TRF2 as novel marker of tumor response to taxane-based therapy: from mechanistic insight to clinical implication.

BACKGROUND: Breast Cancer (BC) can be classified, due to its heterogeneity, into multiple subtypes that differ for prognosis and clinical management. Notably, triple negative breast cancer (TNBC) - the most aggressive BC form - is refractory to endocrine and most of the target therapies. In this view, taxane-based therapy still represents the elective strategy for the treatment of this tumor. However, due variability in patients' response, management of TNBC still represents an unmet medical need. Telomeric Binding Factor 2 (TRF2), a key regulator of telomere integrity that is over-expressed in several tumors, including TNBC, has been recently found to plays a role in regulating autophagy, a degradative process that is involved in drug detoxification. Based on these considerations, we pointed, here, at investigating if TRF2, regulating autophagy, can affect tumor sensitivity to therapy. METHODS: Human TNBC cell lines, over-expressing or not TRF2, were subjected to treatment with different taxanes and drug efficacy was tested in terms of autophagic response and cell proliferation. Autophagy was evaluated first biochemically, by measuring the levels of LC3, and then by immunofluorescence analysis of LC3-puncta positive cells. Concerning the proliferation, cells were subjected to colony formation assays associated with western blot and FACS analyses. The obtained results were then confirmed also in mouse models. Finally, the clinical relevance of our findings was established by retrospective analysis on a cohort of TNBC patients subjected to taxane-based neoadjuvant chemotherapy. RESULTS: This study demonstrated that TRF2, inhibiting autophagy, is able to increase the sensitivity of TNBC cells to taxanes. The data, first obtained in in vitro models, were then recapitulated in preclinical mouse models and in a cohort of TNBC patients, definitively demonstrating that TRF2 over-expression enhances the efficacy of taxane-based neoadjuvant therapy in reducing tumor growth and its recurrence upon surgical intervention. CONCLUSIONS: Based on our finding it is possible to conclude that TRF2, already known for its role in promoting tumor formation and progression, might represents an Achilles' heel for cancer. In this view, TRF2 might be exploited as a putative biomarker to predict the response of TNBC patients to taxane-based neoadjuvant chemotherapy.

Drug repositioning strategy for the identification of novel telomere-damaging agents: A role for NAMPT inhibitors.

Drug repositioning strategy represents a valid tool to accelerate the pharmacological development through the identification of new applications for already existing compounds. In this view, we aimed at discovering molecules able to trigger telomere-localized DNA damage and tumor cell death. By applying an automated high-content spinning-disk microscopy, we performed a screening aimed at identifying, on a library of 527 drugs, molecules able to negatively affect the expression of TRF2, a key protein in telomere maintenance. FK866, resulting from the screening as the best candidate hit, was then validated at biochemical and molecular levels and the mechanism underlying its activity in telomere deprotection was elucidated both in vitro and in vivo. The results of this study allow us to discover a novel role of FK866 in promoting, through the production of reactive oxygen species, telomere loss and deprotection, two events leading to an accumulation of DNA damage and tumor cell death. The ability of FK866 to induce telomere damage and apoptosis was also demonstrated in advanced preclinical models evidencing the antitumoral activity of FK866 in triple-negative breast cancer-a particularly aggressive breast cancer subtype still orphan of targeted therapies and characterized by high expression levels of both NAMPT and TRF2. Overall, our findings pave the way to the development of novel anticancer strategies to counteract triple-negative breast cancer, based on the use of telomere deprotecting agents, including NAMPT inhibitors, that would rapidly progress from bench to bedside.

Canine mortality in Umbria Region (Central Italy): a population-based analysis.

Companion dogs may be valuable sentinels to better understand the environmental determinants of morbidity and mortality in humans. This study aimed to assess the dog population and mortality in Umbria Region. The source of data was the local Canine Registry. Attribute-specific crude mortality rates by sex, age, and breed were produced on a five-year basis (2014-2018). The human ICD-10 was employed to code the causes of deaths. Over 2014-2018, an annual average population of 226,875 specimens and a total of 46,743 deaths were estimated. Mortality rate was higher in young males than in young females. A specific cause of death was reported for 5,209 dogs; the 62.8 per cent (95%CI = 61.4-64.1) was due to external causes. Neoplasms were the fourth cause of death. Differences in mortality between sexes were consistent with human ones. The death registration procedure needs improvement by a systematic coding of the causes. An adjustment of the human ICD could address the lack of a coding system until the introduction of international standards for animals.

The telomeric protein TERF2/TRF2 impairs HMGB1-driven autophagy.

TERF2/TRF2 is a pleiotropic telomeric protein that plays a crucial role in tumor formation and progression through several telomere-dependent and -independent mechanisms. Here, we uncovered a novel function for this protein in regulating the macroautophagic/autophagic process upon different stimuli. By using both biochemical and cell biology approaches, we found that TERF2 binds to the non-histone chromatin-associated protein HMGB1, and this interaction is functional to the nuclear/cytoplasmic protein localization. Specifically, silencing of TERF2 alters the redox status of the cells, further exacerbated upon EBSS nutrient starvation, promoting the cytosolic translocation and the autophagic activity of HMGB1. Conversely, overexpression of wild-type TERF2, but not the mutant unable to bind HMGB1, negatively affects the cytosolic translocation of HMGB1, counteracting the stimulatory effect of EBSS starvation. Moreover, genetic depletion of HMGB1 or treatment with inflachromene, a specific inhibitor of its cytosolic translocation, completely abolished the pro-autophagic activity of TERF2 silencing. In conclusion, our data highlighted a novel mechanism through which TERF2 modulates the autophagic process, thus demonstrating the key role of the telomeric protein in regulating a process that is fundamental, under both physiological and pathological conditions, in defining the fate of the cells.Abbreviations: ALs: autolysosomes; ALT: alternative lengthening of telomeres; ATG: autophagy related; ATM: ATM serine/threonine kinase; CQ: Chloroquine; DCFDA: 2',7'-dichlorofluorescein diacetate; DDR: DNA damage response; DHE: dihydroethidium; EBSS: Earle's balanced salt solution; FACS: fluorescence-activated cell sorting; GFP: green fluorescent protein; EGFP: enhanced green fluorescent protein; GSH: reduced glutathione; GSSG: oxidized glutathione; HMGB1: high mobility group box 1; ICM: inflachromene; IF: immunofluorescence; IP: immunoprecipitation; NAC: N-acetyl-L-cysteine; NHEJ: non-homologous end joining; PLA: proximity ligation assay; RFP: red fluorescent protein; ROS: reactive oxygen species; TIF: telomere-induced foci; TERF2/TRF2: telomeric repeat binding factor 2.

G-quadruplex Stabilization Fuels the ALT Pathway in ALT-positive Osteosarcoma Cells.

Most human tumors maintain telomere lengths by telomerase, whereas a portion of them (10%-15%) uses a mechanism named alternative lengthening of telomeres (ALT). The telomeric G-quadruplex (G4) ligand RHPS4 is known for its potent antiproliferative effect, as shown in telomerase-positive cancer models. Moreover, RHPS4 is also able to reduce cell proliferation in ALT cells, although the influence of G4 stabilization on the ALT mechanism has so far been poorly investigated. Here we show that sensitivity to RHPS4 is comparable in ALT-positive (U2OS; SAOS-2) and telomerase-positive (HOS) osteosarcoma cell lines, unlinking the telomere maintenance mechanism and RHPS4 responsiveness. To investigate the impact of G4 stabilization on ALT, the cardinal ALT hallmarks were analyzed. A significant induction of telomeric doublets, telomeric clusterized DNA damage, ALT-associated Promyelocytic Leukaemia-bodies (APBs), telomere sister chromatid exchanges (T-SCE) and c-circles was found exclusively in RHPS4-treated ALT cells. We surmise that RHPS4 affects ALT mechanisms through the induction of replicative stress that in turn is converted in DNA damage at telomeres, fueling recombination. In conclusion, our work indicates that RHPS4-induced telomeric DNA damage promotes overactivation of telomeric recombination in ALT cells, opening new questions on the therapeutic employment of G4 ligands in the treatment of ALT positive tumors.

Modifications of H3K4 methylation levels are associated with DNA hypermethylation in acute myeloid leukemia.

The 'instructive model' of aberrant DNA methylation in human tumors is based on the observation that CpG islands prone to hypermethylation in cancers are embedded in chromatin enriched in H3K27me3 in human embryonic stem cells (hESC). Recent studies also link methylation of CpG islands to the methylation status of H3K4, where H3K4me3 is inversely correlated with DNA methylation. To provide insight into these conflicting findings, we generated DNA methylation profiles for acute myeloid leukemia samples from patients and leukemic cell lines and integrated them with publicly available ChIp-seq data, containing H3K4me3 and H3K27me3 CpG island occupation in hESC, or hematopoietic stem or progenitor cells (hHSC/MPP). Hypermethylated CpG islands in AML samples displayed H3K27me3 enrichments in hESC and hHSC/MPP; however, ChIp analysis of specific hypermethylated CpG islands revealed a significant reduction in H3K4me3 signal with a concomitant increase in H3K4me0 levels as opposed to a nonsignificant increase in H3K27me3 marks. The integration of AML DNA methylation profiles with the ChIp-seq data in hESC and hHSC/MPP also led to the identification of Iroquois homeobox 2 (IRX2) as a previously unknown factor promoting differentiation of leukemic cells. Our results indicate that in contrast to the 'instructive model', H3K4me3 levels are strongly associated with DNA methylation patterns in AML and have a role in the regulation of critical genes, such as the putative tumor suppressor IRX2.

A web-based tumor registration system for a regional Canine Cancer Registry in Umbria, central Italy.

The Umbria Canine Cancer Registry (CCR) is a web-based platform for cancer registration set up in order to estimate the incidence of spontaneous tumors. It is an integral part of the regional canine demographic registry in which veterinary practitioners and pathologists interact. Veterinary pathologists perform double-blind comparisons and classify neoplasms in an automated classification process using the WHO criteria for canine neoplasms and the ICD-O tumor topographical and morphological keys. Here we describe the organization, on-line procedures and the methods used to assess canine demography, a pre-requisite for accurately estimating the incidence of cancer. In its first 4 years the CCR recruited 4857 cases of suspected tumors, as diagnosed by practitioners, clinics and a veterinary hospital. After the first year the number of enrolled cases increased by 63%, suggesting growing interest from the regional veterinary community.

BRCA2 Deletion Induces Alternative Lengthening of Telomeres in Telomerase Positive Colon Cancer Cells.

BRCA1/2 are tumor suppressor genes controlling genomic stability also at telomeric and subtelomeric loci. Their mutation confers a predisposition to different human cancers but also sensitivity to antitumor drugs including poly(ADP-ribose) polymerase (PARP) inhibitors and G-quadruplex stabilizers. Here we demonstrate that BRCA2 deletion triggers TERRA hyperexpression and alternative lengthening mechanisms (ALT) in colon cancer cells in presence of telomerase activity. This finding opens the question if cancer patients bearing BRCA2 germline or sporadic mutation are suitable for anti-telomerase therapies, or how ALT activation could influence the short or long-term response to anti-PARP inhibitors or anti-G-quadruplex therapies.

Prevalence estimation of Italian ovine cystic echinococcosis in slaughterhouses: A retrospective Bayesian data analysis, 2010-2015.

Cystic echinococcosis (CE) is a complex zoonosis with domestic and sylvatic life-cycles, involving different intermediate and definitive host species. Many previous studies have highlighted the lack of a surveillance system for CE, its persistence in Italy, and endemicity in several Italian regions. Because of the absence of a uniform surveillance program for both humans and animals, disease occurrence is widely underestimated. This study aimed to estimate the prevalence of ovine CE in Italy. Survey data on the prevalence of Echinococcus granulosus complex infections in Italian sheep farms from 2010 to 2015 were obtained in collaboration with Regional Veterinary Epidemiology Observatories (OEVRs). Bayesian analysis was performed to estimate the true CE farm prevalence. The prior true CE prevalence was estimated using data from Sardinia. Second, Bayesian modelling of the observed prevalence in different regions and the true prevalence estimation from the first step were used to ultimately estimate the prevalence of ovine CE in Italy. We obtained survey data from 10 OEVRs, covering 14 Italian regions. We observed that the risk of CE infection decreased over the years, and it was strictly correlated with the density of susceptible species. Using Sardinia as prior distribution, where the disease farm prevalence was approximately 19% (95% CI, 18.82-20.02), we estimated that the highest endemic CE farm prevalence was in Basilicata with a value of 12% (95% BCI: 7.49-18.9%) and in Piemonte 7.64%(95% BCI: 4.12-13.04%). Our results provide spatially relevant data crucial for guiding CE control in Italy. Precise information on disease occurrence location would aid in the identification of priority areas for disease control implementation by the authorities. The current underestimation of CE occurrence should urge the Italian and European governments to become aware of the public health importance of CE and implement targeted interventions for high-risk areas.

Reference values for serum amyloid A, haptoglobin, lysozyme, zinc and iron in healthy lactating Lacaune sheep.

Acute-phase proteins and trace elements are considered biomarkers of the immune response to infection, inflammation, trauma and other pathological conditions, as well as indicators of the health status and productivity of farm animals. Given the scarcity of published data on this topic, the purpose of this study was to determine the serum levels of serum amyloid A (SAA) and haptoglobin, as well as lysozyme, iron and zinc in clinically healthy Lacaune sheep in lactation months 3-7 ranging in age from 2 to 6 years. The mean serum levels for SAA (12.2 µg/mL), lysozyme (1.47 µg/mL), zinc (78.9 µg/dL) and iron (26.6 mmol/L) differed from those found in other sheep breeds and indicate considerable variations depending on breed, age and physiological status of the sheep. Reference values for clinically healthy mid-lactating Lacaune sheep were determined by using well-described and modern analytical and statistical methods. The reference intervals may be used to determine the health and welfare of lactating Lacaune sheep and may serve as a starting point to investigate diseases.

SIRT6 interacts with TRF2 and promotes its degradation in response to DNA damage.

Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response.

Left-sided early-onset vs late-onset colorectal carcinoma: histologic, clinical, and molecular differences.

OBJECTIVES: Carcinomas of the left colon represent a neoplasm of older patients (late onset), but epidemiologic evidence has been showing an increasing incidence in patients 50 years or younger (early onset). In this study, we investigate pathologic and molecular features of early- and late-onset carcinoma of the left colon. METHODS: We selected 22 patients 50 years or younger and 21 patients 70 years or older with left-sided colorectal carcinoma (CRC). All samples were evaluated for pathologic features, microsatellite instability, and KRAS and BRAF mutations. Moreover, both groups were analyzed to identify CpG island methylator phenotype features and assessed with restriction landmark genome scanning (RLGS) to unveil differential DNA methylation patterns. RESULTS: Early-onset patients had advanced pathologic stages compared with late-onset patients (P = .0482). All cases showed a microsatellite stable profile and BRAF wild-type sequence. Early-onset patients (43%) more frequently had mutations at KRAS codon 12 compared with late-onset patients (14%) (P =.0413). RLGS showed that patients younger than 50 years who had CRC had a significantly lower percentage of methylated loci than did patients 70 years or older (P = .04124), and differential methylation of several genomic loci was observed in the two groups. CONCLUSIONS: Our results suggest that left-sided CRCs may present differential patterns of aberrant DNA methylation when they are separated by age.

Polycombs and microRNA-223 regulate human granulopoiesis by transcriptional control of target gene expression.

Epigenetic modifications regulate developmental genes involved in stem cell identity and lineage choice. NFI-A is a posttranscriptional microRNA-223 (miR-223) target directing human hematopoietic progenitor lineage decision: NFI-A induction or silencing boosts erythropoiesis or granulopoiesis, respectively. Here we show that NFI-A promoter silencing, which allows granulopoiesis, is guaranteed by epigenetic events, including the resolution of opposing chromatin "bivalent domains," hypermethylation, recruitment of polycomb (PcG)-RNAi complexes, and miR-223 promoter targeting activity. During granulopoiesis, miR-223 localizes inside the nucleus and targets the NFI-A promoter region containing PcGs binding sites and miR-223 complementary DNA sequences, evolutionarily conserved in mammalians. Remarkably, both the integrity of the PcGs-RNAi complex and DNA sequences matching the seed region of miR-223 are required to induce NFI-A transcriptional silencing. Moreover, ectopic miR-223 expression in human myeloid progenitors causes heterochromatic repression of NFI-A gene and channels granulopoiesis, whereas its stable knockdown produces the opposite effects. Our findings indicate that, besides the regulation of translation of mRNA targets, endogenous miRs can affect gene expression at the transcriptional level, functioning in a critical interface between chromatin remodeling complexes and the genome to direct fate lineage determination of hematopoietic progenitors.