Human Basal Cell Carcinoma Research Articles

Abstract 2021: Ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis through upregulation of keratin-17

Abstract Solar UV irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is 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 cancerr and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic ablation of caspase-7 are significantly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and tumor numbers were increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation biomarkers, 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. 2-DE 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. Furthermore 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 suggests that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17. Citation Format: Mee-Hyun Lee, Do Young Lim, Sung Young Lee, Jung Hyun Shim, Xuejiao Liu, Ran Zhao, Hai Huang, G.Timothy Bowden, Young-Joon Surh, Yong-Yeon Cho, Ann M. Bode, Zigang Dong. Ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis through upregulation of keratin-17 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2021. doi:10.1158/1538-7445.AM2017-2021

INTU is essential for oncogenic Hh signaling through regulating primary cilia formation in basal cell carcinoma.

Inturned (INTU), a cilia and planar polarity effector (CPLANE), performs prominent ciliogenic functions during morphogenesis, such as in the skin. INTU is expressed in adult tissues but its role in tissue maintenance is unknown. Here, we report that the expression of the INTU gene is aberrantly elevated in human basal cell carcinoma (BCC), coinciding with increased primary cilia formation and activated hedgehog (Hh) signaling. Disrupting Intu in an oncogenic mutant Smo (SmoM2)-driven BCC mouse model prevented the formation of BCC through suppressing primary cilia formation and Hh signaling, suggesting that Intu performs a permissive role during BCC formation. INTU is essential for IFT-A complex assembly during ciliogenesis. To further determine whether Intu is directly involved in the activation of Hh signaling downstream of ciliogenesis, we examined the Hh signaling pathway in mouse embryonic fibroblasts, which readily respond to Hh pathway activation. Depleting Intu blocked SAG-induced Hh pathway activation, whereas the expression of Gli2ΔN, a constitutively active Gli2, restored Hh pathway activation in Intu-deficient cells, suggesting that INTU functions upstream of Gli2 activation. In contrast, overexpressing Intu did not promote ciliogenesis or Hh signaling. Taken together, data obtained from this study suggest that INTU is indispensable during BCC tumorigenesis and that its aberrant upregulation is likely a prerequisite for primary cilia formation during Hh-dependent tumorigenesis.

Differing tumor-suppressor functions of Arf and p53 in murine basal cell carcinoma initiation and progression.

Human basal cell carcinomas (BCCs) very frequently carry p53 mutations, and p53 loss markedly accelerates murine BCC carcinogenesis. We report here our studies of the mechanism by which p53 is activated to suppress BCC carcinogenesis. We find that aberrant hedgehog signaling in microscopic BCCs activates p53 in part via Arf (that is, the oncogene-induced stress pathway) but not via the DNA damage response pathway. However, Arf loss and p53 loss produce differing outcomes-loss of p53 promotes both tumor initiation and progression; loss of Arf promotes tumor progression but not initiation. Intriguingly, increased expression of Arf in tumor stromal cells, as in tumor keratinocytes themselves, contributes to suppression of BCC carcinogenesis.

Ultraviolet Radiation-Induced Downregulation of SERCA2 Mediates Activation of NLRP3 Inflammasome in Basal Cell Carcinoma.

Basal cell carcinomas (BCCs) account for majority of skin malignancies in the United States. The incidence of BCCs is strongly associated with exposure of ultraviolet (UV) radiation. Nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome plays an important role in innate immune responses. Different stimuli such as toxins, microorganisms and particles released from injured cells activate the NLRP3 inflammasome. Activated NLRP3 results in activation of caspase-1, which cleaves pro-IL-1β to active IL-1β. In this study, we have shown that NLRP3 is expressed in human basal cell carcinomas. The proximal steps in activation of NLRP3 inflammasome are not well understood. Here, we have attempted to elucidate a critical role for Ca2+ mobilization in activation of the NLRP3 inflammasome by UVB exposure using HaCaT keratinocytes. We have demonstrated that UVB exposure blocks Ca2+ mobilization by downregulating the expression of sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA2), a component of store-operated Ca2+ entry that leads to activation of the NLRP3 inflammasome.

The Concerted Action of Type 2 and Type 3 Deiodinases Regulates the Cell Cycle and Survival of Basal Cell Carcinoma Cells.

Thyroid hormones (THs) mediate pleiotropic cellular processes involved in metabolism, cellular proliferation, and differentiation. The intracellular hormonal environment can be tailored by the type 1 and 2 deiodinase enzymes D2 and D3, which catalyze TH activation and inactivation respectively. In many cellular systems, THs exert well-documented stimulatory or inhibitory effects on cell proliferation; however, the molecular mechanisms by which they control rates of cell cycle progression have not yet been entirely clarified. We previously showed that D3 depletion or TH treatment influences the proliferation and survival of basal cell carcinoma (BCC) cells. Surprisingly, we also found that BCC cells express not only sustained levels of D3 but also robust levels of D2. The aim of the present study was to dissect the contribution of D2 to TH metabolism in the BCC context, and to identify the molecular changes associated with cell proliferation and survival induced by TH and mediated by D2 and D3. We used the CRISPR/Cas9 technology to genetically deplete D2 and D3 in BCC cells and studied the consequences of depletion on cell cycle progression and on cell death. Cell cycle progression was analyzed by fluorescence activated cell sorting analysis of synchronized cells, and the apoptosis rate by annexin V incorporation. Mechanistic investigations revealed that D2 inactivation accelerates cell cycle progression thereby enhancing the proportion of S-phase cells and cyclin D1 expression. Conversely, D3 mutagenesis drastically suppressed cell proliferation and enhanced apoptosis of BCC cells. Furthermore, the basal apoptotic rate was oppositely regulated in D2- and D3-depleted cells. Our results indicate that BCC cells constitute an example in which the TH signal is finely tuned by the concerted expression of opposite-acting deiodinases. The dual regulation of D2 and D3 expression plays a critical role in cell cycle progression and cell death by influencing cyclin D1-mediated entry into the G1-S phase. These findings reinforce the concept that TH is a potential therapeutic target in human BCC.

Notch signaling is significantly suppressed in basal cell carcinomas and activation induces basal cell carcinoma cell apoptosis.

A subset of basal cell carcinomas (BCCs) are directly derived from hair follicles (HFs). In some respects, HFs can be defined as ‘ordered’ skin appendage growths, while BCCs can be regarded as ‘disordered’ skin appendage growths. The aim of the present study was to examine HFs and BCCs to define the expression of common and unique signaling pathways in each skin appendage. Human nodular BCCs, along with HFs and non-follicular skin epithelium from normal individuals, were examined using microarrays, qPCR, and immunohistochemistry. Subsequently, BCC cells and root sheath keratinocyte cells from HFs were cultured and treated with Notch signaling peptide Jagged1 (JAG1). Gene expression, protein levels, and cell apoptosis susceptibility were assessed using qPCR, immunoblotting, and flow cytometry, respectively. Specific molecular mechanisms were found to be involved in the process of cell self-renewal in the HFs and BCCs, including Notch and Hedgehog signaling pathways. However, several key Notch signaling factors showed significant differential expression in BCCs compared with HFs. Stimulating Notch signaling with JAG1 induced apoptosis of BCC cells by increasing Fas ligand expression and downstream caspase-8 activation. The present study showed that Notch signaling pathway activity is suppressed in BCCs, and is highly expressed in HFs. Elements of the Notch pathway could, therefore, represent targets for the treatment of BCCs and potentially in hair follicle engineering.

Investigation of P120catenin Expression in Human Basal Cell Carcinoma of the Skin.

P120(ctn) is a specific membranous adhesion protein, that maintains the stability of intercellular junctions. An altered expression of p120(ctn), either reduced in the cell membrane or increase in the cytoplasm, plays a crucial role in carcinogenesis. No research has analysed the expression of p120(ctn) in basal cell carcinoma (BCC) of the skin so far. Therefore, we immunohistochemically studied p120(ctn) in a set of cutaneous BCCs in order to determine, whether there is difference in the expression pattern related to the histologic subtypes and tumor growth characteristics. The study group consisted of 38 BCCs cathegorized into low-risk (non-infiltrative) subroup (8 superficial and 12 nodular subtypes) and high-risk (infiltrative) subgroup (10 nodular-infiltrative and 8 infiltrative subtypes). Specific monoclonal antibody against p120(ctn) was used for staining. Overall, there were 12 cases (31.6%) with normal preserved and 26 cases (68.4%) with abnormal p120(ctn) expression. In superficial, nodular, nodular-infiltrative and infiltrative subtypes, abnormal p120(ctn) immunoreactivity was found in 37.5% (3/8), 41.7% (5/12), 100% (10/10) and 100% (8/8), respectively. We have confirmed a strong correlation between the expression of p120(ctn) and both given, non-infiltrative and infiltrative BCC growth phenotypes. In the latter subgroup, almost all lesions showed diffusely reduced membranous staining, of which five also manifested an aberrant immunoreactivity in the cytoplasm. This cytoplasmic positivity occurred solely at the invasive front of the infiltrative tumor formations. Our results showed that decreased membranous expression of p120(ctn) was a frequent event in human cutaneous BCC and it was associated with infiltrative growth phenotype. Considering that nearly half of the BCCs with non-infiltrative growth pattern also exhibited reduced membranous expression, aberrant cytoplasmic immunoreactivity of p120(ctn), which was found exclusively in the high-risk BCC variants, can more reliably reflect and predict biological behaviour and malignant potential.

Altered Expression of PRKX, WNT3 and WNT16 in Human Nodular Basal Cell Carcinoma.

Nodular and superficial are the most common subtypes of basal cell carcinoma (BCC). Signaling pathways such as Hedgehog (HH) and Wingless (WNT) signaling are associated with BCC phenotypic variation. The aim of the study was to evaluate of the expression profiles of 84 genes related to the WNT and HH signaling pathways in patients with nodular and superficial BCC. A total of 58 BCCs and 13 samples of normal skin were evaluated by quantitative real-time polymerase chain reaction (qPCR) to detect the gene-expression profile. qPCR array showed segregation in BCC subtypes compared to healthy skin. PRKX, WNT3 and WNT16 were significantly (p<0.05) altered: PRKX was up-regulated, and WNT3 and WNT16 were down-regulated in nodular BCC. PRKX, WNT3 and WNT16 genes, belonging to the WNT signaling pathway, are involved in the tumorigenic process of nodular BCC.

081 Resistant basal cell carcinomas require SRF/MRTF to maintain hedgehog pathway activation and tumor growth

080 Mutational landscape of MCPyV-positive and MCPyV-negative Merkel cell carcinomas with implications for immunotherapy T Walradt, V Makarov, R Doumani, N Riaz, K Stafstrom, A Moshiri, L Yelistratova, J Levinsohn, T Chan, P Nghiem, R Lifton and J Choi 1 Dermatology, Northwestern University, Chicago, IL, 2 Dermatology, Yale University, New Haven, CT, 3 Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, 4 Dermatology, University of Washington, Seattle, WA, 5 Genetics, Yale University, New Haven, CT and 6 Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine carcinoma, associated with the merkel cell polyomavirus (MCPyV) in 80% of cases. To define the genetic basis of MCCs, we performed exome sequencing of 49 MCCs. We show that MCPyV-negative MCCs have a high mutation burden (median of 1121 Somatic Single Nucleotide Variants (SSNVs) per-exome with frequent mutations in RB1 and TP53 and additional damaging mutations in genes in the chromatin modification (ASXL1, MLL2, and MLL3), JNK (MAP3K1 and TRAF7), and DNA-damage pathways (ATM, MSH2, and BRCA1). In contrast, MCPyV-positive MCCs harbor few SSNVs (median of 12.5 SSNVs/tumor) with none in the genes listed above. In both subgroups, there are rare cancer-promoting mutations predicted to activate the PI3K pathway (HRAS, KRAS, PIK3CA, PTEN, and TSC1) and to inactivate the NOTCH pathway (NOTCH1 and NOTCH2). Virus-negative MCCs harbor potentially targetable gain-of-function mutations in TP53 at p.R248 and p.P278. Moreover, TP53 mutational status predicts death in early stage MCC (5-year survival in TP53 mutant vs wild-type stage I and II MCCs is 20% vs. 92%, respectively; P1⁄40.0036). Lastly, we identified the tumor neoantigens in MCPyV-negative and MCPyV-positive MCCs. We found that virusnegative MCCs harbor more tumor neoantigens than melanomas or non-small cell lung cancers (median of 173, 65, and 111 neoantigens/sample, respectively), two cancers for which immune checkpoint blockade can produce durable clinical responses. Collectively, these data support the use of immunotherapies for virus-negative MCCs. 081 Resistant basal cell carcinomas require SRF/MRTF to maintain hedgehog pathway activation and tumor growth R Whitson, A Lee, N Urman, J Li, A Mirza, A Brown, C Yao, G Shankar, M Fry, SX Atwood, E Epstein, J Tang and AE Oro 1 Dermatology, Stanford, Stanford, CA, 2 Childrens Hospital Oakland Research Institute, Oakland, CA and 3 UC Irvine, Irvine, CA Basal cell carcinoma (BCC) and other hedgehog-dependent cancers commonly acquire drug resistance to the Smoothened (Smo) inhibitor vismodegib (Atwood et al., 2014; Sekulic et al., 2015). Resistant BCCs maintain activation of the hedgehog pathway suggesting the presence of mutations at or downstream of Smo. Variants in Smo are found in w50% of resistance cases (Atwood et al., 2015b), however, many variants are non-functional (Atwood et al., 2015a) leaving tumor escape mechanisms for a large proportion of patient tumors unexplored. We recently uncovered a novel resistance mechanism using multi-dimensional genomic analysis in human and mouse resistant BCCs to identify a non-canonical hedgehog activation pathway driven by the transcription factor, serum response factor (SRF). The majority of resistant human and mouse BCCs measured acquire the active nuclear form of SRF, which forms a novel protein complex and shares chromosomal occupancy with the hedgehog transcription factor Gli1. SRF is necessary for hedgehog pathway output, and activation by the SRF coactivator myocardin-related transcription factor (MRTF) is sufficient to potentiate hedgehog activation. Remarkably, pharmacological inhibitors targeting MRTF suppress tumor growth in mice harboring resistant BCCs. Our data uncovers a novel non-canonical mechanism for hedgehog transcriptional activation through SRF/MRTF which is activated in a majority of resistant BCCs. Combined with our in vivo data, we highlight SRF/MRTF-driven transcription as a promising therapeutic target to combat resistant BCC growth.

Ionizing Radiation Exposure and Basal Cell Carcinoma Pathogenesis.

This commentary summarizes studies showing risk of basal cell carcinoma (BCC) development in relationship to environmental, occupational and therapeutic exposure to ionizing radiation (IR). BCC, the most common type of human cancer, is driven by the aberrant activation of hedgehog (Hh) signaling. Ptch, a tumor suppressor gene of Hh signaling pathway, and Smoothened play a key role in the development of radiation-induced BCCs in animal models. Epidemiological studies provide evidence that humans exposed to radiation as observed among the long-term, large scale cohorts of atomic bomb survivors, bone marrow transplant recipients, patients with tinea capitis and radiologic workers enhances risk of BCCs. Overall, this risk is higher in Caucasians than other races. People who were exposed early in life develop more BCCs. The enhanced IR correlation with BCC and not other common cutaneous malignancies is intriguing. The mechanism underlying these observations remains undefined. Understanding interactions between radiation-induced signaling pathways and those which drive BCC development may be important in unraveling the mechanism associated with this enhanced risk. Recent studies showed that Vismodegib, a Smoothened inhibitor, is effective in treating radiation-induced BCCs in humans, suggesting that common strategies are required for the intervention of BCCs development irrespective of their etiology.

Use of Drawing Lithography-Fabricated Polyglycolic Acid Microneedles for Transdermal Delivery of Itraconazole to a Human Basal Cell Carcinoma Model Regenerated on Mice.

Itraconazole is a triazole agent that is routinely used for treatment of nail infections and other fungal infections. Recent studies indicate that itraconazole can also inhibit the growth of basal cell carcinoma (BCC) through suppression of the Sonic Hedgehog (SHH) signaling pathway. In this study, polyglycolic acid microneedle arrays and stainless steel microneedle arrays were used for transdermal delivery of itraconazole to a human BCC model which was regenerated on mice. One-by-four arrays of 642-μm-long polyglycolic acid microneedles with sharp tips were prepared using injection molding and drawing lithography. Arrays of 85 stainless steel 800-μm-tall microneedles attached to syringes were obtained for comparison purposes. Skin grafts containing devitalized split-thickness human dermis that had been seeded with human keratinocytes transduced to express human SHH protein were sutured to the skin of immunodeficient mice. Mice with this human BCC model were treated daily for 2 weeks with itraconazole dissolved in 60% dimethylsulfoxane and 40% polyethylene glycol-400 solution; transdermal administration of the itraconazole solution was facilitated by either four 1 × 4 polyglycolic acid microneedle arrays or stainless steel microneedle arrays. The epidermal tissues treated with polyglycolic acid microneedles or stainless steel microneedles were markedly thinner than that of the control (untreated) graft tissue. These preliminary results indicate that microneedles may be used to facilitate transdermal delivery of itraconazole for localized treatment of BCC.

Immunomodulatory Effectiveness of Fish Oil and omega-3 Fatty Acids in Human Non-melanoma Skin Carcinoma Cells.

Fish oil is composed of various fatty acids among which omega-3 fatty acids are considered as most beneficial. The effects of fish oil on the activity of a topical anticancer drug, imiquimod, and the immunomodulatory activity of omega-3 fatty acids was investigated in human basal and squamous cell carcinoma cell lines. Imiquimod-fish oil mixture exhibited higher carcinoma cell growth inhibition and immunomodulatory activity than imiquimod alone, especially against squamous cell carcinoma cells. Omega-3 fatty acids exhibited growth inhibition of both basal cell and squamous cell carcinoma cell lines and modulated the immune response. Omega-3 fatty acids of fish oil serve as inducers of interleukin-10, an anti-inflammatory cytokine, and as suppressors of interleukin-6 and tumor necrosis factor-alpha, which not only depress tumor growth but also adequately control the inflammatory side effects of imiquimod. Thus, imiquimod administration with fish oil could be beneficial for inhibition of non-melanoma skin carcinoma cells but further in vivo studies are needed to understand their role in skin cancer.

Genetic ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis: the involvement of keratin-17.

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.

Erk1/2 activation in stromal fibroblasts from sporadic basal cell carcinomas.

Constitutive activation of the Erk pathway can lead to oncogenic transformation. However, the Erk pathway is not activated in human basal cell carcinomas (BCCs); although in animal models, this seems to be important. To help understand the role of Erk activity in BCC formation. The authors assayed the specific levels of phosphorylated Erk by immunohistochemistry in BCCs and normal skin biopsies. They have also analyzed Erk activation by immunoblot in fibroblasts isolated from BCC. By immunohistochemical analysis, the authors have observed that 10 of BCCs (56%) did not show phosphor-Erk staining in tumor masses and 7 (40%) showed a gradient staining exhibiting phospho-Erk only in the epidermal side of tumor masses. Remarkably, 15 BCC samples (83%) showed phospho-Erk accumulation in stroma. Six of the 9 independent cultures of dermal fibroblasts isolated from BCC maintained Erk activation "in vitro." The authors propose that there is a specific cell-type regulation of Erk activity in BCC, and this feature may be relevant during BCC formation. Stroma region from BCCs showed Erk activation and reduced proliferation. Conversely, Erk activation is barely detectable in proliferative BCCs.

Th17 cytokines in non-melanoma skin cancer.

T-helper-type 17 cytokines have been implicated in epithelial cancer progression at mucosal sites. In this issue of the European Journal of Immunology, Nardinocchi et al. [Eur. J. Immunol. 2015. 45: 922-931] show that the Th17 cytokines IL-17 and IL-22 can both signal to nonmelanoma skin cancer cells, inducing both cellular proliferation and enhanced migration of human basal cell carcinoma and squamous cell carcinoma cell lines in vitro. These cytokines were also shown to exacerbate tumor growth in mice injected with the squamous cell carcinoma line, CAL27. Thus, IL-17 and IL-22 may be key factors in skin cancer progression and may provide novel prognostic markers in nonmelanoma skin cancer.

Mouse models of nonmelanoma skin cancer.

The skin is the largest organ of the mammalian body, made up of multiple layers, which include the epidermis, dermis, and subcutis (Alam and Ratner, N Engl J Med 344(13):975-983, 2001). The human interfollicular epidermis can be subdivided into five different layers: (1) stratum basale, (2) stratum spinosum, (3) stratum granulosum, (4) stratum lucidum, and (5) stratum corneum, all originating from basal keratinocytes by differentiation (Hameetman et al., BMC cancer 13:58, 2013; Ramirez et al., Differentiation 58(1):53-64, 1994). The epidermis is also able to generate different appendages: hair follicles (HF) and their associated sebaceous glands (Sibilia et al., Cell 102(2):211-220, 2000) as well as sweat glands (Luetteke et al., Genes Dev 8(4):399-413, 1994). The skin has important functions in several biological processes like environmental barrier, tissue regeneration, hair cycling, and wound repair. During these processes, stem cells from the interfollicular epidermis and from the hair follicle bulge are activated to renew the epidermis or hair. The epidermis and hair undergo continuous homeostatic regeneration and mutations, upon mutations which disturb the balance of homeostatic regeneration of epidermis and hair and lead to enhanced proliferation of keratinocytes, development of skin cancer is developed. Tumors that arise in the skin are mainly of three types: malignant melanoma, arising from melanocytes, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC), the latter two both arising from keratinocytes or hair follicle cells. In this chapter, we will describe some genetically engineered mouse models (GEMM) that aim at modeling human BCC and SCC and their respective precancerous lesions. We will describe the experimental approaches used in our laboratory to analyze tumor-bearing mice focusing on methods necessary for the induction of tumor growth as well as for the molecular and histological analysis of tumor tissue.

Tazarotene induces apoptosis in human basal cell carcinoma via activation of caspase-8/t-Bid and the reactive oxygen species-dependent mitochondrial pathway.

Previous studies suggest that tazarotene, a new member of the acetylenic class of RARβ/γ selective retinoids which is approved to treat a variety of skin diseases, exhibits an anti-proliferative effect in human basal cell carcinoma (BCC) by triggering caspase-dependent apoptosis. However, the detailed molecular mechanisms underlying the anti-tumor activity of tazarotene are poorly understood. This study aims at investigating the molecular mechanisms of tazarotene-induced apoptosis in human BCC cells. Our results are the first to demonstrate that tazarotene induces mitochondria-dependent cleavage of caspase-9 and -3 and PARP in BCC cells by producing reactive oxygen species (ROS) and activating caspase-8 through both ROS and death receptor signaling. These events are accompanied by a decrease in BCL-2 and BCL-xl anti-apoptotic proteins as well as by survivin and XIAP, two IAP family members. Furthermore, our results presented for the first time that tazarotene triggers a convergence of the intrinsic and extrinsic apoptotic pathways via the caspase-8-truncated Bid signaling pathway. Collectively, these data provide insights into the molecular mechanisms underlying tazarotene-induced apoptosis in human BCC cells, suggesting that this compound is a potential anti-skin cancer drug.

Role of CRD-BP in the Growth of Human Basal Cell Carcinoma Cells

Although the number of new cases of Basal Cell Carcinoma (BCC) has increased rapidly in the last few decades, the molecular basis of its pathogenesis is not completely understood. Activation of Hedgehog (Hh) signaling pathway has been shown to be a key factor driving the development of BCC. The Wnt/β-catenin signaling pathway was also shown to be activated in BCCs and to perhaps modulate the activity of Hh pathway. We have previously identified a novel mechanism by which Wnt signaling regulates the transcriptional outcome of Hh signaling pathway. We demonstrated that CRD-BP, a direct target of the Wnt/β-catenin signaling, binds to GLI1 mRNA, stabilizes it, and consequently upregulates its levels (mRNA and protein) and activities. We hypothesized that Wnt-induced and CRD-BP-dependent regulation of GLI1 expression and activities is important to the development of BCC. In this study, we show that CRD-BP is over-expressed in BCC and that its expression positively correlates with the activation of both Wnt and Hh signaling pathways. We also describe the generation and characterization of a human BCC cell line. This cell line was utilized to demonstrate the importance of CRD-BP-dependent regulation of GLI1 expression and activities in the development of BCC.

Tazarotene: Randomized, Double-Blind, Vehicle-Controlled, and Open-Label Concurrent Trials for Basal Cell Carcinoma Prevention and Therapy in Patients with Basal Cell Nevus Syndrome

Sporadic human basal cell carcinomas (BCC) are generally well managed with current surgical modalities. However, in the subset of high-risk patients predisposed to developing large numbers of BCCs, there is an unmet need for effective, low-morbidity chemoprevention. This population includes fair-skinned patients with extensive sun exposure and those with genodermatoses such as the basal cell nevus (Gorlin) syndrome (BCNS). Tazarotene (Tazorac, Allergan) is a topical retinoid with relative specificity for RAR-β and RAR-γ receptors. We previously demonstrated tazarotene's robust anti-BCC efficacy in Ptch1(+/-) mice, a murine equivalent of BCNS, and others have found it to have some efficacy against sporadic human BCCs. We report here results of a randomized, double-blind, vehicle-controlled study in patients with BCNS evaluating the efficacy of topically applied tazarotene for BCC chemoprevention (N = 34 subjects), along with an open-label trial evaluating tazarotene's efficacy for chemotherapy of BCC lesions (N = 36 subjects) for a maximum follow-up period of 3 years. We found that only 6% of patients had a chemopreventive response and that only 6% of treated BCC target lesions were clinically cured. Our studies provide no evidence for either chemopreventive or chemotherapeutic effect of tazarotene against BCCs in patients with BCNS.

Open-Label, Exploratory Phase II Trial of Oral Itraconazole for the Treatment of Basal Cell Carcinoma

Itraconazole, a US Food and Drug Administration-approved antifungal drug, inhibits the Hedgehog (HH) signaling pathway, a crucial driver of basal cell carcinoma (BCC) tumorigenesis, and reduces BCC growth in mice. We assessed the effect of itraconazole on the HH pathway and on tumor size in human BCC tumors. Patients with ≥ one BCC tumor > 4 mm in diameter were enrolled onto two cohorts to receive oral itraconazole 200 mg twice per day for 1 month (cohort A) or 100 mg twice per day for an average of 2.3 months (cohort B). The primary end point was change in biomarkers: Ki67 tumor proliferation and HH activity (GLI1 mRNA). Secondary end points included change in tumor size in a subset of patients with multiple tumors. A total of 29 patients were enrolled, of whom 19 were treated with itraconazole. Itraconazole treatment was associated with two adverse events (grade 2 fatigue and grade 4 congestive heart failure). Itraconazole reduced cell proliferation by 45% (P = .04), HH pathway activity by 65% (P = .03), and reduced tumor area by 24% (95% CI, 18.2% to 30.0%). Of eight patients with multiple nonbiopsied tumors, four achieved partial response, and four had stable disease. Tumors from untreated control patients and from those previously treated with vismodegib showed no significant changes in proliferation or tumor size. Itraconazole has anti-BCC activity in humans. These results provide the basis for larger trials of longer duration to measure the clinical efficacy of itraconazole, especially relative to other HH pathway inhibitors.