Ana Bravo

Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Aberrant activation of the phosphoinositide-3-kinase (PI3K)/PTEN/Akt pathway, leading to increased proliferation and decreased apoptosis, has been implicated in several human pathologies including cancer. Our previous data have shown that Akt-mediated signaling is an essential mediator in the mouse skin carcinogenesis system during both the tumor promotion and progression stages. In addition, overexpression of Akt is also able to transform keratinocytes through transcriptional and posttranscriptional processes. Here, we report the consequences of the increased expression of Akt1 (wtAkt) or constitutively active Akt1 (myrAkt) in the basal layer of stratified epithelia using the bovine keratin K5 promoter. These mice display alterations in epidermal proliferation and differentiation. In addition, transgenic mice with the highest levels of Akt expression developed spontaneous epithelial tumors in multiple organs with age. Furthermore, both wtAkt and myrAkt transgenic lines displayed heightened sensitivity to the epidermal proliferative effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and heightened sensitivity to two-stage skin carcinogenesis. Finally, enhanced susceptibility to two-stage carcinogenesis correlated with a more sustained proliferative response following treatment with TPA as well as sustained alterations in Akt downstream signaling pathways and elevations in cell cycle regulatory proteins. Collectively, the data provide direct support for an important role for Akt signaling in epithelial carcinogenesis in vivo, especially during the tumor promotion stage.

Altered skin development and impaired proliferative and inflammatory responses in transgenic mice overexpressing the glucocorticoid receptor

Glucocorticoids (GCs) are potent inhibitors of epidermal proliferation and effective anti‐inflammatory compounds, which make them the drug of choice for a wide range of inflammatory and hyperproliferative skin disorders. GC action is mediated via the glucocorticoid receptor (GR). To study the role of GR in skin development and the molecular mechanisms underlying its action, we generated transgenic mice overexpressing GR in epidermis and other stratified epithelia, under the control of the keratin K5 promoter. Newborn mice show altered skin development, manifested as variable‐sized skin lesions that range from epidermal hypoplasia and underdeveloped dysplastic hair follicles to a complete absence of this tissue. In the most affected individuals, skin was absent at the cranial and umbilical regions, and the vibrissae and eyebrows appear scarce, short, and curly. In addition, as a consequence of transgene expression in other ectodermally derived epithelia, K5‐GR mice exhibited further abnormalities that strikingly resemble the clinical findings in patients with ectodermal dysplasia, which includes aplasia cutis congenita. In adult transgenic skin, topical application of the tumor promoter TPA did not elicit hyperplasia or transcriptional induction of several proinflammatory cytokines. This anti‐inflammatory role of GR was due at least in part to interference with NF‐κB, leading to a strong reduction in the κB‐binding activity without altering the transcriptional levels of the inhibitor IκBα.

Exocrine pancreatic disorders in transsgenic mice expressing human keratin 8

Keratins K8 and K18 are the major components of the intermediate-filament cytoskeleton of simple epithelia. Increased levels of these keratins have been correlated with various tumor cell characteristics, including progression to malignancy, invasive behavior, and drug sensitivity, although a role for K8/K18 in tumorigenesis has not yet been demonstrated. To examine the function of these keratins, we generated mice expressing the human K8 (hk8) gene, which leads to a moderate keratin-content increase in their simple epithelia. These mice displayed progressive exocrine pancreas alterations, including dysplasia and loss of acinar architecture, redifferentiation of acinar to ductal cells, inflammation, fibrosis, and substitution of exocrine by adipose tissue, as well as increased cell proliferation and apoptosis. Histological changes were not observed in other simple epithelia, such as the liver. Electron microscopy showed that transgenic acinar cells have keratins organized in abundant filament bundles dispersed throughout the cytoplasm, in contrast to control acinar cells, which have scarce and apically concentrated filaments. The phenotype found was very similar to that reported for transgenic mice expressing a dominant-negative mutant TGF-beta type II receptor (TGFbetaRII mice). We show that these TGFbetaRII mutant mice also have elevated K8/K18 levels. These results indicate that simple epithelial keratins play a relevant role in the regulation of exocrine pancreas homeostasis and support the idea that disruption of mechanisms that normally regulate keratin expression in vivo could be related to inflammatory and neoplastic pancreatic disorders.

Abnormal epidermal differentiation and impaired epithelial-mesenchymal tissue interactions in mice lacking the retinoblastoma relatives p107 and p130

The functions of p107 and p130, members of the retinoblastoma family, include the control of cell cycle progression and differentiation in several tissues. Our previous studies suggested a role for p107 and p130 in keratinocyte differentiation in vitro. We now extend these data using knockout animal models. We found impaired terminal differentiation in the interfollicular keratinocytes of p107/p130-double-null mice epidermis. In addition, we observed a decreased number of hair follicles and a clear developmental delay in hair, whiskers and tooth germs. Skin grafts of p107/p130-deficient epidermis onto NOD/scid mice showed altered differentiation and hyperproliferation of the interfollicular keratinocytes, thus demonstrating that the absence of p107 and p130 results in the deficient control of differentiation in keratinocytes in a cell-autonomous manner. Besides normal hair formation, follicular cysts, misoriented and dysplastic follicles, together with aberrant hair cycling, were also observed in the p107/p130 skin transplants. Finally, the hair abnormalities in p107/p130-null skin were associated with altered Bmp4-dependent signaling including decreasedΔ Np63 expression. These results indicate an essential role for p107 and p130 in the epithelial-mesenchimal interactions.

Epidermal abnormalities and increased malignancy of skin tumors in human epidermal keratin 8-expressing transgenic mice

Keratins K8 and K18 are the major components of the intermediate filament cytoskeleton of simple epithelia. Increased levels of these keratins have been associated with invasive growth and progression to malignancy in different types of human and murine epithelial tumors (including skin tumors), and even in tumors from nonepithelial origin. However, it has not yet clarified whether K8/K18 expression in tumors is cause or consequence of malignancy. Given the increasing incidence of epidermal cancer in humans (40% of all tumors diagnosed), we generated a mouse model to examine the role of simple epithelium keratins in the establishment and progression of human skin cancer. Transgenic mice expressing human K8 in the epidermis showed severe epidermal and hair follicle dysplasia with concomitant alteration in epidermal differentiation markers. The severity of the skin phenotype of these transgenic mice increases with age, leading to areas of preneoplastic transformation. Skin carcinogenesis assays showed a dramatic increase in the progression of papillomas toward malignancy in transgenic animals. These results support the idea that K8 alters the epidermal cell differentiation, favors the neoplastic transformation of cells, and is ultimately responsible of the invasive behavior of transformed epidermal cells leading of conversion of benign to malignant tumors.

Akt activation synergizes with Trp53 loss in oral epithelium to produce a novel mouse model for head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a common human neoplasia with poor prognosis and survival that frequently displays Akt overactivation. Here we show that mice displaying constitutive Akt activity (myrAkt) in combination with Trp53 loss in stratified epithelia develop oral cavity tumors that phenocopy human HNSCC. The myrAkt mice develop oral lesions, making it a possible model of human oral dysplasia. The malignant conversion of these lesions, which is hampered due to the induction of premature senescence, is achieved by the subsequent ablation of Trp53 gene in the same cells in vivo. Importantly, mouse oral tumors can be followed by in vivo imaging, show metastatic spreading to regional lymph nodes, and display activation of nuclear factor-kappaB and signal transducer and activator of transcription-3 pathways and decreased transforming growth factor-beta type II receptor expression, thus resembling human counterparts. In addition, malignant conversion is associated with increased number of putative tumor stem cells. These data identify activation of Akt and p53 loss as a major mechanism of oral tumorigenesis in vivo and suggest that blocking these signaling pathways could have therapeutic implications for the management of HNSCC.

A Transposon-Based Analysis of Gene Mutations Related to Skin Cancer Development

Nonmelanoma skin cancer (NMSC) is by far the most frequent type of cancer in humans. NMSC includes several types of malignancies with different clinical outcomes, the most frequent being basal and squamous cell carcinomas. We have used the Sleeping Beauty transposon/transposase system to identify somatic mutations associated with NMSC. Transgenic mice bearing multiple copies of a mutagenic Sleeping Beauty transposon T2Onc2 and expressing the SB11 transposase under the transcriptional control of regulatory elements from the keratin K5 promoter were treated with TPA, either in wild-type or Ha-ras mutated backgrounds. After several weeks of treatment, mice with transposition developed more malignant tumors with decreased latency compared with control mice. Transposon/transposase animals also developed basal cell carcinomas. Genetic analysis of the transposon integration sites in the tumors identified several genes recurrently mutated in different tumor samples, which may represent novel candidate cancer genes. We observed alterations in the expression levels of some of these genes in human tumors. Our results show that inactivating mutations in Notch1 and Nsd1, among others, may have an important role in skin carcinogenesis.

An inactivating CYLD mutation promotes skin tumor progression by conferring enhanced proliferative, survival and angiogenic properties to epidermal cancer cells

In this study, we demonstrate that the expression in tumorigenic epidermal cells of a catalytically inactive form of CYLD (CYLDC/S) that mimics the identified mutations of cyld in human tumors and competes with the endogenous CYLD results in enhanced cell proliferation and inhibition of apoptosis; it also stimulates cell migration and induces the expression of angiogenic factors, including vascular endothelial growth factor-A. Altogether, these characteristics indicate an increased oncogenicity of the tumorigenic epidermal CYLDC/S mutant cells in vitro. Moreover, we show the increase in malignancy of epidermal squamous cell carcinomas that express the CYLDC/S transgene in an in vivo xenograft model. Tumors carrying the mutated CYLDC/S exhibit a fast growth, are poorly differentiated and present a robust angiogenesis. CYLDC/S tumors are also characterized by their elevated proliferation rate and decreased apoptosis. In contrast with previous studies showing the development of benign tumors by mutations in the CYLD gene, here we provide evidence that the occurrence of mutations in the CYLD gene in tumorigenic epidermal cells (carrying previous mutations) increases the aggressiveness of carcinomas, mainly through enhancement of the expression of angiogenic factors, having therefore a key role in epidermal cancer malignancy.

IKKβ Leads to an Inflammatory Skin Disease Resembling Interface Dermatitis

IKKbeta is a subunit of the IkappaB kinase (IKK) complex required for NF-kappaB activation in response to pro-inflammatory signals. NF-kappaB regulates the expression of many genes involved in inflammation, immunity, and apoptosis, and also controls cell proliferation and differentiation in different tissues; however, its function in skin physiopathology remains controversial. In this study we report the alterations caused by increased IKKbeta activity in skin basal cells of transgenic mice. These animals suffered chronic inflammation with abundant macrophages and other CD45(+) infiltrating cells in the skin, which resulted in epidermal basal cell injury and degeneration of hair follicles. They showed histological features characteristic of interface dermatitis (ID). This phenotype is accompanied by an increased production of inflammatory cytokines by transgenic keratinocytes. Accordingly, transcriptome studies show upregulation of genes associated with inflammatory responses. The inflammatory phenotype observed as a consequence of IKKbeta overexpression is independent of T and B lymphocytes, as it also arises in mice lacking these cell types. In summary, our data indicate the importance of IKKbeta in the development of ID and in the homeostasis of stratified epithelia. Our results also support the idea that IKKbeta might be a valid therapeutic target for the treatment of skin inflammatory diseases.

IKKα 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 (IκB kinase) α 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 IKKα expression increases both early and terminal differentiation of human keratinocytes through an E-cadherin-dependent mechanism. Increased expression of IKKα 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 IKKα murine tumors, express high levels of both IKKα and E-cadherin, with absence of keratins K1 and K10, usually co-expressed with IKKα and E-cadherin. The tight correlation between the properties of both murine and human ASCC variants strongly suggests that IKKα is responsible for the development of this human SCC variant.