Telomere-to-mitochondria signalling by ZBP1 mediates replicative crisis.

Cancers arise through the accumulation of genetic and epigenetic alterations that enable cells to evade telomere-based proliferative barriers and achieve immortality. One such barrier is replicative crisis-an autophagy-dependent program that eliminates checkpoint-deficient cells with unstable telomeres and other cancer-relevant chromosomal aberrations1,2. However, little is known about the molecular events that regulate the onset of this important tumour-suppressive barrier. Here we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as a regulator of the crisis program. A crisis-associated isoform of ZBP1 is induced by the cGAS-STING DNA-sensing pathway, but reaches full activation only when associated with telomeric-repeat-containing RNA (TERRA) transcripts that are synthesized from dysfunctional telomeres. TERRA-bound ZBP1 oligomerizes into filaments on the outer mitochondrial membrane of a subset of mitochondria, where it activates the innate immune adapter protein mitochondrial antiviral-signalling protein (MAVS). We propose that these oligomerization properties of ZBP1 serve as a signal amplification mechanism, where few TERRA-ZBP1 interactions are sufficient to launch a detrimental MAVS-dependent interferon response. Our study reveals a mechanism for telomere-mediated tumour suppression, whereby dysfunctional telomeres activate innate immune responses through mitochondrial TERRA-ZBP1 complexes to eliminate cells destined for neoplastic transformation.

Autophagic cell death restricts chromosomal instability during replicative crisis.

Replicative crisis is a senescence-independent process that acts as a final barrier against oncogenic transformation by eliminating pre-cancerous cells with disrupted cell cycle checkpoints1. It functions as a potent tumour suppressor and culminates in extensive cell death. Cells rarely evade elimination and evolve towards malignancy, but the mechanisms that underlie cell death in crisis are not well understood. Here we show that macroautophagy has a dominant role in the death of fibroblasts and epithelial cells during crisis. Activation of autophagy is critical for cell death, as its suppression promoted bypass of crisis, continued proliferation and accumulation of genome instability. Telomere dysfunction specifically triggers autophagy, implicating a telomere-driven autophagy pathway that is not induced by intrachromosomal breaks. Telomeric DNA damage generates cytosolic DNA species with fragile nuclear envelopes that undergo spontaneous disruption. The cytosolic chromatin fragments activate the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway and engage the autophagy machinery. Our data suggest that autophagy is an integral component of the tumour suppressive crisis mechanism and that loss of autophagy function is required for the initiation of cancer.

Selective pericentromeric heterochromatin dismantling caused by TP53 activation during senescence.

Cellular senescence triggers various types of heterochromatin remodeling that contribute to aging. However, the age-related mechanisms that lead to these epigenetic alterations remain elusive. Here, we asked how two key aging hallmarks, telomere shortening and constitutive heterochromatin loss, are mechanistically connected during senescence. We show that, at the onset of senescence, pericentromeric heterochromatin is specifically dismantled consisting of chromatin decondensation, accumulation of DNA breakages, illegitimate recombination and loss of DNA. This process is caused by telomere shortening or genotoxic stress by a sequence of events starting from TP53-dependent downregulation of the telomere protective protein TRF2. The resulting loss of TRF2 at pericentromeres triggers DNA breaks activating ATM, which in turn leads to heterochromatin decondensation by releasing KAP1 and Lamin B1, recombination and satellite DNA excision found in the cytosol associated with cGAS. This TP53-TRF2 axis activates the interferon response and the formation of chromosome rearrangements when the cells escape the senescent growth arrest. Overall, these results reveal the role of TP53 as pericentromeric disassembler and define the basic principles of how a TP53-dependent senescence inducer hierarchically leads to selective pericentromeric dismantling through the downregulation of TRF2.

DNA double-strand breaks lead to activation of hypermethylated in cancer 1 (HIC1) by SUMOylation to regulate DNA repair.

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene frequently epigenetically silenced in human cancers. HIC1 encodes a transcriptional repressor involved in the regulation of growth control and DNA damage response. We previously demonstrated that HIC1 can be either acetylated or SUMOylated on lysine 314. This deacetylation/SUMOylation switch is governed by an unusual complex made up of SIRT1 and HDAC4 which deacetylates and thereby favors SUMOylation of HIC1 by a mechanism not yet fully deciphered. This switch regulates the interaction of HIC1 with MTA1, a component of the NuRD complex and potentiates the repressor activity of HIC1. Here, we show that HIC1 silencing in human fibroblasts impacts the repair of DNA double-strand breaks whereas ectopic expression of wild-type HIC1, but not of nonsumoylatable mutants, leads to a reduced number of γH2AX foci induced by etoposide treatment. In this way, we demonstrate that DNA damage leads to (i) an enhanced HDAC4/Ubc9 interaction, (ii) the activation of SIRT1 by SUMOylation (Lys-734), and (iii) the SUMO-dependent recruitment of HDAC4 by SIRT1 which permits the deacetylation/SUMOylation switch of HIC1. Finally, we show that this increase of HIC1 SUMOylation favors the HIC1/MTA1 interaction, thus demonstrating that HIC1 regulates DNA repair in a SUMO-dependent way. Therefore, epigenetic HIC1 inactivation, which is an early step in tumorigenesis, could contribute to the accumulation of DNA mutations through impaired DNA repair and thus favor tumorigenesis.

Telomeres as hotspots for innate immunity and inflammation.

Aging is marked by the gradual accumulation of deleterious changes that disrupt organ function, creating an altered physiological state that is permissive for the onset of prevalent human diseases. While the exact mechanisms governing aging remain a subject of ongoing research, there are several cellular and molecular hallmarks that contribute to this biological process. This review focuses on two factors, namely telomere dysfunction and inflammation, which have emerged as crucial contributors to the aging process. We aim to discuss the mechanistic connections between these two distinct hallmarks and provide compelling evidence highlighting the loss of telomere protection as a driver of pro-inflammatory states associated with aging. By reevaluating the interplay between telomeres, innate immunity, and inflammation, we present novel perspectives on the etiology of aging and its associated diseases.

TZAP overexpression induces telomere dysfunction and ALT-like activity in ATRX/DAXX-deficient cells.

The appropriate regulation of telomere length homeostasis is crucial for the maintenance of genome integrity. The telomere-binding protein TZAP has been suggested to regulate telomere length by promoting t-circle and c-circle excisions through telomere trimming, yet the molecular mechanisms by which TZAP functions at telomeres are not understood. Using a system based on TZAP overexpression, we show that efficient TZAP recruitment to telomeres occurs in the context of open telomeric chromatin caused by loss of ATRX/DAXX independently of H3.3 deposition. Moreover, our data indicate that TZAP binding to telomeres induces telomere dysfunction and ALT-like activity, resulting in the generation of t-circles and c-circles in a Bloom-Topoisomerase IIIα-RMI1-RMI2 (BTR)-dependent manner.

The out-of-field dose in radiation therapy induces delayed tumorigenesis by senescence evasion.

A rare but severe complication of curative-intent radiation therapy is the induction of second primary cancers. These cancers preferentially develop not inside the planning target volume (PTV) but around, over several centimeters, after a latency period of 1-40 years. We show here that normal human or mouse dermal fibroblasts submitted to the out-of-field dose scattering at the margin of a PTV receiving a mimicked patient's treatment do not die but enter in a long-lived senescent state resulting from the accumulation of unrepaired DNA single-strand breaks, in the almost absence of double-strand breaks. Importantly, a few of these senescent cells systematically and spontaneously escape from the cell cycle arrest after a while to generate daughter cells harboring mutations and invasive capacities. These findings highlight single-strand break-induced senescence as the mechanism of second primary cancer initiation, with clinically relevant spatiotemporal specificities. Senescence being pharmacologically targetable, they open the avenue for second primary cancer prevention.

Telomeres and Cancer: Resolving the Paradox.

Decades of study on cell cycle regulation have provided great insight into human cellular life span barriers, as well as their dysregulation during tumorigenesis. Telomeres, the extremities of linear chromosomes, perform an essential role in implementing these proliferative boundaries and preventing the propagation of potentially cancerous cells. The tumor-suppressive function of telomeres relies on their ability to initiate DNA damage signaling pathways and downstream cellular events, ranging from cell cycle perturbation to inflammation and cell death. While the tumor-suppressor role of telomeres is undoubtable, recent advances have pointed to telomeres as a major source of many of the genomic aberrations found in both early- and late-stage cancers, including the most recently discovered mutational phenomenon of chromothripsis. Telomere shortening appears as a double-edged sword that can function in opposing directions in carcinogenesis. This review focuses on the current knowledge of the dual role of telomeres in cancer and suggests a new perspective to reconcile the paradox of telomeres and their implications in cancer etiology.

HIC1 (hypermethylated in cancer 1) SUMOylation is dispensable for DNA repair but is essential for the apoptotic DNA damage response (DDR) to irreparable DNA double-strand breaks (DSBs).

The tumor suppressor gene HIC1 (Hypermethylated In Cancer 1) encodes a transcriptional repressor mediating the p53-dependent apoptotic response to irreparable DNA double-strand breaks (DSBs) through direct transcriptional repression of SIRT1. HIC1 is also essential for DSB repair as silencing of endogenous HIC1 in BJ-hTERT fibroblasts significantly delays DNA repair in functional Comet assays. HIC1 SUMOylation favours its interaction with MTA1, a component of NuRD complexes. In contrast with irreparable DSBs induced by 16-hours of etoposide treatment, we show that repairable DSBs induced by 1 h etoposide treatment do not increase HIC1 SUMOylation or its interaction with MTA1. Furthermore, HIC1 SUMOylation is dispensable for DNA repair since the non-SUMOylatable E316A mutant is as efficient as wt HIC1 in Comet assays. Upon induction of irreparable DSBs, the ATM-mediated increase of HIC1 SUMOylation is independent of its effector kinase Chk2. Moreover, irreparable DSBs strongly increase both the interaction of HIC1 with MTA1 and MTA3 and their binding to the SIRT1 promoter. To characterize the molecular mechanisms sustained by this increased repression potential, we established global expression profiles of BJ-hTERT fibroblasts transfected with HIC1-siRNA or control siRNA and treated or not with etoposide. We identified 475 genes potentially repressed by HIC1 with cell death and cell cycle as the main cellular functions identified by pathway analysis. Among them, CXCL12, EPHA4, TGFßR3 and TRIB2, also known as MTA1 target-genes, were validated by qRT-PCR analyses. Thus, our data demonstrate that HIC1 SUMOylation is important for the transcriptional response to non-repairable DSBs but dispensable for DNA repair.

A novel role for DNA single-strand breaks in senescence and neoplastic escape of epithelial cells.

In contrast to fibroblasts, epithelial cells spontaneously escape from senescence and develop clones of mutated, transformed, and tumorigenic cells. Recently, we revealed that accumulation of unrepaired DNA single-strand breaks is a trigger of the p16 (CDKN2)-dependent cell cycle arrest pathway in senescent epithelial cells and also the mutagenic motor of post-senescence neoplastic escape.

Defective DNA single-strand break repair is responsible for senescence and neoplastic escape of epithelial cells.

The main characteristic of senescence is its stability which relies on the persistence of DNA damage. We show that unlike fibroblasts, senescent epithelial cells do not activate an ATM-or ATR-dependent DNA damage response (DDR), but accumulate oxidative-stress-induced DNA single-strand breaks (SSBs). These breaks remain unrepaired because of a decrease in PARP1 expression and activity. This leads to the formation of abnormally large and persistent XRCC1 foci that engage a signalling cascade involving the p38MAPK and leading to p16 upregulation and cell cycle arrest. Importantly, the default in SSB repair also leads to the emergence of post-senescent transformed and mutated precancerous cells. In human-aged skin, XRCC1 foci accumulate in the epidermal cells in correlation with a decline of PARP1, whereas DDR foci accumulate mainly in dermal fibroblasts. These findings point SSBs as a DNA damage encountered by epithelial cells with aging which could fuel the very first steps of carcinogenesis.