BackgroundhTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research, being used as therapeutically pertinent models of various neuromuscular disorders and in numerous fundamental studies of muscle cell function. However, the cell cycle is linked to other cellular processes such as integrin regulation, the PI3K/Akt pathway, and microtubule stability, raising the question as to whether genetic modification related to the cell cycle results in secondary effects that could undermine the validity of these cell models.ResultsHere we subjected five healthy and disease muscle cell isolates to transcriptomic analysis, comparing immortalized lines with their parent primary populations in both differentiated and undifferentiated states, and testing their myogenic character by comparison with non-myogenic (CD56-negative) cells. Principal component analysis of global gene expression showed tight clustering of immortalized myoblasts to their parent primary populations, with clean separation from the non-myogenic reference. Comparison was made to publicly available transcriptomic data from studies of muscle human pathology, cell, and animal models, including to derive a consensus set of genes previously shown to have altered regulation during myoblast differentiation. Hierarchical clustering of samples based on gene expression of this consensus set showed that immortalized lines retained the myogenic expression patterns of their parent primary populations. Of 2784 canonical pathways and gene ontology terms tested by gene set enrichment analysis, none were significantly enriched in immortalized compared to primary cell populations. We observed, at the whole transcriptome level, a strong signature of cell cycle shutdown associated with senescence in one primary myoblast population, whereas its immortalized clone was protected.ConclusionsImmortalization had no observed effect on the myogenic cascade or on any other cellular processes, and it was protective against the systems level effects of senescence that are observed at higher division counts of primary cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-016-0115-5) contains supplementary material, which is available to authorized users.

TRANSLATIONAL RELEVANCEHead and neck squamous cell carcinoma (HNSCC) is typically characterized by mutation of TP53 gene, associated to therapy resistance and high incidence of local recurrences. However, drugs specifically targeting mutant p53 proteins, frequently presenting gain-of-function activity associated with radioresistance, are not available. We then set out to identify mutant p53-associated functions that might be targeted with drugs currently used in HNSCC trials. This study identifies MYC as a crucial mediator of mutant p53 activity in HNSCC and PI3K inhibitors as compounds able to impinge on mutant p53-MYC dependent gene expression. Of note, down-regulation of mutant p53-MYC dependent genes is associated with response to PI3Kα-selective inhibitor Alpesilib (BYL719) in HNSCC.Research.

Head and neck squamous cell carcinoma is typically characterized by a high incidence of local recurrences. It has been extensively shown that mucosa from head and neck squamous cell carcinoma patients carries both genetic and gene expression alterations, which are mostly attributable to major etiologic agents of head and neck squamous cell carcinoma. We previously identified a signature of microRNAs (miRNAs) whose high expression in tumors is predictive of recurrence. Here, we investigated whether the deregulation of miRNA expression in the tumor-surrounding mucosa is correlated to disease recurrence. Specifically, comparing the miRNA expression in matched tumoral, peritumoral, and normal tissues collected from head and neck squamous cell carcinoma patients, we identified 35 miRNAs that are deregulated in both tumoral and peritumoral tissues as compared with normal matched samples. Four of these composed a miRNA signature that predicts head and neck squamous cell carcinoma local recurrence independently from prognostic clinical variables. The predictive power of the miRNA signature increased when using the expression levels derived from both the peritumoral and the tumoral tissues. The expression signal of the miRNAs composing the predictive signature correlated with the transcriptional levels of genes mostly associated with proliferation. Our results show that expression of miRNAs in tumor-surrounding mucosa may strongly contribute to the identification of head and neck squamous cell carcinoma patients at high risk of local recurrence.

Study approval All the experimental procedures were performed under the ethical approval of the Italian Ministry of Health and the Institutional Animal Care and Use Committee (authorization no. 83/2019-PR). The animals were maintained in an authorized facility at San Raffaele Hospital, Milan (authorization no. 127/2012-A) and Santa Lucia Foundation, Rome (authorization no. 9/2006-A).

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS.