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Supp Table S12. Gene Ontology of Biological processes of the upregulated genes in human recurrent bladder tumors

Supplementary Information. Supplementary Methods Supplementary Figures 1-6. Supp Fig. 1: Identification of bladder tumors by Computerized tomography (CT). Supp Fig 2: Determination of effective elimination of pRb and p130 in bladder tumors. Supp Fig 3: Expression of E2F1, E2F2 and E2F3a genes in mouse bladder tumors. Supp Fig 4: Polycomb-bound genes associate with early recurrence in external datasets of bladder cancer. Supp Fig. 5: Expression of SUZ12 and EED genes in human NMIBC. Supp Fig 6: Expression of E2F1, 2 and 3b genes in human NMIBC. Supplementary Tables 1, 7, 8, 9, 13 and 14. Supp Table 1: Sequence of the Oligonucleotides used in PCR analysis. Supp Table 7: Gene Set Enrichment Analysis of the deregulated genes in mouse bladder tumors. Supp Table 8: Overlap between the downregulated genes in mouse bladder tumors and human bladder cancer datasets from Oncomine database. Supp Table 9: Overlap between the upregulated genes in mouse bladder tumors and human bladder dataset from Oncomine database. Supp Table 13: Gene Set Enrichment Analysis of the deregulated genes in human recurrent bladder tumors. Supp Table 14: Overlap between the upregulated genes in our series of human recurrent bladder tumors and human bladder cancer datasets from Oncomine database. Supplementary References

[Purpose]: Integrins, transmembrane receptors that mediate cell-extracellular matrix and cell-cell interactions, have been linked to several cancer-associated features. A less explored function of integrins in cancer is their role in leukocyte homing and activation. Understanding their relationship with immune cell infiltrates and immune checkpoints is an area of interest in cancer research. [Methods]: The expression of 33 different integrins was evaluated in relation with breast cancer patient outcome using transcriptomic data (Affymetrix dataset, exploratory cohort) and the METABRIC study (validation cohort). The TIMER online tool was used to assess the association of the identified integrin genes with immune cell infiltration, and the TCGA and METABRIC studies to assess correlations between integrin gene expression and genomic signatures of immune activation. [Results]: We identified 7 genes coding for integrin α and β subunits, i.e., ITGA4, ITGB2, ITGAX, ITGB7, ITGAM, ITGAL and ITGA8, which predict a favorable prognosis in Basal-like and HER2+ breast cancers. Their expression positively correlated with the presence of immune cell infiltrates within the tumor (dendritic cells, CD4+ T-cells, neutrophils, CD8+ T-cells and B-cells), with markers of T-cell activation and antigen presentation, and with gene signatures of immune surveillance (cytotoxic T lymphocyte activation and IFN gamma signature). By contrast, we found that genes coding for integrins that predicted a detrimental outcome (IBSP, ITGB3BP, ITGB6, ITGB1 and ITGAV) were not associated with any of these parameters. [Conclusions]: We identified an integrin signature composed of 7 genes with potential to recognize immune infiltrated and activated Basal-like and HER2+ breast cancers with a favorable prognosis., This work was supported by the Instituto de Salud Carlos III (PI19/00808), ACEPAIN, Diputación de Albacete, CIBERONC and CRIS Cancer Foundation (to A. Ocaña) and the Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the scientific foundation of the AECC and the CRIS Foundation (to A. Pandiella). Our laboratories receive support from the European Community through the regional development funding program (FEDER). BG was supported by a 2018 − 2.1.17-TET-KR-00001 grant and by the Higher Education Institutional Excellence Programme (2020 − 4.1.1.-TKP2020) of the Ministry for Innovation and Technology of Hungary, within the framework of the Bionic thematic programme of the Semmelweis University.

Glioblastoma (GBM) is one of the most aggressive forms of cancer. It has been proposed that the presence within these tumors of a population of cells with stem-like features termed Glioma Initiating Cells (GICs) is responsible for the relapses that take place in the patients with this disease. Targeting this cell population is therefore an issue of great therapeutic interest in neuro-oncology. We had previously found that the neurotrophic factor MIDKINE (MDK) promotes resistance to glioma cell death. The main objective of this work is therefore investigating the role of MDK in the regulation of GICs. Methods: Assays of gene and protein expression, self-renewal capacity, autophagy and apoptosis in cultures of GICs derived from GBM samples subjected to different treatments. Analysis of the growth of GICs-derived xenografts generated in mice upon blockade of the MDK and its receptor the ALK receptor tyrosine kinase (ALK) upon exposure to different treatments. Results: Genetic or pharmacological inhibition of MDK or ALK decreases the self-renewal and tumorigenic capacity of GICs via the autophagic degradation of the transcription factor SOX9. Blockade of the MDK/ALK axis in combination with temozolomide depletes the population of GICs in vitro and has a potent anticancer activity in xenografts derived from GICs. Conclusions: The MDK/ALK axis regulates the self-renewal capacity of GICs by controlling the autophagic degradation of the transcription factor SOX9. Inhibition of the MDK/ALK axis may be a therapeutic strategy to target GICs in GBM patients. This work has been funded by the PI18/00442 grant integrated into the State Plan for R & D + I 2017-2020 and funded by the Instituto de Salud Carlos III (ISCIII) and confounded by the European Regional Development Fund (ERDF), “A way to make Europe” and by grants from ISCIII and ERDF, a way to make Europe (PS09/01401; PI12/02248 and PI15/00339 to GV, PI16/01580 to AM, PI16/01278 to JMS) by Ministerio de Economía y Competitividad (grant SAF2015-65175-R/ERDF to PSG), by Fundación Mutua Madrileña (AP101042012 to GV), “Fundació La Marató de TV3” (20134031 to GV), Voices Against Brain Cancer (US) (to GV), and donations by The Medical Cannabis Bike Tour Foundation (The Netherlands to GV) and Jeff Ditchfield (to GV). G Velasco's group is part of the COST Action CA15138 (Transautophagy). Israel López-Valero was supported by a predoctoral P-FIS contract from ISCIII, Cristina Sáiz was supported by a “Juan de la Cierva formación” contract of the Spanish Ministry of Economy and Competitiveness, Ander Matheu was recipient of a Miguel Servet contract (CP16/00039) from ISCIII. Work in JM Sepulveda group is supported by a grant from “Asociación Española contra el Cancer” (AECC) (GCTRA16015SEDA). Part of the work at G Velasco laboratory was funded by LYRAMID and Pfizer. Sí

Epidermal Growth Factor receptor (EGFR) is a tyrosine kinase receptor widely expressed on the surface of numerous cell types, which activates several downstream signalling pathways involved in cell proliferation, migration and survival. EGFR alterations, such as overexpression or mutations, have been frequently observed in several cancers, including glioblastoma (GBM), and are associated to uncontrolled cell proliferation. Here we show that the inhibition of mammalian target of Rapamycin (mTOR) mediates EGFR delivery to lysosomes for degradation in GBM cells, independently of autophagy activation. Coherently with EGFR internalisation and degradation, mTOR blockade negatively affects the mitogen activated protein/extracellular signal-regulated kinase (MAPK)/ERK pathway. Furthermore, we provide evidence that Src kinase activation is required for EGFR internaliation upon mTOR inhibition. Our results further support the hypothesis that mTOR targeting may represent an effective therapeutic strategy in GBM management, as its inhibition results in EGFR degradation and in proliferative signal alteration.

Glioblastoma multiforme (GBM) is the most frequent and aggressive type of brain tumor due, at least in part, to its poor response to current anticancer treatments. These features could be explained, at least partially, by the presence within the tumor mass of a small population of cells termed Glioma Initiating Cells (GICs) that has been proposed to be responsible for the relapses occurring in this disease. Thus, the development of novel therapeutic approaches (and specifically those targeting the population of GICs) is urgently needed to improve the survival of the patients suffering this devastating disease. Previous observations by our group and others have shown that Δ9-Tetrahydrocannabinol (THC, the main active ingredient of marijuana) and other cannabinoids including cannabidiol (CBD) exert antitumoral actions in several animal models of cancer, including gliomas. We also found that the administration of THC (or of THC + CBD at a 1:1 ratio) in combination with temozolomide (TMZ), the benchmark agent for the treatment of GBM, synergistically reduces the growth of glioma xenografts. In this work we investigated the effect of the combination of TMZ and THC:CBD mixtures containing different ratios of the two cannabinoids in preclinical glioma models, including those derived from GICs. Our findings show that TMZ + THC:CBD combinations containing a higher proportion of CDB (but not TMZ + CBD alone) produce a similar antitumoral effect as the administration of TMZ together with THC and CBD at a 1:1 ratio in xenografts generated with glioma cell lines. In addition, we also found that the administration of TMZ + THC:CBD at a 1:1 ratio reduced the growth of orthotopic xenografts generated with GICs derived from GBM patients and enhanced the survival of the animals bearing these intracranial xenografts. Remarkably, the antitumoral effect observed in GICs-derived xenografts was stronger when TMZ was administered together with cannabinoid combinations containing a higher proportion of CBD. These findings support the notion that the administration of TMZ together with THC:CBD combinations - and specifically those containing a higher proportion of CBD - may be therapeutically explored to target the population of GICs in GBM. This work has been funded by the PI15/00339 grant, integrated into the State Plan for R & D + I2013-2016 and funded by the Instituto de Salud Carlos III (ISCIII) (Spain) and the European Regional Development Fund (ERDF) and by grants from Spanish Ministry of Economy and Competitiveness (MINECO)/ISCIII and ERDF (PS09/01401; PI12/02248,to GV), GW Pharma Ltd. (UK), Comunidad de Madrid (Spain) (S2011/BMD-2308 to MG), Fundación Mutua Madrileña (Spain) (AP101042012 to GV), Fundació La Marató de TV3 (Spain) (201334031 to GV), Voices Against Brain Cancer (US), and donations by The Medical Cannabis Bike Tour Foundation (The Netherlands) and Jeff Ditchfield. Israel López-Valero was supported by a predoctoral P-FIS contract from Instituto de Salud Carlos III (ISCIII) and Cristina Sáiz was supported by a “Juan de la Cierva formación” contract of the Spanish Ministry of Economy and Competitiveness. Sí

// 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.

Decreased thyroidal hormone production is found during lipopolysaccharide (LPS)-induced endotoxic shock in animals as well as in critically ill patients. Here we studied the role of the thyroid hormone receptors (TRs) in activation of STAT3, NF-ΰ B and ERK, which play a key role in the response to inflammatory cytokines during sepsis. TR knockout mice showed down-regulation of hepatic inflammatory mediators, including interleukin 6 (IL-6) in response to LPS. Paradoxically, STAT3 and ERK activity were higher, suggesting that TRs could act as endogenous repressors of these pathways. Furthermore, hyperthyroidism increased cytokine production and mortality in response to LPS, despite decreasing hepatic STAT3 and ERK activity. This suggested that TRs could directly repress the response of the cells to inflammatory mediators. Indeed, we found that the thyroid hormone T3 suppresses IL-6 signalling in macrophages and hepatocarcinoma cells, inhibiting STAT3 activation. Consequently, the hormone strongly antagonizes IL-6-stimulated gene transcription, reducing STAT3 recruitment and histone acetylation at IL-6 target promoters. In conclusion, TRs are potent regulators of inflammatory responses and immune homeostasis during sepsis. Reduced responses to IL-6 should serve as a negative feedback mechanism for preventing deleterious effects of excessive hormone signaling during infections., This work was supported by Grants: BFU2011-28058, BFU2014-53610P and SAF2015-71878-REDT from Ministerio de Economía y Competitividad; S2011/BMD-2328 from the Comunidad de Madrid and RD12/0036/0030 from the Instituto de Salud Carlos III

Cyclin-dependent kinase (Cdk)7, the catalytic subunit of the Cdk-activating kinase (CAK) complex has been implicated in the control of cell cycle progression and of RNA polymerase II (RNA pol II)-mediated transcription. Genetic inactivation of the Cdk7 locus revealed that whereas Cdk7 is completely dispensable for global transcription, is essential for the cell cycle via phosphorylation of Cdk1 and Cdk2. In vivo, Cdk7 is also indispensable for cell proliferation except during the initial stages of embryonic development. Interestingly, widespread elimination of Cdk7 in adult tissues with low proliferative indexes had no phenotypic consequences. However, ablation of conditional Cdk7 alleles in tissues with elevated cellular turnover led to the efficient repopulation of these tissues with Cdk7-expressing cells most likely derived from adult stem cells that may have escaped the inactivation of their targeted Cdk7 alleles. This process, a physiological attempt to maintain tissue homeostasis, led to the attrition of adult stem cell pools and to the appearance of age-related phenotypes, including telomere shortening and early death.

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.

The retinoblastoma gene product (pRb) controls proliferation and differentiation processes in stratified epithelia. Importantly, and in contrast to other tissues, Rb deficiency does not lead to spontaneous skin tumor formation. As the cyclin-dependent kinase inhibitor p21 regulates proliferation and differentiation in the absence of pRb, we analyzed the consequences of deleting p21 in pRb-ablated stratified epithelia (hereafter pRb(ΔEpi);p21-/-). These mice display an enhancement of the phenotypic abnormalities observed in pRb(ΔEpi) animals, indicating that p21 partially compensates pRb absence. Remarkably, pRb(ΔEpi);p21-/- mice show an acute skin inflammatory phenotype and develop spontaneous epithelial tumors, particularly affecting tongue and oral tissues. Biochemical analyses and transcriptome studies reveal changes affecting multiple pathways, including DNA damage and p53-dependent signaling responses. Comparative metagenomic analyses, together with the histopathological profiles, indicate that these mice constitute a faithful model for human head and neck squamous cell carcinomas. Collectively, our findings demonstrate that p21, in conjunction with pRb, has a central role in regulating multiple epithelial processes and orchestrating specific tumor suppressor functions.

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.

Background The epidermal specific ablation of Trp53 gene leads to the spontaneous development of aggressive tumors in mice through a process that is accelerated by the simultaneous ablation of Rb gene. Since alterations of p53-dependent pathway are common hallmarks of aggressive, poor prognostic human cancers, these mouse models can recapitulate the molecular features of some of these human malignancies. Results To evaluate this possibility, gene expression microarray analysis was performed in mouse samples. The mouse tumors display increased expression of cell cycle and chromosomal instability associated genes. Remarkably, they are also enriched in human embryonic stem cell gene signatures, a characteristic feature of human aggressive tumors. Using cross-species comparison and meta-analytical approaches, we also observed that spontaneous mouse tumors display robust similarities with gene expression profiles of human tumors bearing mutated TP53, or displaying poor prognostic outcome, from multiple body tissues. We have obtained a 20-gene signature whose genes are overexpressed in mouse tumors and can identify human tumors with poor outcome from breast cancer, astrocytoma and multiple myeloma. This signature was consistently overexpressed in additional mouse tumors using microarray analysis. Two of the genes of this signature, AURKA and UBE2C, were validated in human breast and cervical cancer as potential biomarkers of malignancy. Conclusions Our analyses demonstrate that these mouse models are promising preclinical tools aimed to search for malignancy biomarkers and to test targeted therapies of prospective use in human aggressive tumors and/or with p53 mutation or inactivation.

Continuous cell renewal in mouse epidermis is at the expense of a pool of pluripotent cells that lie in a well defined niche in the hair follicle known as the bulge. To identify mechanisms controlling hair follicle stem cell homeostasis, we developed a strategy to isolate adult bulge stem cells in mice and to define their transcriptional profile. We observed that a large number of transcripts are underexpressed in hair follicle stem cells when compared to non-stem cells. Importantly, the majority of these downregulated genes are involved in cell cycle. Using bioinformatics tools, we identified the E2F transcription factor family as a potential element involved in the regulation of these transcripts. To determine their functional role, we used engineered mice lacking Rb gene in epidermis, which showed increased expression of most E2F family members and increased E2F transcriptional activity. Experiments designed to analyze epidermal stem cell functionality (i.e.: hair regrowth and wound healing) imply a role of the Rb-E2F axis in the control of stem cell quiescence in epidermis. Electronic supplementary material The online version of this article (doi:10.1007/s12015-010-9139-0) contains supplementary material, which is available to authorized users.

// Rafal Zur 1 , Laura Garcia-Ibanez 1 , Angel Nunez-Buiza 1 , Noelia Aparicio 1 , Georgios Liappas 2 , Alejandra Escos 1 , Ana Risco 1 , Angustias Page 3 , Cristina Saiz-Ladera 3 , Dayanira Alsina-Beauchamp 1 , Jose Montans 4 , Jesus M. Paramio 3 and Ana Cuenda 1 1 Department of Immunology and Oncology, Centro Nacional de Biotecnologia/CSIC, Madrid, Spain 2 Molecular Biology Centre Severo Ochoa/CSIC-UAM, Madrid, Spain 3 Molecular Oncology Unit, CIEMAT and I+12 Biomedical Research Institute, University Hospital 12 de Octubre, Madrid, Spain 4 Centro Anatomopatologico, Camino de Vinateros, Madrid, Spain Correspondence to: Ana Cuenda, email: // Keywords : p38γ, p38δ, skin, inflammation-associated cancer, knockout mice Received : April 13, 2015 Accepted : May 25, 2015 Published : May 28, 2015 Abstract The contribution of chronic skin inflammation to the development of squamous cell carcinoma (SCC) is poorly understood. While the mitogen-activated protein kinase p38α regulates inflammatory responses and tumour development, little is known about the role of p38γ and p38δ in these processes. Here we show that combined p38γ and p38δ (p38γ/δ) deletion blocked skin tumour development in a chemically induced carcinogenesis model. p38γ/δ deletion reduced TPA-induced epidermal hyperproliferation and inflammation; it inhibited expression of proinflammatory cytokines and chemokines in keratinocytes in vitro and in whole skin in vivo , resulting in decreased neutrophil recruitment to skin. Our data indicate that p38γ/δ in keratinocytes promote carcinogenesis by enabling formation of a proinflammatory microenvironment that fosters epidermal hyperproliferation and tumourigenesis. These findings provide genetic evidence that p38γ and p38δ have essential roles in skin tumour development, and suggest that targeting inflammation through p38γ/δ offers a therapeutic strategy for SCC treatment and prevention.

e18521 Background: The role of liquid biopsy in diagnosis and therapy monitoring in patients with head and neck cancer has been much less studied compared to other cancers. Our aim was to evaluate the perfomance in the isolation and recovery for molecular characterization of circulating tumour cells (CTC) of a new immunoafinity-based method and to compare it with the molecular diagnostic yield of plasma cell-free DNA. Methods: Patients with recurrent/metastatic (RM) head and neck cancer (HNC) were enrolled prospectively. Forty mililiters (ml) of plasma were collected at one or several time-points. First blood draw was always collected before starting a new therapeutic intervention or at the time of radiologic progression. For CTC detection and isolation, either anti-EpCAM or both anti-EpCAM + anti-EGFR antibodies were used. Digital PCR and castPCR were used to study KRAS and PI3KCA mutations in non-squamous HNC. A 15-gene customized NGS panel was used to characterized both CTC and cfDNA in patients with squamous HNC. Results: Between February 2016 and October 2018, 14 patients with R/M HNC were included (n = 1 local-only disease, n = 10 local and distant disease, n = 3 distant-only disease). Squamous histology (S): n = 9. Non-squamous (NS): n = 5 (1 naso-ethmoidal intestinal-type adenocarcinoma, 1 parotid gland exadenoma pleomorfic carcinoma, 2 parotid-gland salivary duct carcinomas (SDC), 1 parotid-gland high-grade neuroendocrine carcinoma). Twenty-five CTC determinations were performed. In 5 patients serial CTC determinations were performed. Median CTC was 4 (min-max: 0-49). Median CTC among 11 CTC determinations in S-HNC was 4 (min-max: 0-49). Median CTC was 3 CTC (min-max: 0-26) among the 14 determinations performed in NS-HNC. Digital PCR unveiled mutations in CTC and in cfDNA in 2 of 4 patients tested with NS histology (KRAS, PIK3CA), with one of them being concordant for the specific mutation. NGS unveiled mutations in CTC in 7/9 patients and in cfDNA in 6/9 patients, with only one loci-concordant case between CTC and plasma. Conclusions: IsoFlux detected CTC in the majority of patients with R/M HNC, regardless of the histologic type, and allowed for molecular characterization of CTC using different techniques for mutational analysis. Both NGS and digital PCR allowed for the detection in cell-free DNA of commonly mutated genes in HNC. Liquid biopsy should be more actively studied in this disease in order to better define its role in diagnosis and therapeutic monitoring.

Missense mutations in TP53 gene promote metastasis in human tumours. However, little is known about the complete loss of function of p53 in tumour metastasis. Here we show that squamous cell carcinomas generated by the specific ablation of Trp53 gene in mouse epidermis are highly metastatic. Biochemical and genome-wide mRNA and miRNA analyses demonstrated that metastases are associated with the early induction of epithelial-mesenchymal transition (EMT) and deregulated miRNA expression in primary tumours. Increased expression of miR-21 was observed in undifferentiated, prometastatic mouse tumours and in human tumours characterized by p53 mutations and distant metastasis. The augmented expression of miR-21, mediated by active mTOR and Stat3 signalling, conferred increased invasive properties to mouse keratinocytes in vitro and in vivo, whereas blockade of miR-21 in a metastatic spindle cell line inhibits metastasis development. Collectively these data identify novel molecular mechanisms leading to metastasis in vivo originated by p53 loss in epithelia.