Linear unconstrained DNA cannot harbor supercoils since these supercoils can diffuse and be eliminated by free rotation of the DNA strands at the end of the molecule. Mammalian telomeres, despite constituting the ends of linear chromosomes, can hold supercoils and be subjected to topological stress. While negative supercoiling was previously observed, thus proving the existence of telomeric topological constraints, positive supercoils were never probed due to the lack of an appropriate tool. Indeed, the few tools available currently could only investigate unwound (Trioxsalen) or overwound (GapR) DNA topology (variations in twist) but not the variations in writhe (supercoils and plectonemes). To address this question, we have designed innovative tools aimed at analyzing both positive and negative DNA writhe in cells. Using them, we could observe the build-up of positive supercoils following replication stress and inhibition of Topoisomerase 2 on telomeres. TRF2 depletion caused both telomere relaxation and an increase in positive supercoils while the inhibition of Histone Deacetylase I and II by TSA only caused telomere relaxation. Moving outside telomeres, we also observed a build-up of positive supercoils on the FRA3B fragile site following replication stress, suggesting a topological model of DNA fragility for this site.

This article presents an in-depth exploration of the roles of Telomere Repeat-binding Factors 1 and 2 (TRF1 and TRF2), and the shelterin complex, in the context of cancer biology. It emphasizes their emerging significance as potential biomarkers and targets for therapeutic intervention. Central to the shelterin complex, TRF1 and TRF2 are crucial in maintaining telomere integrity and genomic stability, their dysregulation often being a hallmark of cancerous cells. The article delves into the diagnostic and prognostic capabilities of TRF1 and TRF2 across various cancer types, highlighting their sensitivity and specificity. Furthermore, it reviews current strides in drug discovery targeting the shelterin complex, detailing specific compounds and their modes of action. The review candidly addresses the challenges in developing therapies aimed at the shelterin complex, including drug resistance, off-target effects, and issues in drug delivery. By synthesizing recent research findings, the article sheds light on the intricate relationship between telomere biology and cancer development. It underscores the urgency for continued research to navigate the existing challenges and fully leverage the therapeutic potential of TRF1, TRF2, and the shelterin complex in the realm of cancer treatment.

Telomeres, potential biomarkers of aging, are known to shorten with continued cigarette smoke exposure. In order to further investigate this process and its impact on cellular stress and inflammation, we used an in vitro model with cigarette smoke extract (CSE) and observed the downregulation of telomere stabilizing TRF2 and POT1 genes after CSE treatment. hTERT is a subunit of telomerase and a well-known oncogenic marker, which is overexpressed in over 85% of cancers and may contribute to lung cancer development in smokers. We also observed an increase in hTERT and ISG15 expression levels after CSE treatment, as well as increased protein levels revealed by immunohistochemical staining in smokers\' lung tissue samples compared to non-smokers. The effects of ISG15 overexpression were further studied by quantifying IFN-γ, an inflammatory protein induced by ISG15, which showed greater upregulation in smokers compared to non-smokers. Similar changes in gene expression patterns for TRF2, POT1, hTERT, and ISG15 were observed in blood and buccal swab samples from smokers compared to non-smokers. The results from this study provide insight into the mechanisms behind smoking causing telomere shortening and how this may contribute to the induction of inflammation and/or tumorigenesis, which may lead to comorbidities in smokers.

UNASSIGNED: TRF1, TRF2, and TERT (Telomerase reverse transcriptase) are telomere-associated factors that regulate telomere length. Genetic changes in these genes may be associated with cancer pathogenesis; however, this relationship has not yet been comprehensively elucidated in lung cancer.
UNASSIGNED: : Exploring the clinicopathologic and prognostic values of TRF1, TRF2, and TERT mRNA expression in non-small cell lung cancers (NSCLC).
UNASSIGNED: : The clinical significance of TRF1, TRF2, and TERT expression in 141 patients with NSCLC was investigated. Additionally, these findings were supported by the open big data from The Cancer Genome Atlas (TCGA).
UNASSIGNED: : TRF1 and TRF2 expression levels tended to be associated with smoking, and TERT expression was positively correlated with age. The survival analysis showed that TRF1 expression predicted a better prognosis for squamous cell carcinoma (SCC), whereas TRF2 expression was associated with a shorter survival in adenocarcinoma. TCGA data also showed a better prognosis for SCC with TRF1 expression. However, the TRF2 results were not in agreement with our data.
UNASSIGNED: : We present the clinical and prognostic values of TRF1, TRF2, and TERT expression in NSCLC tissues and TCGA. Our findings suggest that TRF1 expression is a possible prognostic marker for NSCLC, particularly SCC.

Biologists aim to explain patterns of growth, reproduction and ageing that characterize life histories, yet we are just beginning to understand the proximate mechanisms that generate this diversity. Existing research in this area has focused on telomeres but has generally overlooked the telomere\'s most direct mediator, the shelterin protein complex. Shelterin proteins physically interact with the telomere to shape its shortening and repair. They also regulate metabolism and immune function, suggesting a potential role in life history variation in the wild. However, research on shelterin proteins is uncommon outside of biomolecular work. Intraspecific analyses can play an important role in resolving these unknowns because they reveal subtle variation in life history within and among populations. Here, we assessed ecogeographic variation in shelterin protein abundance across eight populations of tree swallow (Tachycineta bicolor) with previously documented variation in environmental and life history traits. Using the blood gene expression of four shelterin proteins in 12-day-old nestlings, we tested the hypothesis that shelterin protein gene expression varies latitudinally and in relation to both telomere length and life history. Shelterin protein gene expression differed among populations and tracked non-linear variation in latitude: nestlings from mid-latitudes expressed nearly double the shelterin mRNA on average than those at more northern and southern sites. However, telomere length was not significantly related to latitude. We next assessed whether telomere length and shelterin protein gene expression correlate with 12-day-old body mass and wing length, two proxies of nestling growth linked to future fecundity and survival. We found that body mass and wing length correlated more strongly (and significantly) with shelterin protein gene expression than with telomere length. These results highlight telomere regulatory shelterin proteins as potential mediators of life history variation among populations. Together with existing research linking shelterin proteins and life history variation within populations, these ecogeographic patterns underscore the need for continued integration of ecology, evolution and telomere biology, which together will advance understanding of the drivers of life history variation in nature.

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.

Telomere repeat binding factor 2 (TRF2), a critical element of the shelterin complex, plays a vital role in the maintenance of genome integrity. TRF2 overexpression is found in a wide range of malignant cancers, whereas its down-regulation could cause cell death. Despite its potential role, the selectively small-molecule inhibitors of TRF2 and its therapeutic effects on liver cancer remain largely unknown. Our clinical data combined with bioinformatic analysis demonstrated that TRF2 is overexpressed in liver cancer and that high expression is associated with poor prognosis. Flavokavain B derivative FKB04 potently inhibited TRF2 expression in liver cancer cells while having limited effects on the other five shelterin subunits. Moreover, FKB04 treatment induced telomere shortening and increased the amounts of telomere-free ends, leading to the destruction of T-loop structure. Consequently, FKB04 promoted liver cancer cell senescence without modulating apoptosis levels. In corroboration with these findings, FKB04 inhibited tumor cell growth by promoting telomeric TRF2 deficiency-induced telomere shortening in a mouse xenograft tumor model, with no obvious side effects. These results demonstrate that TRF2 is a potential therapeutic target for liver cancer and suggest that FKB04 may be a selective small-molecule inhibitor of TRF2, showing promise in the treatment of liver cancer.

G-rich sequences have the potential to fold into G-quadruplexes (GQs). G-quadruplexes, particularly those positioned in the regulatory regions of proto-oncogenes, have recently garnered attention in anti-cancer drug design. A thermal FRET assay was employed to conduct preliminary screening of various alkaloids, aiming to identify stronger interactions with a specific set of G-rich double-labeled oligonucleotides in both K + and Na + buffers. These oligonucleotides were derived from regions associated with Kit, Myc, Ceb, Bcl2, human telomeres, and potential G-quadruplex forming sequences found in the Nrf2 and Trf2 promoters. Palmatine generally increased the stability of different G-rich sequences into their folded GQ structures, more or less in a concentration dependent manner. The thermal stability and interaction of palmatine was further studied using transition FRET (t-FRET), CD and UV-visible spectroscopy and molecular dynamics simulation methods. Palmatine showed the strongest interaction with T RF2 in both K+ and Na+ buffers even at equimolar concentration ratio. T-FRET studies revealed that palmatine has the potential to disrupt double-strand formation by the T RF2 sequence in the presence of its complementary strand. Palmatine exhibits a stronger interaction with G-rich strand DNA, promoting its folding into G-quadruplex structures. It is noteworthy that palmatine exhibits the strongest interaction with T RF2, which is the shortest sequence among the G-rich oligonucleotides studied, featuring only one nucleotide for two of its loops. Palmatine represents a suitable structure for drug design to develop more specific ligands targeting G-quadruplexes. Whether palmatine can also affect the expression of the T RF2 gene requires further studies.Communicated by Ramaswamy H. Sarma.

The TRF2 shelterin component is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, causing telomeric DNA damage. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2TRFH domain. We identify APOD53 as our most promising compound, as it consistently induces a telomeric DNA damage response in cancer cell lines. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2TRFH domain and induces phenotypes consistent with TRF2TRFH domain mutants. These include induction of a telomeric DNA damage response, increased telomeric replication stress, and impaired recruitment of RTEL1 and SLX4 to telomeres. We demonstrate that APOD53 impairs cancer cell growth and find that co-treatment with APOD53 can exacerbate telomere replication stress caused by the G4 stabilizer RHPS4 and low dose aphidicolin (APH).

To investigate the effects and underlying molecular mechanisms of the interaction between the non-structural protein 1 (NS1) and nucleolar and coiled-body phosphoprotein 1 (NOLC1) on rRNA synthesis through nucleolar telomeric repeat-binding factor 2 (TRF2) under nucleolar stress in avian influenza A virus infection. The analysis of TRF2 ties into the exploration of ribosomal protein L11 (RPL11) and mouse double minute 2 (MDM2) because TRF2 has been found to interact with NOLC1, and the RPL11-MDM2 pathway plays an important role in nucleolar regulation and cellular processes. Both human embryonic kidney 293T cells and human lung adenocarcinoma A549 cells were transfected with the plasmids pCAGGS-HA and pCAGGS-HA-NS1, respectively. In addition, A549 cells were transfected with the plasmids pEGFP-N1, pEGFP-N1-NS1, and pDsRed2-N1-TRF2. The cell cycle was detected by flow cytometry, and coimmunoprecipitation was applied to examine the interactions between different proteins. The effect of NS1 on TRF2 was detected by immunoprecipitation, and the colocalization of NOLC1 and TRF2 or NS1 and TRF2 was visualized by immunofluorescence. Quantitative real-time PCR was conducted to detect the expression of the TRF2 and p21. There is a strong interaction between NOLC1 and TRF2, and the colocalization of NOLC1 and TRF2 in the nucleus. The protein expression of NOLC1 in A549-HA-NS1 cells was lower than that in A549-HA cells, which was accompanied by the upregulated protein expression of p53 in A549-HA-NS1 cells (all p < .05). TRF2 was scattered throughout the nucleus without clear nucleolar aggregation. RPL11 specifically interacted with MDM2 in the NS1 group, and expression of the p21 gene was significantly increased in the HA-NS1 group compared with the HA group (p < .01). NS1 protein can lead to the reduced aggregation of TRF2 in the nucleolus, inhibition of rRNA expression, and cell cycle blockade by interfering with the NOLC1 protein and generating nucleolar stress.