Transgenic mice expressing constitutively active Akt in oral epithelium validate KLFA as a potential biomarker of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a common human neoplasia, of poor prognosis and survival, which frequently displays Akt overactivation. Previously, we reported that mice expressing high levels of constitutively Akt activity (myrAkt) in oral epithelia develop lesions and tumors in the oral cavity. MATERIALS AND METHODS: Functional genomics of primary keratinocytes from different transgenic mouse lines and immunostaining of mouse and human samples were performed in order to identify and validate putative biomarkers of oral cancer progression. RESULTS: The expression of KLF4 was found to be increased only in tumor prone samples from mice bearing overactivation of Akt. Such increased expression was confirmed in oral dysplasias and tumors arising in those mice. Tissue microarray analysis of human samples confirmed the association between active Akt and increased KLF4 expression. CONCLUSION: These data support the notion that KLF4 is potentially a reliable marker of HNSCC, and that myrAkt transgenic mice are valuable tools for preclinical research of HNSCC.

p107 acts as a tumor suppressor in pRb-deficient epidermis.

The specific deletion of Rb gene in epidermis leads to altered proliferation and differentiation, but not to the development of spontaneous tumors. Our previous data have demonstrated the existence of a functional compensation of Rb loss by Rbl1 (p107) in as the phenotypic differences with respect to controls are intensified. However, the possible evolution of this aggravated phenotype, in particular in relationship with tumorigenesis, has not been evaluated due to the premature death of the double deficient mice. We have now investigated whether p107 can also act as a tumor suppressor in pRb-deficient epidermis using different experimental approaches. We found spontaneous tumor development in doubly-deficient skin grafts. Moreover, Rb-deficient keratinocytes are susceptible to Ha-ras-induced transformation, and this susceptibility is enhanced by p107 loss. Further functional analyses, including microarray gene expression profiling, indicated that the loss of p107, in the absence of pRb, produces the reduction of p53-dependent pro-apoptotic signals. Overall, our data demonstrate that p107 behaves as a tumor suppressor in epidermis in the absence of pRb and suggest novel tumor-suppressive roles for p107 in the context of functional p53 and activated Ras.

Gene profiling approaches help to define the specific functions of retinoblastoma family in epidermis.

The epidermal-specific ablation of Rb gene leads to increased proliferation, aberrant differentiation, and the disengagement of these processes in vivo and in vitro. These differences in phenotype are more severe with the loss of p107, demonstrating the functional compensation between pRb and p107. As p107 and p130 also exert overlapping functions in epidermis, we have generated Rb(F19/F19)K14cre;Rbl2-/- (pRb-;p130-) mice to analyze possible functional redundancies between pRb and p130. The epidermal phenotype was very similar between pRb- and pRb-;p130- mice, suggesting that pRb and p130 activities are not redundant in epidermis. Importantly, we can correlate the proliferation differences with specific changes in gene expression between pRb-, pRb-;p107- and pRb-;p130- primary keratinocytes using microarray analysis, and explain the phenotypes in the context of altered E2F expression and functionality. Our findings support a model in which the distinct retinoblastoma family members, in conjunction with E2F members, play a central role in regulating epidermal homeostasis through specific or overlapping activities.

Susceptibility of pRb-deficient epidermis to chemical skin carcinogenesis is dependent on the p107 allele dosage.

Functional inactivation of the pRb-dependent pathway is a general feature of human cancer. However, only a reduced spectrum of tumors displays inactivation of the Rb gene. This can be attributed, at least partially, to the possible overlapping functions carried out by the related retinoblastoma family members p107 and p130. We observed that loss of pRb in epidermis, using the Cre/LoxP technology, results in proliferation and differentiation defects. These alterations are partially compensated by the elevation in the levels of p107. Moreover, epidermis lacking pRb and p107, but not pRb alone, develops spontaneous tumors, and double deficient primary keratinocytes are highly susceptible to Ha-ras-induced transformation. Two-stage chemical carcinogenesis experiments in mice lacking pRb in epidermis revealed a reduced susceptibility in papilloma formation and an increase in the malignant conversion. We have now explored whether the loss of one p107 allele, inducing a decrease in the levels of p107 up to normal levels could restore the susceptibility of pRb-deficient skin to two-stage protocol. We observed partial restoration in the incidence, number, and size of tumors. However, there is no increased malignancy despite sustained p53 activation. We also observed a partial reduction in the levels of proapoptotic proteins in benign papillomas. These data confirm our previous suggestions on the role of p107 as a tumor suppressor in epidermis in the absence of pRb.

Spontaneous tumor formation in Trp53-deficient epidermis mediated by chromosomal instability and inflammation.

BACKGROUND: The specific ablation of Trp53 gene in mouse epidermis leads to the spontaneous development of aggressive squamous cell carcinoma, a process that is accelerated by the subsequent loss of Rb gene. MATERIALS AND METHODS: The possible mechanisms leading to spontaneous tumor formation in epidermis in the absence of Trp53 were studied focusing on hair cycle defects, inflammation and possible chromosomal instability (CIN). RESULTS: Loss of p53 induces tumorigenesis primarily by mediating early CIN and, to a minor extent, nuclear factor kappaB activation. Notably, CIN occurs not only in p53-deficient skin, but also in epidermis lacking both Rb and Tp53 tumor suppressors, indicating a predominant role of this process in spontaneous tumorigenesis. CONCLUSION: These data identify CIN as a major mechanism in tumorigenesis originated by Trp53 loss in stratified epithelia and imply that therapies aimed to counterbalance CIN might be of relevance for the treatment of human cancer bearing impaired p53 functions.

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.

Spontaneous squamous cell carcinoma induced by the somatic inactivation of retinoblastoma and Trp53 tumor suppressors.

Squamous cell carcinomas (SCC) represent the most aggressive type of nonmelanoma skin cancer. Although little is known about the causal alterations of SCCs, in organ-transplanted patients the E7 and E6 oncogenes of human papillomavirus, targeting the p53- and pRb-dependent pathways, have been widely involved. Here, we report the functional consequences of the simultaneous elimination of Trp53 and retinoblastoma (Rb) genes in epidermis using Cre-loxP system. Loss of p53, but not pRb, produces spontaneous tumor development, indicating that p53 is the predominant tumor suppressor acting in mouse epidermis. Although the simultaneous inactivation of pRb and p53 does not aggravate the phenotype observed in Rb-deficient epidermis in terms of proliferation and/or differentiation, spontaneous SCC development is severely accelerated in doubly deficient mice. The tumors are aggressive and undifferentiated and display a hair follicle origin. Detailed analysis indicates that the acceleration is mediated by premature activation of the epidermal growth factor receptor/Akt pathway, resulting in increased proliferation in normal and dysplastic hair follicles and augmented tumor angiogenesis. The molecular characteristics of this model provide valuable tools to understand epidermal tumor formation and may ultimately contribute to the development of therapies for the treatment of aggressive squamous cancer.

Constitutively active Akt induces ectodermal defects and impaired bone morphogenetic protein signaling.

Aberrant activation of the Akt pathway has been implicated in several human pathologies including cancer. However, current knowledge on the involvement of Akt signaling in development is limited. Previous data have suggested that Akt-mediated signaling may be an essential mediator of epidermal homeostasis through cell autonomous and noncell autonomous mechanisms. Here we report the developmental consequences of deregulated Akt activity in the basal layer of stratified epithelia, mediated by the expression of a constitutively active Akt1 (myrAkt) in transgenic mice. Contrary to mice overexpressing wild-type Akt1 (Akt(wt)), these myrAkt mice display, in a dose-dependent manner, altered development of ectodermally derived organs such as hair, teeth, nails, and epidermal glands. To identify the possible molecular mechanisms underlying these alterations, gene profiling approaches were used. We demonstrate that constitutive Akt activity disturbs the bone morphogenetic protein-dependent signaling pathway. In addition, these mice also display alterations in adult epidermal stem cells. Collectively, we show that epithelial tissue development and homeostasis is dependent on proper regulation of Akt expression and activity.

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.