Iron retardation in lysosomes protects senescent cells from ferroptosis.

Ferroptosis, an iron-triggered modality of cellular death, has been reported to closely relate to human aging progression and aging-related diseases. However, the involvement of ferroptosis in the development and maintenance of senescent cells still remains elusive. Here, we established a doxorubicin-induced senescent HSkM cell model and found that both iron accumulation and lipid peroxidation increase in senescent cells. Moreover, such iron overload in senescent cells has changed the expression panel of the ferroptosis-response proteins. Interestingly, the iron accumulation and lipid peroxidation does not trigger ferroptosis-induced cell death. Oppositely, senescent cells manifest resistance to the ferroptosis inducers, compared to the proliferating cells. To further investigate the mechanism of ferroptosis-resistance for senescent cells, we traced the iron flux in cell and found iron arrested in lysosome. Moreover, disruption of lysosome functions by chloroquine and LLOMe dramatically triggered the senescent cell death. Besides, the ferroitinophagy-related proteins FTH1/FTL and NCOA4 knockdown also increases the senescent cell death. Thus, we speculated that iron retardation in lysosome of senescent cells is the key mechanism for ferroptosis resistance. And the lysosome is a promising target for senolytic drugs to selectively clear senescent cells and alleviate the aging related diseases.

Lysosome passivation triggered by silver nanoparticles enhances subcellular-targeted drug therapy.

Frequently, subcellular-targeted drugs tend to accumulate in lysosomes after cellular absorption, a process termed the lysosomal trap. This accumulation often interferes with the drug's ability to bind to its target, resulting in decreased efficiency. Existing methods for addressing lysosome-induced drug resistance mainly involve improving the structures of small molecules or enveloping drugs in nanomaterials. Nonetheless, these approaches can lead to changes in the drug structure or potentially trigger unexpected reactions within organisms. To address these issues, we introduced a strategy that involves inactivating the lysosome with the use of Ag nanoparticles (Cy3.5@Ag NPs). In this method, the Cy3.5@Ag NPs gradually accumulate inside lysosomes, leading to permeation of the lysosomal membrane and subsequent lysosomal inactivation. In addition, Cy3.5@Ag NPs also significantly affected the motility of lysosomes and induced the occurrence of lysosome passivation. Importantly, coincubating Cy3.5@Ag NPs with various subcellular-targeted drugs was found to significantly increase the efficiency of these treatments. Our strategy illustrates the potential of using lysosomal inactivation to enhance drug efficacy, providing a promising therapeutic strategy for cancer.

Single-cell transcriptomic analysis reveals the landscape of epithelial-mesenchymal transition molecular heterogeneity in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a prevalent and highly lethal malignant disease. The epithelial-mesenchymal transition (EMT) is crucial in promoting ESCC development. However, the molecular heterogeneity of ESCC and the potential inhibitory strategies targeting EMT remain poorly understood. In this study, we analyzed high-resolution single-cell transcriptome data encompassing 209,231 ESCC cells from 39 tumor samples and 16 adjacent samples obtained from 44 individuals. We identified distinct cell populations exhibiting heterogeneous EMT characteristics and identified 87 EMT-associated molecules. The expression profiles of these EMT-associated molecules showed heterogeneity across different stages of ESCC progression. Moreover, we observed that EMT primarily occurred in early-stage tumors, before lymph node metastasis, and significantly promoted the rapid deterioration of ESCC. Notably, we identified SERPINH1 as a potential novel marker for ESCC EMT. By classifying ESCC patients based on EMT gene sets, we found that those with high EMT exhibited poorer prognosis. Furthermore, we predicted and experimentally validated drugs targeting ESCC EMT, including dactolisib, docetaxel, and nutlin, which demonstrated efficacy in inhibiting EMT and metastasis in ESCC. Through the integration of scRNA-seq, RNA-seq, and TCGA data with experimental validation, our comprehensive analysis elucidated the landscape of EMT during the entire course of ESCC development and metastasis. These findings provide valuable insights and a reference for refining ESCC clinical treatment strategies.

Human CST complex restricts excessive PrimPol repriming upon UV induced replication stress by suppressing p21.

DNA replication stress, caused by various endogenous and exogenous agents, halt or stall DNA replication progression. Cells have developed diverse mechanisms to tolerate and overcome replication stress, enabling them to continue replication. One effective strategy to overcome stalled replication involves skipping the DNA lesion using a specialized polymerase known as PrimPol, which reinitiates DNA synthesis downstream of the damage. However, the mechanism regulating PrimPol repriming is largely unclear. In this study, we observe that knockdown of STN1 or CTC1, components of the CTC1/STN1/TEN1 complex, leads to enhanced replication progression following UV exposure. We find that such increased replication is dependent on PrimPol, and PrimPol recruitment to stalled forks increases upon CST depletion. Moreover, we find that p21 is upregulated in STN1-depleted cells in a p53-independent manner, and p21 depletion restores normal replication rates caused by STN1 deficiency. We identify that p21 interacts with PrimPol, and STN1 depletion stimulates p21-PrimPol interaction and facilitates PrimPol recruitment to stalled forks. Our findings reveal a previously undescribed interplay between CST, PrimPol and p21 in promoting repriming in response to stalled replication, and shed light on the regulation of PrimPol repriming at stalled forks.

Human CST complex protects stalled replication forks by directly blocking MRE11 degradation of nascent-strand DNA.

Degradation and collapse of stalled replication forks are main sources of genomic instability, yet the molecular mechanisms for protecting forks from degradation/collapse are not well understood. Here, we report that human CST (CTC1-STN1-TEN1) proteins, which form a single-stranded DNA-binding complex, localize at stalled forks and protect stalled forks from degradation by the MRE11 nuclease. CST deficiency increases MRE11 binding to stalled forks, leading to nascent-strand degradation at reversed forks and ssDNA accumulation. In addition, purified CST complex binds to 5' DNA overhangs and directly blocks MRE11 degradation in vitro, and the DNA-binding ability of CST is required for blocking MRE11-mediated nascent-strand degradation. Our results suggest that CST inhibits MRE11 binding to reversed forks, thus antagonizing excessive nascent-strand degradation. Finally, we uncover that CST complex inactivation exacerbates genome instability in BRCA2 deficient cells. Collectively, our findings identify the CST complex as an important fork protector that preserves genome integrity under replication perturbation.

Genome-wide mapping and profiling of γH2AX binding hotspots in response to different replication stress inducers.

BACKGROUND: Replication stress (RS) gives rise to DNA damage that threatens genome stability. RS can originate from different sources that stall replication by diverse mechanisms. However, the mechanism underlying how different types of RS contribute to genome instability is unclear, in part due to the poor understanding of the distribution and characteristics of damage sites induced by different RS mechanisms. RESULTS: We use ChIP-seq to map γH2AX binding sites genome-wide caused by aphidicolin (APH), hydroxyurea (HU), and methyl methanesulfonate (MMS) treatments in human lymphocyte cells. Mapping of γH2AX ChIP-seq reveals that APH, HU, and MMS treatments induce non-random γH2AX chromatin binding at discrete regions, suggesting that there are γH2AX binding hotspots in the genome. Characterization of the distribution and sequence/epigenetic features of γH2AX binding sites reveals that the three treatments induce γH2AX binding at largely non-overlapping regions, suggesting that RS may cause damage at specific genomic loci in a manner dependent on the fork stalling mechanism. Nonetheless, γH2AX binding sites induced by the three treatments share common features including compact chromatin, coinciding with larger-than-average genes, and depletion of CpG islands and transcription start sites. Moreover, we observe significant enrichment of SINEs in γH2AX sites in all treatments, indicating that SINEs may be a common barrier for replication polymerases. CONCLUSIONS: Our results identify the location and common features of genome instability hotspots induced by different types of RS, and help in deciphering the mechanisms underlying RS-induced genetic diseases and carcinogenesis.