We demonstrate here that the E2F1 induced by DNA damage can bind to and promote the apoptotic function of p53 via the cyclin A binding site of E2F1. This function of E2F1 does not require its DP-1 binding, DNA binding, or transcriptional activity and is independent of mdm2. All the cyclin A binding E2F family members can interact and cooperate with p53 to induce apoptosis. This suggests a novel role for E2F in regulating apoptosis in response to DNA damage. Cyclin A, but not cyclin E, prevents E2F1 from interacting and cooperating with p53 to induce apoptosis. However, in response to DNA damage, cyclin A levels decrease, with a concomitant increase in E2F1-p53 complex formation. These results suggest that the binding of E2F1 to p53 can specifically stimulate the apoptotic function of p53 in response to DNA damage.
The extracellular signals regulating mammary epithelial cell growth are of relevance to understanding the pathophysiology of mammary epithelia, yet they remain poorly characterized. In this study, we applied an unbiased approach to understanding the functional role of signalling molecules in several models of normal physiological growth and translated these results to the biological understanding of breast cancer subtypes. We developed and utilized a cytogenetically normal clonal line of hTERT immortalized human mammary epithelial cells in a fibroblast-enhanced co-culture assay to conduct a genome-wide small interfering RNA (siRNA) screen for evaluation of the functional effect of silencing each gene. Our selected endpoint was inhibition of growth. In rigorous postscreen validation processes, including quantitative RT-PCR, to ensure on-target silencing, deconvolution of pooled siRNAs and independent confirmation of effects with lentiviral short-hairpin RNA constructs, we identified a subset of genes required for mammary epithelial cell growth. Using three-dimensional Matrigel growth and differentiation assays and primary human mammary epithelial cell colony assays, we confirmed that these growth effects were not limited to the 184-hTERT cell line. We utilized the METABRIC dataset of 1,998 breast cancer patients to evaluate both the differential expression of these genes across breast cancer subtypes and their prognostic significance. We identified 47 genes that are critically important for fibroblast-enhanced mammary epithelial cell growth. This group was enriched for several axonal guidance molecules and G protein–coupled receptors, as well as for the endothelin receptor PROCR. The majority of genes (43 of 47) identified in two dimensions were also required for three-dimensional growth, with HSD17B2, SNN and PROCR showing greater than tenfold reductions in acinar formation. Several genes, including PROCR and the neuronal pathfinding molecules EFNA4 and NTN1, were also required for proper differentiation and polarization in three-dimensional cultures. The 47 genes identified showed a significant nonrandom enrichment for differential expression among 10 molecular subtypes of breast cancer sampled from 1,998 patients. CD79A, SERPINH1, KCNJ5 and TMEM14C exhibited breast cancer subtype–independent overall survival differences. Diverse transmembrane signals are required for mammary epithelial cell growth in two-dimensional and three-dimensional conditions. Strikingly, we define novel roles for axonal pathfinding receptors and ligands and the endothelin receptor in both growth and differentiation.
Abstract CX-5461 is a G-quadruplex stabilizer that exhibits synthetic lethality in homologous recombination-deficient models. In this multicentre phase I trial in patients with solid tumors, 40 patients are treated across 10 dose levels (50–650 mg/m 2 ) to determine the recommended phase II dose (primary outcome), and evaluate safety, tolerability, pharmacokinetics (secondary outcomes). Defective homologous recombination is explored as a predictive biomarker of response. CX-5461 is generally well tolerated, with a recommended phase II dose of 475 mg/m 2 days 1, 8 and 15 every 4 weeks, and dose limiting phototoxicity. Responses are observed in 14% of patients, primarily in patients with defective homologous recombination. Reversion mutations in PALB2 and BRCA2 are detected on progression following initial response in germline carriers, confirming the underlying synthetic lethal mechanism. In vitro characterization of UV sensitization shows this toxicity is related to the CX-5461 chemotype, independent of G-quadruplex synthetic lethality. These results establish clinical proof-of-concept for this G-quadruplex stabilizer. Clinicaltrials.gov NCT02719977.
Abstract Chemical biology approaches to the global functions of splicing reactions are gaining momentum, with an increasing repertoire of small molecule probes becoming available. Here we map the association of eIF4A3 with transcript expression, NMD and alternative splicing using a set of selective novel small molecule allosteric helicase inhibitors whose synthesis and chemical properties we have recently described. We show through analysis of dose monotonic transcriptional responses to increasing inhibition that both full length and NMD prone transcripts link eIF4A3 to normal functioning of cell division including chromosome segregation and cell cycle checkpoints, pointing to a conserved role of splicing and transcript quality processing in cell cycle functions. Cell cycle analysis and microscopy of inhibitor treated cells demonstrates chromosome mis-segregation and spindle defects, associated with a G2/M arrest, validating this observation. Through analysis of conserved alternative splicing patterns exhibiting monotonic responses, we find that eIF4A3 dependent alternative splicing involves exons that are longer and introns that are shorter than transcripts not modulated by eIF4A3. Moreover we observe conservation of over/under representation of RBP binding motif density over introns and exons implicated eIF4A3 modulated skipped exon and retained introns. The distribution of motif densities over 5’ and branch intron sites and 5’ exons is consistent with function of the exon-junction complex. Taken together we have defined a fraction of the transcrip-tome dependent on eIF4A3 functions and revealed a link between eIF4A3 and cell cycle regulation. The systems approach described here suggests additional avenues for therapeutic exploitation of eIF4A3 functions in cancer and related diseases.
Datasets for the manuscript Eirew et al Accurate determination of CRISPR-mediated gene fitness in transplantable tumours: - targeted sequencing FASTQ files - sgRNA-UMI count files - sgRNA library content - experiment description
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