XPO7 is a tumor suppressor regulating p21CIP1-dependent senescence.

Senescence is a key barrier to neoplastic transformation. To identify senescence regulators relevant to cancer, we screened a genome-wide shRNA library. Here, we describe exportin 7 (XPO7) as a novel regulator of senescence and validate its function in telomere-induced, replicative, and oncogene-induced senescence (OIS). XPO7 is a bidirectional transporter that regulates the nuclear-cytoplasmic shuttling of a broad range of substrates. Depletion of XPO7 results in reduced levels of TCF3 and an impaired induction of the cyclin-dependent kinase inhibitor p21CIP1 during OIS. Deletion of XPO7 correlates with poorer overall survival in several cancer types. Moreover, depletion of XPO7 alleviated OIS and increased tumor formation in a mouse model of liver cancer. Our results suggest that XPO7 is a novel tumor suppressor that regulates p21CIP1 expression to control senescence and tumorigenesis.

Nonviral CRISPR/Cas9 mutagenesis for streamlined generation of mouse lung cancer models.

Functional analysis in mouse models is necessary to establish the involvement of a set of genetic variations in tumor development. A modeling platform to facilitate and cost-effectively analyze the role of multiple genes in carcinogenesis would be valuable. Here, we present an innovative strategy for lung mutagenesis using CRISPR/Cas9 ribonucleoproteins delivered via cationic polymers. This approach allows the simultaneous inactivation of multiple genes. We validate the effectiveness of this system by targeting a group of tumor suppressor genes, specifically Rb1, Rbl1, Pten, and Trp53, which were chosen for their potential to cause lung tumors, namely small cell lung carcinoma (SCLC). Tumors with histologic and transcriptomic features of human SCLC emerged after intratracheal administration of CRISPR/polymer nanoparticles. These tumors carried loss-of-function mutations in all four tumor suppressor genes at the targeted positions. These findings were reproduced in two different pure genetic backgrounds. We provide a proof of principle for simplified modeling of lung tumorigenesis to facilitate functional testing of potential cancer-related genes.

SWI/SNF regulates a transcriptional program that induces senescence to prevent liver cancer.

Oncogene-induced senescence (OIS) is a potent tumor suppressor mechanism. To identify senescence regulators relevant to cancer, we screened an shRNA library targeting genes deleted in hepatocellular carcinoma (HCC). Here, we describe how knockdown of the SWI/SNF component ARID1B prevents OIS and cooperates with RAS to induce liver tumors. ARID1B controls p16INK4a and p21CIP1a transcription but also regulates DNA damage, oxidative stress, and p53 induction, suggesting that SWI/SNF uses additional mechanisms to regulate senescence. To systematically identify SWI/SNF targets regulating senescence, we carried out a focused shRNA screen. We discovered several new senescence regulators, including ENTPD7, an enzyme that hydrolyses nucleotides. ENTPD7 affects oxidative stress, DNA damage, and senescence. Importantly, expression of ENTPD7 or inhibition of nucleotide synthesis in ARID1B-depleted cells results in re-establishment of senescence. Our results identify novel mechanisms by which epigenetic regulators can affect tumor progression and suggest that prosenescence therapies could be employed against SWI/SNF-mutated cancers.

Fanconi anemia-isogenic head and neck cancer cell line pairs: A basic and translational science resource.

Fanconi anemia (FA) is a heritable malformation, bone marrow failure and cancer predisposition syndrome that confers an exceptionally high risk of squamous carcinomas. These carcinomas originate in epithelia lining the mouth, proximal esophagus, vulva and anus: their origins are not understood, and no effective ways have been identified to prevent or delay their appearance. Many FA-associated carcinomas are also therapeutically challenging: they may be multi-focal and stage-advanced at diagnosis, and most individuals with FA cannot tolerate standard-of-care systemic therapies such as DNA cross-linking drugs or ionizing radiation due to constitutional DNA damage hypersensitivity. We developed the Fanconi Anemia Cancer Cell Line Resource (FA-CCLR) to foster new work on the origins, treatment and prevention of FA-associated carcinomas. The FA-CCLR consists of Fanconi-isogenic head and neck squamous cell carcinoma (HNSCC) cell line pairs generated from five individuals with FA-associated HNSCC, and five individuals with sporadic HNSCC. Sporadic, isogenic HNSCC cell line pairs were generated in parallel with FA patient-derived isogenic cell line pairs to provide comparable experimental material to use to identify cell and molecular phenotypes driven by germline or somatic loss of Fanconi pathway function, and the subset of these FA-dependent phenotypes that can be modified, complemented or suppressed. All 10 FANC-isogenic cell line pairs are available to academic, non-profit and industry investigators via the "Fanconi Anemia Research Materials" Resource and Repository at Oregon Health & Sciences University, Portland OR.

Novel Mouse Cell Lines and In Vivo Models for Human High-Grade Neuroendocrine Lung Carcinoma, Small Cell Lung Carcinoma (SCLC), and Large Cell Neuroendocrine Carcinoma (LCNEC).

There is a clear need to expand the toolkit of adequate mouse models and cell lines available for preclinical studies of high-grade neuroendocrine lung carcinoma (small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC)). SCLC and LCNEC are two highly aggressive tumor types with dismal prognoses and few therapeutic options. Currently, there is an extreme paucity of material, particularly in the case of LCNEC. Given the lack of murine cell lines and transplant models of LCNEC, the need is imperative. In this study, we generated and examined new models of LCNEC and SCLC transplantable cell lines derived from our previously developed primary mouse LCNEC and SCLC tumors. RNA-seq analysis demonstrated that our cell lines and syngeneic tumors maintained the transcriptome program from the original transgenic primary tumor and displayed strong similarities to human SCLC or LCNEC. Importantly, the SCLC transplanted cell lines showed the ability to metastasize and mimic this characteristic of the human condition. In summary, we generated mouse cell line tools that allow further basic and translational research as well as preclinical testing of new treatment strategies for SCLC and LCNEC. These tools retain important features of their human counterparts and address the lack of LCNEC disease models.

Huntington's disease-specific mis-splicing unveils key effector genes and altered splicing factors.

Correction of mis-splicing events is a growing therapeutic approach for neurological diseases such as spinal muscular atrophy or neuronal ceroid lipofuscinosis 7, which are caused by splicing-affecting mutations. Mis-spliced effector genes that do not harbour mutations are also good candidate therapeutic targets in diseases with more complex aetiologies such as cancer, autism, muscular dystrophies or neurodegenerative diseases. Next-generation RNA sequencing (RNA-seq) has boosted investigation of global mis-splicing in diseased tissue to identify such key pathogenic mis-spliced genes. Nevertheless, while analysis of tumour or dystrophic muscle biopsies can be informative on early stage pathogenic mis-splicing, for neurodegenerative diseases, these analyses are intrinsically hampered by neuronal loss and neuroinflammation in post-mortem brains. To infer splicing alterations relevant to Huntington's disease pathogenesis, here we performed intersect-RNA-seq analyses of human post-mortem striatal tissue and of an early symptomatic mouse model in which neuronal loss and gliosis are not yet present. Together with a human/mouse parallel motif scan analysis, this approach allowed us to identify the shared mis-splicing signature triggered by the Huntington's disease-causing mutation in both species and to infer upstream deregulated splicing factors. Moreover, we identified a plethora of downstream neurodegeneration-linked mis-spliced effector genes that-together with the deregulated splicing factors-become new possible therapeutic targets. In summary, here we report pathogenic global mis-splicing in Huntington's disease striatum captured by our new intersect-RNA-seq approach that can be readily applied to other neurodegenerative diseases for which bona fide animal models are available.

A new non-aggregative splicing isoform of human Tau is decreased in Alzheimer's disease.

Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with Tau pathology (FTLD-tau), are a group of neurodegenerative disorders characterized by Tau hyperphosphorylation. Post-translational modifications of Tau such as phosphorylation and truncation have been demonstrated to be an essential step in the molecular pathogenesis of these tauopathies. In this work, we demonstrate the existence of a new, human-specific truncated form of Tau generated by intron 12 retention in human neuroblastoma cells and, to a higher extent, in human RNA brain samples, using qPCR and further confirming the results on a larger database of human RNA-seq samples. Diminished protein levels of this new Tau isoform are found by Westernblotting in Alzheimer's patients' brains (Braak I n = 3; Braak II n = 6, Braak III n = 3, Braak IV n = 1, and Braak V n = 10, Braak VI n = 8) with respect to non-demented control subjects (n = 9), suggesting that the lack of this truncated isoform may play an important role in the pathology. This new Tau isoform exhibits similar post-transcriptional modifications by phosphorylation and affinity for microtubule binding, but more interestingly, is less prone to aggregate than other Tau isoforms. Finally, we present evidence suggesting this new Tau isoform could be linked to the inhibition of GSK3ß, which would mediate intron 12 retention by modulating the serine/arginine rich splicing factor 2 (SRSF2). Our results show the existence of an important new isoform of Tau and suggest that further research on this less aggregation-prone Tau may help to develop future therapies for Alzheimer's disease and other tauopathies.

Pathogenic SREK1 decrease in Huntington's disease lowers TAF1 mimicking X-linked dystonia parkinsonism.

Huntington's disease and X-linked dystonia parkinsonism are two monogenic basal ganglia model diseases. Huntington's disease is caused by a polyglutamine-encoding CAG repeat expansion in the Huntingtin (HTT) gene leading to several toxic interactions of both the expanded CAG-containing mRNA and the polyglutamine-containing protein, while X-linked dystonia parkinsonism is caused by a retrotransposon insertion in the TAF1 gene, which decreases expression of this core scaffold of the basal transcription factor complex TFIID. SRSF6 is an RNA-binding protein of the serine and arginine-rich (SR) protein family that interacts with expanded CAG mRNA and is sequestered into the characteristic polyglutamine-containing inclusion bodies of Huntington's disease brains. Here we report decreased levels of the SRSF6 interactor and regulator SREK1-another SR protein involved in RNA processing-which includes TAF1 as one of its targets. This led us to hypothesize that Huntington's disease and X-linked dystonia parkinsonism pathogeneses converge in TAF1 alteration. We show that diminishing SRSF6 through RNA interference in human neuroblastoma cells leads to a decrease in SREK1 levels, which, in turn, suffices to cause diminished TAF1 levels. We also observed decreased SREK1 and TAF1 levels in striatum of Huntington's disease patients and transgenic model mice. We then generated mice with neuronal transgenic expression of SREK1 (TgSREK1 mice) that, interestingly, showed transcriptomic alterations complementary to those in Huntington's disease mice. Most importantly, by combining Huntington's disease and TgSREK1 mice we verify that SREK1 overexpression corrects TAF1 deficiency and attenuates striatal atrophy and motor phenotype of Huntington's disease mice. Our results therefore demonstrate that altered RNA processing upon SREK1 dysregulation plays a key role in Huntington's disease pathogenesis and pinpoint TAF1 as a likely general determinant of selective vulnerability of the striatum in multiple neurological disorders.

G-protein-coupled receptor kinase 2 safeguards epithelial phenotype in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) arises from the mucosal lining of the upper aerodigestive tract and display few treatment options in advanced stages. Despite increased knowledge of HNSCC molecular biology, the identification of new players involved in triggering HNSCC recurrence and metastatic disease is needed. We uncover that G-protein-coupled receptor kinase-2 (GRK2) expression is reduced in undifferentiated, high-grade human HNSCC tumors, whereas its silencing in model human HNSCC cells is sufficient to trigger epithelial-to-mesenchymal transition (EMT) phenotypic features, an EMT-like transcriptional program and enhanced lymph node colonization from orthotopic tongue tumors in mice. Conversely, enhancing GRK2 expression counteracts mesenchymal cells traits by mechanisms involving phosphorylation and decreased functionality of the key EMT inducer Snail1. Our results suggest that GRK2 safeguards the epithelial phenotype, whereas its downregulation contributes to the activation of EMT programs in HNSCC.

Deciphering the complexity of human non-coding promoter-proximal transcriptome.

MOTIVATION: Long non-coding RNAs (lncRNAs) have gained increasing relevance in epigenetic regulation and nuclear functional organization. High-throughput sequencing approaches have revealed frequent non-coding transcription in promoter-proximal regions. However, a comprehensive catalogue of promoter-associated RNAs (paRNAs) and an analysis of the possible interactions with neighboring genes and genomic regulatory elements are missing. RESULTS: Integrating data from multiple cell types and experimental platforms we identified thousands of paRNAs in the human genome. paRNAs are transcribed in both sense and antisense orientation, are mostly non-polyadenylated and retained in the cell nucleus. Transcriptional regulators, epigenetic effectors and activating chromatin marks are enriched in paRNA-positive promoters. Furthermore, paRNA-positive promoters exhibit chromatin signatures of both active promoters and enhancers. Promoters with paRNAs reside preferentially at chromatin loop boundaries, suggesting an involvement in anchor site recognition and chromatin looping. Importantly, these features are independent of the transcriptional state of neighboring genes. Thus, paRNAs may act as cis-regulatory modules with an impact on local recruitment of transcription factors, epigenetic state and chromatin loop organization. This study provides a comprehensive analysis of the promoter-proximal transcriptome and offers novel insights into the roles of paRNAs in epigenetic processes and human diseases. AVAILABILITY AND IMPLEMENTATION: Genomic coordinates of predicted paRNAs are available at https://figshare.com: https://doi.org/10.6084/m9.figshare.7392791.v1 and https://doi.org/10.6084/m9.figshare.4856630.v2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Tumour-infiltrating Gr-1+ myeloid cells antagonize senescence in cancer.

Aberrant activation of oncogenes or loss of tumour suppressor genes opposes malignant transformation by triggering a stable arrest in cell growth, which is termed cellular senescence. This process is finely tuned by both cell-autonomous and non-cell-autonomous mechanisms that regulate the entry of tumour cells to senescence. Whether tumour-infiltrating immune cells can oppose senescence is unknown. Here we show that at the onset of senescence, PTEN null prostate tumours in mice are massively infiltrated by a population of CD11b(+)Gr-1(+) myeloid cells that protect a fraction of proliferating tumour cells from senescence, thus sustaining tumour growth. Mechanistically, we found that Gr-1(+) cells antagonize senescence in a paracrine manner by interfering with the senescence-associated secretory phenotype of the tumour through the secretion of interleukin-1 receptor antagonist (IL-1RA). Strikingly, Pten-loss-induced cellular senescence was enhanced in vivo when Il1ra knockout myeloid cells were adoptively transferred to PTEN null mice. Therapeutically, docetaxel-induced senescence and efficacy were higher in PTEN null tumours when the percentage of tumour-infiltrating CD11b(+)Gr-1(+) myeloid cells was reduced using an antagonist of CXC chemokine receptor 2 (CXCR2). Taken together, our findings identify a novel non-cell-autonomous network, established by innate immunity, that controls senescence evasion and chemoresistance. Targeting this network provides novel opportunities for cancer therapy.

Bosutinib Inhibits EGFR Activation in Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and although new therapeutic approaches have been recently evaluated, overall patient survival is still poor. Thus, new effective and selective clinical treatments are urgently needed. An analysis of data from large-scale, high-throughput drug screening cell line projects identified Bosutinib, a Src/Abl inhibitor that is currently used for the treatment of chronic myelogenous leukemia, as a candidate drug to treat HNSCC. Using a panel of HNSCC-derived cell lines, we found that treatment with Bosutinib reduced cell proliferation and induced apoptosis of sensitive cell lines. The drug rapidly inhibited Src and EGFR (epidermal growth factor receptor) phosphorylation, and sensitivity to Bosutinib was correlated with the activation status of EGFR. Similar findings were observed in in vivo xenograft assays using HNSCC derived cells. Moreover, in the presence of mutations in PIK3CA, the combination of Bosutinib with the PI3Kα inhibitor Alpelisib showed a synergistic effect. These results suggest that Bosutinib could be a new effective drug for the treatment of HNSCC, particularly in tumors with high EGFR activity. Its combination with Alpelisib could especially benefit patients bearing activating mutations of PIK3CA.

The Rho exchange factors Vav2 and Vav3 favor skin tumor initiation and promotion by engaging extracellular signaling loops.

The catalytic activity of GDP/GTP exchange factors (GEFs) is considered critical to maintain the typically high activity of Rho GTPases found in cancer cells. However, the large number of them has made it difficult to pinpoint those playing proactive, nonredundant roles in tumors. In this work, we have investigated whether GEFs of the Vav subfamily exert such specific roles in skin cancer. Using genetically engineered mice, we show here that Vav2 and Vav3 favor cooperatively the initiation and promotion phases of skin tumors. Transcriptomal profiling and signaling experiments indicate such function is linked to the engagement of, and subsequent participation in, keratinocyte-based autocrine/paracrine programs that promote epidermal proliferation and recruitment of pro-inflammatory cells. This is a pathology-restricted mechanism because the loss of Vav proteins does not cause alterations in epidermal homeostasis. These results reveal a previously unknown Rho GEF-dependent pro-tumorigenic mechanism that influences the biology of cancer cells and their microenvironment. They also suggest that anti-Vav therapies may be of potential interest in skin tumor prevention and/or treatment.

PIK3CA gene alterations in bladder cancer are frequent and associate with reduced recurrence in non-muscle invasive tumors.

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.

Akt signaling leads to stem cell activation and promotes tumor development in epidermis.

Hair follicle stem cells (HF-SCs) alternate between periods of quiescence and proliferation, to finally differentiate into all the cell types that constitute the hair follicle. Also, they have been recently identified as cells of origin in skin cancer. HF-SCs localize in a precise region of the hair follicle, the bulge, and molecular markers for this population have been established. Thus, HF-SCs are good model to study the potential role of oncogenic activations on SC physiology. Expression of a permanently active form of Akt (myrAkt) in basal cells leads to Akt hyperactivation specifically in the CD34(+)Itga6(H) population. This activation causes bulge stem cells to exit from quiescence increasing their response to proliferative stimuli and affecting some functions such as cell migration. HF-SC identity upon Akt activation is preserved; in this sense, increased proliferation does not result in stem cell exhaustion with age suggesting that Akt activation does not affect self-renewal an important aspect for normal tissue maintenance and cancer development. Genome-wide transcriptome analysis of HF-SC isolated from myrAkt and wild-type epidermis underscores changes in metabolic pathways characteristic of cancer cells. These differences manifest during a two-step carcinogenesis protocol in which Akt activation in HF-SCs results in increased tumor development and malignant transformation.

Intron retention as a productive mechanism in human MAPT: RNA species generated by retention of intron 3.

BACKGROUND: Tau is a microtubule-binding protein encoded by the MAPT gene. Tau is essential for several physiological functions and associated with pathological processes, including Alzheimer's disease (AD). Six tau isoforms are typically described in the central nervous system, but current research paints a more diverse landscape and a more nuanced balance between isoforms. Recent work has described tau isoforms generated by intron 11 and intron 12 retention. This work adds to that evidence, proving the existence of MAPT transcripts retaining intron 3. Our aim is to demonstrate the existence of mature MAPT RNA species that retain intron 3 in human brain samples and to study its correlation with Alzheimer's disease across different regions. METHODS: Initial evidence of intron-3-retaining MAPT species come from in silico analysis of RNA-seq databases. We further demonstrate the existence of these mature RNA species in a human neuroepithelioma cell line and human brain samples by quantitative PCR. We also use digital droplet PCR to demonstrate the existence of RNA species that retain either intron 3, intron 12 or both introns. FINDINGS: Intron-3-retaining species are even more prominently present that intron-12-retaining ones. We show the presence of MAPT transcripts that retain both introns 3 and 12. These intron-retaining species are diminished in brain samples of patients with Alzheimer's disease with respect to individuals without dementia. Conversely, relative abundance of intron-3- or intron-12-retaining MAPT species with respect to double-retaining species as well as their percentage of expression with respect to total MAPT are increased in patients with Alzheimer's disease, especially in hippocampal samples. Among these TIR-MAPT species, TIR3+12 double truncation allows better classification potential of Alzheimer's disease samples. Moreover, we find a significant increase in intron-3- or intron-12-retaining species and its relative abundance with respect to double-retaining MAPT species in cerebellum in contrast to frontal lateral cortex and hippocampus in individuals with no signs of dementia. INTERPRETATION: Intron retention constitutes a potential mechanism to generate Tau isoforms whose mature RNA expression levels correlate with Alzheimer's pathology showing its potential as a biomarker associated to the disease. FUNDING: This research was funded by the Spanish Ministry of Science, Innovation and Universities: PGC2018-096177-B-I00 (J.A.); Spanish Ministry of Science and Innovation (MCIN): PID2020-113204GB-I00 (F.H.) and PID2021-123859OB-100 from MCIN/AEI/10.13039/501100011033/FEDER, UE (J.A.). It was also supported by CSIC through an intramural grant (201920E104) (J.A.) and the Centre for Networked Biomedical Research on Neurodegenerative Diseases (J.A.). The Centro de Biología Molecular Severo Ochoa (CBMSO) is a Severo Ochoa Center of Excellence (MICIN, award CEX2021-001154-S).

Restored glyoxylate metabolism after AGXT gene correction and direct reprogramming of primary hyperoxaluria type 1 fibroblasts.

Primary hyperoxaluria type 1 (PH1) is a rare inherited metabolic disorder characterized by oxalate overproduction in the liver, resulting in renal damage. It is caused by mutations in the AGXT gene. Combined liver and kidney transplantation is currently the only permanent curative treatment. We combined locus-specific gene correction and hepatic direct cell reprogramming to generate autologous healthy induced hepatocytes (iHeps) from PH1 patient-derived fibroblasts. First, site-specific AGXT corrected cells were obtained by homology directed repair (HDR) assisted by CRISPR-Cas9, following two different strategies: accurate point mutation (c.731T>C) correction or knockin of an enhanced version of AGXT cDNA. Then, iHeps were generated, by overexpression of hepatic transcription factors. Generated AGXT-corrected iHeps showed hepatic gene expression profile and exhibited in vitro reversion of oxalate accumulation compared to non-edited PH1-derived iHeps. This strategy set up a potential alternative cellular source for liver cell replacement therapy and a personalized PH1 in vitro disease model.

Protein kinase C (PKC)ζ-mediated Gαq stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts.

Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gα(q)-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gα(q) acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gα(q)/PKCζ/ERK5 signaling axis.

Keratinocyte cell lines derived from severe generalized recessive epidermolysis bullosa patients carrying a highly recurrent COL7A1 homozygous mutation: models to assess cell and gene therapies in vitro and in vivo.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by deficiency of type VII collagen due to COL7A1 mutations such as c.6527insC, recurrently found in the Spanish RDEB population. Assessment of clonal correction-based therapeutic approaches for RDEB requires large expansions of cells, exceeding the replication capacity of human primary keratinocytes. Thus, immortalized RDEB cells with enhanced proliferative abilities would be valuable. Using either the SV40 large T antigen or papillomavirus HPV16-derived E6-E7 proteins, we immortalized and cloned RDEB keratinocytes carrying the c.6527insC mutation. Clones exhibited high proliferative and colony-forming features. Cytogenetic analysis revealed important differences between T antigen-driven and E6-E7-driven immortalization. Immortalized cells responded to differentiation stimuli and were competent for epidermal regeneration and recapitulation of the blistering RDEB phenotype in vivo. These features make these cell lines useful to test novel therapeutic approaches including those aimed at editing mutant COL7A1.

Early Diagnosis of Oral Cancer and Lesions in Fanconi Anemia Patients: A Prospective and Longitudinal Study Using Saliva and Plasma.

Fanconi anemia (FA) patients display an exacerbated risk of oral squamous cell carcinoma (OSCC) and oral potentially malignant lesions (OPMLs) at early ages. As patients have defects in their DNA repair mechanisms, standard-of-care treatments for OSCC such as radiotherapy and chemotherapy, give rise to severe toxicities. New methods for early diagnosis are urgently needed to allow for treatment in early disease stages and achieve better clinical outcomes. We conducted a prospective, longitudinal study wherein liquid biopsies from sixteen patients with no clinical diagnoses of OPML and/or OSCC were analyzed for the presence of mutations in cancer genes. The DNA from saliva and plasma were sequentially collected and deep-sequenced, and the clinical evaluation followed over a median time of approximately 2 years. In 9/16 FA patients, we detected mutations in cancer genes (mainly TP53) with minor allele frequencies (MAF) of down to 0.07%. Importantly, all patients that had mutations and clinical follow-up data after mutation detection (n = 6) developed oral precursor lesions or OSCC. The lead-time between mutation detection and tumor diagnosis ranged from 23 to 630 days. Strikingly, FA patients without mutations displayed a significantly lower risk of developing precursor lesions or OSCCs. Therefore, our diagnostic approach could help to stratify FA patients into risk groups, which would allow for closer surveillance for OSCCs or precursor lesions.