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Identification of Genes Controlling Senescence in Human Epithelial Cells: Role in Cancer

Final Report Summary - EPITHELIALSENESCENCE (Identification of genes controlling senescence in human epithelial cells: role in cancer)

Cellular senescence was originally described as a stable cell cycle arrest that accompanies the exhaustion of proliferative potential in cultured primary mammalian cells. Whereas this so-called replicative senescence is triggered by telomere erosion, other stimuli such as activated oncogenes, oxidative stress, deoxyribonucleic acid (DNA) damage and enforced expression of pluripotency-associated factors can trigger premature senescence. Senescent cells remain metabolically active but undergo a cell cycle arrest in G1, and are characterised by a flat and enlarged morphology, senescence-associated beta-galactosidase activity, due to the increased lysosome numbers, and the presence of sescence-associated heterochromatin foci (SAHF). Senescent cells also secret a plethora of extracellular proteins and soluble factors, referred to as the senescence-associated secretory phenotype (SASP) which have multiple and sometimes contradictory functions. This secretome includes extracellular proteases, matrix components (such as MMPs), growth factors, proinflammatory cytokines (IL6, IL1-alpha, IL1-beta) and chemokines (IL8, GRO-alpha, MCP1). Cellular senescence is relevant in multiple physiological and pathological contexts. Importantly, oncogene-induced senescence (OIS) acts as a potent cell-intrinsic tumour suppressor mechanism inhibiting tumour progression. On the other hand, cellular senescence limits the self-renewal potential of stem cells and has been implicated in age-related disorders. A related observation is that senescence impairs the efficiency of reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). Cellular senescence is a genetically-driven programme implemented by the activation of the p53 and RB tumour suppressor pathways. The INK4 / ARF locus (comprising the CDKN2A and CDKN2B genes) has the potential to regulate both pathways, as it encodes for p15INK4b and p16INK4a, two cyclin-dependent kinase inhibitors controlling RB phosphorylation, and the unrelated protein ARF that activates p53. In primary cells, gene expression of the INK4 / ARF locus is kept under control by Polycomb repressive complexes 1 (PRC1) and 2 (PRC2). PRC2 interacts with histone deacetylases (HDAC) and catalyses histone H3 lysine 27 trimethylation (H3K27me3). This epigenetic mark is recognised by PRC1, which catalyses histone H2A lysine 119 monoubiquitination. Upregulation of many PRC target genes is observed during senescence while aberrant silencing of PRC target genes is frequent in tumourigenesis. However, the question of how PRCs are recruited to their target genes is still a matter of investigation. Recent data provided evidence for the role of specific transcription factors and long intergenic non-coding ribonucleic acids (lincRNAs) in PRC recruitment.

We recently identified HLX1 (H2.0-like homeobox 1) as a suppressor of cellular senescence in a genetic screen for transcription factors increasing cellular lifespan of human fibroblasts. HLX1 is part of the Homeobox family of transcription factors, which have important roles in developmental patterning. We observed that HLX1 overexpression both extends replicative lifespan and blunts premature senescence induced by oncogenic Ras. Conversely, HLX1 knockdown induces premature senescence. In order to understand the molecular mechanisms underlying HLX1 function, we used proteomics to monitor changes in proteins levels in response to HLX1 knockdown. Stable isotope labelling with amino acids in cell culture (SILAC) identified p16INK4a as the key target mediating HLX1 effects. We observed that HLX1 directly associates with the INK4a promoter and represses INK4a expression by recruiting HDAC1 and PRC2. A small interfering RNA (siRNA) screen identified 6 other Homeobox proteins (DLX3, HOXA9, HOXB13, HOXC13, HOXD3 and HOXD8) able to repress p16INK4a. We showed that expression of HOXA9 (Homeobox A9), used as an example amongst these factors, is sufficient to delay senescence by recruiting PRCs and HDACs to repress INK4a. Altogether our work added Homeobox proteins to the list of factors (which includes TWIST1, Zfp277 and the lincRNA ANRIL recruiting PRCs to repress INK4a. In addition, gene set enrichment analysis (GSEA) suggested that HLX1 also regulated a subset of PRC targets. We have also identified senescence-associated PRC targets other than INK4a which are also regulated by HLX1 and HOXA9 and we think that there are broad implications in the interplay between Homeobox proteins and PRCs forsenescence and cancer.