as skin cancer chemopreventive agents. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN02-06.

Cutaneous squamous cell carcinoma (cuSCC) is the second most common skin cancer, for which long term UV exposure and chronic wounding are the dominant risk factors. Despite these clinically established connections, little is understood about the early molecular response of human skin to UV exposure and its connection to acute wounding and cuSCC. Thus, our goal is to find common and specific signatures driven by UV-exposure and wounding as a means of developing new approaches for treating and preventing cuSCC. Here, we perform integrated analyses of proteomic, RNA-seq and miR-seq on 3 datasets: (1) UV-unexposed and acute UV-exposed human skin, (2) public dataset on acute wound healing and (3) our previously published dataset on normal skin and cuSCC from humans. We find that biological signatures and processes regulated by acute UV exposure and wounding has profound similarity. miR-seq data shows that miR-223, miR-132 and miR-142 are significantly upregulated in both acute events. Combined gene set enrichment analysis shows that G-protein-coupled-receptors (GPCRs) pathways are upregulated, possibly through Gαi activation. While ECM remodeling is significantly enriched in all three datasets, gene expression regulated by PPARα is suppressed. Interestingly, upregulation of matrisome components is observed among all three datasets. This suggests that these changes are important early events that regulated by both UV-exposure and wounding which eventually can promote cuSCC initiation. Thus, our findings suggest that these common signatures can be potentially validated as chemopreventive targets for cuSCC. Citation Format: Tran N. Nguyen, Kimal Rajapakshe, Stanislav Avdieiev, Courtney Nicholas, Vida Chitsazzadeh, Eric Welsh, Bin Fang, John Koomen, Cristian Coarfa, Janine Einspahr, Kenneth Y. Tsai. A proteome-transcriptome-miRnome integrated analysis identifies similarity between UV-exposed skin and wounding skin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 406.

The chemopreventive and antitumor properties of perillyl alcohol (POH) that were studied preclinically indicate that topical POH inhibits both UVB-induced murine skin carcinogenesis (squamous cell tumor models) and 7,12-dimethylbenz(a)anthracene–induced murine melanoma (transgenic models involving tyrosinase-driven Ras). A previous phase 1 clinical trial in participants with normal-appearing skin showed that topical POH cream was well tolerated at a dose of 0.76% (w/w). Here, we performed a 3-month, double-blind, randomized, placebo-controlled phase 2a trial of two different doses of topical POH in individuals with sun-damaged skin. Participants applied POH cream twice daily to each dorsal forearm. Baseline and end-of-study biopsies were taken from each participant to evaluate whether the topical application of POH was effective in reversing actinic damage as evidenced by normalization of quantitative skin histopathologic scores and change in nuclear chromatin pattern as measured by karyometric analysis. There was a borderline reduction in the histopathologic score of the lower-dose POH group compared with the placebo (P = 0.1), but this was not observed in the high-dose group. However, in the high-dose group, a statistically significant reduction in the proportion of nuclei deviating from normal was observed by the use of karyometric analysis (P < 0.01). There was no statistical significance shown in the lower-dose group. No changes were observed in p53 expression, cellular proliferation (by proliferating cell nuclear antigen expression), or apoptosis in either treatment group compared with the placebo group. These results suggest that whereas our karyometric analyses can detect a modest effect of POH in sun-damaged skin, improved delivery into the epidermis may be necessary. Cancer Prev Res; 3(2); 160–9

Prior research shows that topical application of free, nonfatty acid–conjugated vitamin E (dl-α-tocopherol) prevents skin cancer in mice, as well as immunosuppression induced by UVB radiation. This study investigated the chemopreventive potential of dl-α-tocopherol in humans through monitoring surrogate end point biomarkers in sun-damaged skin. Contralateral arms of healthy human volunteers with actinic keratoses (AK) were randomly assigned to receive either 12.5% dl-α-tocopherol or placebo in a crème base for 6 months. Changes in number of AKs, levels of p53 protein expression, proliferating cell nuclear antigen, and polyamines were assessed along with skin and systemic vitamin E levels. Following treatment, plasma concentration levels of dl-α-tocopherol were unchanged, but skin levels were highly elevated (P < 0.001). Levels of p53 and proliferating cell nuclear antigen did not change significantly, whereas number of AKs declined insignificantly in both placebo and treatment arms. Regression models showed significant decreases in putrescine, spermidine, spermine, and total polyamine concentrations following treatment. Topically applied dl-α-tocopherol was substantially absorbed in skin, but the 6-month application did not significantly reduce numbers of preexisting AKs on moderately to severely sun-damaged forearms. Increases in polyamine synthesis are expected during tumor initiation and promotion; conversely, the significant reductions in polyamine levels resulting from the topical dl-α-tocopherol application are consistent with reductions in tumorigenesis potential. Topical tocopherol did not normalize established sun-induced lesions, but dl-α-tocopherol–induced reductions in polyamine metabolism are consistent with the inhibition of skin squamous cell carcinogenesis as seen in previous human trials and animal models.

Acute UVB irradiation of mouse skin results in activation of phospatidyinositol-3 (PI-3) kinase and mitogen-activated protein kinase (MAPK) pathways leading to altered protein phosphorylation and downstream transcription of genes. We determined whether activation of these pathways also occurs in human skin exposed to 4x minimal erythemic dose of UVB in 23 volunteers. Biopsies were taken prior to, at 30 min, 1 and 24 h post-UVB. In agreement with mouse studies, the earliest UV-induced changes in epidermis were seen in phospho-CREB (two- and five-fold at 30 min and 1 h) and in phospho-MAPKAPK-2 (three-fold at both 30 min and 1 h). At 1 h, phospho-c-JUN and phospho-p38 were increased five- and two-fold, respectively. Moreover, phospho-c-JUN and phospho-p38 were further increased at 24 h (12- and six-fold, respectively). Phospho-GSK-3beta was similarly increased at all time points. Increases in phospho-p53 (12-fold), COX-2 (four-fold), c-FOS (14-fold) and apoptosis were not seen until 24 h. Our data suggest that UVB acts through MAPK p38 and PI-3 kinase with phosphorylation of MAPKAPK-2, CREB, c-JUN, p38, GSK-3beta and p53 leading to marked increases in c-FOS, COX-2 and apoptosis. Validation of murine models in human skin will aid in development of effective skin cancer chemoprevention and prevention strategies.

Background: A preceding exploratory study (J. Clin. Pathol. 57(2004), 1201–1207) had shown that a karyometric assessment of nuclei from papillary urothelial neoplasms of low malignant potential (PUNLMP) revealed subtle differences in phenotype which correlated with recurrence of disease. Aim of the Study: To validate the results from the exploratory study on a larger sample size. Materials: 93 karyometric features were analyzed on haematoxylin and eosin-stained sections from 85 cases of PUNLMP. 45 cases were from patients who had a solitary PUNLMP lesion and were disease-free during a follow-up period of at least 8 years. The other 40 were from patients with a unifocal PUNLMP, with one or more recurrences in the follow-up. A combination of the previously defined classification functions together with a new P-index derived classification method was used in an attempt to classify cases and identify a biomarker of recurrence in PUNLMP lesions. Results: Validation was pursued by a number of separate approaches. First, the exact procedure from the exploratory study was applied to the large validation set. Second, since the discriminant function 2 of the exploratory study had been based on a small sample size, a new discriminant function was derived. The case classification showed a correct classification of 61% for non-recurrent and 74% for recurrent cases, respectively. Greater success was obtained by applying unsupervised learning technologies to take advantage of phenotypical composition (correct classification of 92%). This approach was validated by dividing the data into training and test sets with 2/3 of the cases assigned to the training sets, and 1/3 to the test sets, on a rotating basis, and validation of the classification rate was thus tested on three separate data sets by a leave-k-out process. The average correct classification was 92.8% (training set) and 84.6% (test set). Conclusions: Our validation study detected subvisual differences in chromatin organization state between non-recurrent and recurrent PUNLMP, thus allowing a very stable method of predicting recurrence of papillary urothelial neoplasms of low malignant potential by karyometry.

BACKGROUND. A considerable body of evidence supports the concept that a significant number of cutaneous malignant melanomas progress through a precursor lesion or dysplastic melanocytic nevi (DN). Tumor angiogenesis likely plays a critical role in early development of melanoma, and intermediate biomarkers of angiogenesis could be useful as chemoprevention and prognostic markers. METHODS. Markers of angiogenesis that included expression of the vascular endothelial growth factor A (VEGF-A) and microvessel density counts (MVD) were evaluated in 13 prospectively collected benign nevi (BN) and 19 DN from 16 individuals and in a comparison group of 17 primary melanomas (16 archival samples and 1 prospective melanoma). RESULTS. VEGF expression in melanocytic cells (mean ± standard error [SE]) was low or absent in BN (3.4 ± 1.4), increased significantly in DN (41.0 ± 10.1; P = .0003 for BN vs DN), and increased further in primary melanoma (119.9 ± 28.3; P = .06 for DN vs melanoma). MVD using CD31 (mean ± SE [percentage × intensity]) followed a similar pattern with similarity between BN (2.6 ± 0.7; N = 13) and DN (2.2 ± 0.8; N = 19; P = .4 for BN vs DN), whereas primary melanomas were significantly higher (39.4 ± 6.4; N = 17; P = .0001 for BN or DN vs melanoma). CONCLUSIONS. In a prospective setting, the current data suggested that increased VEGF-A expression in DN may be a good indicator of preneoplastic change in melanocytic lesions with the potential for improving the understanding and prevention of the transformation of DN to melanoma. Cancer 2007. © 2007 American Cancer Society.

Solar ultraviolet (UV) light is a major etiological factor in skin carcinogenesis, with solar UV-stimulated signal transduction inducing pathological changes and skin damage. The primary sensor of solar UV-induced cellular signaling has not been identified. We use an experimental system of solar simulated light (SSL) to mimic solar UV and we demonstrate that Fyn is a primary redox sensor involved in SSL-induced signal transduction. Reactive oxygen species (ROS) generated by SSL exposure directly oxidize Cys488 of Fyn, resulting in increased Fyn kinase activity. Fyn oxidation was increased in mouse skin after SSL exposure, and Fyn knockout (Fyn−/−) mice formed larger and more tumors compared to Fyn wildtype mice when exposed to SSL for an extended period of time. Murine embryonic fibroblasts (MEFs) lacking Fyn as well as cells in which Fyn expression was knocked down were resistant to SSL-induced apoptosis. Furthermore, cells expressing mutant Fyn (C448A) were resistant to SSL-induced apoptosis. These findings suggest that Fyn acts as a regulatory nexus between solar UV, ROS and signal transduction during skin carcinogenesis.

Solar ultraviolet irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is reportedly expressed at reduced levels in many cancers. The present study was designed to examine the role of caspase-7 in solar-simulated light (SSL)-induced skin cancer and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic deficiency of caspase-7 are highly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and the average number of tumors were significantly increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation markers, such as survivin and Ki-67, was elevated in SSL-irradiated skin of caspase-7 KO mice compared with those observed in SSL-exposed wild-type SKH1 mouse skin. Moreover, SSL-induced apoptosis was abolished in skin from caspase-7 KO mice. Two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight analysis of skin tissue lysates from SSL-irradiated SKH1 wild-type and caspase-7 KO mice revealed an aberrant induction of keratin-17 in caspase-7 KO mice. Immunohistochemical analysis of skin tumors also showed an increase of keratin-17 expression in caspase-7 KO mice compared with SKH1 wild-type mice. The expression of keratin-17 was also elevated in SSL-irradiated caspase-7 KO keratinocytes as well as in human basal cell carcinomas. The in vitro caspase activity assay showed keratin-17 as a substrate of caspase-7, but not caspase-3. Overall, our study demonstrates that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17.

In colorectal tumorigenesis, Ki-ras proto-oncogene mutation often occurs early in the adenoma-adenocarcinoma sequence, whereas mutation of the p53 gene is associated with late progression to carcinoma. We evaluated the relationship of demographic and clinicopathologic characteristics to Ki-ras mutation and p53 gene product overexpression in 1,093 baseline sporadic colorectal adenomas from 926 individuals enrolled in a phase III recurrence prevention trial. Ki-ras mutation was found in 14.7% of individuals and p53 overexpression was found in 7.0% of those tested. Multivariate analysis found older age, rectal location, and villous histology to be independently associated with Ki-ras mutation. Individuals with an advanced adenoma (≥1 cm or high-grade dysplasia or villous histology) had a 4-fold higher likelihood of Ki-ras mutation [odds ratios (OR), 3.96; 95% confidence intervals (CI), 2.54-6.18]. Ki-ras mutations in codon 12 and of the G-to-A transition type were more frequent in older individuals, whereas G-to-T transversion was more frequent in rectal adenomas than in the colon. Multivariate analysis showed that previous history of a polyp (P = 0.03) was inversely associated with p53 overexpression. Large adenoma size (≥1 cm), high-grade dysplasia, and villous histology were independently associated with p53 overexpression, with the strongest association for advanced adenomas (OR, 7.20; 95% CI, 3.01-17.22). Individuals with a Ki-ras mutated adenoma were more likely to overexpress p53 (OR, 2.46; 95% CI, 1.36-4.46), and 94.8% of adenomas with both alterations were classified as advanced (P ≤ 0.0001). Our large cross-sectional study supports the role of both Ki-ras and p53 in the progression of adenomas and shows that their molecular pathogenesis differs by anatomic location, age, and mucosal predisposition as evidenced by previous history of a polyp. (Cancer Epidemiol Biomarkers Prev 2006;15(8):1443–50)

Background: Sporadic colorectal cancers often arise from a region of cells characterized by a “ fi eld defect ” that has not been well defi ned molecularly. DNA methylation has been proposed as a candidate mediator of this fi eld defect. The DNA repair gene O 6 -methylguanine-DNA methyltransferase (MGMT) is frequently methylated in colorectal cancer. We hypothesized that MGMT methylation could be one of the mediators of fi eld cancerization in the colon mucosa. Methods: We studied MGMT promoter methylation by three different bisulfi te-based techniques in tumor, adjacent mucosa, and nonadjacent mucosa from 95 colorectal cancer patients and in colon mucosa from 33 subjects with no evidence of cancer. Statistical tests were two-sided. Results: MGMT promoter methylation was present in 46% of the tumors. Patients whose cancer had MGMT promoter methylation also had substantial MGMT promoter methylation in apparently normal adjacent mucosa. This methylation was seen with a quantitative assay in 50% (22/44; 95% confi dence interval [CI] = 34% to 65%) of normal samples with MGMT promoter methylation in the adjacent tumors, 6% (3/51; 95% CI = 1% to 16%) of samples without MGMT methylation in adjacent tumors, and 12% (4/33; 95% CI = 3% to 28%) of control samples ( P

Background: Ursodeoxycholic acid (UDCA) treatment is associated with a reduced incidence of colonic neoplasia in preclinical models and in patients with conditions associated with an increased risk for colon cancer. We conducted a phase III, double-blind placebo-controlled trial of UDCA to evaluate its ability to prevent colorectal adenoma recurrence. Methods: We randomly assigned 1285 individuals who had undergone removal of a colorectal adenoma within the past 6 months to daily treatment with UDCA (8 – 10 mg/kg of body weight; 661 participants) or with placebo (624 participants) for 3 years or until follow-up colonoscopy. Recurrence rates (number of recurrent adenomas per unit time) were compared by use of a Huber – White variance estimator. Proportions of participants with one or more recurrent adenomas were compared with a Pearson chi-square statistic; adjusted odds ratios (ORs) were obtained by logistic regression. All statistical tests were two-sided. Results: We observed a non – statistically signifi cant 12% reduction in the adenoma recurrence rate associated with UDCA treatment, compared with placebo treatment. However, UDCA treatment was associated with a statistically signifi cant reduction ( P = .03) in the recurrence of adenomas with high-grade dysplasia (adjusted OR = 0.61, 95% confi dence interval = 0.39 to 0.96). We observed no statistically signifi cant differences between UDCA and placebo groups in recurrence with regard to adenoma size, villous histology, or location. Conclusions: UDCA treatment was associated with a non – statistically signifi cant reduction in total colorectal adenoma recurrence but with a statistically signifi cant 39% reduction in recurrence of adenomas with high-grade dysplasia. Because severely dysplastic lesions have a high risk of progression to invasive colorectal carcinoma, this fi nding indicates that future chemoprevention trials of UDCA in individuals with such lesions should be considered. [J Natl Cancer Inst 2005;97:846 – 53]

Thioredoxin-1 is a redox protein that, when overexpressed, causes increased cancer-cell growth and inhibited apoptosis. Thioredoxin-1 expression has been reported to be increased in several human primary tumors, but its relationship to tumor progression and patient survival has not been established. We studied the expression of thioredoxin-1 as measured with immunohistochemical staining in paraffin-embedded human normal colonic mucosa, adenomatous polyps, and primary and metastatic colorectal cancer. Thioredoxin-1 expression was not increased in 12 colorectal adenomatous polyps, compared with 8 samples of normal colonic mucosa, but was significantly increased in 12 primary colorectal cancers (P

The positive association of decreased risk of colorectal cancer with nonsteroidal antiinflammatory drug (NSAID) use, combined with the observation that cyclooxygenase(COX)-2 is present in a majority of colorectal tumors, has led to the proposed use of isozyme-specific COX inhibitors as preventive agents in polyp and tumor formation in the colon. However, the exact biochemical mechanisms and disease stage at which reduced risk is mediated remain somewhat controversial, in part because of the complex biochemical changes that occur during the progression from aberrant crypt to polyp to tumor. In this study, COX-1 and COX-2 protein expression levels were determined in sets of tumor and normal colon tissue. Changes were characterized in COX-1 and COX-2 expression within individuals, in relation to such factors as sex, tumor grade, and location in the colorectum. COX-1 expression levels were found to be significantly reduced in tumors compared to matched normal tissues (Dunn's method, P < 0.05). Additionally, COX-1 expression was decreased in stage T3 tumors as compared to stage T2 tumors (Student's t-test, P = 0.009). Similar to previous reports, COX-2 protein expression was present in 73% of the tumors studied and appeared to be independent of tumor grade and sex. Interestingly, decreased COX-2 expression correlated with tumor occurrence in rectal mucosa (Wilcoxon two-sample test, P < 0.05). These results warrant further investigation, especially the identification of determinants that would predict which populations would be most responsive to COX-2 inhibition as a means of colorectal cancer chemoprevention.

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

[corrected] The goal of this study was to examine the relationship between Ki-ras mutations in colorectal adenomas and characteristics of both the subject (age, gender, and family/personal history of colonic neoplasia) and the adenoma (multiplicity, size, location, and histologic features).Ki-ras mutations were detected by direct sequencing in 738 adenomatous polyps removed at baseline from 639 participants in a nutritional trial of adenoma recurrence.Ki-ras mutations were detected in 17.2% of the adenomas. Ki-ras mutations were unrelated to gender, family, or personal history of colonic neoplasia, location within the colorectum, or adenoma multiplicity, but were more common in older subjects (P = 0.01 for trend), in larger adenomas (P0.0001 for trend), in adenomas with villous histology (odds ratio [OR], 3.2; 95% confidence interval [CI], 2.1-4.9 vs. tubular), and in adenomas with high-grade dysplasia (32.0% vs. 13.6%; OR, 3.0; 95% CI, 1.9-4.6 vs. low-grade dysplasia). Multivariate analysis showed Ki-ras mutations to be independently associated with subject age (P = 0.01 for trend), tubulovillous/villous histology (OR, 2.3; 95% CI, 1.5-3.7), and high-grade dysplasia (OR, 1.9; 95% CI, 1.2-3.1). Adenoma size was not independently related to Ki-ras mutation.Ki-ras mutations are associated with the histologic features of adenoma progression (villous histology and high-grade dysplasia) rather than with adenoma growth.

The effects of wheat bran fiber on surrogate endpoint biomarkers for colon cancer risk have been studied in rats and humans. In both species, there is little evidence that wheat bran fiber significantly modifies epithelial cell proliferation. In rat studies, however, dietary supplementation with wheat bran fiber has decreased mucosal formation of aberrant crypt foci, an important marker currently used to estimate the efficacy of colon cancer chemoprevention agents. In humans, wheat bran fiber has been shown to consistently decrease fecal bile acid concentrations, mainly by reducing toxic secondary bile acids.

A biomarker of skin cancer would be beneficial in evaluating the efficacy of potential cancer chemoprevention agents. To this end, we investigated the tumor suppressor gene p53 in precancerous actinic keratosis lesions (AK) and malignant squamous cell carcinomas (SCCs) using polymerase chain reaction and single-strand conformation polymorphism analysis (PCR-SSCP) techniques. In addition, p53 protein expression was evaluated using immunohistochemistical analysis with the PAB 1801 monoclonal antibody. Nine out of 13 (69%) SCCs and 8 of 15 (53%) AKs were positive for p53 mutations. In contrast, normal skin samples were negative for p53 mutations. Sequence analysis of AKs and SCCs showed primarily C to T transition mutations. Nuclear immunochemical staining for p53 was observed in 12/15 (80%) AK and 12/13 (92%) SSCs. These results suggest that p53 mutations may be involved in the malignant conversion of AKs to SCCs and that p53 may be useful as a biomarker to study the potential modulatory effects of cancer chemopreventive agents against skin cancer.

By identifying aggressive cutaneous squamous cell carcinoma (cSCC) in patients who are at high risk for recurrences or second primaries after resection, intensive surveillance and therapy may decrease morbidity and mortality. We investigated the role of nuclear morphometry (karyometry) in differentiating between aggressive and nonaggressive cSCC. We retrospectively analyzed cSCC lesions from 40 male patients. Twenty-two patients had evidence of aggressive cSCC (local/regional recurrence or a second primary cSCC), and 18 patients were identified with similar ages and sites of disease as control patients with nonaggressive cSCC (no evidence of recurrence, metastasis, or second primary). We carried out karyometric analysis to identify nuclear features that discriminate between aggressive and nonaggressive cSCC nuclei. We used statistically significant differences (Kruskal–Wallis test, P < 0.0001) to compose a quantitative aggressive classification score (proportion of aggressive nuclei from 0% to 100%). For comparisons, we used Fisher's exact test or Student's t test. The mean age was 79 ± 7 years for aggressive cSCC and 80 ± 9 years for nonaggressive cSCC (P = 0.66). We analyzed a mean of 96 nuclei in each group. The mean classification score for aggressive cSCC was significantly higher (69% ± 6%) than for nonaggressive cSCC (28% ± 5%, P = 0.00002). Overall, the classification score accurately categorized 80% of our patients (P = 0.0004). In most patients, karyometry differentiated between aggressive and nonaggressive cSCC. We found that classification scores, which provide information on individual lesions, could be used for risk stratification. Cancer Prev Res; 4(11); 1770–7. ©2011 AACR.

Formal studies of skin cancer began more than 200 yr ago. The first skin tumor model to appear in the literature was reported by London surgeon Percivall Pott in 1775 (1). His account of scrotal cancer in chimney sweeps is considered the historical beginning of cancer research, as he delved beyond treatment into the etiology of the disease (2). Pott’s discovery of the linkage between scrotal carcinoma and soot exposure was the first evidence of a cancer cause, and launched research leading to the discovery of chemical carcinogens and their mechanisms of action.

Objective: The study summarizes results of karyometric measurements in epithelial cells of the colorectal mucosa to document evidence of a field effect of preneoplastic development among patients with colorectal adenocarcinoma or adenoma. Methods: Karyometric analyses were done on high-resolution images of histologic sections from 48 patients with colorectal adenocarcinomas and 44 patients with adenomas and on images from matching normal-appearing mucosa directly adjacent to such lesions, at a 1-cm and 10-cm distance from the lesions or from the rectal mucosa of adenoma patients, as well as from 24 healthy normal controls with no family history of colonic disease. Results: The nuclei recorded in the histologically normal-appearing mucosa of patients with either colorectal adenoma or adenocarcinoma exhibited differences in karyometric features in comparison with nuclei recorded in rectal mucosa from patients who were free of a colonic lesion. These differences were expressed to the same extent in tissue adjacent to the lesions and in normal-appearing tissue as distant as the rectum. Conclusions: The nuclear chromatin pattern may serve as an integrating biomarker for a preneoplastic development. The field effect might provide an end point in chemopreventive intervention trials. (Cancer Epidemiol Biomarkers Prev 2007;16(12):2704–16)

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 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

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

To develop a reliable and valid scoring system for grading skin biopsies from actinic keratosis (AK) and sun-damaged skin for use in evaluating the efficacy of skin cancer chemopreventive agents.A panel of dermatopathologists developed histologic criteria and diagnostic definitions for the progression of lesions from early AK to AK. The criteria were then applied to a sample of 335 histologic slides from an ongoing chemoprevention study. A 10% sample of 35 slides was reread in order to assess intrarater reliability.Six of the 7 criteria demonstrated high reliability (85%). The total histologic score, calculated using the 6 criteria, was found to significantly differentiate between (blinded) biopsy location (normal, pre-AK, AK and adjacent to squamous cell carcinoma) and histologic diagnosis (normal, pre- or early AK, AK and squamous cell carcinoma).The total histologic score, having demonstrated reliability on repeated readings and validity in its association with biopsy location and histologic score, is a reliable and valid end point for judging the efficacy of agents in skin cancer chemoprevention studies. Additional interrater reliability tests utilizing larger test sets and a rigorous statistical design should be undertaken to establish its portability.

There are over 1 million cases of skin cancer diagnosed yearly in the United States. The majority of these are nonmelanoma (NMSCs) and are associated with chronic exposure to ultraviolet light (UV). Actinic keratosis (AK) has been identified as a precursor for SCC, but not for BCC. AKs are far more common than SCC, making them excellent targets for chemoprevention. Cancer chemoprevention can prevent or delay the occurrence of cancer in high-risk populations using dietary or chemical interventions. We have developed strategies that have rational mechanisms of action and demonstrate activity in preclinical models of skin cancer. Promising agents proceed to phase I-III trials in subjects at high risk of skin cancer. UV light induces molecular signaling pathways and results in specific genetic alterations (i.e., mutation of p53) that are likely critical to skin cancer development. UVB-induced changes serve as a basis for the development of novel agents. Targets include inhibition of polyamine or prostaglandin synthesis, specific retinoid receptors, and components of the Ras and MAP kinase signaling pathways. Agents under study include: epigallocatechin gallate (EGCG), a green tea catechin with antioxidant and sunscreen activity, as well as UVB signal transduction blocking activity; perillyl alcohol, a monoterpene derived from citrus peel that inhibits Ras farnesylation; difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase and polyamines; retinoids that target retinoid X receptors and AP-1 activity; and nonsteroidal anti-inflammatory agents that inhibit cylooxygenase and prostaglandin synthesis. We performed a series of Phase I-II trials in subjects with multiple AK. For example, a phase II randomized trial of topical DFMO reduced AK number, suppressed polyamines, and reduced p53 protein. Our goal is to develop agents for use in combination and/or incorporation into sunscreens to improve chemoprevention efficacy and reduce skin cancer incidence.

The cyclooxygenase (COX) pathway is important in colorectal carcinogenesis with the majority of cancers overexpressing COX-2; however, the role of COX-2 in the development of colorectal adenomas is less well defined. Accordingly, we analyzed 108 colorectal adenomas for COX-1 and COX-2 transcription in archival formalin-fixed, paraffin-embedded tissue using by real-time PCR and normalized to beta-actin. Neither COX-1 nor COX-2 mRNA expression differed with regard to age or gender of the subject. COX-2 mRNA expression was significantly higher in distal adenomas (2.2 +/- 1.9) compared with proximal (0.7 +/- 0.5) adenomas (P0.0001) and in larger (/=7 mm) compared with smaller (7 mm) adenomas (2.3 +/- 2.2 and 1.7 +/- 1.3, respectively, P = 0.04). COX-2 expression did not differ significantly in tubular compared with tubulovillous adenomas, although there appeared to be a trend toward higher COX-2 expression in tubulovillous adenomas with increasing villous content. Additionally, there was not a significant difference in either COX-1 or COX-2 based on the degree of dysplasia Therefore, if COX-2 inhibitors work through a COX-2 mechanism, these agents may have differential effects on colorectal adenomas that are distal and larger.

Most sporadic colon adenomas acquire mutations in the adenomatous polyposis coli gene ( APC ) and show defects in APC -dependent signaling. APC influences the expression of several genes, including the c- myc oncogene and its antagonist Mad1 . Ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is a transcriptional target of c- myc and a modifier of APC -dependent tumorigenesis. A single-nucleotide polymorphism exists in intron 1 of the human ODC gene, which lies between two myc -binding domains. This region is known to affect ODC transcription, but no data exist on the relationship of this polymorphism to risk of colorectal neoplasia in humans. We show that individuals homozygous for the minor ODC A-allele who reported using aspirin are ≈0.10 times as likely to have an adenoma recurrence as non-aspirin users homozygous for the major G-allele. Mad1 selectively suppressed the activity of the ODC promoter containing the A-allele, but not the G-allele, in a human colon cancer-derived cell line (HT29). Aspirin (≥10 μM) did not affect ODC allele-specific promoter activity but did activate polyamine catabolism and lower polyamine content in HT29 cells. We propose that the ODC polymorphism and aspirin act independently to reduce the risk of adenoma recurrence by suppressing synthesis and activating catabolism, respectively, of colonic mucosal polyamines. These findings confirm the hypothesis that the ODC polymorphism is a genetic marker for colon cancer risk, and support the use of ODC inhibitors and aspirin, or other nonsteroidal antiinflammatory drugs (NSAIDs), in combination as a strategy for colon cancer prevention.

Factors that affect the concentration of secondary bile acids in the aqueous phase of stool may have a greater impact on colon carcinogenesis than those that only modify the total fecal bile acid concentration. This hypothesis was tested using stool samples of a subset of participants enrolled in a Phase III colorectal adenomatous polyp prevention trial, which documented the inability of a 13.5 g/day wheat bran fiber (WBF) supplement to reduce polyp recurrence. Stool was collected from 68 consecutively consented participants who were enrolled in a Phase III clinical trial of WBF for the prevention of adenomatous polyp recurrence. Nineteen (27.9%) of these fecal bile acid substudy participants were on the low fiber (2.0 g/day) intervention group, whereas 49 (72.7%) were on the high fiber (13.5 g/day) intervention group for approximately 3 years. Sixty-four participants had both the aqueous and solid phases of stool samples analyzed for bile acid content. Bile acid concentrations, measured in microg/ml for fecal water and microg/mg for dry feces, were determined for lithochilic, deoxycholic, chenodeoxycholic, cholic, ursodeoxycholic, isodeoxycholic, isoursodeoxycholic, ursocholic, 7-ketolithocholic, and 12-ketolithocholic acids. There were no significant differences between the low and high fiber groups concerning mean or median aqueous phase concentrations of lithocholic or deoxycholic bile acids. In contrast, the median concentrations of deoxycholic acid and other secondary bile acids (including lithochilic, isodeoxycholic, ursodeoxycholic, isoursodeoxycholic, ursocholic, 7-ketolithocholic, and 12-ketolithocholic acids) were significantly lower for the high fiber group in the solid-phase stool (P0.05). These results document that a high WBF intervention, taken for a median of 2.4 years, does not significantly reduce aqueous-phase concentrations of secondary bile acids in stool, although their concentrations in solid-phase stool were suppressed. Thus, the inability of the high WBF intervention to reduce colorectal adenoma recurrence may be a consequence of its lack of effect on fecal aqueous-phase secondary bile acid concentrations.