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© The Author(s) 2020., Regenerative proliferation capacity and poor differentiation are histological features usually linked to poor prognosis in head and neck squamous cell carcinoma (hnSCC). However, the pathways that regulate them remain ill-characterized. Here, we show that those traits can be triggered by the RHO GTPase activator VAV2 in keratinocytes present in the skin and oral mucosa. VAV2 is also required to maintain those traits in hnSCC patient-derived cells. This function, which is both catalysis- and RHO GTPase-dependent, is mediated by c-Myc- and YAP/TAZ-dependent transcriptomal programs associated with regenerative proliferation and cell undifferentiation, respectively. High levels of VAV2 transcripts and VAV2-regulated gene signatures are both associated with poor hnSCC patient prognosis. These results unveil a druggable pathway linked to the malignancy of specific SCC subtypes., We thank M. Blázquez and CIC facilities’ personnel for lab work and core unit technical assistance, respectively. X.R.B. is supported by grants from Worldwide Cancer Research (14-1248), the Castilla-León Government (CSI049U16, CLC-2017-01), the Spanish Ministry of Science and Innovation (MSI) (SAF2015-64556-R, RTI2018-096481-B-I00), the Ramón Areces Foundation, and the Spanish Association against Cancer (GC16173472GARC). X.R.B.’s institution is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Ministry of Education of the Castilla-León Government (CLC-2017-01). J.M.P. is supported by MSI grants (SAF2015-66015-R, PIE15/00076). S.A.B. was supported by the European Research Council, the Spanish MSIU, and the IRB-Barcelona. S.R.-F. and L.F. L.-M. contracts have been supported by funding from the MSI (BES-2013-063573) and the Spanish Ministry of Education, Culture, and Sports (L.F.L.-M., FPU13/02923), respectively. Both Spanish and Castilla-León government-associated funding is partially supported by the European Regional Development Fund.

[Background]: A subpopulation of cancer stem cells (CSCs) with capacity for self-renewal is believed to drive initiation, progression, and relapse of breast tumors., [Methods]: Since the thyroid hormone receptor β (TRβ) appears to suppress breast tumor growth and metastasis, we have analyzed the possibility that TRβ could affect the CSC population using MCF-7 cells grown under adherent conditions or as mammospheres, as well as inoculation into immunodeficient mice., [Results]: Treatment of TRβ-expressing MCF-7 cells (MCF7-TRβ cells) with the thyroid hormone triiodothyronine (T3) decreased significantly CD44+/CD24− and ALDH+ cell subpopulations, the efficiency of mammosphere formation, the self-renewal capacity of CSCs in limiting dilution assays, the expression of the pluripotency factors in the mammospheres, and tumor initiating capacity in immunodeficient mice, indicating that the hormone reduces the CSC population present within the bulk MCF7-TRβ cultures. T3 also decreased migration and invasion, a hallmark of CSCs. Transcriptome analysis showed downregulation of the estrogen receptor alpha (ERα) and ER-responsive genes by T3. Furthermore, among the T3-repressed genes, there was an enrichment in genes containing binding sites for transcription factors that are key determinants of luminal-type breast cancers and are required for ER binding to chromatin., [Conclusion]: We demonstrate a novel role of TRβ in the biology of CSCs that may be related to its action as a tumor suppressor in breast cancer., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., This work was supported by grants BFU2014-53610-P (MINECO, AEI, FEDER, UE), CIBERONC CB/16/00228, and SAF2017-90604-REDT (Spanish Ministry of Economy and Competitiveness), and B2017/BMD-3724 (Comunidad de Madrid). The cost of this publication has been paid, in part, by FEDER funds.

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.

Bladder cancer is lethal in its advanced, muscle-invasive phase with very limited therapeutic advances1,2. Recent molecular characterization has defined new (epi)genetic drivers and potential targets for bladder cancer3,4. The immune checkpoint inhibitors have shown remarkable efficacy but only in a limited fraction of bladder cancer patients5–8. Here, we show that high G9a (EHMT2) expression is associated with poor clinical outcome in bladder cancer and that targeting G9a/DNMT methyltransferase activity with a novel inhibitor (CM-272) induces apoptosis and immunogenic cell death. Using an immunocompetent quadruple-knockout (PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1−/−) transgenic mouse model of aggressive metastatic, muscle-invasive bladder cancer, we demonstrate that CM-272 + cisplatin treatment results in statistically significant regression of established tumors and metastases. The antitumor effect is significantly improved when CM-272 is combined with anti-programmed cell death ligand 1, even in the absence of cisplatin. These effects are associated with an endogenous antitumor immune response and immunogenic cell death with the conversion of a cold immune tumor into a hot tumor. Finally, increased G9a expression was associated with resistance to programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer. In summary, these findings support new and promising opportunities for the treatment of bladder cancer using a combination of epigenetic inhibitors and immune checkpoint blockade. Inhibition of histone and DNA methyltransferase activity enhances sensitivity to platinum-based and immunotherapy in a novel transgenic mouse model of metastatic bladder cancer.

Lung cancer is the leading worldwide cause of cancer mortality, however, neither curative treatments nor substantial prolonged survival has been achieved, highlighting the need for investigating new proteins responsible for its development and progression. IKKα is an essential protein for cell survival and differentiation, which expression is enhanced in human non-small cell lung cancer (NSCLC) and correlates with poor patient survival, appearing as a relevant molecule in lung cancer progression. However, there are not conclusive results about its role in this type of cancer. We have recently found that IKKα performs different functions and activates different signaling pathways depending on its nuclear or cytoplasmic localization in tumor epidermal cells. In this work, we have studied the involvement of IKKα in lung cancer progression through the generation of lung cancer cell lines expressing exogenous IKKα either in the nucleus or in the cytoplasm. We demonstrate that IKKα signaling promotes increased cell malignancy of NSCLC cells as well as lung tumor progression and metastasis in either subcellular localization, through activation of common protumoral proteins, such as Erk, p38 and mTor. But, additionally, we found that depending on its subcellular localization, IKKα has non-overlapping roles in the activation of other different pathways known for their key implication in lung cancer progression: while cytoplasmic IKKα increases EGFR and NF-κB activities in lung tumor cells, nuclear IKKα causes lung tumor progression through c-Myc, Smad2/3 and Snail activation. These results suggest that IKKα may be a promising target for intervention in human NSCLC., Graphical Abstract Unlabelled Image

Currently, bladder cancer (BC) represents a challenging problem in the field of Oncology. The high incidence, prevalence, and progression of BC have led to the exploration of new avenues in its management, in particular in advanced metastatic stages. The recent inclusion of immune checkpoint blockade inhibitors as a therapeutic option for BC represents an unprecedented advance in BC management. However, although some patients show durable responses, the fraction of patients showing benefit is still limited. Notwithstanding, cell-based therapies, initially developed for the management of hematological cancers by infusing immune or trained immune cells or after the engineering of chimeric antigen receptor (CAR) expressing cells, are promising tools to control, or even cure, solid tumors. In this review, we summarize recent cell-based immunotherapy studies, with a special focus on BC.

Background Epigenetic alterations are known contributors to cancer development and aggressiveness. Additional to alterations in cancer cells, aberrant epigenetic marks are present in cells of the tumor microenvironment, including lymphocytes and tumor-associated macrophages, which are often overlooked but known to be a contributing factor to a favorable environment for tumor growth. Therefore, the main aim of this review is to give an overview of the epigenetic alterations affecting immune cells in the tumor microenvironment to provoke an immunosuppressive function and contribute to cancer development. Moreover, immunotherapy is briefly discussed in the context of epigenetics, describing both its combination with epigenetic drugs and the need for epigenetic biomarkers to predict response to immune checkpoint blockage. Main body Combining both topics, epigenetic machinery plays a central role in generating an immunosuppressive environment for cancer growth, which creates a barrier for immunotherapy to be successful. Furthermore, epigenetic-directed compounds may not only affect cancer cells but also immune cells in the tumor microenvironment, which could be beneficial for the clinical response to immunotherapy. Conclusion Thus, modulating epigenetics in combination with immunotherapy might be a promising therapeutic option to improve the success of this therapy. Further studies are necessary to (1) understand in depth the impact of the epigenetic machinery in the tumor microenvironment; (2) how the epigenetic machinery can be modulated according to tumor type to increase response to immunotherapy and (3) find reliable biomarkers for a better selection of patients eligible to immunotherapy.

Bladder cancer (BC) is the tenth most frequent cancer worldwide and is associated with high mortality when diagnosed in its most aggressive form, which is not reverted by the current treatment options. Thus, the development of new therapeutic strategies, either alternative or complementary to the current ones, is of major importance. The disruption of normal epigenetic mechanisms, namely, DNA methylation, is a known early event in cancer development. Consequently, DNA methyltransferase (DNMT) inhibitors constitute a promising therapeutic target for the treatment of BC. Although these inhibitors, mainly nucleoside analogues such as 5-azacytidine (5-aza) and decitabine (DAC), cause re-expression of tumor suppressor genes, inhibition of tumor cell growth, and increased apoptosis in BC experimental models and clinical trials, they also show important drawbacks that prevent their use as a valuable option for the treatment of BC. However, their combination with chemotherapy and/or immune-checkpoint inhibitors could aid in their implementation in the clinical practice. Here, we provide a comprehensive review of the studies exploring the effects of DNA methylation inhibition using DNMTs inhibitors in BC, from in vitro and in vivo studies to clinical trials.

INTRODUCTION AND OBJECTIVE:Identifying biomarkers to predict BCG response before beginning the treatment is an unsolved need in the clinical practice for patients with high risk non-muscle invasive...

This study unravels an unprecedented murine model of lethal metastatic prostate cancer, based on the combined deletion of Pten and Lkb1. Importantly, minimal activity of LKB1 is sufficient to hamper prostate cancer cell aggressiveness, thus redefining the relationship between gene dosage and tumor suppression., Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination.

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.

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.

RAS genes are mutated in 20% of human tumors, but these mutations are very rare in breast cancer. Here, we used a mouse model to generate tumors upon activation of a mutagenic T2Onc2 transposon via expression of a transposase driven by the keratin K5 promoter in a p53+/− background. These animals mainly developed mammary tumors, most of which had transposon insertions in one of two RASGAP genes, neurofibromin1 (Nf1) and RAS p21 protein activator (Rasa1). Immunohistochemical analysis of a collection of human breast tumors confirmed that low expression of RASA1 is frequent in basal (triple-negative) and estrogen receptor negative tumors. Bioinformatic analysis of human breast tumors in The Cancer Genome Atlas database showed that although RASA1 mutations are rare, allelic loss is frequent, particularly in basal tumors (80%) and in association with TP53 mutation. Inactivation of RASA1 in MCF10A cells resulted in the appearance of a malignant phenotype in the context of mutated p53. Our results suggest that alterations in the Ras pathway due to the loss of negative regulators of RAS may be a common event in basal breast cancer. Cancer Res; 77(6); 1357–68. ©2017 AACR.

// Sara Lazaro 1 , Miriam Perez-Crespo 1 , Ana Belen Enguita 2 , Pilar Hernandez 1 , Jesus Martinez-Palacio 1 , Marta Oteo 3 , Julien Sage 4, 5 , Jesus M. Paramio 1, 6, 7 , Mirentxu Santos 1, 6, 7 1 Molecular Oncology Unit Institute of Biomedical Investigation University Hospital “12 de Octubre”, Madrid, Spain 2 Pathology Department Institute of Biomedical Investigation University Hospital “12 de Octubre”, Madrid, Spain 3 Biomedical Applications and Pharmacokinetics CIEMAT (ed 70A), Madrid, Spain 4 Department of Pediatrics, Stanford University, Stanford, USA 5 Department of Genetics. Stanford University, CCSR Rm. 1215a. Stanford, California, USA 6 Molecular Oncology, Institute of Biomedical Investigation University Hospital “12 de Octubre”, Madrid, Spain 7 Centro de Investigaciones Biomedicas en Red de Cancer (CIBERONC), Madrid, Spain Correspondence to: Jesus M. Paramio, email: jesusm.paramio@ciemat.es Mirentxu Santos, email: mirentxu.santos@ciemat.es Keywords: retinoblastoma family, lung cancer, urethane, DHPN Received: January 13, 2016 Accepted: December 05, 2016 Published: December 10, 2016 ABSTRACT Lung cancer is a deadly disease with increasing cases diagnosed worldwide and still a very poor prognosis. While mutations in the retinoblastoma ( RB1 ) tumor suppressor have been reported in lung cancer, mainly in small cell lung carcinoma, the tumor suppressive role of its relatives p107 and p130 is still a matter of debate. To begin to investigate the role of these two Rb family proteins in lung tumorigenesis, we have generated a conditional triple knockout mouse model (TKO) in which the three Rb family members can be inactivated in adult mice. We found that ablation of all three family members in the lung of mice induces tumorlets, benign neuroendocrine tumors that are remarkably similar to their human counterparts. Upon chemical carcinogenesis, DHPN and urethane accelerate tumor development; the TKO model displays increased sensitivity to DHPN, and urethane increases malignancy of tumors. All the tumors developing in TKO mice (spontaneous and chemically induced) have neuroendocrine features but do not progress to fully malignant tumors. Thus, loss of Rb and its family members confers partial tumor susceptibility in neuroendocrine lineages in the lungs of mice. Our data also imply the requirement of other oncogenic signaling pathways to achieve full transformation in neuroendocrine lung lesions mutant for the Rb family.

The bidirectional regulation of epithelial–mesenchymal transitions (EMT) is key in tumorigenesis. Rho GTPases regulate this process via canonical pathways that impinge on the stability of cell-to-cell contacts, cytoskeletal dynamics, and cell invasiveness. Here, we report that the Rho GTPase activators Vav2 and Vav3 utilize a new Rac1-dependent and miR-200c-dependent mechanism that maintains the epithelial state by limiting the abundance of the Zeb2 transcriptional repressor in breast cancer cells. In parallel, Vav proteins engage a mir-200c-independent expression prometastatic program that maintains epithelial cell traits only under 3D culture conditions. Consistent with this, the depletion of endogenous Vav proteins triggers mesenchymal features in epithelioid breast cancer cells. Conversely, the ectopic expression of an active version of Vav2 promotes mesenchymal-epithelial transitions using E-cadherin-dependent and independent mechanisms depending on the mesenchymal breast cancer cell line used. In silico analyses suggest that the negative Vav anti-EMT pathway is operative in luminal breast tumors. Gene signatures from the Vav-associated proepithelial and prometastatic programs have prognostic value in breast cancer patients., XRB is supported by grants from the Castilla-León Government (CSI049U16), the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2015-64556-R), Worldwide Cancer Research (14-1248), the Ramón Areces Foundation, and the Spanish Association against Cancer (GC16173472GARC). MD (BFU2014-52729-P), JMP (SAF2015-66015-R, PIE15/00076), and PMF-S (SAF2014-51813-R) are supported by MINECO grants. Spanish funding is partially supported by the European Regional Development Fund. LFL-M, JC, and SR-F were supported by funding from the Spanish Ministry of Education, Culture and Sports (LFL-M, FPU13/02923) and the MINECO (JC, CD15/00113; SR-F, BES-2013-063573).

Background Exosomes are small membranous vesicles (around 40-130 nm), that have been detected in different biological samples, that play a key role in NSCLC and being relevant in stem cell differentiation as well. The main objective of this study was to analyze the exosomes cargo from NSCLC cell cultures growth in monolayer (2D) and suspension conditions (3D, lung tumorespheres). Methods Cultures were established from NSCLC resected patients and cell lines. Exosomes isolation was performed by ultracentrifugation. Characterization was carried out by NTA, electron microscopy, immunoblot and flow cytometry. Mutational status of EGFR and RAS genes was analyzed by BEAMing dPCR. Transcriptomic analysis has been carried out from exosomal RNA with microarrays, (p ≤ 0.01). Consequently, XAGE1B (significantly expressed gene in exosomes) was analyzed by RTqPCR in 189 paired fresh-frozen tumor and normal tissue samples of resected NSCLC. Prognostic value was assessed by Kaplan‐Meier curves (log rank‐test), considering significant p Results Exosomal characterization through NTA and electron microscopy showed an exosomes size from 108-125 nm. Specific markers were detected by IB and FC. Mutational analysis of EGFR and RAS genes in exosomes shown the same pattern displayed by the origin cells. Transcriptomic analysis showed that the expression of mRNAs, miRNAs and precursors were significantly different between 3D and 2D-derived exosomes. A pathway enrichment was carried out to know in which processes (cancer-related) are involved. Significant differential expression was also found between ADC vs SCC–derived exosomes. Concretely, XAGE1B is overexpressed in ADC-derived exosomes (p = 0.00003). This overexpression in ADC was validated in NSCLC cohort (p = 0.002). Furthermore, it has revealed a significant association with patient prognosis for overall survival in the ADC group (n = 74)(OS 49.8 vs. NR months, p = 0.043). Conclusions Differences in exosomal cargo have been observed between: i) 3D vs. 2D cultures and ii) ADC vs. SCC. In addition, the same mutational pattern was detected in exosomes as compared with parental cells. Therefore, exosomes can be a useful source of biomarkers in NSCLC analysis. Supported by grant GV/2018/026, PI18/00266, & AECC Valencia. Legal entity responsible for the study Fundacion de Investigacion del Hospital General Universitario de Valencia. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Non-muscle invasive bladder cancer (NMIBC) represents a crucial problem for the national health care systems due to its high rates of recurrence and the consequent need of frequent follow-ups. Here, gene expression analyses in patients diagnosed as NMIBC were performed to determine those molecular pathways involved in tumor initiation, finding that both MYC and E2F are up regulated and helps to tumor initiation and progression. Our results also support an important involvement of alternative splicing events, modifying key pathways to favour bladder tumor evolution. Finally, since MDM2 showed differential exon usage, mutations in TP53 and its protein expression have been also studied in the same patients. Our data support that recurrence is epigenetically mediated and favoured by an increase protein expression of TP53, which appears more frequently mutated in advanced stages and grades, being associated to a worse prognosis. Therefore, TP53 mutational status could be used as a potential biomarker in the first stages of NMIBC to predict recurrence and prognosis. We express our deepest acknowledgement to patients and their families. The authors also acknowledge the computing resources and technical support provided by Abel Paz-Gallardo and Alfonso Pardo from Extremadura Research Centre for Advanced Technologies (CETA−CIEMAT). This work was supported FEDER cofounded MINECO grant SAF2015-66015-R, ISCIII-RETIC RD12/0036/0009, and PIE15/00076 and CB/16/00228 (to J.M. Paramio); MMF was supported by a Jose Castillejo Fellowship (CAS16/00115).

Metabolism reprogramming is considered a hallmark of cancer. The study of bladder cancer (BC) metabolism could be the key to developing new strategies for diagnosis and therapy. This work aimed to identify tissue and urinary metabolic signatures as biomarkers of BC and get further insight into BC tumor biology through the study of gene-metabolite networks and the integration of metabolomics and transcriptomics data. BC and control tissue samples (n = 44) from the same patients were analyzed by High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance and microarrays techniques. Besides, urinary profiling study (n = 35) was performed in the same patients to identify a metabolomic profile, linked with BC tissue hallmarks, as a potential non-invasive approach for BC diagnosis. The metabolic profile allowed for the classification of BC tissue samples with a sensitivity and specificity of 100%. The most discriminant metabolites for BC tissue samples reflected alterations in amino acids, glutathione, and taurine metabolic pathways. Transcriptomic data supported metabolomic results and revealed a predominant downregulation of metabolic genes belonging to phosphorylative oxidation, tricarboxylic acid cycle, and amino acid metabolism. The urinary profiling study showed a relation with taurine and other amino acids perturbed pathways observed in BC tissue samples, and classified BC from non-tumor urine samples with good sensitivities (91%) and specificities (77%). This urinary profile could be used as a non-invasive tool for BC diagnosis and follow-up.

// Josefa P. Alameda 1, 2 , Manuel Navarro 1, 2 , Angel Ramirez 1, 2 , Angustias Page 1, 2 , Cristian Suarez-Cabrera 1, 2 , Rodolfo Moreno-Maldonado 3 , Jesus M. Paramio 1, 2 , Maria del Carmen Farina 4 , Marcela Del Rio 5, 6, 7 , Maria Jesus Fernandez-Acenero 8 , Ana Bravo 9 , Maria de los Llanos Casanova 1, 2 1 Molecular Oncology Unit, Centro de Investigaciones Energeticas, Medioambientales y Tecnologicas (CIEMAT), Madrid, Spain 2 Molecular Oncology, Institute of Biomedical Investigation University Hospital “12 de Octubre”, Madrid, Spain 3 Present address: SILO Espana, Madrid, Spain 4 Department of Dermatology, Fundacion Jimenez Diaz, Madrid, Spain 5 Epithelial Biomedicine Division, CIEMAT-CIBERER (U714), Madrid, Spain 6 Department of Bioengineering, Carlos III University (UC3M), Leganes, Madrid, Spain 7 Catedra Fundacion Jimenez Diaz (IIS-FJD) of Regenerative Medicine and Tissue Bioengineer, Madrid, Spain 8 Department of Pathology, Hospital Clinico San Carlos, Madrid, Spain 9 Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, University of Santiago de Compostela, Lugo, Spain Correspondence to: M. Llanos Casanova, email: llanos.casanova@ciemat.es Keywords: IKKα, keratinocyte differentiation, MMP9, skin, skin cancer Received: July 29, 2016 Accepted: September 29, 2016 Published: October 08, 2016 ABSTRACT 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.

TPS598 Background: Intravesical Bacillus Calmette-Guerin (BCG) induction + BCG maintenance after transurethral resection (TURBT) is the current standard of care for patients (pt) with high-risk non-muscle invasive bladder cancer (NMIBC). Recurrence rate at 2 years is around 30%, which is clearly unsatisfactory. Programmed death ligand 1 (PD-L1) is a surface glycoprotein that functions as an inhibitor of T-cells and plays a crucial role in suppression of cellular immune response. Atezolizumab is a humanized IgG1 monoclonal antibody targeting PD-L1 and is associated with long-term durable remissions in pts with metastatic urothelial cancer (Powles T. Lancet. 2018). Atezolizumab in combination with standard BCG could be beneficial in pts with NMIBC. BladderGATE (NCT04134000) is a phase Ib-II study aims to evaluate the clinical impact and safety of the combination of Atezolizumab + intravesical BGC (medac strain), including a translational study (microbioma and urobioma) in pts with NMIBC. Methods: Eligible pt have confirmed histopathology of NMIBC, BCG naïve or stopped >3 years ago, WHO PS 0-1, no prior radiation to bladder and adequate hematologic and end-organ function. We propose a de-escalation design to enroll patients to identify DLT and MTD proposed to safety and efficacy expansion cohort. Pt will receive induction BCG, 1 instillation every week (qw) (dose level 0) or BCG, 1/2 instillation qw (dose level -1) + intravenously atezolizumab 1200 mg every 3 w (q3w), during 6 w. After induction, BCG will be administered as maintenance treatment at weeks 12, 24 and 48 and atezolizumab 1200 mg q3w up to 1 year. Pt will be accrued to each dose level in cohorts of 10 pt until the MTD is achieved (the highest dose at which

757 Background: Immune checkpoint inhibitors have increased survival in mRCC across all risk groups. In this new treatment paradigm, inflammatory markers could add prognostic information to the International metastatic Renal Cell Carcinoma Database Consortium (IMDC) model in patients treated with ICI. Methods: We conducted a multicenter retrospective analysis of mRCC patients treated with ICI from 2013 to 2019. Clinical, pathological and laboratory data including blood cell counts were collected. Multivariate Cox-regression models were performed to evaluate the independent prognostic significance of the IMDC score, the derived neutrophil to lymphocyte ratio (dNLR) and LDH at baseline, as well as the inflammatory prognostic index (IPI). Results: A total of 104 patients were included. Of these, 19% were treatment-naïve, 34% had received one previous line of treatment and 47% had received two or more previous lines of treatment. Distribution of IMDC model for favorable, intermediate and poor risk was 23%,57% and 20%. Median OS was 15 months and the disease control rate (DCR) was 51%. The multivariate analysis identified the IMDC risk score, the dNLR and the IPI as independent predictors of OS. In addition, both inflammatory markers, the IPI and the dNLR, were able to improve the prognostic value of the IMDC risk score (p = 0.01 and p = 0.006, respectively). Specifically, in patients with 0 or 1 IMDC risk factors, both the IPI and the dNLR were able to subclassify additional prognostic subgroups (i.e. dNLR > 3 vs ≤3 HR = 3,16; 95% CI; 1.71-5.76). Conclusions: Adding IPI and/or dNLR may add further information about the benefit of ICIs in mRCC patients with 0 or 1 IMDC risk factors. Inflammatory systemic markers may improve the prognostic performance of the IMDC model.[Table: see text]

CYLD is a deubiquitinating enzyme known for its role as a tumor suppressor whose mutation leads to skin appendages tumors and other cancers. In this manuscript we report that the tumor suppressor CYLD, similarly to other renowned tumor suppressor genes, protects from premature aging and cancer. We have generated transgenic mice expressing the mutant CYLDC/S protein, lacking its deubiquitinase function, under the control of the keratin 5 promoter, the K5-CYLDC/S mice. These mice express the transgene in different organs, including those considered to be more susceptible to aging, such as skin and thymus. Our results show that K5-CYLDC/S mice exhibit epidermal, hair follicle, and sebaceous gland alterations; and, importantly, they show signs of premature aging from an early age. Typically, 3-month-old K5-CYLDC/S mice exhibit a phenotype characterized by alopecia and kyphosis, and, the histological examination reveals that transgenic mice show signs of accelerated aging in numerous organs such as skin, thymus, pancreas, liver and lung. Additionally, they spontaneously develop tumors of diverse origin. Over-activation of the NF-κB pathway, along with hyperactivation of Akt, JNK and c-Myc, and chronic inflammation, appear as the mechanisms responsible for the premature aging of the K5-CYLDC/S mice.

Macrophages are major components of the immune infiltration in cancer where they can affect tumor behavior. In the bladder, they play important roles during the resolution of infectious processes and they have been associated with a worse clinical prognosis in bladder cancer. The present review focused on the characteristics of these important immune cells, not only eliciting an innate immune surveillance, but also on their importance during the cancer immunoediting process. We further discuss the potential of targeting macrophages for anticancer therapy, the current strategies and the state of the art as well as the foreseen role on combined therapies on the near future. This review shows how a comprehensive understanding of macrophages within the tumor should translate to better clinical outcome and new therapeutic strategies focusing especially on bladder cancer.

Although Ras genes are frequently mutated in human tumors, these mutations are uncommon in breast cancer. However, many breast tumors show evidences of Ras pathway activation. In this manuscript, we have analyzed and characterized mouse mammary tumors generated by random Sleeping Beauty transposon mutagenesis and identify ERAS -a member of the RAS family silenced in adult tissues- as a new gene involved in progression and malignancy of breast cancer. Forced expression of ERAS in human non-transformed mammary gland cells induces a process of epithelial-to-mesenchymal transition and an increase in stem cells markers; these changes are mediated by miR-200c downregulation. ERAS expression in human tumorigenic mammary cells leads to the generation of larger and less differentiated tumors in xenotransplant experiments. Immunohistochemical, RT-qPCR and bioinformatics analysis of human samples show that ERAS is aberrantly expressed in 8–10% of breast tumors and this expression is associated with distant metastasis and reduced metastasis-free survival. In summary, our results reveal that inappropriate activation of ERAS may be important in the development of a subset of breast tumors. These findings open the possibility of new specific treatments for this subset of ERAS-expressing tumors., This research was supported partially by funds from Fondo Europeo de Desarrollo Regional (FEDER) and by grants from the Spanish Government PI16/00161, PI16/00134, PIE15/00076, PI17/00578, CB/16/00228, CB16/12/00295 and RD12/0036/0009 from Instituto de Salud Carlos III (Ministerio de Economía y Competitividad) and SAF-2015-66015-R from the Ministerio de Economía y Competitividad. Biobanco 1 + 12 is supported by Instituto de Salud Carlos III.

Background In patients with stage III or IV HGSOC who are not suitable for primary surgery (PDS), 3 cycles of platinum based NACT followed by interval surgery (IDS) and adjuvant chemotherapy is an accepted treatment approach. NACT enhances host immune response by increasing levels of PD1, CTLA4 and PDL1 (Bohm S et al. Clin Cancer Res 2016). Gene expression profiling of tumors has identified prognostic signatures for patient selection with immunotherapy (Ribas A et al. J Clin Oncol 2015). Methods The purpose of this study is to assess the effect of neoadjuvant chemotherapy (NACT) on immune activation in stage IIIC/IV tubo-ovarian high-grade serous carcinoma (HGSOC). We obtained pre- and post-treatment omental biopsies from a total of 45 patients undergoing platinum-based NACT followed by IDS. We measured T-cell density and phenotype, immune activation, and markers of cancer-related inflammation using IHC. Messenger RNA expression was analyzed on the nCounter system using the PanCancer IO360 Panel. Results There was evidence of T-cell activation in omental biopsies after NACT. CD4+ T cells showed enhanced IFNgproduction and antitumor Th1 gene signatures were increased. T-cell activation correlated with therapeutic response to NACT. The CD8 T-cell and CD45RO memory cell density in the tumor microenvironment was unchanged after NACT, but biopsies after a good therapeutic response had significantly fewer FoxP3 + (Treg) cells. Biopsies of good therapeutic responders also showed areduction in a Treg gene signature in post versus pre-NACT samples.Preliminary results according to gene expression of the immune compartment reveal that tumor infiltrating lymphocytes were more abundant in samples from patients showing a good response over those with no or incomplete response to NACT, as well as an increase in gene signatures associated with antigen presentation and Cytokine and Chemokine Signaling. When comparing the expression profiles between samples obtained before or after NACT in patients showing incomplete response,we obtained an expected decreased in cellular proliferation signature. Conclusions NACT may promote an immune modulatory effect that could improve or favour the further use of specific immunotherapy in HGSOC patients. Legal entity responsible for the study Fundacion para la Investigacion 12 de Octubre. Funding Tesaro SL. Disclosure L.M. Manso: Research grant / Funding (self): Tesaro; Advisory / Consultancy: Roche, Novartis, Tesaro, AstraZeneca, Pfizer, Clovis; Speaker Bureau / Expert testimony: Roche, Novartis, Tesaro, AstraZeneca, Pfizer, Clovis; Travel / Accommodation / Expenses: Roche, Novartis, Tesaro, Pfizer. S. Wang: Full / Part-time employment: Tesaro. All other authors have declared no conflicts of interest.

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.

Traditionally, bladder cancer has been classified based on histology features. Recently, some works have proposed a molecular classification of invasive bladder tumors. To determine whether proteomics can define molecular subtypes of muscle invasive urothelial cancer (MIUC) and allow evaluating the status of biological processes and its clinical value. 58 MIUC patients who underwent curative surgical resection at our institution between 2006 and 2012 were included. Proteome was evaluated by high-throughput proteomics in routinely archive FFPE tumor tissue. New molecular subgroups were defined. Functional structure and individual proteins prognostic value were evaluated and correlated with clinicopathologic parameters. 1,453 proteins were quantified, leading to two MIUC molecular subgroups. A protein-based functional structure was defined, including several nodes with specific biological activity. The functional structure showed differences between subtypes in metabolism, focal adhesion, RNA and splicing nodes. Focal adhesion node has prognostic value in the whole population. A 6-protein prognostic signature, associated with higher risk of relapse (5 year DFS 70% versus 20%) was defined. Additionally, we identified two MIUC subtypes groups. Prognostic information provided by pathologic characteristics is not enough to understand MIUC behavior. Proteomics analysis may enhance our understanding of prognostic and classification. These findings can lead to improving diagnosis and treatment selection in these patients.

[Abstract] Background: Several potential predictive markers of efficacy of targeted agents in patients with metastatic renal cell carcinoma (mRCC) have been identified. Interindividual heterogeneity warrants further investigation. Patients and methods: Multicenter, observational, retrospective study in patients with clear-cell mRCC treated with sunitinib. Patients were classified in two groups: long-term responders (LR) (progression-free survival (PFS)≥22 months and at least stable disease), and primary refractory (PR) (progressive disease within 3-months of sunitinib onset). Objectives were to compare baseline clinical factors in both populations and to correlate tumor expression of selected signaling pathways components with sunitinib PFS. Results: 123 patients were analyzed (97 LR, 26 PR). In the LR cohort, overall response rate was 79% and median duration of best response was 30 months. Median PFS and overall survival were 43.2 (95% confidence intervals[CI]:37.2-49.3) and 63.5 months (95%CI:55.1-71.9), respectively. At baseline PR patients had a significantly lower proportion of nephrectomies, higher lactate dehydrogenase and platelets levels, lower hemoglobin, shorter time to and higher presence of metastases, and increased Fuhrman grade. Higher levels of HEYL, HEY and HES1 were observed in LR, although only HEYL discriminated populations significantly (AUC[ROC]=0.704; cut-off=34.85). Increased levels of hsa-miR-27b, hsa-miR-23b and hsa-miR-628-5p were also associated with prolonged survival. No statistical significant associations between hsa-miR-23b or hsa-miR-27b and the expression of c-Met were found. Conclusions: Certain mRCC patients treated with sunitinib achieve extremely long-term responses. Favorable baseline hematology values and longer time to metastasis may predict longer PFS. HEYL, hsa-miR-27b, hsa-miR-23b and hsa-miR-628-5p could be potentially used as biomarkers of sunitinib response. PPfizer; SOGUG-2011-05 Ministerio de Economía, Industria y Competitividad; SAF2015-66015-R Ministerio de Economía, Industria y Competitividad; ISCIII-RETIC RD12/0036/0009 Ministerio de Economía, Industria y Competitividad; PIE 15/00076

Purpose: Bladder cancer is a current clinical and social problem. At diagnosis, most patients present with nonmuscle-invasive tumors, characterized by a high recurrence rate, which could progress to muscle-invasive disease and metastasis. Bone morphogenetic protein (BMP)–dependent signaling arising from stromal bladder tissue mediates urothelial homeostasis by promoting urothelial cell differentiation. However, the possible role of BMP ligands in bladder cancer is still unclear. Experimental Design: Tumor and normal tissue from 68 patients with urothelial cancer were prospectively collected and analyzed for expression of BMP and macrophage markers. The mechanism of action was assessed in vitro by experiments with bladder cancer cell lines and peripheral blood monocyte–derived macrophages. Results: We observed BMP4 expression is associated and favored type II macrophage differentiation. In vitro experiments showed that both recombinant BMP4 and BMP4-containing conditioned media from bladder cancer cell lines favored monocyte/macrophage polarization toward M2 phenotype macrophages, as shown by the expression and secretion of IL10. Using a series of human bladder cancer patient samples, we also observed increased expression of BMP4 in advanced and undifferentiated tumors in close correlation with epithelial–mesenchymal transition (EMT). However, the p-Smad 1,5,8 staining in tumors showing EMT signs was reduced, due to the increased miR-21 expression leading to reduced BMPR2 expression. Conclusions: These findings suggest that BMP4 secretion by bladder cancer cells provides the M2 signal necessary for a protumoral immune environment. In addition, the repression of BMPR2 by miR-21 makes the tumor cells refractory to the prodifferentiating actions mediated by BMP ligands, favoring tumor growth. Clin Cancer Res; 23(23); 7388–99. ©2017 AACR.

BackgroundLong non-coding RNAs (lncRNAs) have emerged as key players in a remarkably variety of biological processes and pathologic conditions, including cancer. Next-generation sequencing technologies and bioinformatics procedures predict the existence of tens of thousands of lncRNAs, from which we know the functions of only a handful of them, and very little is known in cancer types such as head and neck squamous cell carcinomas (HNSCCs).ResultsHere, we use RNA-seq expression data from The Cancer Genome Atlas (TCGA) and various statistic and software tools in order to get insight about the lncRNome in HNSCC. Based on lncRNAs expression across 426 samples, we discover five distinct tumor clusters that we compare with reported clusters based on various genomic/genetic features. Results demonstrate significant associations between lncRNA-based clustering and DNA-methylation, TP53 mutation, and human papillomavirus infection. Using “guilt by association” procedures, we infer the possible biological functions of representative lncRNAs of each cluster. Furthermore, we found that lncRNA clustering is correlated with some important clinical and pathologic features, including patient survival after treatment, tumor grade or sub-anatomical location.ConclusionsWe present a landscape of lncRNAs in HNSCC, and provide associations with important genotypic and phenotypic features that may help to understand the disease.

// Cristina Segovia 1, 2, 3, * , Monica Martinez-Fernandez 1, 2, 3, * , Marta Duenas 1, 2, 3, * , Carolina Rubio 2, 3 , Fernando F. Lopez-Calderon 2 , Clotilde Costa 1, 4 , Cristina Saiz-Ladera 1, 5 , Maria Fernandez-Grajera 1 , Jose Duarte 2, 3 , Huberto Garcia Munoz 6 , Federico de la Rosa 2, 3 , Felipe Villacampa 2, 3 , Daniel Castellano 2, 3 , Jesus M. Paramio 1, 2, 3 1 Unidad de Oncologia Molecular, CIEMAT (ed70A), Madrid, Spain 2 Grupo de Oncologia celular y Molecular, Hospital Universitario 12 de Octubre, Madrid, Spain 3 Centro de Investigacion Biomedica en Red de Cancer (CIBER ONC), Spain 4 Unidad Mixta Roche-CHUS, Hospital Universitario de Santiago de Compostela, Travesia de Choupana, s/n, Santiago de Compostela, A Coruna, Spain 5 Departamento de Bioquimica y Biologia Molecular I, Facultad de Biologia, Universidad Complutense, Madrid, Spain 6 Servicio de Anatomia Patologica, Hospital Universitario 12 de Octubre, Instituto de Investigacion 12 de Octubre i+12, UCM, Av Cordoba s/n, Madrid, Spain * These authors have contributed equally to this work Correspondence to: Jesus M. Paramio, email: jesusm.paramio@ciemat.es Keywords: bladder cancer, PIK3CA, genomics, Ezh2, recurrence progression Received: July 20, 2016 Accepted: December 12, 2016 Published: January 02, 2017 ABSTRACT The high rates of tumor recurrence and progression represent a major clinical problem in non-muscle invasive bladder cancer. Previous data showed that EZH2-dependent signaling mediates these processes, whereas the frequent alterations of PIK3CA gene (copy gains and mutations) are predictive of reduced recurrence. Here we show, using clinical samples and bladder cancer cell lines, a functional interaction between EZH2- and PIK3CA-dependent signaling pathways. PIK3CA alterations mediated, on the one hand, the increased expression of two miRNAs, miR-101 and miR-138, which posttranscriptionally downregulate EZH2 expression. On the other hand, PIK3CA alterations facilitate the activation of Akt which phosphorylates EZH2 on Ser21, precluding the trimethylation of histone H3 in K27. Remarkably the increased expression of miR101 or miR138 and the expression of Ser21-phosphorylated EZH2 are good prognostic factors regarding non-muscle invasive bladder cancer recurrence and progression. Collectively, this study provides molecular evidences indicating that the gene expression rewiring occurring in primary bladder tumors, associated with increased EZH2 expression and activity and mediating the increased recurrence and progression risk, are prevented by PIK3CA-dependent signaling. This molecular process may have deep implications in the management of bladder cancer patients and in the design of novel molecularly targeted therapeutic approaches.

Bladder cancer (BC) is the fifth most common cancer in the world, being the non-muscle invasive tumors (NMIBC) the most frequent. NMIBC shows a very high frequency of recurrence and, in certain cases, tumor progression. The phosphatidylinositol 3-kinase (PI3K) pathway, which controls cell growth, tumorigenesis, cell invasion and drug response, is frequently activated in numerous human cancers, including BC, in part through alterations of PIK3CA gene. However, the significance of PIK3CA gene alterations with respect to clinicopathological characteristics, and in particular tumor recurrence and progression, remains elusive. Here, we analyzed the presence of mutations in FGFR3 and PIK3CA genes and copy number alterations of PIK3CA gene in bladder tumor and their correspondent paired normal samples from 87 patients. We observed an extremely high frequency of PIK3CA gene alterations (mutations, copy gains, or both) in tumor samples, affecting primarily T1 and T2 tumors. A significant number of normal tissues also showed mutations and copy gains, being coincident with those found in the corresponding tumor sample. In low-grade tumors PIK3CA mutations associated with FGFR3 mutations. Alterations in PIK3CA gene resulted in increased Akt activity in tumors. Interestingly, the presence of PIK3CA gene alterations, and in particular gene mutations, is significantly associated with reduced recurrence of NMIBC patients. Importantly, the presence of FGFR3 mutations may influence the clinical outcome of patients bearing alterations in PIK3CA gene, and increased recurrence was associated to FGFR3 mutated, PIK3CA wt tumors. These findings may have high relevance in terms of using PI3K-targeted therapies for BC treatment.

// Josefa P. Alameda 1, 2 , Miriam Gaspar 1 , Angel Ramirez 1, 2 , Manuel Navarro 1, 2 , Angustias Page 1, 2 , Cristian Suarez-Cabrera 1, 2 , M. Guadalupe Fernandez 3 , Jose R. Merida 3 , Jesus M. Paramio 1, 2 , Rosa A. Garcia-Fernandez 4 , M. Jesus Fernandez-Acenero 5 , M. Llanos Casanova 1, 2 1 Molecular Oncology Unit, Centro de Investigaciones Energeticas, Medioambientales y Tecnologicas (CIEMAT), 28040 Madrid, Spain 2 Molecular Oncology, Institute of Biomedical Investigation University Hospital “12 de Octubre”, 28041 Madrid, Spain 3 Department of Human Anatomy and Embriology, Facultad de Medicina, UCM, 28040 Madrid, Spain 4 Department of Animal Medicine and Surgery, Facultad de Veterinaria, UCM, 28040 Madrid, Spain 5 Department of Pathology, Hospital Clinico San Carlos, 28040 Madrid, Spain Correspondence to: M. Llanos Casanova, email: llanos.casanova@ciemat.es Keywords: nuclear IKKα, cytoplasmic IKKα, skin cancer, Maspin, c-Myc Received: November 06, 2015 Accepted: March 28, 2016 Published: April 18, 2016 SUMMARY Nonmelanoma skin cancers (NMSC) are the most common human malignancies. IKKα is an essential protein for skin development and is also involved in the genesis and progression of NMSC, through mechanisms not fully understood. While different studies show that IKKα protects against skin cancer, others indicate that it promotes NMSC. To resolve this controversy we have generated two models of transgenic mice expressing the IKKα protein in the nucleus (N-IKKα mice) or the cytoplasm (C-IKKα mice) of keratinocytes. Chemical skin carcinogenesis experiments show that tumors developed by both types of transgenic mice exhibit histological and molecular characteristics that make them more prone to progression and invasion than those developed by Control mice. However, the mechanisms through which IKKα promotes skin tumors are different depending on its subcellular localization; while IKKα of cytoplasmic localization increases EGFR, MMP-9 and VEGF-A activities in tumors, nuclear IKKα causes tumor progression through regulation of c-Myc, Maspin and Integrin-α6 expression. Additionally, we have found that N-IKKα skin tumors mimic the characteristics associated to aggressive human skin tumors with high risk to metastasize. Our results show that IKKα has different non-overlapping roles in the nucleus or cytoplasm of keratinocytes, and provide new targets for intervention in human NMSC progression.

p53 is a protein that causes cell cycle arrest, apoptosis or senescence, being crucial in the process of tumor suppression in several cell types. Different in vitro and animal models have been designed for the study of p53 role in skin cancer. These models have revealed opposing results, as in some experimental settings it appears that p53 protects against skin cancer, but in others, the opposite conclusion emerges. We have generated cohorts of mice with efficient p53 deletion restricted to stratified epithelia and control littermates expressing wild type p53 and studied their sensitivity to both chemically-induced and spontaneous tumoral transformation, as well as the tumor types originated in each experimental group. Our results indicate that the absence of p53 in stratified epithelia leads to the appearance, in two-stage skin carcinogenesis experiments, of a higher number of tumors that grow faster and become malignant more frequently than tumors arisen in mice with wild type p53 genotype. In addition, the histological diversity of the tumor type is greater in mice with epidermal p53 loss, indicating the tumor suppressive role of p53 in different epidermal cell types. Aging mice with p53 inactivation in stratified epithelia developed spontaneous carcinomas in skin and other epithelia. Overall, these results highlight the truly protective nature of p53 functions in the development of cancer in skin and in other stratified epithelia.

Tumor cells arise as a consequence of the acquisition of defined properties, due to gene alterations, which control the equilibrium of cell proliferation and survival. Most targeted therapeutic approaches are focused on the inhibition of those alterations leading to gain of function. This situation is different when these acquired properties are used to loss of function, which usually affect tumor suppressor genes. This accounts for a limited use of these alterations as therapeutic targets. However, this potential problem can be overcome by the use of synthetic lethality approaches (see below).

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.

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.

International audience; NOTCH signaling suppresses tumor growth and proliferation in several types of stratified epithelia. Here, we show that missense mutations in NOTCH1 and NOTCH2 found in human bladder cancers result in loss of function. In murine models, genetic ablation of the NOTCH pathway accelerated bladder tumorigenesis and promoted the formation of squamous cell carcinomas, with areas of mesenchymal features. Using bladder cancer cells, we determined that the NOTCH pathway stabilizes the epithelial phenotype through its effector HES1 and, consequently, loss of NOTCH activity favors the process of epithelial-mesenchymal transition. Evaluation of human bladder cancer samples revealed that tumors with low levels of HES1 present mesenchymal features and are more aggressive. Together, our results indicate that NOTCH serves as a tumor suppressor in the bladder and that loss of this pathway promotes mesenchymal and invasive features.

Background Long non-coding RNAs (lncRNAs) have been claimed as key molecular players in gene expression regulation, being involved in diverse epigenetic processes. They are aberrantly expressed in various tumors, but their exact role in bladder cancer is still obscure. We have recently found a major role of the Polycomb repression complex in recurrence of non-muscle-invasive bladder cancer. Here, we report the xpression of Polycomb-related lncRNAs:antisense noncoding RNA in the INK4 locus (ANRIL) and HOX antisense intergenic RNA (HOTAIR) in these tumors. Findings We studied a dataset of non-invasive bladder cancer samples by quantitative reverse transcription PCR (RT-qPCR) and analyzed also invasive bladder cancer samples using TCGA data. Our results showed that, while ANRIL seemed not to have a determining role, an increased HOTAIR expression appeared in recurrent and high-graded tumors associated with poor prognosis. In addition, through genome-wide transcriptome analyses, we observed that HOTAIR-EZH2-complex-regulated genes can efficiently discriminate between non-tumoral, recurrent, and non-recurrent bladder cancer samples. We also observed a significant correlation between EZH2 and HOTAIR expression levels. Using overexpression, knockdown, and pharmacological approaches in bladder cancer cell lines, we also observed that EZH2 regulates HOTAIR expression. Conclusions Our findings indicate that HOTAIR expression has prognostic value for bladder cancer progression, recurrence, and survival and suggest that HOTAIR plays active roles in modulating the cancer epigenome, becoming an interesting candidate as a target for cancer diagnosis and therapy. The observed HOTAIR regulation by EZH2 and the possibility of modulating EZH2 activity with specific inhibitors open new possible paths to be explored in bladder cancer therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13148-015-0141-x) contains supplementary material, which is available to authorized users.

Bladder Cancer (BC) represents a current clinical and social challenge. The recent studies aimed to describe the genomic landscape of BC have underscored the relevance of epigenetic alterations in the pathogenesis of these tumors. Among the epigenetic alterations, histone modifications occupied a central role not only in cancer, but also in normal organism homeostasis and development. EZH2 (Enhancer of Zeste Homolog 2) belongs to the Polycomb repressive complex 2 as its catalytic subunit, which through the trimethylation of H3 (Histone 3) on K27 (Lysine 27), produces gene silencing. EZH2 is frequently overexpressed in multiple tumor types, including BC, and plays multiple roles besides the well-recognized histone mark generation. In this review, we summarize the present knowledge on the oncogenic roles of EZH2 and its potential use as a therapeutic target, with special emphasis on BC pathogenesis and management.

Bladder cancer (BC) is a highly prevalent disease, ranking fifth in the most common cancers worldwide. Various miRNAs have recently emerged as potential prognostic biomarkers in cancer. The miR-200 family, which repressed the epithelial-to-mesenchymal transition (EMT), is repressed in multiple advanced cancers. However, its expression and function in BC is still poorly understood. Here we show that miR-200 family displays increased expression, probably due to the activation of specific oncogenic signaling pathways, and reduced promoter methylation, in BC compared to normal bladder samples. Furthermore, we show that the expression of these miRNAs is decreased in high grade and stage tumors, and the down-regulation is associated with patient's poor clinical outcome. Our data indicate that the miR-200 family plays distinct roles in Non-Muscle (NMIBC) and Muscle-Invasive BC (MIBC). In MIBC, miR-200 expression post transcriptionally regulates EMT-promoting transcription factors ZEB1 and ZEB2, whereas suppresses BMI1 expression in NMIBC. Interestingly, we show that increased EZH2 and/or BMI1 expression repress the expression of miR-200 family members. Collectively, these findings support a model of BC progression through a coordinated action between the Polycomb Repression Complex (PRC) members repressing the miR-200 expression, which ultimately favors invasive BC development. Since pharmacological inhibition of EZH2 in BC cell lines lead to increased miR-200 expression, our findings may support new therapeutic strategies for BC clinical management.

The pRb pathway is inactivated in most, if not all, human and mouse tumors, including skin tumors.However, a relatively low frequency of Rb gene alterations is found. The embryonic lethality of pRbdeficientanimals restricts the analysis of these mice to midgestation and precludes the analysis of the rolesof pRb in mouse cancer models. To solve this problem, we used the Cre/LoxP technology to induce thetissue-specific deletion of pRb. In epidermis, pRb deletion leads to altered proliferation and differentiationbut these alterations do not induce the development of spontaneous skin tumors. To gain insight in thepossible roles of pRb in mouse skin carcinogenesis, we have performed chemical tumorigenesisexperiments in mice bearing epidermal-specific inactivation of Rb gene. Unexpectedly, these mice developfewer and smaller tumors than control animals, but showing increased malignant conversion to squamouscell carcinomas. Detailed biochemical analysis demonstrates that, in the absence of pRb, multiple pathways...

Previous studies have demonstrated that keratin K10 plays an important role in mediating cell signaling processes, since the ectopic expression of this keratin induces cell cycle arrest in proliferating cells in vitro and in vivo. However, apart from its well known function of providing epithelial cells with resilience to mechanical trauma, little is known about its possible roles in nondividing cells. To investigate what these might be, transgenic mice were generated in which the expression of K10 was driven by bovine K6beta gene control elements (bK6(beta)hK10). The transgenic mice displayed severe abnormalities in the tongue and palate but not in other K6-expressing cells such as those of the esophagus, nails, and hair follicles. The lesions in the tongue and palate included the cytolysis of epithelial suprabasal cells associated with an acute inflammatory response and lymphocyte infiltration. The alterations in the oral mucosa caused the death of transgenic pups soon after birth, probably because suckling was impaired. These anomalies, together with others found in the teeth, are reminiscent of the lesions observed in some patients with pachyonychia congenita, an inherited epithelial fragility associated with mutations in keratins K6 and K16. Although no epithelial fragility was observed in the bK6(beta)hK10 oral epithelia of the experimental mice, necrotic processes were seen. Collectively, these data show that the carefully regulated tissue- and differentiation-specific patterns displayed by the keratin genes have dramatic consequences on the biological behavior of epithelial cells and that changes in the specific composition of the keratin intermediate filament cytoskeleton can affect their physiology, in particular those of the oral mucosa.

Nonmelanoma skin cancers (NMSC) are the most common human malignancies. IKKα is an essential protein for skin development and is also involved in the genesis and progression of NMSC, through mechanisms not fully understood. While different studies show that IKKα protects against skin cancer, others indicate that it promotes NMSC. To resolve this controversy we have generated two models of transgenic mice expressing the IKKα protein in the nucleus (N-IKKα mice) or the cytoplasm (C-IKKα mice) of keratinocytes. Chemical skin carcinogenesis experiments show that tumors developed by both types of transgenic mice exhibit histological and molecular characteristics that make them more prone to progression and invasion than those developed by Control mice. However, the mechanisms through which IKKα promotes skin tumors are different depending on its subcellular localization; while IKKα of cytoplasmic localization increases EGFR, MMP-9 and VEGF-A activities in tumors, nuclear IKKα causes tumor progression through regulation of c-Myc, Maspin and Integrin-α6 expression. Additionally, we have found that N-IKKα skin tumors mimic the characteristics associated to aggressive human skin tumors with high risk to metastasize. Our results show that IKKα has different non-overlapping roles in the nucleus or cytoplasm of keratinocytes, and provide new targets for intervention in human NMSC progression.

Bladder cancer is a highly prevalent human disease in which retinoblastoma (Rb) pathway inactivation and epigenetic alterations are common events. However, the connection between these two processes is still poorly understood. Here, we show that the in vivo inactivation of all Rb family genes in the mouse urothelium is sufficient to initiate bladder cancer development. The characterization of the mouse tumors revealed multiple molecular features of human bladder cancer, including the activation of E2F transcription factor and subsequent Ezh2 expression and the activation of several signaling pathways previously identified as highly relevant in urothelial tumors. These mice represent a genetically defined model for human high-grade superficial bladder cancer. Whole transcriptional characterizations of mouse and human bladder tumors revealed a significant overlap and confirmed the predominant role for Ezh2 in the downregulation of gene expression programs. Importantly, the increased tumor recurrence and progression in human patients with superficial bladder cancer is associated with increased E2F and Ezh2 expression and Ezh2-mediated gene expression repression. Collectively, our studies provide a genetically defined model for human high-grade superficial bladder cancer and demonstrate the existence of an Rb–E2F–Ezh2 axis in bladder whose disruption can promote tumor development. Cancer Res; 74(22); 6565–77. ©2014 AACR.