Eleonora Zonta

Splicing switch of an epigenetic regulator by RNA helicases promotes tumor-cell invasiveness

Both epigenetic and splicing regulation contribute to tumor progression, but the potential links between these two levels of gene-expression regulation in pathogenesis are not well understood. Here, we report that the mouse and human RNA helicases Ddx17 and Ddx5 contribute to tumor-cell invasiveness by regulating alternative splicing of several DNA- and chromatin-binding factors, including the macroH2A1 histone. We show that macroH2A1 splicing isoforms differentially regulate the transcription of a set of genes involved in redox metabolism. In particular, the SOD3 gene that encodes the extracellular superoxide dismutase and plays a part in cell migration is regulated in an opposite manner by macroH2A1 splicing isoforms. These findings reveal a new regulatory pathway in which splicing factors control the expression of histone variant isoforms that in turn drive a transcription program to switch tumor cells to an invasive phenotype.

Dual role of the ddx5/ddx17 RNA helicases in the control of the pro-migratory NFAT5 transcription factor.

Ddx5 and ddx17 are two highly related RNA helicases involved in both transcription and splicing. These proteins coactivate transcription factors involved in cancer such as the estrogen receptor alpha, p53 and beta-catenin. Ddx5 and ddx17 are part of the splicing machinery and can modulate alternative splicing, the main mechanism increasing the proteome diversity. Alternative splicing also has a role in gene expression level regulation when it is coupled to the nonsense-mediated mRNA decay (NMD) pathway. In this work, we report that ddx5 and ddx17 have a dual role in the control of the pro-migratory NFAT5 transcription factor. First, ddx5 and ddx17 act as transcriptional coactivators of NFAT5 and are required for activating NFAT5 target genes involved in tumor cell migration. Second, at the splicing level, ddx5 and ddx17 increase the inclusion of NFAT5 exon 5. As exon 5 contains a pre-mature translation termination codon, its inclusion leads to the regulation of NFAT5 mRNAs by the NMD pathway and to a decrease in NFAT5 protein level. Therefore, we demonstrated for the first time that a transcriptional coregulator can simultaneously regulate the transcriptional activity and alternative splicing of a transcription factor. This dual regulation, where ddx5 and ddx17 enhance the transcriptional activity of NFAT5 although reducing its protein expression level, suggests a critical role for ddx5 and ddx17 in tumor cell migration through the fine regulation of NFAT5 pathway.

A Study of Hypermethylated Circulating Tumor DNA as a Universal Colorectal Cancer Biomarker

BACKGROUND Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

The RNA helicase DDX5/p68 is a key factor promoting c-fos expression at different levels from transcription to mRNA export

It is widely accepted that pre-mRNA maturation, including splicing, is tightly coupled to both transcription and mRNA export, but factors linking the three processes are less understood. By analysing the estrogen-regulated expression of the c-fos mRNA that is processed during transcription, we show that the ddx5 RNA helicase, is required throughout the major nuclear steps of the expression of the c-fos gene, from transcription to mRNA export. Indeed, ddx5, whose recruitment on the c-fos gene was increased upon estrogen treatment, was required for the full transcriptional activation of the c-fos gene. In addition, ddx5 was required for c-fos co-transcriptional RNA splicing. When splicing occurred post-transcriptionally in the absence of ddx5, the c-fos mRNA was poorly exported into the cytosol because of inefficient recruitment of the TAP mRNA export receptor. Finally, ddx5 was present in the c-fos messenger ribonucleoprotein together with mRNA export factors, which further supports that ddx5 is a key operator in the c-fos ‘mRNA factory’.

Base-Position Error Rate Analysis of Next-Generation Sequencing Applied to Circulating Tumor DNA in Non-Small Cell Lung Cancer: A Prospective Study.

Background Circulating tumor DNA (ctDNA) is an approved noninvasive biomarker to test for the presence of EGFR mutations at diagnosis or recurrence of lung cancer. However, studies evaluating ctDNA as a noninvasive “real-time” biomarker to provide prognostic and predictive information in treatment monitoring have given inconsistent results, mainly due to methodological differences. We have recently validated a next-generation sequencing (NGS) approach to detect ctDNA. Using this new approach, we evaluated the clinical usefulness of ctDNA monitoring in a prospective observational series of patients with non-small cell lung cancer (NSCLC). Methods and Findings We recruited 124 patients with newly diagnosed advanced NSCLC for ctDNA monitoring. The primary objective was to analyze the prognostic value of baseline ctDNA on overall survival. ctDNA was assessed by ultra-deep targeted NGS using our dedicated variant caller algorithm. Common mutations were validated by digital PCR. Out of the 109 patients with at least one follow-up marker mutation, plasma samples were contributive at baseline (n = 105), at first evaluation (n = 85), and at tumor progression (n = 66). We found that the presence of ctDNA at baseline was an independent marker of poor prognosis, with a median overall survival of 13.6 versus 21.5 mo (adjusted hazard ratio [HR] 1.82, 95% CI 1.01–3.55, p = 0.045) and a median progression-free survival of 4.9 versus 10.4 mo (adjusted HR 2.14, 95% CI 1.30–3.67, p = 0.002). It was also related to the presence of bone and liver metastasis. At first evaluation (E1) after treatment initiation, residual ctDNA was an early predictor of treatment benefit as judged by best radiological response and progression-free survival. Finally, negative ctDNA at E1 was associated with overall survival independently of Response Evaluation Criteria in Solid Tumors (RECIST) (HR 3.27, 95% CI 1.66–6.40, p < 0.001). Study population heterogeneity, over-representation of EGFR-mutated patients, and heterogeneous treatment types might limit the conclusions of this study, which require future validation in independent populations. Conclusions In this study of patients with newly diagnosed NSCLC, we found that ctDNA detection using targeted NGS was associated with poor prognosis. The heterogeneity of lung cancer molecular alterations, particularly at time of progression, impairs the ability of individual gene testing to accurately detect ctDNA in unselected patients. Further investigations are needed to evaluate the clinical impact of earlier evaluation times at 1 or 2 wk. Supporting clinical decisions, such as early treatment switching based on ctDNA positivity at first evaluation, will require dedicated interventional studies.

Assessment of DNA Integrity, Applications for Cancer Research

Many methods have been developed for DNA integrity assessment including electrophoresis-based procedures, quantitative PCR, and, more recently, microfluidics-based procedures. DNA integrity evaluation can be employed for characterizing biological samples quality before extensive genomic analysis and also finds applications in reproductive medicine, prenatal diagnostics, or cancer research. In this chapter, we will focus on the assessment of DNA integrity in cancer research. In particular, we will present the application of the determination of DNA integrity for tracking of circulating tumor DNA. Finally, we will conclude by illustrating the potential innovative application of DNA integrity as a biomarker in clinical research, especially for prognostic purposes, patient follow-up, or early diagnosis.

High throughput single cell counting in droplet-based microfluidics

Droplet-based microfluidics is extensively and increasingly used for high-throughput single-cell studies. However, the accuracy of the cell counting method directly impacts the robustness of such studies. We describe here a simple and precise method to accurately count a large number of adherent and non-adherent human cells as well as bacteria. Our microfluidic hemocytometer provides statistically relevant data on large populations of cells at a high-throughput, used to characterize cell encapsulation and cell viability during incubation in droplets.

Digital PCR compartmentalization II. Contribution for the quantitative detection of circulating tumor DNA

Genetic markers are now widely used in the clinics, particularly in cancer patient management. Indeed, these tumor markers can help in the diagnosis and prognosis of the disease, and provide valuable information for treatment orientation in the context of personalized medicine. The presence of circulating cell-free tumor DNA (cftDNA) and thus of tumor markers in the blood can be considered to partly avoid the use of solid biopsies. The use of blood samples, as liquid biopsies, is less invasive and described as more representative of tumor heterogeneity. However, cftDNA can be found in blood in low proportion that can vary according to the nature and the progression of the tumor. For these reasons, the use of highly sensitive, specific and ideally quantitative methods for its detection are required. These requirements constituted until recently a technological limit, which now can be overcome thanks to digital PCR. This technology could now become a very efficient and non-invasive tool in oncology, complementary to conventional diagnostic techniques.