Knockdown of Rad9A enhanced DNA damage induced by trichostatin A in esophageal cancer cells.

Histone deacetylase (HDAC) inhibitors have recently emerged as a new class of anticancer agents. As a classical HDAC inhibitor, trichostatin A (TSA) has been shown to possess many anticancer activities such as induction of cell cycle arrest, promotion of cell death, and enhancement of radiosensitity. In our previous work, we found that TSA treatment induced Rad9 gene expression, which suggested that Rad9 might play a role in TSA-induced biological effects. As Rad9 is involved in maintaining genomic integrity, we further analyzed the DNA damage induced by TSA and combined with Rad9 knockdown in esophageal cancer cells (ESCCs). Our results showed that TSA treatment alone induced significantly DNA damage in ESCC cells. Simultaneously, TSA also induced Rad9 gene expression both at transcriptional and translational levels in EC109 cells, but not in KYSE150 cells. Further, the induction of Rad9 by TSA was accompanied with increased level of histone H3K9 acetylation in Rad9 promoter region. To understand the role of Rad9 in TSA-induced DNA damage, Rad9 gene expression was efficiently knocked down by small interfering RNA (siRNA), which led to enhanced DNA damage and cell death induced by TSA. Our data suggested that Rad9 plays an important role in DNA damage, which is related to the biological effects of TSA.

The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse.

In mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whether RAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the male germ line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specific ablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male mice were infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested in meiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of γH2AX and DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIß-binding protein 1 (TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meiotic prophase I.

Unveiling an anoikis-related risk model and the role of RAD9A in colon cancer.

OBJECTIVE: Colorectal cancer (CRC), specifically colon adenocarcinoma, is the third most prevalent and the second most lethal form of cancer. Anoikis is found to be specialized form of programmed cell death (PCD), which plays a pivotal role in tumor progression. This study aimed to investigate the role of the anoikis related genes (ARGs) in colon cancer. METHODS: Consensus unsupervised clustering, differential expression analysis, tumor mutational burden analysis, and analysis of immune cell infiltration were utilized in the study. For the analysis of RNA sequences and clinical data of COAD patients, data from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were obtained. A prognostic scoring system for overall survival (OS) prediction was developed using Cox regression and LASSO regression analysis. Furthermore, loss-of-function assay was utilized to explore the role of RAD9A played in the progression of colon cancer. RESULTS: The prognostic value of a risk score composed of NTRK2, EPHA2, RAD9A, CDC25C, and SNAI1 genes was significant. Furthermore, these findings suggested potential mechanisms that may influence prognosis, supporting the development of individualized treatment plans and management of patient outcomes. Further experiments confirmed that RAD9A could promote proliferation and metastasis of colon cancer cells. These effects may be achieved by affecting the phosphorylation of AKT. CONCLUSION: Differences in survival time and the tumor immune microenvironment (TIME) were observed between two gene clusters associated with ARGs. In addition, a prognostic risk model was established and confirmed as an independent risk factor. Furthermore, our data indicated that RAD9A promoted tumorigenicityby activating AKT in colon cancer.

Hypermethylation of RAD9A intron 2 in childhood cancer patients, leukemia and tumor cell lines suggest a role for oncogenic transformation.

Most childhood cancers occur sporadically and cannot be explained by an inherited mutation or an unhealthy lifestyle. However, risk factors might trigger the oncogenic transformation of cells. Among other regulatory signals, hypermethylation of RAD9A intron 2 is responsible for the increased expression of RAD9A protein, which may play a role in oncogenic transformation. Here, we analyzed the RAD9A intron 2 methylation in primary fibroblasts of 20 patients with primary cancer in childhood and second primary cancer (2N) later in life, 20 matched patients with only one primary cancer in childhood (1N) and 20 matched cancer-free controls (0N), using bisulfite pyrosequencing and deep bisulfite sequencing (DBS). Four 1N patients and one 2N patient displayed elevated mean methylation levels (≥ 10 %) of RAD9A. DBS revealed ≥ 2 % hypermethylated alleles of RAD9A, indicative for constitutive mosaic epimutations. Bone marrow samples of NHL and AML tumor patients (n=74), EBV (Epstein Barr Virus) lymphoblasts (n=6), tumor cell lines (n=5) and FaDu subclones (n=13) were analyzed to substantiate our findings. We find a broad spectrum of tumor entities with an aberrant methylation of RAD9A. We detected a significant difference in mean methylation of RAD9A for NHL versus AML patients (p ≤0.025). Molecular karyotyping of AML samples during therapy with hypermethylated RAD9A showed an evolving duplication of 1.8 kb on Chr16p13.3 including the PKD1 gene. Radiation, colony formation assays, cell proliferation, PCR and molecular karyotyping SNP-array experiments using generated FaDu subclones suggest that hypermethylation of RAD9A intron 2 is associated with genomic imbalances in regions with tumor-relevant genes and survival of the cells. In conclusion, this is the very first study of RAD9A intron 2 methylation in childhood cancer and Leukemia. RAD9A epimutations may have an impact on leukemia and tumorigenesis and can potentially serve as a biomarker.

Genome-Scale CRISPR-Cas9 Transcriptional Activation Screening in Metformin Resistance Related Gene of Prostate Cancer.

Metformin is a classic type II diabetes drug which possesses anti-tumor properties for various cancers. However, different cancers do not respond to metformin with the same effectiveness or acquire resistance. Thus, searching for vulnerabilities of metformin-resistant prostate cancer is a promising strategy to improve the therapeutic efficiency of the drug. A genome-scale CRISPR-Cas9 activation library search targeting 23,430 genes was conducted to identify the genes that confer resistance to metformin in prostate cancer cells. Candidate genes were selected by total reads of sgRNA and sgRNA diversity, and then a CCK8 assay was used to verify their resistance to metformin. Interestingly, we discovered that the activation of ECE1, ABCA12, BPY2, EEF1A1, RAD9A, and NIPSNAP1 contributed to in vitro resistance to metformin in DU145 and PC3 cell lines. Notably, a high level of RAD9A, with poor prognosis in PCa, was the most significant gene in the CCK8 assay. Furthermore, we discerned the tumor immune microenvironment with RAD9A expression by CIBERSORT. These results suggested that a high level of RAD9A may upregulate regulatory T cells to counterbalance metformin in the tumor immune microenvironment.

Identification and characterization of a novel nuclear structure containing members of the homologous recombination and DNA damage response pathways.

The human RAD9A protein is required for successful execution of the G2/M DNA damage checkpoint. Along with RAD1 and HUS1, RAD9A exists in a heterotrimeric ring-shaped complex which is necessary for activation of the CHK1 checkpoint kinase. RAD9A is also required for proper localization of both TopBP1 and the Claspin adaptor protein during the DNA damage response. We have shown large, RAD9A-dense nuclear foci containing several members of the homologous recombination pathway as well as BRCA1 and the DNA damage marker γH2AX. This RAD9A-dense body is closely associated with the inactive X in HeLa cells but not in other cell types analyzed including a Klinefelter's syndrome-derived line containing multiple Xi. We have also shown these foci disappear after cell synchronization but are enriched after treatment with the homologous recombination inhibitor pentoxifylline. We conclude these foci are the result of an active process, suspended in perturbed cells, that involves interaction between the cell cycle checkpoint and homologous recombination machinery.

Rad9a is required for spermatogonia differentiation in mice.

Spermatogenesis in testes requires precise spermatogonia differentiation. Spermatocytes lacking the Rad9a gene are arrested in pachytene prophase, implying a possible role for RAD9A in spermatogonia differentiation. However, numerous RAD9A-positive pachytene spermatocytes are still observed in mouse testes following Rad9a excision using the Stra8-Cre system, and it is unclear whether Rad9a deletion in spermatogonia interrupts differentiation. Here, we generated a mouse model in which Rad9a was specifically deleted in spermatogonial stem cells (SSCs) using Cre recombinase expression driven by the germ cell-specific Vasa promoter. Adult Rad9a-null male mice were infertile as a result of completely blocked spermatogonia differentiation. No early spermatocytes were detected in mutant testicular cords of 9-day-old mice. Mutant spermatogonia were prone to apoptosis, although proliferation rates were unaffected. Rad9a deletion also resulted in malformation of seminiferous tubules, in which cells assembled irregularly into clusters, and malformation led to testicular cord disruption. Our findings suggest that Rad9a is indispensable for spermatogonia differentiation and testicular development in mice.

Clamping down on mammalian meiosis.

The RAD9A-RAD1-HUS1 (9-1-1) complex is a PCNA-like heterotrimeric clamp that binds damaged DNA to promote cell cycle checkpoint signaling and DNA repair. While various 9-1-1 functions in mammalian somatic cells have been established, mounting evidence from lower eukaryotes predicts critical roles in meiotic germ cells as well. This was investigated in 2 recent studies in which the 9-1-1 complex was disrupted specifically in the mouse male germline through conditional deletion of Rad9a or Hus1. Loss of these clamp subunits led to severely impaired fertility and meiotic defects, including faulty DNA double-strand break repair. While 9-1-1 is critical for ATR kinase activation in somatic cells, these studies did not reveal major defects in ATR checkpoint pathway signaling in meiotic cells. Intriguingly, this new work identified separable roles for 9-1-1 subunits, namely RAD9A- and HUS1-independent roles for RAD1. Based on these studies and the high-level expression of the paralogous proteins RAD9B and HUS1B in testis, we propose a model in which multiple alternative 9-1-1 clamps function during mammalian meiosis to ensure genome maintenance in the germline.