A genetic screen identifies novel regulators of the senescence-associated secretory phenotype

Cellular senescence is a stable cell-cycle arrest induced by ageing or many stresses such as oncogene activation. Oncogene-Induced Senescence (OIS) is an intrinsic tumour suppressive mechanism but also has numerous extrinsic functions mediated by the senescence-associated secretory phenotype (SASP). Although the SASP attracts the immune system to clear damaged cells it can also promote cancer growth and age-associated systemic inflammation. Consequently, the SASP has been considered a potential therapeutic target. However, a better understanding of the molecular pathways regulating the complex secretome of senescent cells is necessary before designing therapeutic interventions. In this work, we set up and carried out an siRNA screen for genes regulating the SASP during OIS. By screening siRNAs targeting 8,352 genes, 84 novel regulators of IL-8 and IL-6 were found. We further interrogated their effect on levels of p16INK4a, p21Cip1 and BrdU incorporation. 49 genes were found to specifically regulate the SASP without affecting other senescence features. Combining the siRNA screening system with global transcriptome profiling for 38 of these genes revealed common pathways with a potential role in regulating the SASP. One of the candidates was the splicing factor PTBP1. PTBP1 depletion blunted the induction of several SASP factors without affecting the senescence growth arrest. The altered SASP produced upon PTBP1 depletion had distinct properties. For example, it decreased the ability to transmit senescence but retained the ability to attract natural killer cells. Mechanistically, during senescence, PTBP1 promoted exon skipping of several mRNAs involved in intracellular protein trafficking. Future work will aim at fully understanding the role of PTBP1 during senescence. Our data so far suggest the feasibility of targeting PTBP1 to specifically manipulate the SASP while maintaining the ability of inducing cell cycle arrest as a potential strategy to target inflammation-driven tumourigenesis.Open Acces

Novel regulation of PLCζ activity via its XY-linker

The XY-linker region of somatic cell PLC (phospholipase)-β, -γ, -δ and -ϵ isoforms confers potent catalytic inhibition, suggesting a common auto-regulatory role. Surprisingly, the sperm PLCζ XY-linker does not mediate auto-inhibition. Unlike for somatic PLCs, the absence of the PLCζ XY-linker significantly diminishes both in vitro PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis and in vivo Ca2+-oscillation-inducing activity, revealing evidence for a novel PLCζ enzymatic mechanism

Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence

Expression of the transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) reprograms somatic cells into induced pluripotent stem cells (iPSCs). Reprogramming is a slow and inefficient process, suggesting the presence of safeguarding mechanisms that counteract cell fate conversion. One such mechanism is senescence. To identify modulators of reprogramming-induced senescence, we performed a genome-wide shRNA screen in primary human fibroblasts expressing OSKM. In the screen, we identified novel mediators of OSKM-induced senescence and validated previously implicated genes such as CDKN1A. We developed an innovative approach that integrates single-cell RNA sequencing (scRNA-seq) with the shRNA screen to investigate the mechanism of action of the identified candidates. Our data unveiled regulation of senescence as a novel way by which mechanistic target of rapamycin (mTOR) influences reprogramming. On one hand, mTOR inhibition blunts the induction of cyclin-dependent kinase (CDK) inhibitors (CDKIs), including p16INK4a, p21CIP1, and p15INK4b, preventing OSKM-induced senescence. On the other hand, inhibition of mTOR blunts the senescence-associated secretory phenotype (SASP), which itself favors reprogramming. These contrasting actions contribute to explain the complex effect that mTOR has on reprogramming. Overall, our study highlights the advantage of combining functional screens with scRNA-seq to accelerate the discovery of pathways controlling complex phenotypes

A complex secretory program orchestrated by the inflammasome controls paracrine senescence

Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumorigenic properties. Here, we present evidence that the SASP can also induce paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small-molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGF-β family ligands, VEGF, CCL2 and CCL20. Amongst them, TGF-β ligands play a major role by regulating p15INK4b and p21CIP1. Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo

PTBP1-Mediated alternative splicing regulates the inflammatory secretome and the pro-tumorigenic effects of senescent cells

Oncogene-induced senescence is a potent tumor-suppressive response. Paradoxically, senescence also induces an inflammatory secretome that promotes carcinogenesis and age-related pathologies. Consequently, the senescence-associated secretory phenotype (SASP) is a potential therapeutic target. Here, we describe an RNAi screen for SASP regulators. We identified 50 druggable targets whose knockdown suppresses the inflammatory secretome and differentially affects other SASP components. Among the screen candidates was PTBP1. PTBP1 regulates the alternative splicing of genes involved in intracellular trafficking, such as EXOC7, to control the SASP. Inhibition of PTBP1 prevents the pro-tumorigenic effects of the SASP and impairs immune surveillance without increasing the risk of tumorigenesis. In conclusion, our study identifies SASP inhibition as a powerful and safe therapy against inflammation-driven cancer. By performing a genetic screen for regulators of the senescence-associated secretory phenotype (SASP), Georgilis et al. identify PTBP1, which controls SASP by regulating alternative splicing of genes involved in intracellular trafficking such as EXOC7. PTBP1 knockdown blocks the tumor-promoting functions of SASP.</p

PTBP1-Mediated Alternative Splicing Regulates the Inflammatory Secretome and the Pro-tumorigenic Effects of Senescent Cells

Altres ajuts: We are grateful to A.J. Innes and members of J.G.'s laboratory for reagents, comments, and other contributions to this project. We thank S. Vernia, J. Ule, and R. Faraway for advice and members of the Proteomics (P. Faull and A. Montoya) and Genomics (L. Game, K. Rekopoulou, and A. Ivan) LMS facilities for help with the proteomics and RNA-seq, respectively. We thank T.-W. Kang and C. Fellmeth for technical support and Life Science Editors for editorial assistance. J.A. is funded by the Breast Cancer Research Foundation (BCRF, grant BCRF-17-008) and Instituto de Salud Carlos III. N.L.B.-M.'s laboratory is supported by EMBO (Installation grant 3057) and Fundação para a Ciência e a Tecnologia, Portugal (FCT Investigator Starting grant IF/00595/2014). M.H. was supported by an ERC consolidator grant (HepatoMetabopath). Core support from MRC (grants MC-A652-5PZ00 and MC_U120085810) funded the research in J.G.'s laboratory.Oncogene-induced senescence is a potent tumor-suppressive response. Paradoxically, senescence also induces an inflammatory secretome that promotes carcinogenesis and age-related pathologies. Consequently, the senescence-associated secretory phenotype (SASP) is a potential therapeutic target. Here, we describe an RNAi screen for SASP regulators. We identified 50 druggable targets whose knockdown suppresses the inflammatory secretome and differentially affects other SASP components. Among the screen candidates was PTBP1. PTBP1 regulates the alternative splicing of genes involved in intracellular trafficking, such as EXOC7, to control the SASP. Inhibition of PTBP1 prevents the pro-tumorigenic effects of the SASP and impairs immune surveillance without increasing the risk of tumorigenesis. In conclusion, our study identifies SASP inhibition as a powerful and safe therapy against inflammation-driven cancer. By performing a genetic screen for regulators of the senescence-associated secretory phenotype (SASP), Georgilis et al. identify PTBP1, which controls SASP by regulating alternative splicing of genes involved in intracellular trafficking such as EXOC7. PTBP1 knockdown blocks the tumor-promoting functions of SASP

A complex secretory program orchestrated by the inflammasome controls paracrine senescence

Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumorigenic properties. Here, we present evidence that the SASP can also induce paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small-molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGF-β family ligands, VEGF, CCL2 and CCL20. Amongst them, TGF-β ligands play a major role by regulating p15(INK4b) and p21(CIP1). Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo.Core support from the MRC and grants from MRCT, CRUK and the AICR financially supported the research in J.G's laboratory. J.G. is also supported by the EMBO Young Investigator Programm