Measurements Methods for the Development of MicroRNA-Based Tests for Cancer Diagnosis.

Studies investigating microRNAs as potential biomarkers for cancer, immune-related diseases, or cardiac pathogenic diseases, among others, have exponentially increased in the last years. In particular, altered expression of specific miRNAs correlates with the occurrence of several diseases, making these molecules potential molecular tools for non-invasive diagnosis, prognosis, and response to therapy. Nonetheless, microRNAs are not in clinical use yet, due to inconsistencies in the literature regarding the specific miRNAs identified as biomarkers for a specific disease, which in turn can be attributed to several reasons, including lack of assay standardization and reproducibility. Technological limitations in circulating microRNAs measurement have been, to date, the biggest challenge for using these molecules in clinical settings. In this review we will discuss pre-analytical, analytical, and post-analytical challenges to address the potential technical biases and patient-related parameters that can have an influence and should be improved to translate miRNA biomarkers to the clinical stage. Moreover, we will describe the currently available methods for circulating miRNA expression profiling and measurement, underlining their advantages and potential pitfalls

Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex, a sequence-selective N3-adenine methylating agent.

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild typedeltamag1deltaapn1apn2 = deltaapn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in deltamag1, and 23 in deltaapn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in deltamag1, and 23 in deltaapn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in deltamag1; and 14/23, 61%, in deltaapn1apn2). The Mag1 deletion was associated with a significant decrease of GCAT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity

Proline Dehydrogenase (PRODH) Is Expressed in Lung Adenocarcinoma and Modulates Cell Survival and 3D Growth by Inducing Cellular Senescence

The identification of markers for early diagnosis, prognosis, and improvement of therapeutic options represents an unmet clinical need to increase survival in Non-Small Cell Lung Cancer (NSCLC), a neoplasm still characterized by very high incidence and mortality. Here, we investigated whether proline dehydrogenase (PRODH), a mitochondrial flavoenzyme catalyzing the key step in proline degradation, played a role in NSCLC tumorigenesis. PRODH expression was investigated by immunohistochemistry; digital PCR, quantitative PCR, immunoblotting, measurement of reactive oxygen species (ROS), and functional cellular assays were carried out. PRODH expression was found in the majority of lung adenocarcinomas (ADCs). Patients with PRODH-positive tumors had better cancer-free specific and overall survival compared to those with negative tumors. Ectopic modulation of PRODH expression in NCI-H1299 and the other tested lung ADC cell lines decreased cell survival. Moreover, cell proliferation curves showed delayed growth in NCI-H1299, Calu-6 and A549 cell lines when PRODH-expressing clones were compared to control clones. The 3D growth in soft agar was also impaired in the presence of PRODH. PRODH increased reactive oxygen species production and induced cellular senescence in the NCI-H1299 cell line. This study supports a role of PRODH in decreasing survival and growth of lung ADC cells by inducing cellular senescence

Systematic review and critique of circulating miRNAs as biomarkers of stage I-II non-small cell lung cancer

Selected circulating microRNAs (miRNAs) have been suggested for non-invasive screening of non-small cell lung cancer (NSCLC), however the numerous proposed miRNA signatures are inconsistent. Aiming to identify miRNAs suitable specifically for stage I-II NSCLC screening in serum/plasma samples, we searched the databases \u201cPubmed\u201d, \u201cMedline\u201d, \u201cScopus\u201d, \u201cEmbase\u201d and \u201cWOS\u201d and systematically reviewed the publications reporting quantitative data on the efficacy [sensitivity, specificity and/or area under the curve (AUC)] of circulating miRNAs as biomarkers of NSCLC stage I and/or II. The 20 studies fulfilling the search criteria included 1110 NSCLC patients and 1009 controls, and were of medium quality according to Quality Assessment of Diagnostic Accuracy Studies checklist. In these studies, the patient cohorts as well as the control groups were heterogeneous for demographics and clinicopathological characteristics; moreover, numerous pre-analytical and analytical variables likely influenced miRNA determinations, and potential bias of hemolysis was often underestimated. We identified four circulating miRNAs scarcely influenced by hemolysis, each featuring high sensitivity (> 80%) and AUC (> 0.80) as biomarkers of stage I-II NSCLC: miR- 223, miR-20a, miR-448 and miR-145; four other miRNAs showed high specificity (> 90%): miR-628-3p, miR-29c, miR-210 and miR-1244. In a model of two-step screening for stage I-II NSCLC using first the above panel of serum miRNAs with high sensitivity and high AUC, and subsequently the panel with high specificity, the estimated overall sensitivity is 91.6% and overall specificity is 93.4%. These and other circulating miRNAs suggested for stage I-II NSCLC screening require validation in multiple independent studies before they can be proposed for clinical application

PROLINE DEHYDROGENASE EXPRESSION, REGULATION AND FUNCTION IN NON-SMALL CELL LUNG CARCINOMA

Non-Small Cell Lung Cancer (NSCLC) is one of the most frequent and deadliest cancers and comprises two main histotypes, adenocarcinoma (ADC) and squamocellular carcinoma (SCC). Identification of markers to better define the diagnosis, prognosis and therapeutic options of NSCLC is needed. We investigated if proline dehydrogenase (PRODH), a mitochondrial flavoenzyme catalyzing the key step in proline degradation, and involved in the regulation of cell survival, autophagy and apoptosis, may be one such marker. Materials and methods We characterized PRODH expression in NSCLC by immunohistochemistry and qPCR and tested if there was correlation between expression of PRODH and clinical features of the tumors or expression of other markers. We aimed to test what cellular processes are influenced by PRODH in lung ADC cell lines. To do so, we tested the effect of regulating PRODH expression in ADC tumour cell lines on their behaviour, by performing a panel of phenotypic assays. Results and discussion We found PRODH immunostaining in the majority (70%) of lung ADCs. Patients with PRODH positive tumors had better overall survival than those with negative tumors. Protein staining was paralleled by high transcript levels, suggesting transcriptional regulation. In A549 and H1437 ADC cell lines, ectopic modulation of PRODH expression suggested that PRODH favoured survival of these ADC cells, whereas in NCI-H1650 cells overexpression led to a decrease in clonogenic ability. In the latter cell line, PRODH expressing clones also showed a reduced 3D growth in soft agar compared to control clones. Conclusion Our immunohistochemistry data support a possible use of PRODH immunostaining as a prognostic marker. However, further research is necessary to 1) identify molecular interactors that can influence the outcome and 2) to better define the downstream processes activated by PRODH in lung cancer cells

Abstract 3402: ΔN-p63α and TA-p63α exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites

9noneTP63 is a member of the p53 gene family that encodes for up to ten different TA- and ΔN- isoforms through differential promoter usage and alternative C-terminal splicing. The TA isoforms (TA-p63α, β, γ, δ and ϵ) contain the N-terminal transactivation domain (TA1), whereas the ΔN isoforms (ΔN-p63α, β, γ, δ and ϵ) are transcribed from an internal promoter (P2) and lack the TA1 domain. A second C-terminal transactivation domain (TA2) present in all p63α and β isoforms has been reported. Besides being a master regulator of gene expression for squamous epithelial proliferation, differentiation and maintenance (germline TP63 mutations are causative for a subset of human ectodermal dysplasia syndromes -EDs-), TA- and ΔN-p63 isoforms play an important role in tumorigenesis. More recently, p63 was shown to modulate apoptosis in the female and male germ line in response to DNA damage. All isoforms share a large, immunoglobulin-like folded DNA binding domain that is responsible for binding to sequence-specific response elements (REs), whose overall consensus sequence is similar to the p53 RE. Since the ΔN-p63 isoforms lack the N-terminal transactivation domain, it was originally proposed that these proteins might act primarily as oncogenes through dominant-negative mechanisms. However, different studies indicate that ΔN-p63 protein itself can be transcriptionally active. For example, ΔN-p63α may directly contribute to tumorigenesis by up-regulating the chaperone protein Hsp70, which displays proliferative and anti-apoptotic functions or by repressing pro-apoptotic genes. Transcriptional activation of specific genes by ΔN-p63α, namely VDR and Id-3, has been also associated with an anti-tumorigenic role, i.e. a decrease in cell invasion. Using a defined functional assay in yeast where p63 isoform and RE sequence are the only variables (more than 80 different REs were tested), as well as mammalian-transcription assays (gene reporter assays, qPCR measurements, western blotting), we demonstrated that human TA- and ΔN-p63α proteins exhibited differences in transactivation specificity. In fact 21 REs were identified that exhibited higher or selective responsiveness to ΔN-p63α. These differences were not observed with the related p73 and p53 proteins isoforms and were dependent on specific features of the RE sequences. Based on gene annotations, we propose that cis-element sequence features might have been selected along with evolutionarily conserved, intrinsic differences in cooperative DNA binding of p63 proteins, to establish tighter control of the apoptotic processes.Ciribilli, Yari; Bisio, Alessandra; Monti, Paola; Foggetti, Giorgia; Raimondi, Ivan; Campomenosi, Paola; Menichini, Paola; Fronza, Gilberto; Inga, AlbertoCiribilli, Yari; Bisio, Alessandra; Monti, Paola; Foggetti, Giorgia; Raimondi, Ivan; Campomenosi, Paola; Menichini, Paola; Fronza, Gilberto; Inga, Albert

P53 family members modulate the expression of PRODH, but not PRODH2, via intronic p53 response elements.

The tumor suppressor p53 was previously shown to markedly up-regulate the expression of the PRODH gene, encoding the proline dehydrogenase (PRODH) enzyme, which catalyzes the first step in proline degradation. Also PRODH2, which degrades 4-hydroxy-L-proline, a product of protein (e.g. collagen) catabolism, was recently described as a p53 target. Here, we confirmed p53-dependent induction of endogenous PRODH in response to genotoxic damage in cell lines of different histological origin. We established that over-expression of TAp73β or TAp63β is sufficient to induce PRODH expression in p53-null cells and that PRODH expression parallels the modulation of endogenous p73 by genotoxic drugs in several cell lines. The p53, p63, and p73-dependent transcriptional activation was linked to specific intronic response elements (REs), among those predicted by bioinformatics tools and experimentally validated by a yeast-based transactivation assay. p53 occupancy measurements were validated in HCT116 and MCF7 human cell lines. Conversely, PRODH2 was not responsive to p63 nor p73 and, at best, could be considered a weak p53 target. In fact, minimal levels of PRODH2 transcript induction by genotoxic stress was observed exclusively in one of four p53 wild-type cell lines tested. Consistently, all predicted p53 REs in PRODH2 were poor matches to the p53 RE consensus and showed very weak responsiveness, only to p53, in the functional assay. Taken together, our results highlight that PRODH, but not PRODH2, expression is under the control of p53 family members, specifically p53 and p73. This supports a deeper link between proteins of the p53-family and metabolic pathways, as PRODH modulates the balance of proline and glutamate levels and those of their derivative alpha-keto-glutarate (α-KG) under normal and pathological (tumor) conditions

Metasomatic horizon sealing serpentinite-metasediments pair in the Zermatt-Saas metaophiolite (Northwestern Alps): record of a channel for focussed fluid flow during subduction

A metasomatic horizon (MH) occurs between the metaophiolite (serpentinite and metaophicarbonates) basement and metasedimentary sequence (chaotic rocks and calcschists) of the Lake Miserin Ophiolite, in the high pressure Zermatt-Saas Zone of the Northwestern Alps. Macro- and microstructural analyses combined with petrological and geochemical investigations of the MH and surrounding lithologies unravelled a polyphase blastesis-deformation history, which led to the formation of a complex fabric and minero-chemical alteration of the serpentinite basement-metasediments interface. Dehydration, decarbonation and carbonation interplayed from early Alpine subduction up to HP-LT metamorphic peak (T=550-630 °C, P=1.8-2.5 GPa), to produce a distinctive, pervasive amphibole (tremolite/actinolite) replacement both in carbonate-rich and serpentinite-rich domains pertaining to the MH protoliths, i.e. serpentinite and carbonate-bearing metabreccia of the chaotic rock unit. This characteristic amphibole metasomatism is more pronounced toward the contact with the metaophicarbonates, and the average δ18OVSMOW and δ13CVPDB values of dolomite within the MH (+14.4‰ and +0.7‰ respectively) lie between those of the metaophicarbonates and of calcschist. These results suggest that Mg- H2O-rich fluids from the dehydrating slab, CO2 released by decarbonation and SiO2-rich fluids evolved in calcschists mixed together and circulated mostly along the metaophiolite basement/metasediments interface, where the MH developed and recorded a preferential channel for mixed metamorphic fluid flow. These findings highlight and confirm that the study of metasomatic rocks in convergent systems is crucial to comprehend the behaviour of different fluids circulating, mixing and interacting with lithologies along slab-parallel discontinuities, which act as major fluid conduits for deep volatile recycling

A comparison between quantitative PCR and droplet digital PCR technologies for circulating microRNA quantification in human lung cancer

Background. Selected microRNAs (miRNAs) that are abnormally expressed in the serum of patients with lung cancer have recently been proposed as biomarkers of this disease. The measurement of circulating miRNAs, however, requires a highly reliable quantification method. Quantitative real-time PCR (qPCR) is the most commonly used method, but it lacks reliable endogenous reference miRNAs for normalization of results in biofluids. When used in absolute quantification, it must rely on the use of external calibrators. Droplet digital PCR (ddPCR) is a recently introduced technology that overcomes the normalization issue and may facilitate miRNA measurement. Here we compared the performance of absolute qPCR and ddPCR techniques for quantifying selected miRNAs in the serum. Results. In the first experiment, three miRNAs, proposed in the literature as lung cancer biomarkers (miR-21, miR-126 and let-7a), were analyzed in a set of 15 human serum samples. Four independent qPCR and four independent ddPCR amplifications were done on the same samples and used to estimate the precision and correlation of miRNA measurements obtained with the two techniques. The precision of the two methods was evaluated by calculating the Coefficient of Variation (CV) of the four independent measurements obtained with each technique. The CV was similar or smaller in ddPCR than in qPCR for all miRNAs tested, and was significantly smaller for let-7a (p = 0.028). Linear regression analysis of the miRNA values obtained with qPCR and ddPCR showed strong correlation (p < 0.001). To validate the correlation obtained with the two techniques in the first experiment, in a second experiment the same miRNAs were measured in a larger cohort (70 human serum samples) by both qPCR and ddPCR. The correlation of miRNA analyses with the two methods was significant for all three miRNAs. Moreover, in our experiments the ddPCR technique had higher throughput than qPCR, at a similar cost-per-sample. Conclusions: Analyses of serum miRNAs performed with qPCR and ddPCR were largely concordant. Both qPCR and ddPCR can reliably be used to quantify circulating miRNAs, however, ddPCR revealed similar or greater precision and higher throughput of analysis

Human Primary Dermal Fibroblasts Interacting with 3-Dimensional Matrices for Surgical Application Show Specific Growth and Gene Expression Programs

Several types of 3-dimensional (3D) biological matrices are employed for clinical and surgical applications, but few indications are available to guide surgeons in the choice among these materials. Here we compare the in vitro growth of human primary fibroblasts on different biological matrices commonly used for clinical and surgical applications and the activation of specific molecular pathways over 30 days of growth. Morphological analyses by Scanning Electron Microscopy and proliferation curves showed that fibroblasts have different ability to attach and proliferate on the different biological matrices. They activated similar gene expression programs, reducing the expression of collagen genes and myofibroblast differentiation markers compared to fibroblasts grown in 2D. However, differences among 3D matrices were observed in the expression of specific metalloproteinases and interleukin-6. Indeed, cell proliferation and expression of matrix degrading enzymes occur in the initial steps of interaction between fibroblast and the investigated meshes, whereas collagen and interleukin-6 expression appear to start later. The data reported here highlight features of fibroblasts grown on different 3D biological matrices and warrant further studies to understand how these findings may be used to help the clinicians choose the correct material for specific applications