Search

Genome-wide loss of DNA methylation is commonly observed in human cancers, but its impact on the tumor transcriptome remains ill-defined. Previous studies demonstrated that this epige-netic alteration causes aberrant activation of a germline-specific gene expression program. Here, we examined if DNA hypomethylation in tumors also leads to de-repression of gene clusters with other tissue specificities. To this end, we explored transcriptomic and methylomic datasets from human lung adenocarcinoma (LUAD) cell lines, normal lung, and lung alveolar type II cells, con-sidered as the origin of LUAD. Interestingly, DNA demethylation in LUAD cell lines was associ-ated with activation of not only germline-specific (CG) genes, but also gene clusters displaying specific expression in the gastrointestinal tract (GI), or in stratified epithelia (SE). Consistently, genes from all three clusters showed highly specific patterns of promoter methylation among normal tissues and cell types, and were generally sensitive to induction by a DNA demethylating agent. Analysis of TCGA datasets confirmed that demethylation and activation of CG, GI and SE genes also occurs in vivo in LUAD tumor tissues, in association with global genome hypomethyl-ation. For genes of the GI cluster, we demonstrated that HNF4A is a necessary factor for tran-scriptional activation following promoter demethylation. Interestingly, expression of several SE genes, in particular FAM83A, correlated with both tumor grade and reduced patient survival. Together, our study uncovers novel cell-type specific gene clusters that become aberrantly acti-vated in LUAD tumors in association with genome-wide hypomethylation.

Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment.

Tumor development is characterized by both genetic and epigenetic alterations that lead to gene expression changes. One of such transcriptomic changes is the aberrant activation of a group of germline-specific genes in a variety of tumors of somatic origin. The spurious activation of this group of genes is due to the DNA demethylation of their promoter region in tumor cells, a phenomenon associated with the global loss of DNA methylation that often accompanies tumorigenesis. Several of these “cancer-germline” genes were found to exert pro-tumoral functions, or evoke an immune response. Cancer-germline genes are therefore attracting much interest, as they constitute promising targets for anti-cancer therapies with the hope of limited side effects due to their highly restricted pattern of expression. Whether additional tissue-specific gene clusters become activated in tumors, be it by DNA demethylation or not, remains unanswered. Most studies, indeed, have used transcriptomic approaches that were biased towards testis-specific genes. In the present work, we aimed to fill this gap by combining omics data of lung adenocarcinoma (LUAD) tissues and cell lines, as well as normal lung and alveolar type II cells (AT2), the cells of origin of LUAD, without imposing any criteria of expression specificity to the ectopically activated genes we were looking for. We divided our work in two complementary axes. In the first part, we asked whether the global loss of DNA demethylation in tumors is also associated with the ectopic activation of somatic-specific gene clusters in LUAD. We found that, indeed, promoter DNA demethylation in these cells leads to the transcriptional induction of genes displaying selective expression in gastro-intestinal tissues (colon and small intestine), and of genes specific to stratified squamous epithelia (esophagus, skin and vagina). Interestingly, our data show that ectopic activation of this latter group of genes in LUAD is associated with poor progno, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAFV600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy and tumor progression. Histological follow-up by anatomo-pathologists reveals that 2/3 of surgically-removed thyroids do not present malignant lesions. Continued fundamental research into the molecular mechanisms of TC downstream of BRAFV600E remains thus central to better understand the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAFV600E is specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAFV600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAFV600E dependent TC. Finally, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages a PTC mouse model and the relevance of these observations in human thyroid tissues.

In this issue of Molecular Cell, Kaminski et al. and Yadav et al. demonstrate that RAD51AP1 and TERRA non-coding RNA positively regulate the alternative mechanism of telomere maintenance by promoting D-loop formation and chromatin-directed mechanisms to suppress transcription-collision events during ALT telomere synthesis.

Telomeres protect the ends of our chromosomes. The replicative immortality of cancer cells depends on the activation of a telomere maintenance mechanism: the telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT is activated in about 10% of tumors but this prevalence increases greatly in pediatric tumors. The latter are in need of targeted therapies but, to date, there is still no therapy targeting ALT. In this thesis, we identified TSPYL5 (testis-specific Y-encoded-like protein 5) as a critical protein for ALT+ cell viability, opening new perspectives for anti-ALT targeted therapies. We also established a mouse xenograft model with human ALT+ cancer cells to test the efficiency of future anti-ALT compounds. The lack of good tools for the diagnosis of ALT also led us to the development of a new robust test on tumor sections. Finally, we provided additional evidence that replicative stress induces some characteristics of ALT telomeres., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Telomere elongation is coupled with genome replica- tion, raising the question of the repair of short telom- eres in post-mitotic cells. We investigated the fate of a telomere-repeat capped end that mimics a single short telomere in quiescent fission yeast cells. We show that telomerase is able to elongate this single short telomere during quiescence despite the bind- ing of Ku to the proto-telomere. While Taz1 and Rap1 repress telomerase in vegetative cells, both shelterin proteins are required for efficient telomere extension in quiescent cells, underscoring a distinct mode of telomerase control. We further show that Rad3ATR and Tel1ATM are redundantly required for telomere elongation in quiescence through the phosphoryla- tion of Ccq1 and that Rif1 and its associated-PP1 phosphatases negatively regulate telomerase activ- ity by opposing Ccq1 phosphorylation. The distinct mode of telomerase regulation in quiescent fission yeast cells may be relevant to that in human stem and progenitor cells.

More than two decades ago, the G-rich sequences of telomeric DNA were proposed to be preferential targets of oxidative damage with potential detrimental impact on telomere length and integrity. If not breathing is not an option (Markkanen, 2017), understanding how cellular respiration impacts on telomere regulation remains a challenging question that has, so far, not been addressed in the context of naturally-induced oxidative stress. ATP production, through oxidative phosphorylation in mitochondria, is the major endogenous source of ROS in cells. Both oxygen consumption and the proton gradient strength across the inner mitochondrial membrane regulate ROS production. Here, using a transmitochondrial cybrid approach with mitochondria isolated from donors with either very long (>P90), average or short telomeres, we evaluated the impact of the mitochondrial genome on oxidative stress and telomere length in Rho0 recipient cells. Our data revealed that specific mtDNA-encoded mitochondrial protein variants regulate oxidative stress and telomere length. We will discuss how these variants may impact on ROS production and offer a maternal inheritance of telomere length.

An inappropriate response to progestogens in the human endometrium can result in fertility issues and jeopardize progestin-based treatments against pathologies such as endometriosis. PGRMC1 can mediate progesterone response in the breast and ovaries but its endometrial functions remain unknown. AG-205 is an alleged PGRMC1 inhibitor but its specificity was recently questioned. We added AG-205 in the cultures of two endometrial cell lines and performed a transcriptomic comparison. AG-205 significantly increased expression of genes coding enzymes of the cholesterol biosynthetic pathway or of steroidogenesis. However, these observations were not reproduced with cells transfected with siRNA against PGRMC1 or its related proteins (MAPRs). Furthermore, AG-205 retained its ability to increase expression of selected target genes even when expression of PGRMC1 or all MAPRs was concomitantly downregulated, indicating that neither PGRMC1 nor any MAPR is required to mediate AG-205 effect. In conclusion, although AG-205 has attractive effects encouraging its use to develop therapeutic strategies, for instance against breast cancer, our study delivers two important warning messages. First, AG-205 is not specific for PGRMC1 or other MAPRs and its mechanisms of action remain unclear. Second, due to its effects on genes involved in steroidogenesis, its use may increase the risk for endometrial pathologies resulting from imbalanced hormones concentrations.

About 10% of cancer cells employ the Alternative Lengthening of Telomeres (ALT) pathway instead of re-activating the hTERT subunit of telomerase. The hTR RNA subunit is also abnormally silenced in some ALT+ cells not expressing hTERT, suggesting a possible negative impact of hTR on ALT, independently of telomerase. Accordingly, ectopically-expressed hTR reduces p-RPAS33 levels at ALT telomeres by promoting the hnRNPA1- and DNA-PK-dependent depletion of telomere-bound RPA. The resulting defective ATR signaling at telomeres impairs the recruitment of homologous recombination protein, RAD51. This induces ALT telomere fragility, increases POLD3-dependent C-circle production and promotes the recruitment of 53BP1 DNA damage marker at telomeres. In ALT+ cells that naturally retain hTR expression, NHP2 H/ACA ribonucleoprotein levels are downregulated, likely to restrain DNA damage response (DDR) activation at telomeres through a reduced recruitment of 53BP1. This unexpected role of NHP2 is independent from hTR’s non-canonical function in modulating telomeric p-RPAS33. Collectively, our study shines new light on the interference between telomerase- and ALT-pathways and unravels a crucial role for hTR and NHP2 in regulating DDR at ALT telomeres.

DNA methylation is an epigenetic mark associated with gene repression. It is now well established that tumor development involves alterations in DNA methylation patterns, which include both gains (hypermethylation) and losses (hypomethylation) of methylation marks in different genomic regions. The mechanisms underlying these two opposite, yet co-existing, alterations in tumors remain unclear. While studying the human MAGEA6/GABRA3 gene locus, we observed that DNA hypomethylation in tumor cells can lead to the activation of a long transcript (CT-GABRA3) that overlaps downstream promoters (GABRQ and GABRA3) and triggers their hypermethylation. Overlapped promoters displayed increases in H3K36me3, a histone mark known to be deposited during progression of the transcription machinery and to stimulate de novo DNA methylation. Consistent with such a processive mechanism, increases in H3K36me3 and DNA methylation were observed over the entire region covered by the CT-GABRA3 overlapping transcript. Importantly, experimental induction of CT-GABRA3 by depletion of DNMT1 DNA methyltransferase, resulted in a similar pattern of increased DNA methylation in the MAGEA6/GABRA3 locus. Bioinformatics analyses in lung cancer datasets identified other genomic loci displaying this process of coupled DNA hypo- and hypermethylation. In several of these loci, DNA hypermethylation affected tumor suppressor genes, e.g. RERG and PTPRO. Together, our work reveals that focal DNA hypomethylation in tumors can indirectly contribute to hypermethylation of nearby promoters through activation of overlapping transcription, and establishes therefore an unsuspected connection between these two opposite epigenetic alterations.

Human telomerase progressively emerged as a multifaceted ribonucleoprotein complex with additional functions beyond telomeric repeat synthesis. Both the hTERT catalytic subunit and the hTR long non-coding RNA (lncRNA) subunit are engaged in highly regulated cellular pathways that, together, contribute to cell fitness and protection against apoptosis. We recently described a new role for hTR in regulating the abundance of replication protein A at telomeres, adding to the growing repertoire of hTR's functions. Here, we focus on the non-canonical roles of hTR and discuss them in the context of the structural elements of the lncRNA. We propose that some functions of hTR may compete amongst each other through distinct interactions with its partners, proteins or mRNAs. We postulate that hTR's non-canonical functions may be highly relevant in the context of normal somatic cells that naturally silence hTERT gene, while keeping hTR expression.

OBJECTIVES: Because a significant fraction of patients with lupus nephritis (LN) develops renal impairment, there is a need to better understand the mechanisms underlying disease progression. Here, we assessed for cellular senescence in the LN kidney, and its association with disease severity and outcome. METHODS: We enumerated the number of cells positive for p16INK4a protein, a marker of cellular senescence, by immunohistochemistry followed by digital quantification, on renal biopsies from 40 patients with active LN. We tested for an association of p16INK4a with renal fibrosis, CD8+ T cell infiltration, systemic disease and renal function at baseline and at 5 years. RESULTS: The presence of p16INK4a-positive cells was significantly associated with lower estimated glomerular filtration rate at baseline and 5 years post-treatment, independently of patient demographics and systemic disease parameters. It was also associated with higher baseline renal fibrosis and CD8+ T cell infiltration. Interestingly, we observed marked spatial co-distribution of glomerular p16INK4a-positive cells with CD8+ T cells. CONCLUSION: We demonstrate, for the first time, that LN biopsies characterised by renal impairment display increased p16INK4a-positive cells, associated with higher fibrosis and CD8+ T cell infiltration. Cellular senescence may represent a kidney-intrinsic disease mechanism and potentially, a novel therapeutic target in LN.

The vast majority of adult cancer cells achieve cellular immortality by activating a telomere maintenance mechanism (TMM). While this is mostly achieved by the de-silencing of hTERT telomerase gene expression, an alternative homologous recombination-based and telomerase-independent mechanism, known as ALT (Alternative Lengthening of Telomeres), is frequently activated in a subset of tumors, including paediatric cancers. Being absent from normal cells, the ALT mechanism offers interesting perspectives for new targeted cancer therapies. To date, however, the development of better translationally applicable tools for ALT detection in tumor sections is still needed. Here, using a newly derived ALT-positive cancer cell mouse xenograft model, we extensively examined how the previously known ALT markers could be used as reliable tools for ALT diagnosis in tumor sections. We found that, together with the detection of ultra-bright telomeric signals (UBS), an ALT hallmark, native telomeric FISH, that detects single-stranded C-rich telomeric DNA, provides a very sensitive and robust tool for ALT diagnosis in tissues. We applied these assays to paediatric tumor samples and readily identified three ALT-positive tumors for which the TMM was confirmed by the gold-standard C-circle amplification assay. Although the latter offers a robust assay for ALT detection in the context of research laboratories, it is more difficult to set up in histopathological laboratories and could therefore be conveniently replaced by the combination of UBS detection and native telomeric FISH.

Telomere anchoring to nuclear envelope (NE) is a key feature of nuclear genome architecture. Peripheral localization of telomeres is important for chromatin silencing, telomere replication and for the control of inappropriate recombination. Here, we report that fission yeast quiescent cells harbor predominantly a single telomeric cluster anchored to the NE. Telomere cluster association to the NE relies on Rap1-Bqt4 interaction, which is impacted by the length of telomeric sequences. In quiescent cells, reducing telomere length or deleting bqt4, both result in an increase in transcription of the telomeric repeat-containing RNA (TERRA). In the absence of Bqt4, telomere shortening leads to deep increase in TERRA level and the concomitant formation of subtelomeric rearrangements (STEEx) that accumulate massively in quiescent cells. Taken together, our data demonstrate that Rap1-Bqt4-dependent telomere association to NE preserves telomere integrity in post-mitotic cells, preventing telomeric transcription and recombination. This defines the nuclear periphery as an area where recombination is restricted, creating a safe zone for telomeres of post-mitotic cells.

Despite the condensed nature of terminal sequences, the telomeres are transcribed into a group of noncoding RNAs, including the TElomeric Repeat-containing RNA (TERRA). Since the discovery of TERRA, its evolutionary conserved function has been confirmed, and its involvement in telomere length regulation, heterochromatin establishment, and telomere recombination has been demonstrated. We previously reported that TERRA is upregulated in quiescent fission yeast cells, although the global transcription is highly reduced. Elevated telomeric transcription was also detected when telomeres detach from the nuclear periphery. These intriguing observations unveil unexpected facets of telomeric transcription in arrested cells. In this review, we present the different aspects of TERRA transcription during quiescence and discuss their implications for telomere maintenance and cell fate.

Since the discovery of the anticancer properties of cisplatin, using new metals has progressively gained interest for the development of new therapeutic agents. Among them, polyazaaromatic complexes of Ru(II) have shown interesting photochemical and photophysical properties. Indeed, upon light irradiation, the oxidation and reduction potential of these compounds are enhanced and for some of them, photocleavage and/or photoadduct formation with DNA have been demonstrated [1]. In this context, we aim at designing new photo-oxidizing complexes of Ru(II) complexes for the targeting of G-quadruplex DNA. G-quadruplex (G4) consist in secondary structures adopted by guanines-rich nucleic acids. These structures are formed through the stacking of guanine tetrads (G-quartet), the latter resulting from the coplanar arrangement of four guanines. They have been found in the telomeres where they appear to play crucial biological roles [2]. Therefore, designing ligands able to selectively target G-quadruplex versus duplex DNA is of great interest. We report therein on the synthesis of new photo-oxidizing ruthenium(II) complexes (Figure 1). They are able to photo-induce oxidative damage to telomeric G-quadruplex DNA through photo-induced electron transfer (PET) with guanine. The affinity and selectivity for G-quadruplex DNA have been evidenced thanks to steady-state luminescence studies, BLI and molecular modelling. Finally, we demonstrated the cell internalization and the photo-cytotoxicity towards human osteosarcoma cells [3]. [1] B. Elias, A. Kirsch-De Mesmaeker, Coordination Chem. Review. 2006, 1627-1641. [2] H. Han, LH. Hurley, Trends in Pharmacological Sciences. 2000, 21, 136-142. [3] J. Weynand, A. Diman, M. Abraham, L. Marcélis, H. Jamet, A. Decottignies, J. Dejeu, E. Defrancq, B. Elias, Chem. Eur. J. 2018, 24, 19216-19227.

The human genome harbors many duplicated segments, which sometimes show very high sequence identity. This may complicate assignment during genome assembly. One such example is in Xq28, where the arrangement of 2 recently duplicated segments varies between genome assembly versions. The duplicated segments comprise highly similar genes, including MAGEA3 and MAGEA6, which display specific expression in testicular germline cells, and also become aberrantly activated in a variety of tumors. Recently, a new gene was identified, CT-GABRA3, the transcription of which initiates inside the segmental duplication but extends far outside. According to the latest genome annotation, CT- GABRA3 starts near MAGEA3, with which it shares a bidirectional promoter. In an earlier annotation, however, the duplicated segment was positioned in the opposite orientation, and CT-GABRA3 was instead coupled with MAGEA6. To resolve this discrepancy, and based on the contention that genes connected by a bidirectional promoter are almost always co-expressed, we decided to compare the expression profiles of CT-GABRA3, MAGEA3, and MAGEA6. We found that in tumor tissues and cell lines of different origins, the expression of CT-GABRA3 was better correlated with that of MAGEA6. Moreover, in a cellular model of experimental induction with a DNA demethylation agent, activation CT-GABRA3 was associated with that of MAGEA6, but not with that of MAGEA3. Together these results support a connection between CT-GABRA3 and MAGEA6 and illustrate how promoter-sharing genes can be exploited to resolve genome assembly uncertainties.

PARN, poly(A)-specific ribonuclease, regulates the turnover of mRNAs and the maturation and stabilization of the hTR RNA component of telomerase. Biallelic PARN mutations were associated with Høyeraal–Hreidarsson (HH) syndrome, a rare telomere biology disorder that, because of its severity, is likely not exclusively due to hTR down-regulation. Whether PARN deficiency was affecting the expression of telomere-related genes was still unclear. Using cells from two unrelated HH individuals carrying novel PARN mutations and a human PARN knock-out (KO) cell line with inducible PARN complementation, we found that PARN deficiency affects both telomere length and stability and down-regulates the expression of TRF1, TRF2, TPP1, RAP1, and POT1 shelterin transcripts. Down-regulation of dyskerin-encoding DKC1 mRNA was also observed and found to result from p53 activation in PARN-deficient cells. We further showed that PARN deficiency compromises ribosomal RNA biogenesis in patients’ fibroblasts and cells from heterozygous Parn KO mice. Homozygous Parn KO however resulted in early embryonic lethality that was not overcome by p53 KO. Our results refine our knowledge on the pleiotropic cellular consequences of PARN deficiency

A significant fraction (~10%) of cancer cells maintain their telomere length via a telomerase-independent mechanism known as Alternative Lengthening of Telomeres (ALT). Currently, there are no known molecular, ALT-specific, therapeutic target. We have identified TSPYL5 (Testis Specific Y-encoded-Like Protein 5) as a PML body component, co-localizing with ALT telomeres and critical for ALT+ cell viability. TSPYL5 was described as an inhibitor of the USP7 deubiquitinase. We report that TSPYL5 prevents the poly-ubiquitination of POT1 – a shelterin component – and protects POT1 from proteasomal degradation, exclusively in ALT+ cells. USP7 depletion rescued POT1 poly-ubiquitination and loss, suggesting that the deubiquitinase activates POT1 E3 ubiquitin ligase(s). Similarly, PML depletion suppressed POT1 poly-ubiquitination, suggesting an interplay between USP7 and PML to trigger POT1 degradation in TSPYL5-depleted ALT+ cells. We demonstrate that ALT telomeres need to be protected from POT1 degradation in ALT-associated PML bodies, and identify TSPYL5 as a ALT+ cancer-specific therapeutic target.

An estimated 10% of cancer cells maintain their telomeres with a telomerase-independent mechanism known as Alternative Lengthening of Telomeres, or ALT, for which no specific molecular target has been identified yet. Here, we unraveled novel roles for TSPYL5 (Testis Specific Y-encoded-Like Protein 5) in preventing proteasomal degradation of the shelterin component POT1 in ALT+ cells, but not in telomerase-expressing cells or normal fibroblasts. We found that TSPYL5 is a novel PML body component and protects ALT+ cells from telomere deprotection and cell death by titrating away the USP7 deubiquitinase, a TSPYL5-inhibited and PML body-associated protein, from the POT1 interaction partner, TPP1. Co-localization of ALT telomeres with PML bodies is responsible for the sensitivity to TSPYL5 depletion as PML depletion fully rescued POT1 loss. Our discovery that TSPYL5 is required for ALT+ cell viability by keeping POT1 offers novel therapeutic perspectives for ALT+ cancers.

An estimated 10% of cancer cells maintain their telomeres with a telomerase-independent mechanism known as Alternative Lengthening of Telomeres, or ALT, for which no specific molecular target has been identified yet. Here, we unraveled novel roles for TSPYL5 (Testis Specific Y-encoded-Like Protein 5) in preventing proteasomal degradation of the shelterin component POT1 in ALT+ cells, but not in telomerase-expressing cells or normal fibroblasts. We found that TSPYL5 is a novel PML body component and protects ALT+ cells from telomere deprotection and cell death by titrating away the USP7 deubiquitinase, a TSPYL5-inhibited and PML body-associated protein, from the POT1 interaction partner, TPP1. Co-localization of ALT telomeres with PML bodies is responsible for the sensitivity to TSPYL5 depletion as PML depletion fully rescued POT1 loss. Our discovery that TSPYL5 is required for ALT+ cell viability by keeping POT1 offers novel therapeutic perspectives for ALT+ cancers.

As the world demographic structure is getting older, highlighting strategies to counteract age-related diseases is a major public health concern. Telomeres are nucleoprotein structures that serve as guardians of genome stability by ensuring protection against both cell death and senescence. A hallmark of biological aging, telomere health is determined throughout the lifespan by a combination of both genetic and non-genetic influences. This review summarizes data from recently published studies looking at the effect of lifestyle variables such as nutrition and physical activity on telomere dynamics.

Among all small mols. that interfere with DNA replication, photodynamic therapeutic agents are of key interest in developing new strategies to design more specific and efficient anti-cancer treatments. Compared to classical chemotherapeutic agents, their activity can be finely controlled thanks to the light-triggering of their photoreactivity. Usually, the mechanism of photodynamic therapy relies on the generation of singlet oxygen by energy transfer from a photosensitizer. However, the activity of these type-II photosensitizers is significantly limited in hypoxic tumors. Therefore, the development of Type-I photosensitizing agents, which do not rely on the prodn. of ROS, is highly desirable. In this context, we developed new iridium(III) complexes which are able to photoreact with biomols.; namely, our Ir(III) complexes can oxidize guanine residues under visible light irradn. We report the synthesis and the extensive photophys. characterization of four new Ir(III) complexes. In addn. to an extensive exptl. and theor. study of the photophysics of these complexes, we characterize their photoreactivity towards model redox-active targets and the relevant biol. target, the guanine base. We demonstrate that photo-induced electron transfer takes place between the excited Ir(III) complex and guanine which leads to the formation of stable photoproducts, indicating that the targeted guanine is irreversibly damaged. These results pave the way to the elaboration of new Type-I photosensitizers for targeting cancerous cells.

Since the discovery of the anticancer properties of cisplatin, using new metals has progressively gained interest for the development of new therapeutic agents [1,2]. Among them, polyazaaromatic complexes of Ru(II) have shown interesting photochemical and photophysical properties. Indeed, upon light irradiation, the oxidation and reduction potential of these compounds are enhanced and for some of them, photocleavage and/or photoadduct formation with DNA have been demonstrated [3]. In this context, we aim at designing new photo-oxidizing complexes of Ru(II) complexes for the targeting of G-quadruplex DNA. G-quadruplex (G4) consist in secondary structures adopted by guanines-rich nucleic acids. These structures are formed through the stacking of guanine tetrads (G-quartet), the latter resulting from the coplanar arrangement of four guanines. They have been found in the telomeres where they appear to play crucial biological roles. Therefore, designing ligands able to selectively target G-quadruplex versus duplex DNA is of great interest. We report therein on the synthesis of new photo-oxidizing ruthenium(II) complexes [4]. They are able to photo-induce oxidative damage to telomeric G-quadruplex DNA through photo-induced electron transfer (PET) with guanine. The affinity and selectivity for G-quadruplex DNA have been evidenced thanks to steady-state luminescence studies, BLI and molecular modelling. Finally, we demonstrated the cell internalization and the photo-cytotoxicity towards human osteosarcoma cells. References [1] B. Rosenberg et al., Nature 2017, 222, 385-386. [2] MR. Gill et al., Chem. Soc. Rev. 2012, 41, 3179-3192. [3] B. Elias et al., Coordination Chem. Review. 2006, 1627-1641. [4] J. Weynand et al., Chem. Eur. J. 2018, in press.

The use of metal in medicine began with the serendipitous discovery of the anticancer properties of cisplatin.1 Since then, using new metals has progressively gained interest for the development of new therapeutic agents.2 Among them, polyazaaromatic complexes of Ru(II) have shown interesting photochemical and photophysical properties. Indeed, upon light irradiation, the oxidation and reduction potential of these compounds are enhanced and for some of them, photocleavage and/or photoadduct formation with DNA have been demonstrated.3 In this context, we aim at designing new photo-oxidizing complexes of Ru(II) complexes for the targeting of G-quadruplex DNA. G-quadruplex (G4) consist in particular secondary structures adopted by guanines-rich nucleic acids These structures are formed through the stacking of guanine tetrads (G-quartet), the latter resulting from the coplanar arrangement of four guanines.4 They have been found in the telomeres where they appear to play crucial biological roles. Therefore, designing ligands able to selectively target G-quadruplex versus duplex DNA is of great interest. We report therein on the synthesis of new photo-oxidizing ruthenium complexes. They are able to photo-induce oxidative damage to telomeric G-quadruplex DNA through photo-induced electron transfer (PET) with guanine. The affinity and selectivity for G-quadruplex DNA have been evidenced thanks to steady-state luminescence studies, BLI and molecular modelling. Finally, we demonstrated the cell internalization and the photo-cytotoxicity towards human osteosarcoma cells, which do not express telomerase.

Among all small molecules that interfere with DNA replication, photodynamic therapeutic agents are of key interest in developing new strategies to design more specific and efficient anti-cancer treatments. Compared to classical chemotherapeutic agents, their activity can be finely controlled thanks to the light-triggering of their photoreactivity. Usually, the mechanism of photodynamic therapy relies on the generation of singlet oxygen by energy transfer from a photosensitizer. However, the activity of these type-II photosensitizers is significantly limited in hypoxic tumors. Therefore, the development of Type-I photosensitizing agents, which do not rely on the production of ROS, is highly desirable. In this context, we developed new iridium(III) complexes which are able to photoreact with biomolecules; namely, our Ir(III) complexes can oxidize guanine residues under visible light irradiation. We report the synthesis and the extensive photophysical characterization of four new Ir(III) complexes. In addition to an extensive experimental and theoretical study of the photophysics of these complexes, we characterize their photoreactivity towards model redox-active targets and the relevant biological target, the guanine base. We demonstrate that photo-induced electron transfer takes place between the excited Ir(III) complex and guanine which leads to the formation of stable photoproducts, indicating that the targeted guanine is irreversibly damaged. These results pave the way to the elaboration of new Type-I photosensitizers for targeting cancerous cells.

Mammalian CST (CTC1-STN1-TEN1) complex fulfills numerous functions including rescue of the stalled replication forks and termination of telomerase action. In fission yeast lacking the CTC1 ortholog, the Stn1-Ten1 complex restricts telomerase action via its sumoylation-mediated interaction with Tpz1TPP1. We identify a small ubiquitin-like modifier (SUMO)-interacting motif (SIM) in the carboxyl-terminal part of Stn1 and show that this domain is crucial for SUMO and Tpz1-SUMO interactions. Point mutations in the SIM (Stn1-226) lead to telomere elongation, impair Stn1-Ten1 recruitment to telomeres, and enhance telomerase binding, revealing that Stn1 SIM domain contributes to the inhibition of telomerase activity at chromosome ends. Our results suggest that Stn1-Ten1 promotes DNA synthesis at telomeres to limit single-strand DNA accumulation. We further demonstrate that Stn1 functions in the replication of telomeric and subtelomeric regions in a Taz1-independent manner. Genetic analysis reveals that misregulation of origin firing and/or telomerase inhibition circumvents the replication defects of the stn1-226 mutant. Together, our results show that the Stn1-Ten1 complex has a dual function at telomeres by limiting telomerase action and promoting chromosome end replication.

Global loss of DNA methylation is frequently observed in the genome of human tumors. Although this epigenetic alteration is clearly associated with cancer progression, the way it exerts its pro-tumoral effect remains incompletely understood. A remarkable consequence of DNA hypomethylation in tumors is the aberrant activation of “cancer-germline” genes (also known as “cancer-testis“ genes), which comprise a diverse group of germline-specific genes that use DNA methylation as a primary mechanism for repression in normal somatic tissues. Here we review the evidence that such cancer-germline genes contribute to key processes of tumor development. Notably, several cancer-germline genes were found to stimulate oncogenic pathways involved in cell proliferation (SSX, DDX43, MAEL, PIWIL1), angiogenesis (DDX53), immortality (BORIS/CTCFL), and metastasis (CT-GABRA3). Others appear to inhibit tumor suppressor pathways, including those controlling growth inhibition signals (MAGEA11, MAGEB2), apoptosis (MAGEA2, MAGEC2), and genome integrity (HORMAD1, NXF2). Cancer-germline genes were also implicated in the regulation of tumor metabolism (MAGEA3/MAGEA6). Together, our survey substantiates the concept that DNA hypomethylation promotes tumorigenesis via transcriptional activation of oncogenes. Importantly, considering their highly restricted pattern of expression, cancer-germline genes may represent valuable targets for the development of anti-cancer therapies with limited side effects.

In response to metabolic or environmental stress, cells activate powerful defense mechanisms to prevent the formation and accumulation of toxic protein aggregates. The main orchestrator of this cellular response is HSF1 (heat shock factor 1), a transcription factor involved in the up-regulation of protein-coding genes with protective roles. It has become very clear that HSF1 has a broader function than initially expected. Indeed, our previous work demonstrated that, upon stress, HSF1 activates the transcription of a non-coding RNA, named Satellite III, at pericentromeric heterochromatin. Here, we observe that the function of HSF1 extends to telomeres and identify subtelomeric DNA as a new genomic target of HSF1. We show that the binding of HSF1 to subtelomeric regions plays an essential role in the upregulation of non-coding TElomeric Repeat containing RNA (TERRA) transcription upon heat shock. Importantly, our data show that telomere integrity is impacted by heat shock and that telomeric DNA damages are markedly enhanced in HSF1 deficient cells. Altogether, our findings reveal a new direct and essential function of HSF1 in the transcriptional activation of TERRA and in telomere protection upon stress.

It is now well established that alterations in DNA methylation patterns contribute to tumor development. Both gains (DNA hypermethylation) and losses (DNA hypomethylation) of this repressive mark are observed in tumors. Our group demonstrated that DNA hypomethylation in tumors induces transcriptional activation of a defined group of genes, which normally show specific expression in the germline. These genes were grouped under the term "cancer-germline" genes (CG genes). The main goal of the laboratory is to characterize the epigenetic regulation and functional roles of CG genes. Our work focused on the GABRA3 gene locus, where we discovered the existence of two overlapping transcripts: BT-GABRA3, which is expressed specifically in brain and testis; and CT-GABRA3, starting ~250 kb upstream, which is expressed exclusively in testis. CT-GABRA3 (but not BT-GABRA3) exhibits typical features of a CG gene, as it shows promoter hypomethylation and transcriptional activation in various tumors. CT-GABRA3 carries a clustered pair of miRNAs (miR-105 and miR-767), which show concurrent expression in tumors. Interestingly, an independent group identified miR-105 as a crucial promoter of cancer metastasis, due to its ability to weaken vascular endothelial barriers following exosomal secretion. On the other hand, we demonstrated that miR-767 inhibits expression of TET1, a gene with tumor suppressive functions involved in epigenetic processes of DNA demodification. Moreover, our studies revealed that CT-GABRA3 hypomethylation/activation in tumors is correlated with hypermethylation of the downstream BT-GABRA3 promoter. The mechanism underlying this interdependent epigenetic alteration appears to involve deposition of the histone mark H3K36me3 on the entire CT-GABRA3 transcribed region, which includes the BT-GABRA3 promoter. Finally, we observed that in tumor cells where the BT-GABRA3 promoter is hypermethylated, the gene becomes sensitive to DNA demethylation, suggesting a process o, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2017

Unlimited replicative potential is one of the hallmarks of cancer cells. In melanoma, hTERT (telomerase reverse transcriptase) is frequently overexpressed because of activating mutations in its promoter, suggesting that telomerase is necessary for melanoma development. We observed, however, that a subset of melanoma metastases and derived cell lines had no telomere maintenance mechanism. Early passages of the latter displayed long telomeres that progressively shortened and fused before cell death. We propose that, during melanoma formation, oncogenic mutations occur in precursor melanocytes with long telomeres, providing cells with sufficient replicative potential, thereby bypassing the need to re-activate telomerase. Our data further support the emerging idea that long telomeres promote melanoma formation. These observations are important when considering anticancer therapies targeting telomerase.

Unlimited replicative potential is one of the hallmarks of cancer cells. In melanoma, hTERT (Telomerase Reverse Transcriptase) is frequently overexpressed because of activating mutations in its promoter, suggesting that telomerase is necessary for melanoma development. We observed however that a subset of melanoma metastases and derived cell lines had no telomere maintenance mechanism. Early passages of the latter displayed long telomeres that progressively shortened and fused before cell death. We propose that, during melanoma formation, oncogenic mutations occur in precursor melanocytes with long telomeres, providing cells with sufficient replicative potential, thereby bypassing the need to re-activate telomerase. Our data further support the emerging idea that long telomeres promote melanoma formation. These observations are important when considering anticancer therapies targeting telomerase.