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CD34+ cell chaacterization in K5-IKKbeta skin.

Primer sequences.

Legends to Supplementary Figures and supplementary Material and Methods.

Purpose:Bladder cancer is a clinical and social problem due to its high incidence and recurrence rates. It frequently appears in elderly patients showing other medical comorbidities that hamper the use of standard chemotherapy. We evaluated the activity of CDK4/6 inhibitor as a new therapy for patients unfit for cisplatin (CDDP).Experimental Design:Bladder cancer cell lines were tested for in vitro sensitivity to CDK4/6 inhibition. A novel metastatic bladder cancer mouse model was developed and used to test its in vivo activity.Results:Cell lines tested were sensitive to CDK4/6 inhibition, independent on RB1 gene status. Transcriptome analyses and knockdown experiments revealed a major role for FOXM1 in this response. CDK4/6 inhibition resulted in reduced FOXM1 phosphorylation in vitro and in vivo and showed synergy with CDDP, allowing a significant tumor regression. FOXM1 exerted important oncogenic roles in bladder cancer.Conclusions:CDK4/6 inhibitors, alone or in combination, are a novel therapeutic strategy for patients with advanced bladder cancer previously classified as unfit for current treatment options.

Supplementary Table 4: Spreadsheet of transcriptomic data

Includes Supplementary Figures 1-13; Supplementary Tables 1, 2, 3, 7 and 8; and Supplementary References

Supplementary Table 5: Spreadsheet of transcriptomic data

Supplementary Figures 1-4 from Spontaneous Squamous Cell Carcinoma Induced by the Somatic Inactivation of Retinoblastoma and Trp53 Tumor Suppressors

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-κB and signal transducer and activator of transcription-3 pathways and decreased transforming growth factor-β 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. [Cancer Res 2009;69(3):1099–108]

Supplementary Figure 1 from Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Supplementary Legends 1-2 from Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Supplementary Figure 2 from 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. [Cancer Res 2007;67(22):10879–88]

Supplementary Table 1 from Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

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. [Cancer Res 2008;68(3):683–92]

Supplementary Figures 1-10 from Akt Activation Synergizes with Trp53 Loss in Oral Epithelium to Produce a Novel Mouse Model for Head and Neck Squamous Cell Carcinoma

Supplementary Data - Gene List from Spontaneous Squamous Cell Carcinoma Induced by the Somatic Inactivation of Retinoblastoma and Trp53 Tumor Suppressors

This study aimed to investigate the clinical and prognostic relevance of the Hippo-YAP transactivators YAP1 and TAZ in oral squamous cell carcinoma, and their possible relationship with PI3K/mTOR pathway activation.Immunohistochemical analysis of YAP1, TAZ, PIK3CA (p110α), p-AKT (Ser473), and p-S6 (Ser235) was performed in paraffin-embedded tissue specimens from 165 OSCC patients. Correlations between protein expression and clinical data were further assessed.YAP1 expression was detected in both cytoplasm and nucleus of tumor cells, whereas TAZ expression was only found in the nucleus. Nuclear YAP1 was significantly associated with tumor size (p = 0.03), neck lymph node metastasis (p = 0.02), TNM stage (p = 0.02), and poor differentiation (p = 0.04). Nuclear TAZ was associated with tobacco (p = 0.03) and alcohol consumption (p = 0.04), and poor tumor differentiation (p = 0.04). There was a positive significant correlation between nuclear and cytoplasmic YAP1, nuclear TAZ, p110α expression, and mTORC1 activation p-S6 (S235). Combined expression of nuclear and cytoplasmic YAP1 was prognostic in both univariate and multivariate analyses. Active nuclear YAP1 was significantly and independently associated with poor disease-specific (p = 0.005, HR = 2.520; 95% CI = 1.319-4.816) and overall survival (p = 0.015, HR = 2.126; 95% CI = 1.155-3.916).Nuclear YAP1 is an independent predictor of poor survival in oral squamous cell carcinoma.

Head and neck squamous cell carcinoma; Mouse model; Oral mucosa Carcinoma de células escamosas de cabeza y cuello; Modelo de ratón; Mucosa oral Carcinoma de cèl·lules escamoses de cap i coll; Model de ratolí; Mucosa oral Fanconi anemia (FA) patients frequently develop oral squamous cell carcinoma (OSCC). This cancer in FA patients is diagnosed within the first 3–4 decades of life, very often preceded by lesions that suffer a malignant transformation. In addition, they respond poorly to current treatments due to toxicity or multiple recurrences. Translational research on new chemopreventive agents and therapeutic strategies has been unsuccessful partly due to scarcity of disease models or failure to fully reproduce the disease. Here we report that Fanca gene knockout mice (Fanca-/-) frequently display pre-malignant lesions in the oral cavity. Moreover, when these animals were crossed with animals having conditional deletion of Trp53 gene in oral mucosa (K14cre;Trp53F2-10/F2-10), they spontaneously developed OSCC with high penetrance and a median latency of less than ten months. Tumors were well differentiated and expressed markers of squamous differentiation, such as keratins K5 and K10. In conclusion, Fanca and Trp53 genes cooperate to suppress oral cancer in mice, and Fanca-/-;K14cre;Trp53F2-10/F2-10 mice constitute the first animal model of spontaneous OSCC in FA. This study has been funded by Instituto de Salud Carlos III (ISCIII) through the projects CB16/12/00228/CIBERONC, PI18/00263 and P121/00208 and co-funded by FEDER and the European Union; and grants from the Spanish Fundacion Anemia de Fanconi and Fanconi Anemia Research Fund USA. J.P. was supported by a FEDER co-funded grant (ref PEJ2018-002040-A) from the Ministerio de Ciencia, Innovación y Universidades. J.O. was supported by a FEDER co-funded grant (ref PEJ-2019-TL_BMD-12905) from the Comunidad de Madrid. The funding sources were not involved in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

Cancer is the second leading cause of death worldwide, just behind cardiovascular disease. It accounts for nearly 10 million deaths annually, and new strategies to improve early detection and drug delivery are urgently needed. Nanoparticles are small structures within the nanometer range (1 billionth of a meter) that can be used to deliver either an imaging probe (tracer) to allow the detection of a tumor or drugs to kill tumor cells. There are many types of nanoparticles; those based on plant viruses are especially appealing for biomedical purposes because they are biodegradable and noninfectious to humans. Also, their physicochemical properties, such as symmetry, uniformity and loading capacity, make them excellent nanocarriers. We report here for the first time the ability of nanoparticles derived from the turnip mosaic virus (TuMV), a well-known virus naturally infecting cruciferous plants (e.g., broccoli, turnip, radish, cabbage) but not humans, to deliver a fluorescent imaging probe that allows tumor detection

Simple Summary Neuroendocrine lung tumors are a heterogeneous group of malignancies that share a common neuroendocrine nature. They range from low- and intermediate-grade typical and atypical carcinoma, to the highly malignant large cell neuroendocrine lung carcinoma and small cell carcinoma, with marked differences in incidences and prognosis. This review delineates the current knowledge of the genetic landscape of the human tumors, its influence in the development of genetically engineered mouse models (GEMMs) and the molecular imaging tools available to detect and monitor these diseases. While small cell lung carcinoma is one of the diseases best represented by GEMMs, there is a worrying lack of animal models for the other members of the group, these being understudied diseases. Regardless of the incidence and material available, they all are in urgent need of effective therapies. Abstract Neuroendocrine lung tumors comprise a range of malignancies that extend from benign tumorlets to the most prevalent and aggressive Small Cell Lung Carcinoma (SCLC). They also include low-grade Typical Carcinoids (TC), intermediate-grade Atypical Carcinoids (AC) and high-grade Large Cell Neuroendocrine Carcinoma (LCNEC). Optimal treatment options have not been adequately established: surgical resection when possible is the choice for AC and TC, and for SCLC chemotherapy and very recently, immune checkpoint inhibitors. Some mouse models have been generated based on the molecular alterations identified in genomic analyses of human tumors. With the exception of SCLC, there is a limited availability of (preclinical) models making their development an unmet need for the understanding of the molecular mechanisms underlying these diseases. For SCLC, these models are crucial for translational research and novel drug testing, given the paucity of human material from surgery. The lack of early detection systems for lung cancer point them out as suitable frameworks for the identification of biomarkers at the initial stages of tumor development and for testing molecular imaging methods based on somatostatin receptors. Here, we review the relevant models reported to date, their impact on the understanding of the biology of the tumor subtypes and their relationships, as well as the effect of the analyses of the genetic landscape of the human tumors and molecular imaging tools in their development.

Lung cancer remains the leading cause of cancer deaths worldwide. Among the Non-Small Cell Carcinoma (NSCLC) category, Adenocarcinoma (ADC) represents the most common type, with different reported driver mutations, a bunch of models described and therapeutic options. Meanwhile, Pulmonary Sarcomatoid Carcinoma (PSC) is one of the rarest, with very poor outcomes, scarce availability of patient material, no effective therapies and no models available for preclinical research. Here, we describe that the combined deletion of Pten and Trp53 in the lungs of adult conditional mice leads to the development of both ADC and PSC irrespective of the lung targeted cell type after naphthalene induced airway epithelial regeneration. Although this model shows long latency periods and incomplete penetrance for tumor development, it is the first PSC mouse model reported so far, and sheds light on the relationships between ADC and PSC and their cells of origin. Moreover, human ADC show strong transcriptomic similarities to the mouse PSC, providing a link between both tumor types and the human ADC.

The Hippo-YAP (Yes-associated protein) pathway is a key regulator of tissue growth, organ size and stem cell function. More recently, a fundamental role for this pathway has emerged in stem cell function and tumorigenesis. Activation of the transcriptional co-activator YAP promotes cell-contact independent proliferation, epithelial to mesenchymal transition (EMT), cancer stem cell features and drug resistance. In this review, we describe the main components of the pathway, the microenvironment and the cell-intrinsic cues governing its activation, the downstream players of the pathway and the biological implications of their activation in the context of cancer. We will focus on the existing knowledge of this pathway in head and neck squamous carcinoma (HNSCC), its clinical value in this type of cancer as a marker of poor prognosis and resistance to therapy, as well as the most encouraging therapeutic strategies targeting the pathway.

Objective Epithelial-mesenchymal transition (EMT) is considered the initial step in the invasion-metastasis cascade. The aim of this systematic review was to study the signature of genes involved in the EMT process in oral cancer (OC) confirmed by protein expression and its possible relationship with oral squamous cell carcinoma (OSCC) prognostic variables. Materials and methods A search of the scientific literature was carried out with no start date restriction until 17 September 2020 in the electronic databases Pubmed/MEDLINE, Web of Science, Cochrane Library and Scopus, following specific eligibility criteria. The methodological quality of the included studies was assessed using the Newcastle-Ottawa tool. Results A total of 8 retrospective cohort studies were included, all of them performed in China and with low risk of bias. Overexpression of the genes HNRNPC, ITGA5, HMGA2 and SRSF3, and low expression of ALDH3A1 and ARID2 promote EMT in OC. The more advanced clinical stages of the TNM classification were significantly associated with overexpression of HNRNPC, ITGA5, HMGA2 and SRSF3, and low expression of ARID2. Conclusions HNRNPC, ITGA5, HMGA2, SRSF3, ALDH3A1 and ARID2 genes were associated with EMT process. Over- or under-expression of these genes is associated with worse stages of OSCC and/or worse prognosis of the tumor. Further studies on this topic are needed in different countries to be able to confirm these results, since the detection of these genes can help to know which tumors have a worse prognosis.

IKKβ (encoded by IKBKB) is a protein kinase that regulates the activity of numerous proteins important in several signaling pathways, such as the NF-κB pathway. IKKβ exerts a protumorigenic role in several animal models of lung, hepatic, intestinal, and oral cancer. In addition, genomic and proteomic studies of human tumors also indicate that IKBKB gene is amplified or overexpressed in multiple tumor types. Here, the relevance of IKKβ in skin cancer was determined by performing carcinogenesis studies in animal models overexpressing IKKβ in the basal skin layer. IKKβ overexpression resulted in a striking resistance to skin cancer development and an increased expression of several tumor suppressor proteins, such as p53, p16, and p19. Mechanistically, this skin tumor–protective role of IKKβ is independent of p53, but dependent on the activity of the Ink4a/Arf locus. Interestingly, in the absence of p16 and p19, IKKβ-increased expression favors the appearance of cutaneous spindle cell–like squamous cell carcinomas, which are highly aggressive tumors. These results reveal that IKKβ activity prevents skin tumor development, and shed light on the complex nature of IKKβ effects on cancer progression, as IKKβ can both promote and prevent carcinogenesis depending on the cell type or molecular context. Implications: The ability of IKKβ to promote or prevent carcinogenesis suggests the need for further evaluation when targeting this protein. Mol Cancer Res; 15(9); 1255–64. ©2017 AACR.

Epidermal stem cells are responsible for normal tissue homeostasis and contribute to tissue regeneration during injury. Several assays measuring stem cell frequency and function can be used to assess epidermal stem cell potential. However, the ultimate assay that accounts for stemness is the capacity to sustain in vivo long-term tissue regeneration and maintenance. We can use this type of analysis to interrogate whether a specific genetic alteration (e.g., activation or inactivation of any gene thought to be involved in stem cell quiescence or proliferation) confers increased or decreased stem cell potential.

The annexin protein superfamily has been implicated in multiple physiological and pathological processes, including carcinogenesis. Altered expression of various annexins has frequently been observed and linked to the development and progression of various human malignancies. However, information is lacking on the expression and clinical significance of annexin A9 (ANXA9) and A10 (ANXA10) in head and neck squamous cell carcinomas (HNSCC). ANXA9 and ANXA10 expression was evaluated in a large cohort of 372 surgically treated HPV-negative HNSCC patients and correlated with the clinicopathologic parameters and disease outcomes. Down-regulation of ANXA9 expression was found in 42% of HNSCC tissue samples, compared to normal epithelia. ANXA9 expression in tumors was significantly associated with oropharyngeal location and histological differentiation grade (P &lt, 0.001). In marked contrast, ANXA10 expression was absent in normal epithelium, but variably detected in the cytoplasm of cancer cells. Positive ANXA10 expression was found in 64% of tumors, and was significantly associated with differentiation grade (P &lt, 0.001), being also more frequent in oropharyngeal tumors (P = 0.019). These results reveal that the expression of both ANXA9 and ANXA10 is frequently altered in HNSCC and associated to the tumor differentiation grade, suggesting that they could be implicated in the pathogenesis of these cancers.

Epidermal stem cells are responsible for normal tissue homeostasis and contribute to tissue regeneration during injury. Several assays measuring stem cell frequency and function can be used to assess epidermal stem cell potential. However, the ultimate assay that accounts for stemness is the capacity to sustain in vivo long-term tissue regeneration and maintenance. We can use this type of analysis to interrogate whether a specific genetic alteration (e.g., activation or inactivation of any gene thought to be involved in stem cell quiescence or proliferation) confers increased or decreased stem cell potential.

// Clotilde Costa 1, 3 , Mirentxu Santos 1, 2 , Monica Martinez-Fernandez 1, 2 , Corina Lorz 1, 2 , Sara Lazaro 1 , Jesus M. Paramio 1, 2 1 Unidad de Oncologia Molecular, CIEMAT (ed70A), 28040 Madrid, Spain 2 Unidad de Oncologia Molecular y Celular, Instituto de Investigaciones Biomed, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain 3 Present address: Unidad Mixta Roche-Chus, Hospital Universitario, 15706 Santiago de Compostela, Spain Correspondence to: Jesus M. Paramio, email: jesusm.paramio@ciemat.es Mirentxu Santos, email: mirentxu.santos@ciemat.es Keywords: epidermis, Rb, E2F4, mouse, cancer Received: July 16, 2016 Accepted: September 19, 2016 Published: September 30, 2016 ABSTRACT E2F/RB activity is altered in most human tumors. The retinoblastoma family of proteins plays a key role in regulating the progression of the cell cycle from the G1 to S phases. This is achieved through negative regulation of E2F transcription factors, important positive regulators of cell cycle entry. E2F family members are divided into two groups: activators (E2F1-E2F3a) and repressors (E2F3b-E2F8). E2F4 accounts for a large part of the E2F activity and is a main E2F repressor member in vivo . Perturbations in the balance from quiescence towards proliferation contribute to increased mitotic gene expression levels frequently observed in cancer. We have previously reported that combined Rb1 - Rbl1 or Rb1 - E2f1 ablation in epidermis produces important alterations in epidermal proliferation and differentiation, leading to tumor development. However, the possible roles of E2F4 in this context are still to be determined. Here, we show the absence of any discernible phenotype in the skin of mice lacking of E2f4 . In contrast, the inducible loss of Rb1 in the epidermis of E2F4-null mice produced multiple skin abnormalities including altered differentiation and proliferation, spontaneous wounds, carcinoma in situ development and stem cell perturbations. All these phenotypic alterations are associated with extensive gene expression changes, the induction of c-myc and the Akt activation. Moreover the whole transcriptome analyses in comparison with previous models generated also revealed extensive changes in multiple repressive complexes and in transcription factor activity. These results point to E2F4 as a master regulator in multiple steps of epidermal homeostasis in Rb1 absence.

Head and neck cancer affects the upper aerodigestive tract and is the sixth leading cancer worldwide by incidence and the seventh by cause of death. Despite significant advances in surgery and chemotherapy, molecularly targeted therapeutic options for this type of cancer are scarce and long term survival rates remain low. Recently, comprehensive genomic studies have highlighted the most commonly altered genes and signaling pathways in this cancer. The Hippo-YAP pathway has been identified as a key oncogenic pathway in multiple tumors. Expression of genes controlled by the Hippo downstream transcriptional coactivators YAP (Yes-associated protein 1) and TAZ (WWTR1, WW domain containing transcription regulator 1) is widely deregulated in human cancer including head and neck squamous cell carcinoma (HNSCC). Interestingly, YAP/TAZ signaling might not be as essential for the normal homeostasis of adult tissues as for oncogenic growth, altogether making the pathway an amenable therapeutic target in cancer. Recent advances in the role of Hippo-YAP pathway in HNSCC have provided evidence that genetic alterations frequent in this type of cancer such as PIK3CA (phosphatidylinositide 3-kinase catalytic subunit alpha) overexpression or FAT1 (FAT atypical cadherin 1) functional loss can result in YAP activation. We discuss current therapeutic options targeting this pathway which are currently in use for other tumor types.

Bladder cancer is a clinical and social problem due to its high incidence and recurrence rates. It frequently appears in elderly patients showing other medical comorbidities that hamper the use of standard chemotherapy. We evaluated the activity of CDK4/6 inhibitor as a new therapy for patients unfit for cisplatin (CDDP).Bladder cancer cell lines were tested forCell lines tested were sensitive to CDK4/6 inhibition, independent onCDK4/6 inhibitors, alone or in combination, are a novel therapeutic strategy for patients with advanced bladder cancer previously classified as unfit for current treatment options.

Objectives Phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) is commonly altered in many human tumors, leading to the activation of p110α enzymatic activity that stimulates growth factor-independent cell growth. PIK3CA alterations such as mutation, gene amplification and overexpression are common in head and neck squamous cell carcinoma (HNSCC) and. We aim to explore how these alterations and clinical outcome are associated, as well as the molecular mechanisms involved. Material and methods Mutation and copy-number variation in PIK3CA, and whole-genome expression profiles, were analyzed in primary HNSCC tumors from The Cancer Genome Atlas (TCGA) cohort (n = 243). The results were validated in an independent cohort form the University Hospital of A Coruna (UHAC, n = 62). Expression of the PIK3CA gene protein product (PI3K p110α) and nuclear YAP were assessed in tissue microarrays in a cohort from the University Hospital 12 de Octubre (UH12O, n = 91). Results Only high expression of the PIK3CA gene was associated with poor clinical outcome. The study of gene expression, transcription factor and protein signatures suggested that the activation of the Hippo-YAP pathway, involved in organ size, stem cell maintenance and tumorigenesis, could underlie tumor progression in PI3KCA overexpressing tumors. Tissue arrays showed that PI3K p110α levels correlated with YAP nuclear localization in HNSCC tumors. Conclusions High expression of PIK3CA in HNSCC primary tumors identifies patients at high risk for recurrence. In these tumors, progression could rely on the Hippo-YAP pathway instead of the canonical Akt/mTOR pathway. This observation could have important implications in the therapeutic options for patients.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., The TP63 gene codes for two major isoform types, TAp63 and ΔNp63, with probable opposite roles in tumorigenesis. The ΔNp63α protein is frequently amplified and overexpressed in different epithelial tumors. Accordingly, it has been considered a potential oncogene. Nonetheless, a possible metastatic suppressor activity has also been suggested based on the experimental observation that its expression is reduced or even absent in advanced invasive tumors. Such metastatic suppressor activities are often related to tumors bearing point mutated TP53 gene. However, its potential roles in TP53-deficient tumors are poorly characterized. Here we show that in spontaneous tumors, induced by the epidermal-specific Trp53 ablation, the reduction of ΔNp63 expression is an early event, whereas it is re-expressed in the lung metastatic lesions. Using knock down and ectopic expression approaches, we show that ΔNp63 expression opposes the epithelial-mesenchymal transition and reduces the metastatic potential of the cells. This process occurs through the modulation of ΔNp63-dependent downstream targets (including transcription factors and microRNAs) likely to play metastatic roles. Further, ΔNp63 also favors the expression of factors involved in iPS reprogramming, thus suggesting that it can also modulate specific stem cell traits in mouse epidermal tumor cells. Overall, our data assign antimetastatic roles to ΔNp63 in the context of p53 deficiency and epidermis., The study was funded by the following: MINECO grant SAF2012–34378; Comunidad Autónoma de Madrid grant S2010/BMD-2470 (Oncocycle Program); AES grants ISCIII-RETIC RD06/0020/0029 and RD12/0036/0009 to J.M. Paramio. Grants AP99782012 from MMA Foundation to M. Dueñas. AES grant ISCIIIFIS PI12/01959 to M. Santos.

IKKβ (encoded by

Hair follicle stem cells (HF-SCs) alternate between periods of quiescence and proliferation, to finally differentiate into all the cell types that constitute the hair follicle. Also, they have been recently identified as cells of origin in skin cancer. HF-SCs localize in a precise region of the hair follicle, the bulge, and molecular markers for this population have been established. Thus, HF-SCs are good model to study the potential role of oncogenic activations on SC physiology. Expression of a permanently active form of Akt (myrAkt) in basal cells leads to Akt hyperactivation specifically in the CD34+Itga6H population. This activation causes bulge stem cells to exit from quiescence increasing their response to proliferative stimuli and affecting some functions such as cell migration. HF-SC identity upon Akt activation is preserved; in this sense, increased proliferation does not result in stem cell exhaustion with age suggesting that Akt activation does not affect self-renewal an important aspect for normal tissue maintenance and cancer development. Genome-wide transcriptome analysis of HF-SC isolated from myrAkt and wild-type epidermis underscores changes in metabolic pathways characteristic of cancer cells. These differences manifest during a two-step carcinogenesis protocol in which Akt activation in HF-SCs results in increased tumor development and malignant transformation. Stem Cells 2014;32:1917–1928

Squamous cell carcinoma (SCC) or epidermoid cancer is a frequent and aggressive malignancy. However in apparent paradox it retains the squamous differentiation phenotype except for very dysplastic lesions. We have shown that cell cycle stress in normal epidermal keratinocytes triggers a squamous differentiation response involving irreversible mitosis block and polyploidisation. Here we show that cutaneous SCC cells conserve a partial squamous DNA damage-induced differentiation response that allows them to overcome the cell division block. The capacity to divide in spite of drug-induced mitotic stress and DNA damage made well-differentiated SCC cells more genomically instable and more malignant in vivo. Consistently, in a series of human biopsies, non-metastatic SCCs displayed a higher degree of chromosomal alterations and higher expression of the S phase regulator Cyclin E and the DNA damage signal γH2AX than the less aggressive, non-squamous, basal cell carcinomas. However, metastatic SCCs lost the γH2AX signal and Cyclin E, or accumulated cytoplasmic Cyclin E. Conversely, inhibition of endogenous Cyclin E in well-differentiated SCC cells interfered with the squamous phenotype. The results suggest a dual role of cell cycle stress-induced differentiation in squamous cancer: the resulting mitotic blocks would impose, when irreversible, a proliferative barrier, when reversible, a source of genomic instability, thus contributing to malignancy. AG is grateful to Jean-Jeaques Guilhou, Jean-Claude Rossi and the INSERM for professional support and to Renata Polakowska for the generous gift of precious BCCP cell line. We thank Lucía Barbier, Tania Lobato, Evelyn Andrades, Alicia Noriega and María Aramburu for technical assistance and Natalia Sanz for critical reading of the MS. To AG: National grants from Instituto de Salud Carlos III, Fondo de Investigación Sanitaria (ISCIII-FIS/FEDER, Spain): PI08/0890, PI11/02070, PI14/00900; Ligue Nationale Contre la Cancer (La Ligue; France). To AT: ISCIII-FIS PI10/00785. To JP: MINECO grant SAF2015-66015-R; AES grant ISCIII-RETIC RD12/0036/0009. VC was funded by a fellowship from La Ligue (France), PA by IDIVAL (Spain), RM and IdP by AG lab and ISCIII-FIS-FEDER PI11/02070 (Spain).

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-κB and signal transducer and activator of transcription-3 pathways and decreased transforming growth factor-β 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. [Cancer Res 2009;69(3):1099–108]

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. © 2008 Wiley-Liss, Inc.

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 (Aktwt), 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.

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. © 2007 Wiley-Liss, Inc., This work was partially supported by grants: SAF2002-01037 (MCYT), Oncocycle (CAM), ISCIII-RETIC RD06/0020 (MSC), SAF2005-00033 (MCYT) and Oncology Program from La Caixa Foundation to JMP. SR is recipient of a Juan de la Cierva Fellowship.

This article is distributed under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License., Observations in thyroid patients and experimental animals show that the skin is an important target for the thyroid hormones. We previously showed that deletion in mice of the thyroid hormone nuclear receptors TRα1 and TRβ (the main thyroid hormone-binding isoforms) results in impaired epidermal proliferation, hair growth, and wound healing. Stem cells located at the bulges of the hair follicles are responsible for hair cycling and contribute to the regeneration of the new epidermis after wounding. Therefore a reduction in the number or function of the bulge stem cells could be responsible for this phenotype. Bulge cells show increased levels of epigenetic repressive marks, can retain bromodeoxyuridine labeling for a long time, and have colony-forming efficiency (CFE) in vitro. Here we demonstrate that mice lacking TRs do not have a decrease of the bulge stem cell population. Instead, they show an increase of label-retaining cells (LRCs) in the bulges and enhanced CFE in vitro. Reduced activation of stem cells leading to their accumulation in the bulges is indicated by a strongly reduced response to mobilization by 12-O-tetradecanolyphorbol-13-acetate. Altered function of the bulge stem cells is associated with aberrant activation of Smad signaling, leading to reduced nuclear accumulation of β-catenin, which is crucial for stem cell proliferation and mobilization. LRCs of TR-deficient mice also show increased levels of epigenetic repressive marks. We conclude that thyroid hormone signaling is an important determinant of the mobilization of stem cells out of their niche in the hair bulge. These findings correlate with skin defects observed in mice and alterations found in human thyroid disorders., This work was supported by grants from the Ministerio de Economía y Competitividad (BFU2011-28058 to A.A. and SAF2012-34378 to J.M.P.), the Instituto de Salud Carlos III (RD12 /0036/0030 to A.A. and RD12/0036/0009 to J.M.P.), and the Comunidad de Madrid (S2011/BMD-2328 TIRONET to A.A. and S2010/BMD-2470 to J.M.P.).

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the TNF superfamily. TWEAK activates the Fn14 receptor, and may regulate apoptosis, proliferation, and inflammation, processes that play a significant role in pathological conditions. However, there is little information on the function and regulation of this system in the kidney. Therefore, TWEAK and Fn14 expression were studied in cultured murine tubular epithelial MCT cells and in mice in vivo. The effect of TWEAK on cell death was determined. We found that TWEAK and Fn14 expression was increased in experimental acute renal failure induced by folic acid. Cultured tubular cells express both TWEAK and the Fn14 receptor. TWEAK did not induce cell death in non-stimulated tubular cells. However, in cells costimulated with TNFalpha/interferon-gamma, TWEAK induced apoptosis through the activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-8, caspase-9, and caspase-3, Bid cleavage, and evidence of mitochondrial injury. There was no evidence of endoplasmic reticulum stress. A pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp prevented TWEAK-induced apoptosis, but it sensitized cells to necrosis via generation of reactive oxygen species. In conclusion, cooperation between inflammatory cytokines results in tubular cell death. TWEAK and Fn14 may play a role in renal tubular cell injury.

Fas-associated death domain (FADD) is an adaptor protein that is required for the transmission of the death signal from lethal receptors of the tumor necrosis factor superfamily. FADD contains a death domain (DD) and a death effector domain (DED). As death receptors contribute to renal tubular injury and tubular cell FADD increases in acute renal failure, we have studied the function of FADD in tubular epithelium. FADD expression was studied in kidney samples from mice. In order to study the contribution of FADD to renal tubular cell survival, FADD or FADD-DD were overexpressed in murine tubular epithelium. FADD is expressed in renal tubules of the healthy kidney. Both FADD and FADD-DD induce apoptosis in primary cultures of murine tubular epithelium and in the murine cortical tubular cell line. Death induced by FADD-DD has apoptotic morphology, but differs from death receptor-induced apoptosis in that it is not blocked by inhibitors of caspases. Neither an inhibitor of serine proteases nor overexpression of antiapoptotic BclxL prevented cell death. However, the combination of caspase and serine protease inhibition was protective. FADD and FADD-DD overexpression decreased nuclear factor kappa B activity. These data suggest that FADD has a death regulatory function in renal tubular cells that is independent of death receptors. FADD-DD is sufficient to induce apoptosis in these cells. This information is relevant to understanding the role of FADD in tubular injury.

Role of Bcl-xL in paracetamol-induced tubular epithelial cell death. Background Paracetamol overdose causes acute renal failure and chronic exposure to paracetamol has been linked to chronic renal failure. Recently, apoptosis induction has been identified as a possible mechanism of paracetamol nephrotoxicity. Methods Murine proximal tubular epithelial MCT cells were cultured in the presence of paracetamol. The effects of Bcl-xL overexpression, Bax antisense oligodeoxynucleotides, and different caspase inhibitors on cell death were studied. Results While paracetamol did not change the mRNA expression of the antiapoptotic gene bcl-xL, it decreased Bcl-xL protein levels. The decrease in Bcl-xL was prevented by lactacystin, but not by caspase inhibitors. Addition to the culture media of the survival factors present in fetal calf serum (FCS) increased Bcl-xL expression and decreased paracetamol-induced apoptosis. Overexpression of a human bcl-xL transgene decreased apoptosis induced by paracetamol by 60% at 24 hours and increased long-term cell survival. The constitutive expression of the viral caspase inhibitor CrmA decreased the rate of apoptosis by 60% at 24 hours and the broad-specific caspase inhibitor zVAD-fmk prevented paracetamol-induced features of apoptosis. However, caspase inhibitors did not prevent eventual cell death. Bax did not translocate to mitochondria and Bax antisense oligodeoxynucleotides were not protective. Conclusion Our results suggest that induction of apoptosis may underlie the nephrotoxic potential of paracetamol and identify Bcl-xL as a player in toxic tubular cell injury.

Tubular cell apoptosis contributes to the pathogenesis of renal injury. However, the intracellular pathways that are active in tubular epithelium are poorly understood. The lethal pathways activated by cyclosporin A (CsA), a nephrotoxin that induces caspase-dependent apoptosis in tubular epithelium, were explored. Fas expression, caspase activation, and mitochondrial injury were assessed by Western blot, flow cytometry, and microscopy in cultured murine tubular epithelial cells exposed to CsA. The influence of FasL antagonists, Bax antisense oligodeoxynucleotides, and caspase inhibitors on cell survival was explored. Tubular cells constitutively express FasL. CsA increased the expression of Fas. However, Fas had no role in CsA-induced apoptosis, as CsA did not sensitize to FasL-induced apoptosis, caspase-8 activity was not increased, and neither blocking anti-FasL antibodies nor caspase-8 inhibition prevented CsA-induced apoptosis. Apoptosis induced by CsA is associated with the translocation of Bax to the mitochondria and Bax antisense oligodeoxynucleotides protected from CsA-induced apoptosis. CsA promoted a caspase-independent release of cytochrome c and Smac/Diablo from mitochondria. CsA also led to a caspase-dependent loss of mitochondrial membrane potential. Caspase-2, caspase-3, and caspase-9 were activated, and specific caspase inhibitor prevented apoptosis and increased long-term survival. Evidence for endoplasmic reticulum stress, such as induction of GADD153, was also uncovered. However, endoplasmic reticulum-specific caspase-12 was not activated. CsA induces changes in several apoptotic pathways. However, the main lethal apoptotic pathway in CsA-exposed tubular epithelial cells involves mitochondrial injury.

Expression of Smac/Diablo in tubular epithelial cells and during acute renal failure. Background Apoptosis contributes to tubular cell loss in the course of renal injury. However, the mechanisms regulating tubular cell apoptosis are not well understood. Smac/Diablo is a mitochondrial protein that is released to the cytosol during apoptosis, where it blocks the antiapoptotic activity of inhibitor of apoptosis proteins (IAPs). Methods We have studied the regulation of Smac/Diablo mRNA and protein expression in murine toxic acute tubular necrosis, and in cultured tubular epithelial cells exposed to the lethal cytokine tumor necrosis factor (TNF). Results Folic acid–induced acute renal failure was associated with tubular cell apoptosis. Smac/Diablo mRNA and protein levels increased by 50% at 24 hours. TNF, a cytokine whose renal expression increases in folic acid nephropathy, induced apoptosis in cultured tubular epithelial cells in a time-dependent manner. In addition, TNF increased the mRNA and protein expression of Smac/Diablo. Conclusion These findings support the concept that regulation of Smac/Diablo mRNA and protein expression is a mechanism by which lethal stimuli amplify their lethal potential in renal cells.