Keqian Xu

CD8⁺ T-cell exhaustion is a pivotal driver of resistance to melanoma immunotherapy. In preclinical models, alternate-day fasting (ADF) has shown considerable promise for reinvigorating antitumor immune responses. Yet, the molecular mediators that connect metabolic stress to T-cell functional reprogramming remain poorly defined. Here, we hypothesize that ADF counteracts T-cell exhaustion via a synergistic KMT2B-miR-760 regulatory axis—one where chromatin remodeling and post-transcriptional silencing converge to restore CD8⁺ T-cell functionality. Using both melanoma-bearing mouse models and ex vivo-expanded human CD8+ T lymphocytes, we demonstrate that ADF not only boosts intratumoral CD8⁺ T-cell accumulation but also dampens transcriptional programs linked to T-cell exhaustion. Mechanistically, ADF induces the upregulation of KMT2B while simultaneously suppressing miR-760; this, in turn, derepresses genes such as GZMA and those that control effector function and exhaustion in CD8⁺ T cells. Collectively, these findings establish a mechanistic link between the immunometabolic advantages of ADF and an epigenetic-microRNA circuit, providing a rational dietary adjuvant approach to overcome immunotherapy resistance in melanoma.

Background: Circulating tumor cell (CTC) clusters exhibit significantly greater metastatic potential than single CTCs and are associated with poorer overall survival in cancers. However, the molecular mechanisms driving CTC cluster formation remain unclear. p21-activated kinase 2 (PAK2) plays a critical role in cytoskeletal remodeling and is frequently associated with advanced tumor progression and poor prognosis. In this study, we explored the role of PAK2 in CTC cluster formation in breast cancer. Methods: We performed an integrated bioinformatics analysis of transcriptomic profiles from single CTCs and CTC clusters via GEO datasets to identify differentially expressed genes (DEGs) and candidate hub genes associated with CTC clustering. Functional enrichment analyses and gene set enrichment analysis were subsequently conducted to explore relevant pathways. The biological function of the identified hub gene PAK2 was validated via in vitro CTC cluster formation cell models and in vivo orthotopic in situ breast cancer mouse models. Mechanistic studies focused on PAK2-mediated phosphorylation of E-cadherin. Additionally, the therapeutic potential of targeting PAK2 was evaluated via the use of the selective PAK inhibitor FRAX597 in vivo. Results: Bioinformatics analyses revealed that CTC clusters are characterized by enhanced cell-cell adhesion, increased proliferative capacity and survival advantages. Among the identified hub genes, PAK2 was significantly upregulated in breast cancer tissues and cell lines, and its elevated expression was associated with poor patient prognosis. Functional experiments demonstrated that PAK2 promotes CTC cluster formation by increasing E-cadherin phosphorylation at Ser840, thereby strengthening cell-cell adhesion. Pharmacologic inhibition of PAK2 with FRAX597 impaired CTC cluster formation, suppressed tumor growth, reduced metastasis and decreased CTC cluster numbers in vivo. Conclusions: This study revealed that PAK2 promotes CTC cluster formation and breast cancer metastasis by enhancing E-cadherin-mediated cell-cell adhesion. These results provide novel insights into the molecular mechanisms underlying CTC cluster formation and highlight PAK2 as a potential therapeutic target and diagnostic marker for preventing breast cancer metastasis.

Background: DNA double‐strand breaks (DSBs) are the most lethal and dangerous type of lesions with significant implications for both cellular function and organismal health. The number of DSBs (N _DSBs ) across the genome reflects DNA damage severity. However, current quantification methods mainly rely on next‐generation sequencing, which is laborious and expensive. This study aims to provide a simple, low‐cost, and high‐throughput standard curve‐based method for quantifying genome‐wide DSBs. Method: Genomic DNA from human, mouse, Arabidopsis , Saccharomyces cerevisiae , and Escherichia coli was digested by seven blunt‐end restriction enzymes to generate DSB standards. Theoretical N _DSBs for each standard were calculated based on restriction site frequency. Ligation‐mediated quantitative PCR (LM‐qPCR) was performed to obtain the Ct values, which were plotted against log‐transformed N _DSBs to construct standard curves. Method reliability was assessed by comparing results with neutral single‐cell gel electrophoresis and γ‐H2AX flow cytometry. Results: All genomes were successfully digested by seven blunt‐end restriction enzymes to produce standard DSB fragments. Standard curves demonstrated high linearity ( R ² > 0.95), with intra‐ and inter‐assay coefficients of variation of 1.101% and 2.528%, respectively. The detection limit was below 100 DSBs. Quantification results strongly correlated with traditional DSB detection methods (| r | > 0.9). Conclusion: This standard curve‐based method enables accurate, reproducible quantification of genome‐wide DSBs in various organisms. It is simple, low‐cost, and easily standardized, offering a promising tool for applications in genotoxicity testing, environmental exposure monitoring, and DNA damage research.

Background: As one of the most common chromosomal causes, chromosome translocation leads to T-cell acute lymphoblastic leukemia (T-ALL). Ku70 is one of the key factors of error-prone DNA repair and it may end in translocation. So far, the direct correlation between Ku70 and translocation has not been assessed. This study aimed to investigate the association between Ku70 and translocation in human lymphocytes after radiation and T-ALL. Methods: Peripheral blood lymphocytes (PBLs) from volunteers and human lymphocyte cell line AHH-1 were irradiated with X-rays to form the chromosome translocations. Phytohemagglutinin (PHA) was used to stimulate lymphocytes. The frequency of translocation was detected by fluorescence in situ hybridization (FISH). Meanwhile, the expression of Ku70 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. Furthermore, Ku70 interference, overexpression and chemical inhibition were used in AHH-1 cell lines to confirm the correlation. Finally, the expression of Ku70 in T-ALL samples with or without translocation was detected. Results: The expression of Ku70 and frequencies of translocation were both significantly increased in PBLs after being irradiated by X-rays, and a positive correlation between the expression (both mRNA and protein level) of Ku70 and the frequency of translocation was detected (r = 0.4877, P = 0.004; r = 0.3038, P = 0.0358 respectively). Moreover, Ku70 interference decreased the frequency of translocations, while the frequency of translocations was not significantly affected after Ku70 overexpression. The expression of Ku70 and frequencies of translocation were both significantly increased in cells after irradiation, combined with chemical inhibition (P < 0.01). The protein level and mRNA level of Ku70 in T-ALL with translocation were obviously higher than T-ALL with normal karyotype (P = 0.009, P = 0.049 respectively). Conclusions: Ku70 is closely associated with the frequency of chromosome translocation in human lymphocytes after radiation and T-ALL. Ku70 might be a radiation damage biomarker and a potential tumor therapy target.

Background: Structural chromosome abnormality (SCA) is an important cause of human diseases, including recurrent pregnancy loss (RPL). DNA double-strand breaks (DSBs) repair-related genes play critical roles in SCA. The present study aims to investigate the potential contribution of DSBs repair-related gene polymorphisms to SCA.Methods: Fifty-four affected RPL individuals with SCA, 88 affected RPL individuals without SCA, and 84 controls were analyzed. Targeted whole-exome sequencing (WES) was used for screening single nucleotide polymorphisms in six DSBs repair-related genes (EP300, XRCC6, LIG4, XRCC4, PRKDC, and DCLRE1C), and validation was performed by Sanger sequencing. Finally, we detected the frequency of radiation-induced chromosome translocations in no SCA samples with significant polymorphisms by fluorescence in situ hybridization (FISH).Results: A total of 35 polymorphisms have been identified and confirmed. Frequencies of EP300 rs20551, XRCC6 rs132788, and LIG4 rs1805388 were significantly different between SCA RPL and no SCA RPL (p = 0.030, 0.031, and 0.040 respectively). Frequencies of those three gene polymorphisms between SCA RPL and controls also were significantly different (p = 0.017, 0.028, and 0.029 respectively). Moreover, the frequency of the G allele at rs20551 locus, the T allele at rs132788 locus and the A allele at rs1805388 locus was significantly higher in SCA RPL than no SCA RPL (OR = 3.227, p = 0.005; OR = 1.978, p = 0.008 and OR = 1.769, p = 0.036 respectively) and controls (OR = 7.130, p = 0.000; OR = 2.157, p = 0.004; OR = 2.397, p = 0.003 respectively). Additionally, the frequency of radiation-induced translocation in no SCA samples with rs20551, rs132788 or rs1805388 was significantly higher compared with the wild type samples (p = 0.015, 0.012, and 0.007 respectively).Conclusion: Our results suggest that rs20551, rs132788, and rs1805388 might be associated with the risk of SCA. Larger scales of genetic variations studies and functional experiments are necessary to further confirm these findings.

Background: Chromosome translocation is one of the most common chromosomal causes to T-cell acute lymphoblastic leukemia (T-ALL). Ku70 is one of the key factors of error-prone DNA repair that may end in translocation. So far, the direct correlation between Ku70 and translocation has not been assessed. Our study aimed to investigate the association of Ku70 and translocation in human lymphocytes after radiation and T-ALL. Methods: Peripheral blood lymphocytes (PBLs) from volunteers and human lymphocyte cell line AHH-1were irradiated with X-rays to form chromosome translocations. The frequency of translocation was detected by fluorescence in situ hybridization (FISH), meanwhile, the expression of Ku70 was also detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. Furthermore, Ku70 interference, overexpression and chemical inhibition were used in AHH-1 cell lines to confirm the correlation. Finally, we detected the expression of Ku70 in T-ALL samples with or without translocation. Results: Expression of Ku70 and frequencies of translocation were both significantly increased in PBLs after irradiated by X-rays, and a positive correlation between the expression (both mRNA and protein level) of Ku70 and the frequency of translocation was detected (r = 0.4877, P = 0.004; r = 0.3038, P = 0.0358 respectively). Moreover, Ku70 interference decreased the frequency of translocations while the frequency of translocations was not significantly affected after Ku70 overexpression. The expression of Ku70 and frequencies of translocation were both significantly increased in cells after irradiated combined with chemical inhibition (P P = 0.009, P = 0.049 respectively). Conclusions: Ku70 is closely associated with the frequency of chromosome translocation in human lymphocytes after radiation and T-ALL. Ku70 might be a radiation damage biomarker and a potential tumor therapy target.

Background: Melanoma is a malignant tumor of melanocytes, and the incidence has increased faster than any other cancer over the past half century. Most primary melanoma can be cured by local excision, but metastatic melanoma has a poor prognosis. Cutaneous melanoma (CM) is prone to metastasis, so the research on the mechanism of melanoma occurrence and metastasis will be beneficial to diagnose early, improve treatment, and prolong life survival. In this study, we compared the gene expression of normal skin (N), primary cutaneous melanoma (PM) and metastatic cutaneous melanoma (MM) in the Gene Expression Omnibus (GEO) database. Then we identified the key genes and molecular pathways that may be involved in the development and metastasis of cutaneous melanoma, thus to discover potential markers or therapeutic targets. Methods: Three gene expression profiles (GSE7553, GSE15605 and GSE46517) were downloaded from the GEO database, which contained 225 tissue samples. R software identified the differentially expressed genes (DEGs) between pairs of N, PM and MM samples in the three sets of data. Subsequently, we analyzed the gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of the DEGs, and constructed a protein-protein interaction (PPI) network. MCODE was used to seek the most important modules in PPI network, and then the GO function and KEGG pathway of them were analyzed. Finally, the hub genes were calculated by the cytoHubba in Cytoscape software. The Cancer Genome Atlas (TCGA) data were analyzed using UALCAN and GEPIA to validate the hub genes and analyze the prognosis of patients. Results: A total of 134, 317 and 147 DEGs were identified between N, PM and MM in pair. GO functions and KEGG pathways analysis results showed that the upregulated DEGs mainly concentrated in cell division, spindle microtubule, protein kinase activity and the pathway of transcriptional misregulation in cancer. The downregulated DEGs occurred in epidermis development, extracellular exosome, structural molecule activity, metabolic pathways and p53 signaling pathway. The PPI network obtained the most important module, whose GO function and KEGG pathway were enriched in oxidoreductase activity, cell division, cell exosomes, protein binding, structural molecule activity, and metabolic pathways. 14, 18 and 18 DEGs were identified respectively as the hub genes between N, PM and MM, and TCGA data confirmed the expression differences of hub genes. In addition, the overall survival curve of hub genes showed that the differences in these genes may lead to a significant decrease in overall survival of melanoma patients. Conclusions: In this study, several hub genes were found from normal skin, primary melanoma and metastatic melanoma samples. These hub genes may play an important role in the production, invasion, recurrence or death of CM, and may provide new ideas and potential targets for its diagnosis or treatment.

Background: The hMSH5 C85T polymorphism, which encodes hMSH5 P29S, is associated with individual differences in spermatogenic abnormalities caused by ionizing radiation (IR), but the molecular mechanisms remain unclear.Objectives: This manuscript aims to explore the role of hMSH5 C85T polymorphism in IR‐induced individual differences in spermatogenic abnormalities.Material and methods: We transfected pcDNA‐hMSH5^P29S vector into mouse spermatogonia GC‐1, mouse spermatocytes GC‐2, mouse testicular mesenchymal cells TM3, and mouse testicular support cells TM4. After radiation, we evaluated cell survival with colony formation assay, apoptosis with TUNEL assay and caspase‐3 activity assay, DNA damage with comet assay and an in vivo NHEJ activity assay.Results: Results showed that only spermatocytes GC‐2 transfected with pcDNA‐hMSH5^P29S vector had significant differences in IR‐induced cell survival and apoptosis when compared to that transfected with pcDNA empty vector and pcDNA‐wild‐hMSH5 vector, while there was no statistical difference in GC‐1, TM3, and TM4. In addition, comet assay showed that the DNA damage of GC‐2 transfected with pcDNA‐hMSH5^P29S vector increased significantly compared to that transfected with pcDNA empty vector and pcDNA‐wild‐hMSH5 vector after IR. And in vivo NHEJ activity assay showed that the NHEJ activity of GC‐2 transfected with pcDNA‐hMSH5^P29S vector was statistically higher than that transfected with pcDNA empty vector and pcDNA‐wild‐hMSH5 vector.Conclusion: Our study indicates that the hMSH5 C85T polymorphism leads to an abnormal increase in apoptosis and lessen the control on error‐prone NHEJ of spermatocyte GC‐2, thereby altering the difference of radiation sensitivity of spermatogenesis.