Xiuwei Tang

Inhibition of smoothened signaling prevents ultraviolet B-induced basal cell carcinomas through regulation of Fas expression and apoptosis

Abnormal activation of the hedgehog-signaling pathway is the pivotal abnormality driving the growth of basal cell carcinomas (BCCs), the most common type of human cancer. Antagonists of this pathway such as cyclopamine may therefore be useful for treatment of basal cell carcinomas and other hedgehog-driven tumors. We report here that chronic oral administration of cyclopamine dramatically reduces (∼66%) UVBinduced basal cell carcinoma formation in Ptch1+/− mice. Fas expression is low in human and murine basal cell carcinomas but is up-regulated in the presence of the smoothened (SMO) antagonist, cyclopamine, both in vitro in the mouse basal cell carcinoma cell line ASZ001 and in vivo after acute treatment of mice with basal cell carcinomas. This parallels an elevated rate of apoptosis. Conversely, expression of activated SMO in C3H10T1/2 cells inhibits Fas expression. Fas/Fas ligand interactions are necessary for cyclopamine-mediated apoptosis in these cells, a process involving caspase-8 activation. Our data provide strong evidence that cyclopamine and perhaps other SMO antagonists are potent in vivo inhibitors of UVB-induced basal cell carcinomas in Ptch1+/− mice and likely in humans because the majority of human basal cell carcinomas manifest mutations in PTCH1 and that a major mechanism of their inhibitory effect is through up-regulation of Fas, which augments apoptosis.

Cyclooxygenase-2 Expression in Murine and Human Nonmelanoma Skin Cancers: Implications for Therapeutic Approaches¶

Abstract Inflammatory stimuli result in the production of cutaneous eicosanoids, which are known to contribute to the process of tumor promotion. Cyclooxygenase (COX), the rate-limiting enzyme for the production of prostaglandins (PG) from arachidonic acid, exists in at least two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and plays various physiological roles, whereas increased COX-2 expression is known to occur in several types of epithelial neoplasms. Enhanced PG synthesis is a potential contributing factor in UVB-induced nonmelanoma skin cancers (NMSC). Increased COX-2 staining occurs in murine skin neoplasms after chronic exposure to carcinogenic doses of UVB. In this study, immunohistochemical and Western blot analyses were employed to assess longitudinally COX-2 expression in a standard mouse UVB complete carcinogenesis protocol and in human basal cell carcinomas (BCC) and squamous cell carcinomas (SCC). During UVB irradiation of mice, COX-2 expression consistently increased in the hyperplastic skin, the benign papillomas and the SCC. COX-2 expression was also increased in human actinic keratoses, SCC and BCC as well as in murine SCC and BCC. The pattern of COX-2 expression was quite variable, occurring in a patchy distribution in some lesions with staining confined mainly to suprabasal cell layers. In general, COX-2 expression progressively became more extensive in benign papillomas and well-differentiated murine SCC. The staining was predominantly cytoplasmic and perinuclear in some focal areas in tissue stroma around both murine and human tumors. Western blot analysis confirmed negative COX-2 expression in normal skin, whereas acute UVB exposure resulted in increased enzyme expression, which continued to increase in developing papillomas and SCC. Because of the evidence indicating a pathogenic role for eicosanoids in murine and human skin neoplasms, we performed studies to assess the anti-inflammatory and anticarcinogenic effects of green tea extracts, which are potent antioxidants. Acute exposure of the human skin to UVB (minimum erythema dose × 4) caused a transient enhancement of the COX-2 expression, which reverted to baseline within hours; however, in murine skin the expression persisted for several days. Pretreatment with the topically applied green tea extract (1 mg/cm2) largely abrogated the acute COX-2 response to UVB in mice or humans. In summary, enhanced COX-2 expression serves as a marker of epidermal UVB exposure for murine and human NMSC. These results suggest that COX-2 inhibitors could have potent anticarcinogenic effects in UVB-induced skin cancer.

Hedgehog signalling in skin development and cancer

Abstract:  Basal cell carcinoma (BCC) is the most common human malignancy, affecting 750 000 Americans each year. The understanding of mutations that are known to activate hedgehog (Hh) signalling pathway genes, including PATCHED (PTCH), sonic hedgehog (Shh) and smoothened (Smo), has substantially expanded our current understanding of the genetic basis of BCC development. The Hh signalling pathway is one of the most fundamental signal transduction pathways in embryonic development. In skin, the Shh pathway is crucial for maintaining stem cell population, and for regulating hair follicle and sebaceous gland development. This pathway plays a minimal role in adult tissues, but is known to be activated in many neoplasms, including those arising in the skin. In this review, we attempt to summarize the results of published studies on some important aspects of the Shh pathway and its involvement in skin development and carcinogenesis. We also provide a description of various animal models that have been developed, based on our knowledge of the Shh pathway in human skin cancers. Additionally, we include a brief description of studies conducted in our laboratory and by others on the chemoprevention of BCCs. This review therefore provides a current understanding of the role of the Shh pathway in skin development and neoplasia. It also provides a basis for the molecular target‐based chemoprevention and therapeutic management of skin cancer.

Mitochondrial Damage Mediates Genotoxicity of Arsenic in Mammalian Cells

Arsenic is an important environmental carcinogen that affects millions of people worldwide through contaminated water supplies. For decades, arsenic was considered a nongenotoxic carcinogen. Using the highly sensitive A(L) mutation assay, we previously showed that arsenic is, indeed, a potent gene and chromosomal mutagen and that its effects are mediated through the induction of reactive oxygen species. However, the origin of these radicals and the pathways involved are not known. Here we show that mitochondrial damage plays a crucial role in arsenic mutagenicity. Treatment of enucleated cells with arsenic followed by rescue fusion with karyoplasts from controls resulted in significant mutant induction. In contrast, treatment of mitochondrial DNA-depleted (rho(0)) cells produced few or no mutations. Mitochondrial damage can lead to the release of superoxide anions, which then react with nitric oxide to produce the highly reactive peroxynitrites. The mutagenic damage was dampened by the nitric oxide synthase inhibitor, N(G)-methyl-L-arginine. These data illustrate that mitochondria are a primary target in arsenic-induced genotoxic response and that a better understanding of the mutagenic/carcinogenic mechanism of arsenic should provide a basis for better interventional approach in both treatment and prevention of arsenic-induced cancer.

CP-31398 restores mutant p53 tumor suppressor function and inhibits UVB-induced skin carcinogenesis in mice

Mutations in the tumor suppressor p53 are detectable in over 50% of all human malignancies. Mutant p53 protein is incapable of transactivating its downstream target genes that are required for DNA repair and apoptosis. Chronic exposure to UVB induces p53 mutations and is carcinogenic in both murine and human skin. CP-31398, a styrylquinazoline compound, restores the tumor suppressor functions of mutant forms of p53 in tumor cells. However, its effectiveness in vivo remains unclear. Here, we demonstrate that CP-31398 blocked UVB-induced skin carcinogenesis and was associated with increases in p53, p21, and BclXs. CP-31398 downregulated Bcl2, proliferating nuclear cell antigen, and cyclin D1. Activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase also occurred in both tumor and perilesional skin following treatment. CP-31398 induced the expression of p53-dependent target proteins, and this was followed by apoptosis in UVB-irradiated wild-type mice but not in their p53-deficient littermates. Similar effects were observed in human skin carcinoma A431 cells expressing mutant p53. In addition, CP-31398 induced mitochondrial translocation of p53, leading to changes in mitochondrial membrane permeability pore transition (MPT) and consequent cytochrome c release in these cells. Blocking MPT diminished p53 translocation and apoptosis. These studies indicate that reconstituting p53 tumor suppressor functions in vivo by small molecular weight compounds may block the pathogenesis and progression of skin cancer.

Basal Cell Carcinoma Chemoprevention with Nonsteroidal Anti-inflammatory Drugs in Genetically Predisposed PTCH1+/− Humans and Mice

In vitro and epidemiologic studies favor the efficacy of nonsteroidal anti-inflammatory drugs (NSAID) in preventing skin squamous photocarcinogenesis, but there has been relatively little study of their efficacy in preventing the more common skin basal cell carcinoma (BCC) carcinogenesis. We first compared the relative anti-BCC effects of genetic deletion and NSAID pharmacologic inhibition of cyclooxygenase (COX) enzymes in the skin of Ptch1+/− mice. We then assessed the effects of celecoxib on the development of BCCs in a 3-year, double-blinded, randomized clinical trial in 60 (PTCH1+/−) patients with the basal cell nevus syndrome. In Ptch1+/− mice, genetic deletion of COX1 or COX2 robustly decreased (75%; P < 0.05) microscopic BCC tumor burden, but pharmacologic inhibition with celecoxib reduced microscopic BCCs less efficaciously (35%; P < 0.05). In the human trial, we detected a trend for oral celecoxib reducing BCC burden in all subjects (P = 0.069). Considering only the 60% of patients with less severe disease (<15 BCCs at study entry), celecoxib significantly reduced BCC number and burden: subjects receiving placebo had a 50% increase in BCC burden per year, whereas subjects in the celecoxib group had a 20% increase (Pdifference = 0.024). Oral celecoxib treatment inhibited BCC carcinogenesis in PTCH1+/− mice and had a significant anti-BCC effect in humans with less severe disease. Cancer Prev Res; 3(1); OF1–11

Ornithine decarboxylase is a target for chemoprevention of basal and squamous cell carcinomas in Ptch1+/– mice

Solar ultraviolet B (UVB) radiation induces cutaneous ornithine decarboxylase (ODC), the first enzyme in the polyamine-biosynthesis pathway, which drives continued proliferation and clonal expansion of initiated (mutated) cells, leading to tumorigenesis. Therefore ODC is a potentially important target for chemoprevention of basal cell carcinomas (BCCs), the majority of which have mutations in the tumor-suppressor gene known as patched (PTCH). To assess this possibility, we first overexpressed ODC in the skin of Ptch1+/- mice using a keratin 6 (K6) promoter that directs constitutive ODC expression in the outer root sheath of the hair follicle. UVB irradiation of these mice accelerated induction of BCCs as compared with their Ptch1+/- littermates. To further verify the role of ODC in BCC tumorigenesis, we used an antizyme (AZ) approach to inhibit ODC activity in the Ptch1+/- mice. Ptch1+/- mice with AZ overexpression driven by the K6 promoter were resistant to the induction of BCCs by UVB. Furthermore, oral administration of the suicidal ODC inhibitor alpha-difluoromethylornithine reduced UVB-induced BCCs in Ptch1+/- mice. These results demonstrate the crucial importance of ODC for the induction of BCCs and indicate that chemopreventive strategies directed at inhibiting this enzyme may be useful in reducing BCCs in human populations.

Cyclooxygenase-2 Inhibitor Nimesulide Blocks Ultraviolet B-induced Photocarcinogenesis in SKH-1 Hairless Mice†

Cyclooxygenase‐2 (COX‐2) inhibition can inhibit UVB‐induced carcinogenesis in the skin. We have shown that COX‐2 is overexpressed in UVB‐induced squamous cell carcinomas (SCCs). Celecoxib, a specific inhibitor of COX‐2, blocks UVB‐induced papillomas and carcinomas in murine skin. However, as COX‐2 inhibitors of this type are associated with an increased risk of adverse cardiovascular events, we decided to study nimesulide, a different class of COX‐2 inhibitor, an N‐arylmethanesulfonamide derivative not known to have these untoward effects. To assess the antitumor‐promoting effects of nimesulide, 90 mice were equally divided into three groups. Group I animals received no test agent or UVB and served as age‐matched controls; group II animals were irradiated with UVB (180 mJ cm−2, twice weekly for 35 weeks) and group III animals received 300 p.p.m. nimesulide in drinking water and were irradiated with UVB as described for group‐II. Nimesulide treatment reduced the growth of UVB‐induced tumors both in terms of tumor number and tumor volume. By weeks 25, 30 and 35, the tumor numbers in the nimesulide‐treated group were 79%, 49% and 53% less than the number occurring in UVB‐treated animals whereas tumor volume was reduced 69%, 54% and 53%, respectively, compared to the UVB‐irradiated control group. Nimesulide also inhibited the malignant progression of SCCs. The reduction in tumorigenesis was paralleled by a decrease in cell cycle regulatory proteins (cyclins A, B1, D1, E, CDK2/4/6) and the antiapoptotic protein (Bcl2); concomitantly there was an increase in proapoptotic markers, poly (ADP‐ribose) polymerase (PARP) and caspase‐3. Nimesulide also decreased ornithine decarboxylase expression and the nuclear accumulation of nuclear factor kappa B transcriptionally active protein complexes. These results show that alternative classes of COX‐2 inhibitors may likely be efficacious as cancer chemopreventive agents and may have an improved therapeutic index.

Shh and p50/Bcl3 signaling crosstalk drives pathogenesis of BCCs in Gorlin syndrome

Nevoid basal cell carcinoma syndrome (NBCCS) is a rare autosomal dominant disorder that is due, in large measure, to aberrant Shh signaling driven by mutations in the tumor suppressor gene Ptch1. Here, we describe the development of Ptch1+/−/SKH-1 mice as a novel model of this disease. These animals manifest many features of NBCCS, including developmental anomalies and are remarkably sensitive to both ultraviolet (UVB) and ionizing radiation that drive the development of multiple BCCs. Just as in patients with NBCCS, Ptch1+/−/SKH-1 also spontaneously develops BCCs and other neoplasms such as rhabdomyomas/rhabdomyosarcomas. Administration of smoothened inhibitors (vismodegib/itraconazole/cyclopamine) or non-steroidal anti-inflammatory drug (sulindac/sulfasalazine) each result in partial resolution of BCCs in these animals. However, combined administration of these agents inhibits the growth of UVB-induced BCCs by >90%. Employing small molecule- and decoy-peptide-based approaches we further affirm that complete remission of BCCs could only be achieved by combined inhibition of p50-NFκB/Bcl3 and Shh signaling. We posit that Ptch1+/−/SKH-1 mice are a novel and relevant animal model for NBCCS. Understanding mechanisms that govern genetic predisposition to BCCs should facilitate our ability to identify and treat NBCCS gene carriers, including those at risk for sporadic BCCs while accelerating development of novel therapeutic modalities for these patients.

Hair follicle disruption facilitates pathogenesis to UVB-induced cutaneous inflammation and basal cell carcinoma development in Ptch(+/-) mice.

Hairless mice carrying homozygous mutations in hairless gene manifest rudimentary hair follicles (HFs), epidermal cysts, hairless phenotype, and enhanced susceptibility to squamous cell carcinomas. However, their susceptibility to basal cell carcinomas (BCCs), a neoplasm considered originated from HF-localized stem cells, is unknown. To demonstrate the role of HFs in BCC development, we bred Ptch(+/-)/C57BL6 with SKH-1 hairless mice, followed by brother-sister cross to get F2 homozygous mutant (hairless) or wild-type (haired) mice. UVB-induced inflammation was less pronounced in shaved haired than in hairless mice. In hairless mice, inflammatory infiltrate was found around the rudimentary HFs and epidermal cysts. Expression of epidermal IL1f6, S100a8, vitamin D receptor, repetin, and major histocompatibility complex II, biomarkers depicting susceptibility to cutaneous inflammation, was also higher. In these animals, HF disruption altered susceptibility to UVB-induced BCCs. Tumor onset in hairless mice was 10 weeks earlier than in haired littermates. The incidence of BCCs was significantly higher in hairless than in haired animals; however, the magnitude of sonic hedgehog signaling did not differ significantly. Overall, 100% of hairless mice developed >12 tumors per mouse after 32 weeks of UVB therapy, whereas haired mice developed fewer than three tumors per mouse after 44 weeks of long-term UVB irradiation. Tumors in hairless mice were more aggressive than in haired littermates and manifested decreased E-cadherin and enhanced mesenchymal proteins. These data provide novel evidence that disruption of HFs in Ptch(+/-) mice enhances cutaneous susceptibility to inflammation and BCCs.