Regulation of the Cell-Intrinsic DNA Damage Response by the Innate Immune Machinery

Maintenance of genomic integrity is crucial for cell survival. As such, elegant DNA damage response (DDR) systems have evolved to ensure proper repair of DNA double-strand breaks (DSBs) and other lesions that threaten genomic integrity. Towards this end, most therapeutic studies have focused on understanding of the canonical DNA DSB repair pathways to enhance the efficacy of DNA-damaging therapies. While these approaches have been fruitful, there has been relatively limited success to date and potential for significant normal tissue toxicity. With the advent of novel immunotherapies, there has been interest in understanding the interactions of radiation therapy with the innate and adaptive immune responses, with the ultimate goal of enhancing treatment efficacy. While a substantial body of work has demonstrated control of the immune-mediated (extrinsic) responses to DNA-damaging therapies by several innate immune pathways (e.g., cGAS–STING and RIG-I), emerging work demonstrates an underappreciated role of the innate immune machinery in directly regulating tumor cell-intrinsic/cell-autonomous responses to DNA damage.     

Crosstalk between Pancreatic Cancer Cells and Cancer-Associated Fibroblasts in the Tumor Microenvironment Mediated by Exosomal MicroRNAs

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant digestive tumors, characterized by a low rate of early diagnosis, strong invasiveness, and early metastasis. The abundant stromal cells, dense extracellular matrix, and lack of blood supply in PDAC limit the penetration of chemotherapeutic drugs, resulting in poor efficacy of the current treatment regimens. Cancer-associated fibroblasts (CAFs) are the major stromal cells in the tumor microenvironment. Tumor cells can secrete exosomes to promote the generation of activated CAFs, meanwhile exosomes secreted by CAFs help promote tumor progression. The aberrant expression of miRNAs in exosomes is involved in the interaction between tumor cells and CAFs, which provides the possibility for the application of exosomal miRNAs in the diagnosis and treatment of PDAC. The current article reviews the mechanism of exosomal miRNAs in the crosstalk between PDAC cells and CAFs in the tumor microenvironment, in order to improve the understanding of TME regulation and provide evidence for designing diagnostic and therapeutic targets against exosome miRNA in human PDAC.     

Evaluation of the Comet Assay for Assessing the Dose-Response Relationship of DNA Damage Induced by Ionizing Radiation

Dose- and time-response curves were combined to assess the potential of the comet assay in radiation biodosimetry. The neutral comet assay was used to detect DNA double-strand breaks in lymphocytes caused by γ-ray irradiation. A clear dose-response relationship with DNA double-strand breaks using the comet assay was found at different times after irradiation (p < 0.001). A time-response relationship was also found within 72 h after irradiation (p < 0.001). The curves for DNA double-strand breaks and DNA repair in vitro of human lymphocytes presented a nice model, and a smooth, three-dimensional plane model was obtained when the two curves were combined.     

Type I Interferons Enhance the Repair of Ultraviolet Radiation-Induced DNA Damage and Regulate Cutaneous Immune Suppression

Type I interferons (IFNs) are important enhancers of immune responses which are downregulated in human cancers, including skin cancer. Solar ultraviolet (UV) B radiation is a proven environmental carcinogen, and its exposure contributes to the high prevalence of skin cancer. The carcinogenic effects of UV light can be attributed to the formation of cyclobutane pyrimidine dimers (CPD) and errors in the repair and replication of DNA. Treatment with a single dose of UVB (100 mJ/cm²) upregulated IFNα and IFNβ in the skin of C57BL/6 mice. IFNα and IFNβ were predominantly produced by CD11b+ cells. In mice lacking the type I IFN receptor 1 (IFNAR1), the repair of CPD following cutaneous exposure to a single dose of UVB (100 mJ/cm²) was decreased. UVB induced the expression of the DNA repair gene xeroderma pigmentosum A (XPA) in wild-type (WT) mice. In contrast, such treatment in IFNAR1 (IFNAR1-/-) mice downregulated XPA. A local UVB regimen consisting of UVB radiation (150 mJ/cm²) for 4 days followed by sensitization with hapten 2,4, dinitrofluorobenzene (DNFB) resulted in significant suppression of immune responses in both WT and IFNAR1-/- mice. However, there were significantly higher CD4+CD25+Foxp3+ regulatory T-cells in the draining lymph nodes of IFNAR1-/- mice in comparison to WT mice. Overall, our studies reveal a previously unknown action of type I IFNs in the repair of photodamage and the prevention of UVB-induced immune suppression.     

多表位DNA壳聚糖微球疫苗的体液免疫应答

目的研究多表位DNA壳聚糖微球疫苗的体液免疫应答。方法制备多表位DNA壳聚糖微球疫苗pcD-NA3.1-HME-3C3d,经鼻腔免疫小鼠,蛋白检测微孔试剂盒检测小鼠特异性IgG抗体水平。结果经免疫的小鼠均能产生针对各表位的特异性IgG抗体,DNA壳聚糖微球疫苗的抗体水平明显高于DNA疫苗。结论壳聚糖微球疫苗投递系统可提高多表位DNA疫苗的免疫应答效果。     

A Novel Molecular Signature of Cancer-Associated Fibroblasts Predicts Prognosis and Immunotherapy Response in Pancreatic Cancer

Cancer-associated fibroblasts (CAFs), a prominent population of stromal cells, play a crucial role in tumor progression, prognosis, and treatment response. However, the relationship among CAF-based molecular signatures, clinical outcomes, and tumor microenvironment infiltration remains largely elusive in pancreatic cancer (PC). Here, we collected multicenter PC data and performed integrated analysis to investigate the role of CAF-related genes (CRGs) in PC. Firstly, we demonstrated that α-SMA⁺ CAFs were the most prominent stromal components and correlated with the poor survival rates of PC patients in our tissue microarrays. Then, we discriminated two diverse molecular subtypes (CAF clusters A and B) and revealed the significant differences in the tumor immune microenvironment (TME), four reported CAF subpopulations, clinical characteristics, and prognosis in PC samples. Furthermore, we analyzed their association with the immunotherapy response of PC patients. Lastly, a CRG score was constructed to predict prognosis, immunotherapy responses, and chemosensitivity in pancreatic cancer patients. In summary, these findings provide insights into further research targeting CAFs and their TME, and they pave a new road for the prognosis evaluation and individualized treatment of PC patients.     

Sensitivity to Cisplatin in Head and Neck Cancer Cells Is Significantly Affected by Patient-Derived Cancer-Associated Fibroblasts

Cancer-associated fibroblasts (CAFs) are one of the most abundant and critical components of the tumor stroma. CAFs can impact many important steps of cancerogenesis and may also influence treatment resistance. Some of these effects need the direct contact of CAFs and cancer cells, while some involve paracrine signals. In this study, we investigated the ability of head and neck squamous cell carcinomas (HNSCC) patient-derived CAFs to promote or inhibit the colony-forming ability of HNSCC cells. The effect of cisplatin on this promoting or inhibiting influence was also studied. The subsequent analysis focused on changes in the expression of genes associated with cancer progression. We found that cisplatin response in model HNSCC cancer cells was modified by coculture with CAFs, was CAF-specific, and different patient-derived CAFs had a different “sensitizing ratio”. Increased expression of VEGFA, PGE2S, COX2, EGFR, and NANOG in cancer cells was characteristic for the increase of resistance. On the other hand, CCL2 expression was associated with sensitizing effect. Significantly higher amounts of cisplatin were found in CAFs derived from patients who subsequently experienced a recurrence. In conclusion, our results showed that CAFs could promote and/or inhibit colony-forming capability and cisplatin resistance in HNSCC cells via paracrine effects and subsequent changes in gene expression of cancer-associated genes in cancer cells.     

Molecular Link between DNA Damage Response and Microtubule Dynamics

Microtubules are major components of the cytoskeleton that play important roles in cellular processes such as intracellular transport and cell division. In recent years, it has become evident that microtubule networks play a role in genome maintenance during interphase. In this review, we highlight recent advances in understanding the role of microtubule dynamics in DNA damage response and repair. We first describe how DNA damage checkpoints regulate microtubule organization and stability. We then highlight how microtubule networks are involved in the nuclear remodeling following DNA damage, which leads to changes in chromosome organization. Lastly, we discuss how microtubule dynamics participate in the mobility of damaged DNA and promote consequent DNA repair. Together, the literature indicates the importance of microtubule dynamics in genome organization and stability during interphase.     

Targeting DNA Damage Response and Immune Checkpoint for Anticancer Therapy

Deficiency in DNA damage response (DDR) genes leads to impaired DNA repair functions that will induce genomic instability and facilitate cancer development. However, alterations of DDR genes can serve as biomarkers for the selection of suitable patients to receive specific therapeutics, such as immune checkpoint blockade (ICB) therapy. In addition, certain altered DDR genes can be ideal therapeutic targets through adapting the mechanism of synthetic lethality. Recent studies indicate that targeting DDR can improve cancer immunotherapy by modulating the immune response mediated by cGAS-STING-interferon signaling. Investigations of the interplay of DDR-targeting and ICB therapies provide more effective treatment options for cancer patients. This review introduces the mechanisms of DDR and discusses their crucial roles in cancer therapy based on the concepts of synthetic lethality and ICB. The contemporary clinical trials of DDR-targeting and ICB therapies in breast, colorectal, and pancreatic cancers are included.     

Cell Type-Specific Patterns in the Accumulation of DNA Damage Following Multifractional Radiation Exposure

Predicting the risk of second malignant neoplasms is complicated by uncertainties regarding the shape of the dose–response relationship at high doses. Limited understanding of the competitive relationship between cell killing and the accumulation of DNA lesions at high doses, as well as the effects of other modulatory factors unique to radiation exposure during radiotherapy, such as dose heterogeneity across normal tissue and dose fractionation, contribute to these uncertainties. The aim of this study was to analyze the impact of fractionated irradiations on two cell systems, focusing on the endpoints relevant for cancer induction. To simulate the heterogeneous dose distribution across normal tissue during radiotherapy, exponentially growing VH10 fibroblasts and AHH-1 lymphoblasts were irradiated with 9 and 12 fractions (VH10) and 10 fractions (AHH-1) at 0.25, 0.5, 1, or 2 Gy per fraction. The effects on cell growth, cell survival, radiosensitivity and the accumulation of residual DNA damage lesions were analyzed as functions of dose per fraction and the total absorbed dose. Residual γH2AX foci and other DNA damage markers (micronuclei, nuclear buds, and giant nuclei) were accumulated at high doses in both cell types, but in a cell type-dependent manner. The competitive relationship between cell killing and the accumulation of carcinogenic DNA damage following multifractional radiation exposure is cell type-specific.     

Zinc Prevents DNA Damage in Normal Cells but Shows Genotoxic and Cytotoxic Effects in Acute Myeloid Leukemia Cells

Genomic instability is prevented by the DNA damage response (DDR). Micronutrients, like zinc (Zn), are cofactors of DDR proteins, and micronutrient deficiencies have been related to increased cancer risk. Acute myeloid leukemia (AML) patients commonly present Zn deficiency. Moreover, reports point to DDR defects in AML. We studied the effects of Zn in DDR modulation in AML. Cell lines of AML (HEL) and normal human lymphocytes (IMC) were cultured in standard culture, Zn depletion, and supplementation (40 μM ZnSO₄) conditions and exposed to hydrogen peroxide (H₂O₂) or ultraviolet (UV) radiation. Chromosomal damage, cell death, and nuclear division indexes (NDI) were assessed through cytokinesis-block micronucleus assay. The phosphorylated histone H2AX (yH2AX) expression was monitored at 0 h, 1 h, and 24 h after exposure. Expression of DDR genes was evaluated by quantitative real time polymerase chain reaction (qPCR). Zn supplementation increased the genotoxicity of H₂O₂ and UV radiation in AML cells, induced cytotoxic and antiproliferative effects, and led to persistent yH2AX activation. In contrast, in normal lymphocytes, supplementation decreased damage rates, while Zn depletion favored damage accumulation and impaired repair kinetics. Gene expression was not affected by Zn depletion or supplementation. Zn presented a dual role in the modulation of genome damage, preventing damage accumulation in normal cells and increasing genotoxicity and cytotoxicity in AML cells.     

Interplay between the DNA Damage Response and Immunotherapy Response in Cancer

Genome instability and immune evasion are both defining hallmarks of cancer. Tumorigenesis is frequently initiated when there is DNA damage to a proto-oncogene or tumor suppressor gene and DNA repair mechanisms are lost or insufficient to correct the damage; immune evasion then prevents the host immune system from recognizing these transformed cells. Therapies targeting genomic instability and immune evasion have been effectively used to treat cancer. Genotoxic therapies such as chemoradiation have been employed in cancer treatments for several decades, while immunotherapy is a relatively new class of cancer therapy that has led to disease regression even in patients with advanced cancer. Several recent studies have shown synergy between both classes of therapy targeting these two defining hallmarks of cancer, and different mechanisms are proposed to be involved. Here, we review the different classes of DNA damage, their links to cancer, and their contribution to immunotherapy responses, as well as the different models that are currently being used to study tumor–immune interactions.     

DNA Damage Response Regulation by Histone Ubiquitination

Cells are constantly exposed to numerous genotoxic stresses that induce DNA damage. DNA double-strand breaks (DSBs) are among the most serious damages and should be systematically repaired to preserve genomic integrity. The efficiency of repair is closely associated with chromatin structure, which is regulated by posttranslational modifications of histones, including ubiquitination. Recent evidence shows crosstalk between histone ubiquitination and DNA damage responses, suggesting an integrated model for the systematic regulation of DNA repair. There are two major pathways for DSB repair, viz., nonhomologous end joining and homologous recombination, and the choice of the pathway is partially controlled by posttranslational modifications of histones, including ubiquitination. Histone ubiquitination changes chromatin structure in the vicinity of DSBs and serves as a platform to select and recruit repair proteins; the removal of these modifications by deubiquitinating enzymes suppresses the recruitment of repair proteins and promotes the convergence of repair reactions. This article provides a comprehensive overview of the DNA damage response regulated by histone ubiquitination in response to DSBs.     

骨髓间充质干细胞对电离辐射诱发的小鼠胸腺瘤的抑制作用

目的观察骨髓间充质干细胞(MSCs)对电离辐射诱发的小鼠胸腺瘤的抑制作用。方法采用经典Kaplan法复制电离辐射诱发的小鼠胸腺瘤模型。应用全骨髓贴壁法分离培养C57BL/6小鼠MSCs,DAPI标记,经尾静脉注入荷瘤小鼠后,分别于1、5、10d处死小鼠,取胸腺组织,激光共聚焦显微镜下观察MSCs在胸腺瘤组织中的定位;第1次全身大剂量照射后6个月取胸腺组织,HE染色观察胸腺组织的病理变化,并判断成瘤情况。结果激光共聚焦显微镜下观察可见,MSCs经尾静脉输注后可迁徙至小鼠胸腺组织内;病理观察显示,胸腺组织皮髓质结构清楚,淋巴样肿瘤细胞较少,细胞形态、大小不一,偶见核分裂象;MSCs输注使辐射诱导的胸腺瘤成瘤率由57.00%±9.78%降低至37.50%±7.55%。结论已成功建立辐射诱发的小鼠胸腺瘤模型;输注的MSCs可迁徙至胸腺组织中,并降低胸腺瘤的成瘤率。     

汉坦病毒DNA疫苗pVAX/G2诱导小鼠的免疫应答

目的探讨含汉坦病毒L99株G2糖蛋白基因的DNA疫苗诱导BALB/c小鼠的免疫应答。方法重组质粒pVAX/G2转染cos-7细胞,用间接免疫荧光法(IFA)检测瞬时表达产物。经碱裂解法提取质粒pVAX/G2,Sepharose CL-4B柱层析纯化后,免疫BALB/c小鼠(SPF),并采用含有人IL-2基因的重组质粒pVAX/IL-2及重组人IL-2蛋白作为DNA疫苗的佐剂,检测小鼠的体液免疫和细胞免疫应答。结果重组质粒pVAX/G2能够在真核细胞中表达。免疫小鼠血清中能够检测到抗汉坦病毒中和抗体、酶标抗体和荧光抗体。淋转刺激指数和脾淋巴细胞上清液中IL-2活性,与空载体pVAX1阴性对照比较差异有显著意义。质粒佐剂pVAX/IL-2能显著提高DNA疫苗的细胞免疫水平。结论含汉坦病毒L99株G2糖蛋白基因的DNA疫苗能诱导BALB/c小鼠产生一定强度的体液免疫和细胞免疫应答。     

Cancer-Associated Fibroblasts Modify the Response of Prostate Cancer Cells to Androgen and Anti-Androgens in Three-Dimensional Spheroid Culture

Androgen receptor (AR) targeting remains the gold standard treatment for advanced prostate cancer (PCa); however, treatment resistance remains a major clinical problem. To study the therapeutic effects of clinically used anti-androgens we characterized herein a tissue-mimetic three-dimensional (3D) in vitro model whereby PCa cells were cultured alone or with PCa-associated fibroblasts (CAFs). Notably, the ratio of PCa cells to CAFs significantly increased in time in favor of the tumor cells within the spheroids strongly mimicking PCa in vivo. Despite this loss of CAFs, the stromal cells, which were not sensitive to androgen and even stimulated by the anti-androgens, significantly influenced the sensitivity of PCa cells to androgen and to the anti-androgens bicalutamide and enzalutamide. In particular, DuCaP cells lost sensitivity to enzalutamide when co-cultured with CAFs. In LAPC4/CAF and LNCaP/CAF co-culture spheroids the impact of the CAFs was less pronounced. In addition, 3D spheroids exhibited a significant increase in E-cadherin and substantial expression of vimentin in co-culture spheroids, whereas AR levels remained unchanged or even decreased. In LNCaP/CAF spheroids we further found increased Akt signaling that could be inhibited by the phosphatidyl-inositol 3 kinase (PI3K) inhibitor LY294002, thereby overcoming the anti-androgen resistance of the spheroids. Our data show that CAFs influence drug response of PCa cells with varying impact and further suggest this spheroid model is a valuable in vitro drug testing tool.     

Overcoming Radiation Resistance in Gliomas by Targeting Metabolism and DNA Repair Pathways

Gliomas represent a wide spectrum of brain tumors characterized by their high invasiveness, resistance to chemoradiotherapy, and both intratumoral and intertumoral heterogeneity. Recent advances in transomics studies revealed that enormous abnormalities exist in different biological layers of glioma cells, which include genetic/epigenetic alterations, RNA expressions, protein expression/modifications, and metabolic pathways, which provide opportunities for development of novel targeted therapeutic agents for gliomas. Metabolic reprogramming is one of the hallmarks of cancer cells, as well as one of the oldest fields in cancer biology research. Altered cancer cell metabolism not only provides energy and metabolites to support tumor growth, but also mediates the resistance of tumor cells to antitumor therapies. The interactions between cancer metabolism and DNA repair pathways, and the enhancement of radiotherapy sensitivity and assessment of radiation response by modulation of glioma metabolism are discussed herein.