Judith A. Riggenbach

Accurate Molecular Characterization of Formalin-Fixed, Paraffin-Embedded Tissues by microRNA Expression Profiling

Formalin-fixed, paraffin-embedded tissues are an invaluable tool for biomarker discovery and validation. As these archived specimens are not always compatible with modern genomic techniques such as gene expression arrays, we assessed the use of microRNA (miRNA) as an alternative means for the reliable molecular characterization of formalin-fixed, paraffin-embedded tissues. Expression profiling using two different microarray platforms and multiple mouse and human formalin-fixed, paraffin-embedded tissue types resulted in the correlation ratios of miRNA expression levels between frozen and fixed tissue pairs ranging from 0.82 to 0.99, depending on the cellular heterogeneity of the tissue type. The same miRNAs were identified as differentially expressed between tissues using both fixed and frozen specimens. While formalin fixation time had only marginal effects on microarray performance, extended storage times for tissue blocks (up to 11 years) resulted in a gradual loss of detection of miRNAs expressed at low levels. Method reproducibility and accuracy were also evaluated in two different tissues stored for different lengths of time. The technical variation between full process replicates, including independent RNA isolation methods, was approximately 5%, and the correlation of expression levels between microarray and real-time quantitative reverse transcriptase polymerase chain reaction was 0.98. Together, these data demonstrate that miRNA expression profiling is an accurate and robust method for the molecular analysis of archived clinical specimens, potentially extending the use of miRNAs as new diagnostic, prognostic, and treatment response biomarkers.

UV Light B–Mediated Inhibition of Skin Catalase Activity Promotes Gr-1+CD11b+ Myeloid Cell Expansion

Skin cancer incidence and mortality are higher in men compared to women, but the causes of this sex discrepancy remain largely unknown. Ultraviolet light exposure induces cutaneous inflammation and neutralizes cutaneous antioxidants. Gr-1+CD11b+ myeloid cells are heterogeneous bone marrow-derived cells that promote inflammation-associated carcinogenesis. Reduced activity of catalase, an antioxidant present within skin, has been associated with skin carcinogenesis. We utilized the outbred, immune competent Skh-1 hairless mouse model of ultraviolet light B (UVB)-induced inflammation and non-melanoma skin cancer to further define sex discrepancies in UVB-induced inflammation. Our results demonstrated that male skin had relatively lower baseline catalase activity, which was inhibited following acute UVB exposure in both sexes. Further analysis revealed that skin catalase activity inversely correlated with splenic Gr-1+CD11b+ myeloid cell percentage. Acute UVB exposure induced Gr-1+CD11b+ myeloid cell skin infiltration, which was inhibited to a greater extent in males by topical catalase treatment. In chronic UVB studies, we demonstrated that the percentage of splenic Gr-1+CD11b+ myeloid cells was 55% higher in male tumor-bearing mice compared to their female counterparts. Together, our findings indicate that lower skin catalase activity in male mice may at least in part contribute to increased UVB-induced Gr-1+CD11b+ myeloid cells and subsequent skin carcinogenesis.

Preventative topical diclofenac treatment differentially decreases tumor burden in male and female Skh-1 mice in a model of UVB-induced cutaneous squamous cell carcinoma

Ultraviolet B (UVB) light is the major environmental carcinogen contributing to non-melanoma skin cancer (NMSC) development. There are over 3.5 million NMSC diagnoses in two million patients annually, with men having a 3-fold greater incidence of squamous cell carcinoma (SCC) compared with women. Chronic inflammation has been linked to tumorigenesis, with a key role for the cyclooxygenase-2 (COX-2) enzyme. Diclofenac, a COX-2 inhibitor and non-steroidal anti-inflammatory drug, currently is prescribed to patients as a short-term therapeutic agent to induce SCC precursor lesion regression. However, its efficacy as a preventative agent in patients without evidence of precursor lesions but with significant UVB-induced cutaneous damage has not been explored. We previously demonstrated in a murine model of UVB-induced skin carcinogenesis that when exposed to equivalent UVB doses, male mice had lower levels of inflammation but developed increased tumor multiplicity, burden and grade compared with female mice. Because of the discrepancy in the degree of inflammation between male and female skin, we sought to determine if topical treatment of previously damaged skin with an anti-inflammatory COX-2 inhibitor would decrease tumor burden and if it would be equally effective in the sexes. Our results demonstrated that despite observed sex differences in the inflammatory response, prolonged topical diclofenac treatment of chronically UVB-damaged skin effectively reduced tumor multiplicity in both sexes. Unexpectedly, tumor burden was significantly decreased only in male mice. Our data suggest a new therapeutic use for currently available topical diclofenac as a preventative intervention for patients predisposed to cutaneous SCC development before lesions appear.

Differential Effects of Topical Vitamin E and C E Ferulic® Treatments on Ultraviolet Light B-Induced Cutaneous Tumor Development in Skh-1 Mice

Because of the ever-increasing incidence of ultraviolet light B (UVB)-induced skin cancer, considerable attention is being paid to prevention through the use of both sunscreens and after sun treatments, many of which contain antioxidants. Vitamin E is included as an antioxidant in many sunscreens and lotions currently on the market. Studies examining the efficacy of vitamin E as a topical preventative agent for UVB-induced skin cancer have yielded conflicting results. A likely contributor to differences in study outcome is the stability of vitamin E in the particular formulation being tested. In the current study we examined the effects of topical vitamin E alone as well as vitamin E combined with vitamin C and ferulic acid in a more stable topical formula (C E Ferulic®). Mice were exposed to UVB for 10 weeks in order to induce skin damage. Then, before the appearance of any cutaneous lesions, mice were treated for 15 weeks with a topical antioxidant, without any further UVB exposure. We found that topical C E Ferulic decreased tumor number and tumor burden and prevented the development of malignant skin tumors in female mice with chronically UVB-damaged skin. In contrast, female mice chronically exposed to UVB and treated topically with vitamin E alone showed a trend towards increased tumor growth rate and exhibited increased levels of overall DNA damage, cutaneous proliferation, and angiogenesis compared to vehicle-treated mice. Thus, we have demonstrated that topical 5% alpha tocopherol may actually promote carcinogenesis when applied on chronically UVB-damaged skin while treating with a more stable antioxidant compound may offer therapeutic benefits.

MIF Antagonist (CPSI-1306) Protects against UVB-Induced Squamous Cell Carcinoma

Macrophage migration inhibitory factor (MIF) is a homotrimeric proinflammatory cytokine implicated in chronic inflammatory diseases and malignancies, including cutaneous squamous cell carcinomas (SCC). To determine whether MIF inhibition could reduce UVB light–induced inflammation and squamous carcinogenesis, a small-molecule MIF inhibitor (CPSI-1306) was utilized that disrupts homotrimerization. To examine the effect of CPSI-1306 on acute UVB-induced skin changes, Skh-1 hairless mice were systemically treated with CPSI-1306 for 5 days before UVB exposure. In addition to decreasing skin thickness and myeloperoxidase (MPO) activity, CPSI-1306 pretreatment increased keratinocyte apoptosis and p53 expression, decreased proliferation and phosphohistone variant H2AX (γ-H2AX), and enhanced repair of cyclobutane pyrimidine dimers. To examine the effect of CPSI-1306 on squamous carcinogenesis, mice were exposed to UVB for 10 weeks, followed by CPSI-1306 treatment for 8 weeks. CPSI-1306 dramatically decreased the density of UVB-associated p53 foci in non–tumor-bearing skin while simultaneously decreasing the epidermal Ki67 proliferation index. In addition to slowing the rate of tumor development, CPSI-1306 decreased the average tumor burden per mouse. Although CPSI-1306–treated mice developed only papillomas, nearly a third of papillomas in vehicle-treated mice progressed to microinvasive SCC. Thus, MIF inhibition is a promising strategy for prevention of the deleterious cutaneous effects of acute and chronic UVB exposure. Implications: Macrophage migration inhibitory factor is a viable target for the prevention of UVB-induced cutaneous SSCs. Mol Cancer Res; 12(9); 1292–302. ©2014 AACR.

Extended UVB Exposures Alter Tumorigenesis and Treatment Efficacy in a Murine Model of Cutaneous Squamous Cell Carcinoma

Epidemiological studies support a link between cumulative sun exposure and cutaneous squamous cell carcinoma (SCC) development. However, the presumed effects of extended ultraviolet light B (UVB) exposure on tumorigenesis in the sexes have not been formally investigated. We examined differences in ultimate tumorigenesis at 25 weeks in mice exposed to UVB for either 10 or 25 weeks. Additionally, we investigated the effect of continued UVB exposure on the efficacy of topical treatment with anti-inflammatory (diclofenac) or antioxidant (C E Ferulic or vitamin E) compounds on modulating tumorigenesis. Vehicle-treated mice in the 25-week UVB exposure model exhibited an increased tumor burden and a higher percentage of malignant tumors compared to mice in the 10-week exposure model, which correlated with increases in total and mutant p53-positive epidermal cells. Only topical diclofenac decreased tumor number and burden in both sexes regardless of UVB exposure length. These data support the commonly assumed but not previously demonstrated fact that increased cumulative UVB exposure increases the risk of UVB-induced SCC development and can also affect therapeutic efficacies. Our study suggests that cessation of UVB exposure by at-risk patients may decrease tumor development and that topical NSAIDs such as diclofenac may be chemopreventive.

Ultraviolet Light B-Mediated Inhibition of Skin Catalase Activity Promotes Gr-1+CD11b+ Myeloid Cell Expansion

Skin cancer incidence and mortality are higher in men compared to women, but the causes of this sex discrepancy remain largely unknown. Ultraviolet light exposure induces cutaneous inflammation and neutralizes cutaneous antioxidants. Gr-1+CD11b+ myeloid cells are heterogeneous bone marrow-derived cells that promote inflammation-associated carcinogenesis. Reduced activity of catalase, an antioxidant present within skin, has been associated with skin carcinogenesis. We utilized the outbred, immune competent Skh-1 hairless mouse model of ultraviolet light B (UVB)-induced inflammation and non-melanoma skin cancer to further define sex discrepancies in UVB-induced inflammation. Our results demonstrated that male skin had relatively lower baseline catalase activity, which was inhibited following acute UVB exposure in both sexes. Further analysis revealed that skin catalase activity inversely correlated with splenic Gr-1+CD11b+ myeloid cell percentage. Acute UVB exposure induced Gr-1+CD11b+ myeloid cell skin infiltration, which was inhibited to a greater extent in males by topical catalase treatment. In chronic UVB studies, we demonstrated that the percentage of splenic Gr-1+CD11b+ myeloid cells was 55% higher in male tumor-bearing mice compared to their female counterparts. Together, our findings indicate that lower skin catalase activity in male mice may at least in part contribute to increased UVB-induced Gr-1+CD11b+ myeloid cells and subsequent skin carcinogenesis.

UV Light B|[ndash]|Mediated Inhibition of Skin Catalase Activity Promotes Gr-1|[plus]|CD11b|[plus]| Myeloid Cell Expansion

Skin cancer incidence and mortality are higher in men compared to women, but the causes of this sex discrepancy remain largely unknown. Ultraviolet light exposure induces cutaneous inflammation and neutralizes cutaneous antioxidants. Gr-1+CD11b+ myeloid cells are heterogeneous bone marrow-derived cells that promote inflammation-associated carcinogenesis. Reduced activity of catalase, an antioxidant present within skin, has been associated with skin carcinogenesis. We utilized the outbred, immune competent Skh-1 hairless mouse model of ultraviolet light B (UVB)-induced inflammation and non-melanoma skin cancer to further define sex discrepancies in UVB-induced inflammation. Our results demonstrated that male skin had relatively lower baseline catalase activity, which was inhibited following acute UVB exposure in both sexes. Further analysis revealed that skin catalase activity inversely correlated with splenic Gr-1+CD11b+ myeloid cell percentage. Acute UVB exposure induced Gr-1+CD11b+ myeloid cell skin infiltration, which was inhibited to a greater extent in males by topical catalase treatment. In chronic UVB studies, we demonstrated that the percentage of splenic Gr-1+CD11b+ myeloid cells was 55% higher in male tumor-bearing mice compared to their female counterparts. Together, our findings indicate that lower skin catalase activity in male mice may at least in part contribute to increased UVB-induced Gr-1+CD11b+ myeloid cells and subsequent skin carcinogenesis.

Abstract 4202: Sex disparities in the cutaneous response to acute ultraviolet light exposure

Epidemiological evidence has identified a 3-fold increase in squamous cell skin cancer incidence in men compared to women. The historical explanation for this sex-bias is increased occupational exposure to ultraviolet (UV) light. Our studies demonstrated, however, that when chronically exposed to equivalent doses of UV light, male mice developed more skin tumors that were larger and more aggressive than those arising in female mice. Additionally, the skin of male mice acutely exposed to UV light had higher levels of oxidative DNA damage and decreased total antioxidant capacity compared to females (Thomas-Ahner, JM Cancer Research 2007). The hypothesis for the current study is that the topical application of anti-inflammatory and/or antioxidant compounds may differentially affect the acute UVB induced inflammatory response in the sexes. To test this hypothesis, female and male Skh-1 mice were exposed to 2240J/m2 UVB light followed immediately by topical application of either an anti-inflammatory compound (Solaraze), an antioxidant (Vitamin E), or a combination of the 2 compounds. Animals were sacrificed at 24 and 48 hrs following a single exposure and topical treatment. Skin was analyzed for myeloperoxidase (MPO) activity as a measure of neutrophil infiltration, catalase activity, and immunohistochemically for the presence of p53 protein in the epidermis. In response to UVB exposure, vehicle treated female mice had significantly increased MPO activity compared to male mice. Topical treatments with Solaraze, Vitamin E or the combination significantly reduced MPO levels in female mice but had no effects on the already low levels of UV-induced MPO in male skin. Catalase is a key enzyme used for detoxifying hydrogen peroxide in the skin. Baseline levels of catalase were lower in male skin compared to female skin and UVB exposure significantly reduced catalase in both sexes. Topical treatment with Solaraze significantly increased male catalase activity to levels similar to female unirradiated skin while vitamin E partially restored catalase activity in female mice but had no effect in male skin. The single exposure to UVB induced higher numbers of p53 positive epidermal cells in male skin, however at this time point topical treatment had no effect on UVB-induced p53 levels in either sex. Our data suggests that UVB differentially effects inflammation in the sexes and that topical application of anti-inflammatory or anti-oxidant compounds have differential effects on markers of an acute inflammatory response in the skin. Future studies will evaluate the effects of additional compounds and delivery vehicles following both acute and chronic UVB exposure. This work was supported by NIH NCI CA133629. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4202.

Abstract 3378: E-cadherin suppresses breast cancer cell growth and interleukin-6 production

Carcinomas arise from epithelial tissues and represent the most common type of human cancer. E-cadherin is an epithelial-specific intercellular adhesion molecule, which localizes to epithelial cell adherens junctions and is a critical mediator of carcinoma cell invasion and metastasis. Although impaired E-cadherin expression can be clinically utilized to predict breast cancer cell invasiveness, metastasis, and patient survival, E-cadherin has received less attention for its role as a putative regulator of epithelial cell growth. We have previously shown that interleukin-6 (IL-6), a pleiotropic inflammatory cytokine associated with poor clinical outcome in breast cancer patients, is a potent growth factor and repressor of E-cadherin in human breast cancer cells. Consistent with clinical reports and our previous findings, we hypothesized that E-cadherin repression may be responsible for IL-6-induced breast cancer cell growth. The current study demonstrated that IL-6 enhanced the growth rates of estrogen receptor-alpha (ERα)-positive breast cancer cells in a three-dimensional (3D) tumor growth assay (TGA) in a dose-dependent manner. We showed that IL-6 also repressed E-cadherin protein levels in ERα-positive breast cancer cells in a 3D TGA in a dose-dependent manner. We have previously shown that IL-6 treatment does not affect E-cadherin-negative MDA-MB-231 cell growth, and the current study showed similar results in MCF-7 cells which express Twist and therefore lack E-cadherin. MDA-MB-231 cells lack E-cadherin and express IL-6, thus allowing us to express stable ectopic E-cadherin to evaluate its impact on cell growth and IL-6 production. Ectopic E-cadherin expression significantly suppressed MDA-MB-231 cell growth (P-value Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3378.