CYLD Inhibits the Development of Skin Squamous Cell Tumors in Immunocompetent Mice.

Cylindromatosis (CYLD) is a deubiquitinase (DUB) enzyme that was initially characterized as a tumor suppressor of adnexal skin tumors in patients with CYLD syndrome. Later, it was also shown that the expression of functionally inactive mutated forms of CYLD promoted tumor development and progression of non-melanoma skin cancer (NMSC). However, the ability of wild-type CYLD to inhibit skin tumorigenesis in vivo in immunocompetent mice has not been proved. Herein, we generated transgenic mice that express the wild type form of CYLD under the control of the keratin 5 (K5) promoter (K5-CYLDwt mice) and analyzed the skin properties of these transgenic mice by WB and immunohistochemistry, studied the survival and proliferating characteristics of primary keratinocytes, and performed chemical skin carcinogenesis experiments. As a result, we found a reduced activation of the nuclear factor kappa B (NF-κB) pathway in the skin of K5-CYLDwt mice in response to tumor necrosis factor-α (TNF-α); accordingly, when subjected to insults, K5-CYLDwt keratinocytes are prone to apoptosis and are protected from excessive hyperproliferation. Skin carcinogenesis assays showed inhibition of tumor development in K5-CYLDwt mice. As a mechanism of this tumor suppressor activity, we found that a moderate increase in CYLD expression levels reduced NF-κB activation, which favored the differentiation of tumor epidermal cells and inhibited its proliferation; moreover, it decreased tumor angiogenesis and inflammation. Altogether, our results suggest that increased levels of CYLD may be useful for anti-skin cancer therapy.

Correction: IKKα regulates the stratification and differentiation of the epidermis: implications for skin cancer development

[This corrects the article DOI: 10.18632/oncotarget.12527.]

IKKα regulates the stratification and differentiation of the epidermis: implications for skin cancer development.

IKKα plays a mandatory role in keratinocyte differentiation and exerts an important task in non-melanoma skin cancer development. However, it is not fully understood how IKKα exerts these functions. To analyze in detail the role of IKKα in epidermal stratification and differentiation, we have generated tridimensional (3D) cultures of human HaCaT keratinocytes and fibroblasts in fibrin gels, obtaining human skin equivalents that comprise an epidermal and a dermal compartments that resembles both the structure and differentiation of normal human skin. We have found that IKKα expression must be strictly regulated in epidermis, as alterations in its levels lead to histological defects and promote the development of malignant features. Specifically, we have found that the augmented expression of IKKα results in increased proliferation and clonogenicity of human keratinocytes, and leads to an accelerated and altered differentiation, augmented ability of invasive growth, induction of the expression of oncogenic proteins (Podoplanin, Snail, Cyclin D1) and increased extracellular matrix proteolytic activity. All these characteristics make keratinocytes overexpressing IKKα to be at a higher risk of developing skin cancer. Comparison of genetic profile obtained by analysis of microarrays of RNA of skin equivalents from both genotypes supports the above described findings.

RNAi-mediated knockdown of IKK1 in transgenic mice using a transgenic construct containing the human H1 promoter.

Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models.

Increased IKKα expression in the basal layer of the epidermis of transgenic mice enhances the malignant potential of skin tumors.

Non-melanoma skin cancer is the most frequent type of cancer in humans. In this study we demonstrate that elevated IKKα expression in murine epidermis increases the malignancy potential of skin tumors. We describe the generation of transgenic mice overexpressing IKKα in the basal, proliferative layer of the epidermis and in the outer root sheath of hair follicles. The epidermis of K5-IKKα transgenic animals shows several alterations such as hyperproliferation, mislocalized expression of integrin-α6 and downregulation of the tumor suppressor maspin. Treatment of the back skin of mice with the mitogenic agent 12-O-tetradecanoylphorbol-13-acetate causes in transgenic mice the appearance of different preneoplastic changes such as epidermal atypia with loss of cell polarity and altered epidermal tissue architecture, while in wild type littermates this treatment only leads to the development of benign epidermal hyperplasia. Moreover, in skin carcinogenesis assays, transgenic mice carrying active Ha-ras (K5-IKKα-Tg.AC mice) develop invasive tumors, instead of the benign papillomas arising in wild type-Tg-AC mice also bearing an active Ha-ras. Therefore we provide evidence for a tumor promoter role of IKKα in skin cancer, similarly to what occurs in other neoplasias, including hepatocarcinomas and breast, prostate and colorectal cancer. The altered expression of cyclin D1, maspin and integrin-α6 in skin of transgenic mice provides, at least in part, the molecular bases for the increased malignant potential found in the K5-IKKα skin tumors.

IKKalpha enhances human keratinocyte differentiation and determines the histological variant of epidermal squamous cell carcinomas.

Squamous cell carcinomas (SCCs) of the skin display different clinical features according to their epithelial differentiation grade and histological variant. Understanding the causes of these divergences might increase the curability of SCCs. Therefore, it is important to study the mechanisms of differentiation in keratinocytes. IKK (IkappaB kinase) alpha is an important protein for epidermal morphogenesis, although the pathways through which it exerts its function are unknown and controversy exists about its role in cancer development. We show that enhanced IKKalpha expression increases both early and terminal differentiation of human keratinocytes through an E-cadherin-dependent mechanism. Increased expression of IKKalpha in mouse tumorigenic epidermal cells leads to changes in the differentiation pattern of the resulting SCCs, originating a distinct histological variant that resembles the human acantholytic SCC (ASCC) variant. Although human ASCCs have an aggressive clinical course and high risk of metastasis, nothing is known about their etiology. We show that human ASCCs, as observed in the counterpart IKKalpha murine tumors, express high levels of both IKKalpha and E-cadherin, with absence of keratins K1 and K10, usually co-expressed with IKKalpha and E-cadherin. The tight correlation between the properties of both murine and human ASCC variants strongly suggests that IKKalpha is responsible for the development of this human SCC variant.