Senescence, defined by the cessation of cell proliferation, plays a critical and multifaceted role in breast cancer progression and treatment. Senescent cells produce senescence-associated secretory phenotypes (SASP) comprising inflammatory cytokines, chemokines, and small molecules, which actively shape the tumor microenvironment, influencing cancer development, progression, and metastasis. This review provides a comprehensive analysis of the types and origins of senescent cells in breast cancer, alongside their markers and detection methods. Special focus is placed on pharmacological strategies targeting senescence, including drugs that induce or inhibit senescence, their molecular mechanisms, and their roles in therapeutic outcomes when combined with chemotherapy and radiotherapy. By exploring these pharmacological interventions and their impact on breast cancer treatment, this review underscores the potential of senescence-targeting therapies to revolutionize breast cancer management.

Human exonuclease 1 (EXO1), a member of the structure-specific nuclease family, plays a critical role in maintaining genome stability by processing DNA double-strand breaks (DSBs), nicks, and replication intermediates during DNA replication and repair. As its exonuclease activity is essential for homologous recombination (HR) and replication fork processing, EXO1 has emerged as a compelling therapeutic target, especially in cancers marked by heightened DNA damage and replication stress. Through high-throughput screening of 45,000 compounds, we identified seven distinct chemical scaffolds that demonstrated effective and selective inhibition of EXO1. Representative compounds from two of the most potent scaffolds, C200 and F684, underwent a comprehensive docking analysis and subsequent site-directed mutagenesis studies to evaluate their binding mechanisms. Biochemical assays further validated their potent and selective inhibition of the EXO1 nuclease activity. Tumor cell profiling experiments revealed that these inhibitors exploit synthetic lethality in BRCA1-deficient cells, emphasizing their specificity and therapeutic potential for targeting genetically HR-deficient (HRD) cancers driven by deleterious mutations in HR genes like BRCA1/2. Mechanistically, EXO1 inhibition suppressed DNA end resection, stimulated the accumulation of DNA double-strand breaks, and triggered S-phase PARylation, effectively disrupting DNA repair pathways that are essential for cancer cell survival. These findings establish EXO1 inhibitors as promising candidates for the treatment of HRD cancers and lay the groundwork for the further optimization and development of these compounds as targeted therapeutics.

Pancreatic cancer (PC) is becoming one of the most serious health problems at present, but its causes and risk factors are still unclear. One of the drivers in pancreatic carcinogenesis is altered genomic (DNA) integrity with subsequent genomic instability in cancer cells. The latter comprises a) DNA damage response and DNA repair mechanisms, b) DNA replication and mitosis, c) epigenetic regulation, and d) telomere maintenance. In our review we addressed the above aspects in relation to the most abundant and severe form of PC, pancreatic ductal adenocarcinoma (PDAC). In summary, the interactions between the DNA damage response, telomere homeostasis and mitotic regulation are not comprehensively understood at present, including the epigenetic factors entering the trait of genomic stability maintenance. In addition, the complexity of telomere homeostasis in relation to PDAC risk, prognosis and prediction also warrants further investigations.

Polycomb repressive complex 1 (PRC1) is a multisubunit, evolutionarily conserved epigenetic regulator critical to numerous biological processes. Being a core component of the canonical PRC1 subunit within the Polycomb group protein complex, Chromobox4 (CBX4), a SUMO E3 ligase, can bind to H3K27me3 and recruit PRC1. This ligase regulates the SUMOylation of various proteins and permits their post-translational modification under different physiological conditions. CBX4 has been reported to regulate the development of senescence and various diseases in vivo. This review delves into the physiological functions and action mechanisms of CBX4 across different tissues and cells, particularly focusing on its primarily roles in migration, cellular senescence, metabolic dysregulation, inflammation development, and tumor proliferation. Targeting CBX4 offers a therapeutic potential for delaying cell senescence and suppressing tumor growth.

Aging is characterized by a steady loss of physiological integrity, leading to impaired function and increased vulnerability to death. Cell senescence is a biological process that progresses with aging and is believed to be a key driver of age-related diseases. Senescence, a hallmark of aging, also demonstrates its beneficial physiological aspects as an anti-cancer, pro-regenerative, homeostatic, and developmental mechanism. A transitory response in which the senescent cells are quickly formed and cleared may promote tissue regeneration and organismal fitness. At the same time, senescence-related secretory phenotypes associated with extended senescence can have devastating effects. The fact that the interaction between senescent cells and their surroundings is very context-dependent may also help to explain this seemingly opposing pleiotropic function. Further, mitochondrial dysfunction is an often-unappreciated hallmark of cellular senescence and figures prominently in multiple feedback loops that induce and maintain the senescent phenotype. This review summarizes the mechanism of cellular senescence and the significance of acute senescence. We concisely introduced the context-dependent role of senescent cells and SASP, aspects of mitochondrial biology altered in the senescent cells, and their impact on the senescent phenotype. Finally, we conclude with recent therapeutic advancements targeting cellular senescence, focusing on acute injuries and age-associated diseases. Collectively, these insights provide a future roadmap for the role of senescence in organismal fitness and life span extension.

Disinfection byproducts (DBPs), such as haloacetamides (HAMs), have been associated with adverse health outcomes, including bladder cancer. The potential for DBPs to exacerbate cellular senescence, thereby linking exposure to health impacts, remains underexplored. In this study, MRC-5 cells were exposed to HAMs at concentrations of 2, 5, and 8 μg/L for 30 days to simulate long-term exposure to levels found in drinking water. All six tested HAMs significantly increased the cellular senescence degree and enriched the cellular senescence pathway at the proteomic-wide level. Specifically, HAMs upregulated microRNA-24 expression, which increased p16 mRNA levels and decreased p16 protein levels, thereby activating oncogene-induced senescence pathways. Additionally, HAMs were found to covalently bind to TNRC6A, activating the p53/p21 pathway. Principal component analysis highlighted the critical role of functional groups in activating senescence, and the interaction between HAMs and TNRC6A could extend to at least 27 other amide-containing DBPs. Prolonged exposure to HAMs at environmentally relevant levels notably exacerbates cellular senescence, shedding light on a commonly overlooked phenomenon. Given the widespread presence of DBPs in drinking water and their continuous exposure in humans, their role in cellular senescence represents an ongoing public health concern.

Homologous recombination repair (HRR) is crucial for maintaining genomic stability by repairing DNA damage. Despite its importance, HRR's role in cancer progression is not fully elucidated. Here, this work shows that nuclear-localized branched-chain α-ketoacid dehydrogenase kinase (BCKDK) acts as a modulator of HRR, promoting cell resistance against DNA damage-inducing therapy in breast cancer. Mechanistically, this work demonstrates that BCKDK is localized in the nucleus and phosphorylates RNF8 at Ser157, preventing the ubiquitin-mediated degradation of RAD51, thereby facilitating HRR-mediated DNA repair under replication stress. Notably, aberrant expression of the BCKDK/p-RNF8/RAD51 axis correlates with breast cancer progression and poor patient survival. Furthermore, this work identifies a small molecule inhibitor of BCKDK, GSK180736A, that disrupts its HRR function and exhibits strong tumor suppression when combined with DNA damage-inducing drugs. Collectively, this study reveals a new role of BCKDK in regulating HRR, independent of its metabolic function, presenting it as a potential therapeutic target and predictive biomarker in breast cancer.

Glioblastoma (GBM) is a highly malignant brain tumor with extensive cellular heterogeneity and plasticity. Bone morphogenetic protein 4 (BMP4) has shown potential as a therapeutic agent by promoting differentiation, but its effects are complex and context dependent. While BMP4's role in differentiation is well established, its impact on senescence remains unclear. This study investigates BMP4's ability to induce senescence in GBM cells. Primary GBM cultures were treated with BMP4 and analyzed for senescence markers, including cell enlargement, p21 expression, senescence-related gene enrichment, and senescence-associated-β-galactosidase activity. A p21 knockout model was used to determine its role in BMP4-induced senescence, and sensitivity to the senolytic agent navitoclax was evaluated. BMP4 induced senescence in the GBM cultures, particularly in mesenchymal (MES)-like GBM cells with high baseline p21 levels. The knockout of p21 nearly abolished BMP4-induced senescence, maintaining cell size and proliferation. Furthermore, navitoclax effectively eliminated BMP4-induced senescent cells through apoptosis, while sparing cells with normal p21 expression. Our findings highlight BMP4 as an inducer of p21-dependent senescence in GBM, particularly in MES-like cells. This study clarifies BMP4's dual roles in differentiation and senescence, emphasizing their context dependence. Given the strong link between MES-like cells and therapy resistance, their heightened susceptibility to senescence may aid in developing targeted therapies for GBM and potentially other cancers with similar cellular dynamics.

In recent years, synthetic lethality has become an important theme in the field of targeted cancer therapy. Synthetic lethality refers to simultaneous defects in two or more genes leading to cell death, whereas defects in any single gene do not lead to cell death. Taking advantage of the genetic vulnerability that exists within cancer cells, it theoretically has no negative impact on healthy cells and has fewer side effects than non-specific chemotherapy. Currently, targeted cancer therapies focus on inhibiting key pathways in cancer. However, it has been found that over-activation of oncogenic-related signaling pathways can also induce cancer cell death, which is a major breakthrough in the new field of targeted therapies. In this review, we summarize the conventional gene targets in synthetic lethality (PARP, ATR, ATM, WEE1, PRMT) and provide an in-depth analysis of their latest potential mechanisms. We explore the impact of over-activation of pathways such as PI3K/AKT, MAPK, and WNT on cancer cell survival, and present the technical challenges of current research. Important theoretical foundations and insights are provided for the application of synthetic lethal strategies in cancer therapy, as well as future research directions.

Head and neck squamous cell carcinoma (HNSCC) is characterized by a high rate of locoregional or distant relapse among patients. It is well established that resistance to chemotherapeutic drugs has an important role in the emergence of the recurrent and/or metastatic type of this malignancy which is associated with poor prognosis. Therefore, understanding the molecular basis of chemoresistance in head and neck cancer is required for the development of effective therapeutic strategies. Activating mutations in the HRAS gene are driver events in human cancer. Although numerous studies have demonstrated that oncogenic HRAS mutations promote chemoresistance in HNSCC, the molecular profile of HNSCC tumors that overexpress wild-type HRAS (wtHRASov) and their response to chemotherapy is poorly investigated. To gain deeper insights into the characteristics of wtHRASov tumors, we conducted a gene expression analysis using transcriptome data from The Cancer Genome Atlas (TCGA). This analysis revealed a distinct signature of overexpressed nucleotide excision repair (NER) genes in wtHRASov tumors, which are associated with chemoresistance. We further explored the role of these NER components in response to genotoxic stress, utilizing a diverse panel of HNSCC cell lines and patient-derived xenografts. Our findings indicate that in a specific cluster of head and neck tumors, ERK/cJun signaling activation is strongly reliant on HRAS activity. Inhibiting HRAS in these tumors results in a significant downregulation of the NER signature components, re-sensitizing cancer cells to platinum-based chemotherapy.

The ability of organisms to effectively respond to challenges is critical for survival. We investigated how an acute stressor affected corticosterone, mitochondrial function, and DNA oxidative damage in a wild population of Leach's storm-petrels (Hydrobates leucorhous). We conducted a standardized 20-min handling procedure on storm-petrel chicks and collected baseline and post-handling blood samples. We measured plasma corticosterone and red blood cell DNA oxidative damage levels through the detection of a mutated DNA base 8-Hydroxy-2'-deoxyguanosine (8-OHdG). In addition, we quantified six measures of mitochondrial aerobic metabolism from red blood cells. Overall, the handling stressor increased plasma corticosterone levels and decreased mitochondrial efficiency to produce ATP. Although the increase in corticosterone was inversely related to the change in DNA oxidative damage, the decrease in mitochondrial efficiency was positively correlated with the change in DNA oxidative damage. Thus, over an acute stress response, individuals who had the largest increase in corticosterone also had the least amount of oxidative damage. In addition, individuals who prioritized ATP production during the acute stress also showed higher levels of oxidative damage. This work highlights the complex pathways by which corticosterone and mitochondrial efficiency affect oxidative damage during acute stress, providing new insights into the trade-offs underlying physiological responses in wild animals.

Cellular senescence is a state of permanent loss of proliferative capacity. Therefore, cells that reach a senescent state prevent tumor initiation, acting as an anti-tumor mechanism. However, despite not being proliferative, senescent cells have high secretory activity, constituting the Senescence-Associated Secretory Phenotype (SASP). SASP includes thousands of soluble molecules and extracellular vesicles, through which senescent cells can affect other cells and the extracellular matrix. In advanced tumors, the enrichment of senescent cells can have anti- or pro-tumor effects depending on features like SASP composition, tumor microenvironment (TME) composition, the anatomic site, histopathologic characteristics of malignancy, and tumor molecular background. We reviewed the studies assessing the impact of the senescence status, measured by mRNA or lncRNA molecular signatures, in the prognosis and other clinically relevant information in cancer, including anti-tumor immunity and response to therapy. We discussed the pros and cons of different strategies to define those molecular signatures and the main limitations of the studies. Finally, we also raised clinical challenges regarding the crossroad between cellular senescence and cancer prognosis, including some therapeutic opportunities in the field.

Background: Tumor cells engage in continuous self-replication by utilizing a large number of resources and capabilities, typically within an aberrant metabolic regulatory network to meet their own demands. This metabolic dysregulation leads to the formation of the tumor microenvironment (TME) in most solid tumors. Nanomedicines, due to their unique physicochemical properties, can achieve passive targeting in certain solid tumors through the enhanced permeability and retention (EPR) effect, or active targeting through deliberate design optimization, resulting in accumulation within the TME. The use of nanomedicines to target critical metabolic pathways in tumors holds significant promise. However, the design of nanomedicines requires the careful selection of relevant drugs and materials, taking into account multiple factors. The traditional trial-and-error process is relatively inefficient. Artificial intelligence (AI) can integrate big data to evaluate the accumulation and delivery efficiency of nanomedicines, thereby assisting in the design of nanodrugs. Methods: We have conducted a detailed review of key papers from databases, such as ScienceDirect, Scopus, Wiley, Web of Science, and PubMed, focusing on tumor metabolic reprogramming, the mechanisms of action of nanomedicines, the development of nanomedicines targeting tumor metabolism, and the application of AI in empowering nanomedicines. We have integrated the relevant content to present the current status of research on nanomedicines targeting tumor metabolism and potential future directions in this field. Results: Nanomedicines possess excellent TME targeting properties, which can be utilized to disrupt key metabolic pathways in tumor cells, including glycolysis, lipid metabolism, amino acid metabolism, and nucleotide metabolism. This disruption leads to the selective killing of tumor cells and disturbance of the TME. Extensive research has demonstrated that AI-driven methodologies have revolutionized nanomedicine development, while concurrently enabling the precise identification of critical molecular regulators involved in oncogenic metabolic reprogramming pathways, thereby catalyzing transformative innovations in targeted cancer therapeutics. Conclusions: The development of nanomedicines targeting tumor metabolic pathways holds great promise. Additionally, AI will accelerate the discovery of metabolism-related targets, empower the design and optimization of nanomedicines, and help minimize their toxicity, thereby providing a new paradigm for future nanomedicine development.

In recent decades, millions of patients with cancer have been cured by chemotherapy alone. By 'cure', we mean that patients with cancers that would be fatal if left untreated receive a time-limited course of chemotherapy and their cancer disappears, never to return. In an era when hundreds of thousands of cancer genomes have been sequenced, a remarkable fact persists: in most patients who have been cured, we still do not fully understand the mechanisms underlying the therapeutic index by which the tumour cells are killed, but normal cells are somehow spared. In contrast, in more recent years, patients with cancer have benefited from targeted therapies that usually do not cure but whose mechanisms of therapeutic index are, at least superficially, understood. In this Perspective, we will explore the various and sometimes contradictory models that have attempted to explain why chemotherapy can cure some patients with cancer, and what gaps in our understanding of the therapeutic index of chemotherapy remain to be filled. We will summarize principles which have benefited curative conventional chemotherapy regimens in the past, principles which might be deployed in constructing combinations that include modern targeted therapies.

Oncogene activation in normal untransformed cells induces DNA replication stress and creates a dependency on DNA damage response (DDR) mechanisms for cell survival. Different oncogenic stimuli signal via distinct mechanisms in every cancer setting. The DDR is also pathologically reprogrammed and deployed in diverse ways in different cancers. Because mutant KRAS is the driver oncogene in 90% of pancreatic ductal adenocarcinomas (PDACs), here we have investigated DDR mechanisms by which KRAS-induced DNA replication stress is tolerated in normal human pancreatic epithelial cells [human pancreatic nestin-expressing (HPNE) cells]. Using a candidate screening approach, we identify TRIP13 as a KRASG12V-induced messenger RNA that is also expressed at high levels in PDAC relative to normal tissues. Using genetic and pharmacological tools, we show that TRIP13 is necessary to sustain ongoing DNA synthesis and viability specifically in KRASG12V-expressing cells. TRIP13 promotes survival of KRASG12V-expressing HPNE cells in a homologous recombination (HR)-dependent manner. KRASG12V-expressing HPNE cells lacking TRIP13 acquire hallmark HR deficiency phenotypes, including sensitivity to inhibitors of translesion synthesis and poly-ADP ribose polymerase. Established PDAC cell lines are also sensitized to intrinsic DNA damage and therapy-induced genotoxicity following TRIP13 depletion. Taken together, our results expose TRIP13 as an attractive new and therapeutically tractable vulnerability of KRAS-mutant PDAC.

In eukaryotic cells, DNA replication is organised both spatially and temporally, as evidenced by the stage-specific spatial distribution of replication foci in the nucleus. Despite the genetic association of aberrant DNA replication with numerous human diseases, the labour-intensive methods employed to study DNA replication have hindered large-scale analyses of its roles in pathological processes. In this study, we employ two distinct methodologies. We first apply supervised machine learning, successfully classifying S-phase patterns in wild-type mouse embryonic stem cells (mESCs), while additionally identifying altered replication dynamics in Rif1-deficient mESCs. Given the constraints imposed by a classification-based approach, we then develop an unsupervised method for large-scale detection of aberrant S-phase cells. Such a method, which does not aim to classify patterns based on pre-defined categories but rather detects differences autonomously, closely recapitulates expected differences across genotypes. We therefore extend our approach to a well-characterised cellular model of inducible deregulated origin firing, involving cyclin E overexpression. Through parallel EdU- and PCNA-based analyses, we demonstrate the potential applicability of our method to patient samples, offering a means to identify the contribution of deregulated DNA replication to a plethora of pathogenic processes.

Cancers that retain wild type TP53 presumably harbor other clonal alterations that permitted their precursors to bypass p53-mediated growth suppression. Consequently, studies that employ TP53-wild type cancer cells and their isogenic derivatives may systematically fail to appreciate the full scope of p53 functionality. Several TP53 phenotypes are known to be absent in the widely used isogenic HCT116 colorectal cancer (CRC) model, which originated from a tumor that had retained wild type TP53. In contrast, we show that restoration of p53 in the TP53-mutant CRC cell line DLD-1 impeded cell proliferation, increased levels of senescence and sensitized cells to ionizing radiation (IR). To study p53 in a non-cancer context, we disrupted TP53 in hTERT-RPE1 cells. Derived from primary cells that were immortalized in vitro, hTERT-RPE1 expressed striking p53-dependent phenotypes and appeared to select for p53 loss during routine culture. hTERT-RPE1 expressed a p53-responsive transcriptome that was highly representative of diverse experimental systems. We discovered several novel downstream p53 targets of potential clinical relevance including ALDH3A1, which is involved in the detoxification of aldehydes and the metabolism of reactive oxygen species, and nectin cell adhesion molecule 4 (NECTIN4) which encodes a secreted surface protein that is overexpressed in many tumors.

Cellular senescence is an essentially irreversible cell cycle arrest associated with upregulated inflammatory responses that contribute to various pathological and physiological processes, including aging, cancer, and cancer prevention. However, the underlying mechanisms are not fully understood. Here, we show that the downregulation of CNOT3, a subunit of the CCR4-NOT complex that deadenylates mRNA poly(A) tails, promotes cellular senescence in subpopulation of A549 human non-small cell lung cancer cells. Previous work has shown that CNOT3 knockdown upregulates p21 (CDKN1A), a cyclin-dependent kinase inhibitor. Since p21 is one of the key regulators of cellular senescence, we hypothesized that CNOT3 downregulation might lead to cellular senescence. Indeed, CNOT3 knockdown upregulates several key cellular senescence hallmarks, including p53, senescence-associated β-galactosidase activity, and senescence-associated secretory phenotype in subpopulation of A549 cell culture. The senescent cell hallmarks were more prominent in the culture after additional treatment with BI 2536, a polo-like kinase 1 inhibitor. These results suggest that CNOT3 downregulation followed by BI 2536 treatment upregulates the hallmarks of cellular senescence in A549 cell culture.

Oncogene activation in normal untransformed cells induces DNA replication stress and creates a dependency on DNA Damage Response (DDR) mechanisms for cell survival. Different oncogenic stimuli signal via distinct mechanisms in every cancer setting. The DDR is also pathologically re-programmed and deployed in diverse ways in different cancers. Because mutant KRAS is the driver oncogene in 90% of Pancreatic Ductal Adenocarcinomas (PDAC), here we have investigated DDR mechanisms by which KRAS-induced DNA replication stress is tolerated in normal human pancreatic epithelial cells (HPNE). Using a candidate screening approach, we identify TRIP13 as a KRASG12V-induced mRNA that is also expressed at high levels in PDAC relative to normal tissues. Using genetic and pharmacological tools, we show that TRIP13 is necessary to sustain ongoing DNA synthesis and viability specifically in KRASG12V-expressing cells. TRIP13 promotes survival of KRASG12V-expressing HPNE cells in a Homologous Recombination (HR)-dependent manner. KRASG12V-expressing HPNE cells lacking TRIP13 acquire hallmark HR-deficiency (HRD) phenotypes including sensitivity to inhibitors of Trans-Lesion Synthesis (TLS) and Poly-ADP Ribose Polymerase (PARP). Established PDAC cell lines are also sensitized to intrinsic DNA damage and therapy-induced genotoxicity following TRIP13-depletion. Taken together our results expose TRIP13 as an attractive new and therapeutically-tractable vulnerability of KRAS-mutant PDAC.

Oncogene-Induced Senescence (OIS) is a form of senescence that occurs as a consequence of oncogenic overstimulation and possibly infection by oncogenic viruses. Whether senescence plays a role in the pathogenesis of cervical cancer (CC) is not well understood. Moreover, whether cervical epithelial cells that are part of the premalignant cervical intraepithelial neoplasia (CIN), exhibit markers of OIS in Human Papillomavirus (HPV)-infected tissue, has not been investigated.

We utilized a set of patient-derived premalignant and malignant tissue samples to investigate the protein (Ki67 and Lamin B1) and gene (TP53, IL1A, CCL2, and MMP9) expression of several OIS-associated biomarkers using immunohistochemistry (IHC) and qRT-PCR, respectively. Furthermore, we characterized the HPV status of all tissue samples.

Most of the CC samples (34/37) were positive for HPV, mainly HPV-16 which was observed in 62.2% of the CC samples. Among CINs, HPV infection was found in 60.2% of the 32 samples with HPV-16 as the dominant genotype in 58.5% of the CINs. IHC analysis revealed a significant increase in the expression levels of both Ki67 and Lamin B1 proteins in CC tissue compared to CIN. On average, 93% of tumor cells were positive for Ki67 in comparison to only 25% of premalignant cells in CIN samples. Similarly, Lamin B1 expression was observed in 89% of tumor cells in malignant tissue on average, compared to 60% in CIN samples. Importantly, Lamin B1 expression was elevated in nonmalignant cervical tissue suggesting that its downregulation is more predominant in the premalignant state. Furthermore, RT-PCR revealed a significant decrease in the expression of TP53, IL1a, CCL2, and MMP9 markers in CC samples compared to CINs. Specifically, 84% of CC samples showed reduced TP53 expression, 90% showed reduced IL1a expression, 74% showed reduced CCL2 expression, and 76% showed reduced MMP9 expression when compared with their premalignant baseline. Infection of HPV was confirmed in 61% of the tumor tissues while only 25% of the CINs were positive for HPV.

This work shall provide an opportunity to further examine the role of OIS in the process of HPV-driven CC development.

The DNA damage response (DDR) mechanisms that allow cells to tolerate DNA replication stress are critically important for genome stability and cell viability. Using an unbiased genetic screen we identify a role for the RING finger E3 ubiquitin ligase RNF25 in promoting DNA replication stress tolerance. In response to DNA replication stress, RNF25-deficient cells generate aberrantly high levels of single-stranded DNA (ssDNA), accumulate in S-phase and show reduced mitotic entry. Using single-molecule DNA fiber analysis, we show that RNF25 protects reversed DNA replication forks generated by the fork remodeler HLTF from nucleolytic degradation by MRE11 and CtIP. Mechanistically, RNF25 interacts with the replication fork protection factor REV7 and recruits REV7 to nascent DNA after replication stress. The role of RNF25 in protecting replication forks is fully separable from its canonical functions in ubiquitin conjugation. This work reveals the RNF25-REV7 signaling axis as an important protective mechanism in cells experiencing replication stress.

Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ spatiotemporally controlled oncogene activation and tumor suppressor inhibition together with multiomics to unveil the processes underlying oral epithelial progenitor cell reprogramming into tumor initiating cells at single cell resolution. Tumor initiating cells displayed a distinct stem-like state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal invasive gene programs. YAP-mediated tumor initiating cell programs included activation of oncogenic transcriptional networks and mTOR signaling, and recruitment of myeloid cells to the invasive front contributing to tumor infiltration. Tumor initiating cell transcriptional programs are conserved in human head and neck cancer and associated with poor patient survival. These findings illuminate processes underlying cancer initiation at single cell resolution, and identify candidate targets for early cancer detection and prevention.

Tandem duplications (TD) are among the most frequent type of structural variant (SV) in the cancer genome. They are characterized by a single breakpoint junction that defines the boundaries and the size of the duplicated segment. Cancer-associated TDs often increase oncogene copy number or disrupt tumor suppressor gene function, and thus have important roles in tumor evolution. TDs in cancer genomes fall into three classes, defined by the size of duplications, and are associated with distinct genetic drivers. In this review, we survey key features of cancer-related TDs and consider possible underlying mechanisms in relation to stressed DNA replication and the 3D organization of the S phase genome.

Cellular senescence, a state of permanent cell cycle arrest, recapitulates the aging process at the cellular level. It can be triggered by intrinsic or extrinsic factors including telomere shortening (replicative senescence) and in response to various types of stresses such as oncogenic stress (oncogene-induced senescence, OIS). Senescence has been detected in vitro and in premalignant lesions in mice and humans expressing mutant oncogenes. MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression at the posttranscriptional level, and have been involved in both replicative senescence and OIS. Several methods have been used to identify miRNAs and compare their expression in normal versus oncogene-induced senescent cells, as well as to analyze their role and their targets in senescence. Here, we describe several methods that can be employed to identify miRNAs in cells undergoing OIS, including miRNA-sequencing, RT-qPCR-based detection and quantification of miRNAs and Nanostring miRNA analysis (nCounter miRNA Expression Assay). Moreover, we perform a meta-analysis of studies employing the above methodologies, pinpoint miRNAs with consistent expression changes across senescence models, and predict their target genes and the pathways in which they partake.

Cellular senescence plays a critical role in cancer, acting as both a tumor-suppressive and tumor-promoting mechanism. Senescent cells undergo stable cell-cycle arrest in response to various stressors, including DNA damage and oncogenic signaling, and exhibit a complex secretory phenotype known as the senescence-associated secretory phenotype (SASP), which can impact the tumor microenvironment. The hallmarks of senescence include cell-cycle arrest, secretion of pro-inflammatory factors, structural changes, and metabolic alterations. These features, while initially suppressing tumorigenesis, can later contribute to cancer progression under certain conditions. Methods for studying senescence in preclinical models include in vitro assays, ex vivo tissue analysis, and in vivo detection techniques. Emerging therapeutic strategies focus on exploiting senescence for cancer treatment, particularly through the use of senolytic agents that selectively eliminate senescent cells and senomorphic compounds that modulate SASP activity. However, the identification of reliable and universal biomarkers for senescence remains a challenge, necessitating a multimarker approach to accurately detect and characterize senescent cells in various contexts.

Hepatocellular carcinoma (HCC) originates from differentiated hepatocytes undergoing compensatory proliferation in livers damaged by viruses or metabolic-dysfunction-associated steatohepatitis (MASH)1. While increasing HCC risk2, MASH triggers p53-dependent hepatocyte senescence3, which we found to parallel hypernutrition-induced DNA breaks. How this tumour-suppressive response is bypassed to license oncogenic mutagenesis and enable HCC evolution was previously unclear. Here we identified the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) as a p53 target that is elevated in senescent-like MASH hepatocytes but suppressed through promoter hypermethylation and proteasomal degradation in most human HCCs. FBP1 first declines in metabolically stressed premalignant disease-associated hepatocytes and HCC progenitor cells4,5, paralleling the protumorigenic activation of AKT and NRF2. By accelerating FBP1 and p53 degradation, AKT and NRF2 enhance the proliferation and metabolic activity of previously senescent HCC progenitors. The senescence-reversing and proliferation-supportive NRF2-FBP1-AKT-p53 metabolic switch, operative in mice and humans, also enhances the accumulation of DNA-damage-induced somatic mutations needed for MASH-to-HCC progression.

Senescence is an inherent cellular mechanism triggered as a response to stressful insults. It associates with several aspects of cancer progression and therapy. Senescent cells constitute a highly heterogeneous cellular population and their identification can be very challenging. In fact, the term "senescence" has been often misused. This is also true in the case of immune cells. While several studies indicate the presence of senescent-like features (mainly in T cells), senescent immune cells are poorly described. Under this prism, we herein review the current literature on what has been characterized as T cell senescence and provide insights on how to accurately discriminate senescent cells against exhausted or anergic ones. We also summarize the major metabolic and epigenetic modifications associated with T cell senescence and underline the role of senescent T cells in the tumor microenvironment (TME). Moreover, we discuss how these cells associate with standard clinical therapeutic interventions and how they impact their efficacy. Finally, we underline the importance of precise identification and thorough characterization of "truly" senescent T cells in order to design successful therapeutic manipulations that would delay cancer incidence and maximize efficacy of immunotherapy.

Selective targeting of senescent cells has been thus far considered a widespread preventive strategy, as well as a main or adjuvant therapy for age-associated diseases, fueling the research on the discovery of senotherapeutics (i.e., senolytic or senomorphic compounds). Given that until now no single senotherapeutic has been reported to exert a universal anti-senescence action due to the cell- /tissue-, and context-dependent specificity of such compounds, seeking novel selective senotherapeutics remains of great importance. In this chapter, a research strategy that could be followed to screen natural product collections for putative senotherapeutics with enhanced specificity and reduced toxicity is presented, from the extraction of the source material and the isolation and chemical characterization of the compounds of interest to their biological evaluation in vitro and in vivo.

The emerging field of senolytics is centered on eliminating senescent cells to block their contribution to the progression of age-related diseases, including cancer, and to facilitate healthy aging. Enhancing the selectivity of senolytic treatments toward senescent cells stands to reduce the adverse effects associated with existing senolytic interventions. Taking advantage of lipofuscin accumulation in senescent cells, we describe here the development of a highly efficient senolytic platform consisting of a lipofuscin-binding domain scaffold, which can be conjugated with a senolytic drug via an ester bond. As a proof of concept, we present the generation of GL392, a senolytic compound that carries a dasatinib senolytic moiety. Encapsulation of the GL392 compound in a micelle nanocarrier (termed mGL392) allows for both in vitro and in vivo (in mice) selective elimination of senescent cells via targeted release of the senolytic agent with minimal systemic toxicity. Our findings suggest that this platform could be used to enhance targeting of senotherapeutics toward senescent cells.

Understanding the molecular landscape of nonmuscle-invasive bladder cancer (NMIBC) is essential to improve risk assessment and treatment regimens. We performed a comprehensive genomic analysis of patients with NMIBC using whole-exome sequencing (n = 438), shallow whole-genome sequencing (n = 362) and total RNA sequencing (n = 414). A large genomic variation within NMIBC was observed and correlated with different molecular subtypes. Frequent loss of heterozygosity in FGFR3 and 17p (affecting TP53) was found in tumors with mutations in FGFR3 and TP53, respectively. Whole-genome doubling (WGD) was observed in 15% of the tumors and was associated with worse outcomes. Tumors with WGD were genomically unstable, with alterations in cell-cycle-related genes and an altered immune composition. Finally, integrative clustering of multi-omics data highlighted the important role of genomic instability and immune cell exhaustion in disease aggressiveness. These findings advance our understanding of genomic differences associated with disease aggressiveness in NMIBC and may ultimately improve patient stratification.

FISH (fluorescence in situ hybridization) is one of the most reliable cytogenetic techniques for detecting changes in DNA integrity, including copy number gains and losses as well as telomeric erosion. Oncogene-induced senescence (OIS) is characterized by the persistence of DNA damage at telomeres. Immunofluorescence with γH2AX, a double-strand break marker, and PML, a tumor suppressor gene involved in OIS activation, can identify DNA lesions that activate the DNA repair machinery. Here, we describe a protocol by combining the two immuno-FISH approaches to detect dysfunctional telomeres in cell cultures, which can be used to monitor OIS under experimental conditions and potentially applied to ex vivo isolated senescent cells.

DNA double-strand breaks (DSBs) are a major source of genomic instability. Physiological DSBs are naturally occurring breaks that happen during normal cellular processes. Unlike DNA breaks resulting from DNA damage due to external factors like radiation or chemicals, physiological DSBs play critical roles in various normal biological functions. Some key processes involving physiological DSBs include V(D)J recombination, transcription, and replication. These breaks are typically tightly controlled and are part of the cellular machinery designed to maintain and enhance genomic integrity and diversity. However, if these breaks are misrepaired or left unrepaired, they can contribute to genomic instability, potentially leading to senescence and diseases such as cancer. Here, we outline various methods commonly employed to detect physiological DSBs and introduce a detailed, step-by-step protocol for mapping these breaks using the in-suspension break labeling in situ and sequencing (sBLISS) technique. sBLISS offers single nucleotide resolution and is versatile enough to be applied to any cell type amenable to single-cell suspension. This comprehensive approach not only enhances our understanding of DSBs but also aids in the exploration of their roles in genomic instability.

Cellular senescence is a stress response that restrains the growth of aged, damaged or abnormal cells. Thus, senescence has a crucial role in development, tissue maintenance and cancer prevention. However, lingering senescent cells fuel chronic inflammation through the acquisition of a senescence-associated secretory phenotype (SASP), which contributes to cancer and age-related tissue dysfunction. Recent progress in understanding senescence has spurred interest in the development of approaches to target senescent cells, known as senotherapies. In this Review, we evaluate the status of various types of senotherapies, including senolytics that eliminate senescent cells, senomorphics that suppress the SASP, interventions that mitigate senescence and strategies that harness the immune system to clear senescent cells. We also summarize how these approaches can be combined with cancer therapies, and we discuss the challenges and opportunities in moving senotherapies into clinical practice. Such therapies have the potential to address root causes of age-related diseases and thus open new avenues for preventive therapies and treating multimorbidities.

Classical radiation biology as we understand it clearly identifies genomic DNA as the primary target of ionizing radiation. The evidence appears rock-solid: ionizing radiation typically induces DSBs with a yield of ~30 per cell per Gy, and unrepaired DSBs are a very cytotoxic lesion. We know very well the kinetics of induction and repair of different types of DNA damage in different organisms and cell lines. And yet, higher organisms differ in their radiation sensitivity-humans can be unpredictably radiosensitive during radiotherapy; this can be due to genetic defects (e.g., ataxia telangiectasia (AT), Fanconi anemia, Nijmegen breakage syndrome (NBS), and the xeroderma pigmentosum spectrum, among others) but most often is unexplained. Among other mammals, goats (Capra hircus) appear to be very radiosensitive (LD50 = 2.4 Gy), while Mongolian gerbils (Meriones unguiculatus) are radioresistant and withstand quadruple that dose (LD50 = 10 Gy). Primary radiation lethality in mammals is due most often to hematopoietic insufficiency, which is, in the words of Dr. Theodor Fliedner, one of the pioneers of radiation hematology, "a disturbance in cellular kinetics". And yet, what makes one cell type, or one particular organism, more sensitive to ionizing radiation? The origins of radiosensitivity go above and beyond the empirical evidence and models of DNA damage and repair-as scientists, we must consider other phenomena: the radiation-induced bystander effect (RIBE), abscopal effects, and, of course, genomic instability and immunomodulation. It seems that radiosensitivity is not entirely determined by the mathematics of DNA damage and repair, and it is conceivable that radiation biology may benefit from an informed enquiry into physiology and organism-level signaling affecting radiation responses. The current article is a review of several key aspects of radiosensitivity beyond DNA damage induction and repair; it presents evidence supporting new potential venues of research for radiation biologists.

Uncontrolled degradation and collapse of stalled replication forks (RFs) are primary sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse remain to be fully elaborated. Here, we show that polynucleotide kinase-phosphatase (PNKP) localizes at stalled forks and protects stalled forks from excessive degradation. The loss of PNKP results in nucleolytic degradation of nascent DNA at stalled RFs. This mechanism is different from the BRCA2-dependent fork protection pathway, which protects stalled forks from excessive MRE11-dependent nucleolytic degradation. Our research shows that hydroxyurea treatment leads to increased misincorporation of ribonucleotides in DNA, which in turn traps TOP1 on stalled RFs. We have also found that reducing the levels of TOP1 or TDP1 in cells can reverse the degradation of nascent DNA observed in PNKP-deficient cells. In summary, our data suggest that PNKP plays a role in maintaining the stability of stalled RFs.

Cell division cycle 6 (CDC6) is essential for the initiation of DNA replication in eukaryotic cells and contributes to the development of various human tumors. Polycystic ovarian syndrome (PCOS) is a reproductive endocrine disease in women of childbearing age, with a significant risk of endometrial cancer (EC). However, the role of CDC6 in the progression of PCOS to EC is unclear. Therefore, we examined CDC6 expression in patients with PCOS and EC. We evaluated the relationship between CDC6 expression and its prognostic value, potential biological functions, and immune infiltrates in patients with EC. In vitro analyses were performed to investigate the effects of CDC6 knockdown on EC proliferation, migration, invasion, and apoptosis. CDC6 expression was significantly upregulated in patients with PCOS and EC. Moreover, this protein caused EC by promoting the aberrant infiltration of macrophages into the immune microenvironment in patients with PCOS. A functional enrichment analysis revealed that CDC6 exerted its pro-cancer and pro-immune cell infiltration functions via the PI3K-AKT pathway. Moreover, it promoted EC proliferation, migration, and invasion but inhibited apoptosis. This protein significantly reduced EC survival when mutated. These findings demonstrate that CDC6 regulates the progression of PCOS to EC and promotes immune infiltration.