[Effect of WRAP53 ß Targeted Co-Inhibitory Pathways Based on Comprehensive Bioinformatics Analysis in Treating Squamous Cell Carcinoma of the Head and Neck].

Objective: To investigate the association between WD40-encoding RNA antisense to p53 ( WRAP53 ß), a telomerase new core subunit, and the clinical, genomic and immune infiltration characteristics of squamous cell carcinoma of the head and neck (HNSC), and to explore for potential joint targeted therapy of HNSC. Methods: Tumor IMmune Estimation Resource (TIMER) online modules were adopted to predict the association between WRAP53 ß expression and the clinical features, oncogene, and immune infiltration of HNSC in the Cancer Genome Atlas (TCGA) cohort. Tumor Immune Single-cell Hub (TISCH) was used to analyze WRAP53 ß expression at the single cell level. Analysis of the small molecule inhibitors potentially targeting WRAP53 ß was carried out by Computational Analysis of REsistance (CARE). In the in vitro verification experiment, recombinant lentiviral particles with the sh WRAP53 ß sequence were synthesized. Then, the oral squamous cell carcinoma cell line Cal27 (the sh WRAP53 ßgroup) stably expressing sh WRAP53 ß were constructed, and two control groups were set up (the shNC group consisting of Cal27 cells added with lentiviral particles containing non-specific control sequences and the Con group consisting of untreated Cal27 cells). MTT assay was done to examine the proliferation of cells in the three groups. Cellular immunofluorescence assay was done for further qualitative examination of the expression of P53 protein in the cells of the sh WRAP53 ß group and the shNC group. Western blot was done to measure the expression of WRAP53ß and γ-H2AX, a DNA damage protein, in the 18 th, 23 rd and 28 th passages of the sh WRAP53 ß group and the shNC group. Finally, specimens of 13 cases of oral squamous cell carcinoma and 7 cases of oral mucosal inflammation were collected, and the expression of WRAP53ß and γ-H2AX in the clinical specimens of oral squamous cell carcinoma was verified with immunohistochemistry. Resluts: TIMER analysis revealed that the expression level of WRAP53 ß in HNSC tissues was significantly higher than that in normal tissues. There was a significant positive correlation between WRAP53 ß expression and multiple genes in the p53 pathway, including CCNB1, CCNB2 and CDK1. Although no significant correlation between WRAP53 ß expression and infiltrating immune cells was found, WRAP53 ß was significantly positively correlated with the inflammatory factors IFN-γ and IL23A, and negatively correlated with IL-1A and IL-6 in HPV-positive carcinoma of the head and neck. TISCH single cell sequencing datasets also showed higher expression of WRAP53 ß in malignant cells, and very low or zero expression in immune cells. According to the CARE scores, the most potent WRAP53 ß co-inhibitory drugs were ATM, CDK1 and MDM4 targeted inhibitors. In vitro cell experiments showed that the proliferation ability of Cal27 cells decreased significantly in the sh WRAP53 ß group as compared with that of the control group between Day 5 and Day 7 ( P<0.05). Furthermore, the expression of P53 decreased significantly in the sh WRAP53 ß group. As compared with the control group, the expression of WRAP53ß in sh WRAP53 ß group significantly decreased in the 18 th, 23 rd and 28 th passages ( P<0.05), while γ-H2AX expression only decreased in the 18 th and 28 th passages ( P<0.05) according to the results of Western blot. Clinical specimens showed rather high positive expression rate of γ-H2AX in oral squamous cell carcinoma tissues (12/13), while the expression of WRAP53ß was not detected in oral mucositis samples (0/7). Conclusions: WRAP53 ß showed significantly higher expression level in HSNC, and was significantly associated with p53 pathway genes. ATM, CDK1 and MDM4 inhibitors may be potential WRAP53 ß co-inhibitory agents. RNA interference of WRAP53 ß expression may cause inhibition of DNA damage, thereby indicating therapeutic potential for HNSC.

Evaluation of dyskerin expression and the Cajal body protein WRAP53ß as potential prognostic markers for patients with primary vaginal carcinoma.

Primary vaginal cancer (PVC) is a rare gynaecological malignancy, which, at present, lacks appropriate biomarkers for prognosis. The proteins dyskerin and WD repeat containing antisense to TP53 (WRAP53ß), both of which exert their functions in the telomerase holoenzyme complex, have been shown to be upregulated in different cancer types. These proteins have also been proposed as prognostic markers in some types of cancer. The aim of the present study was to examine the expression patterns of dyskerin and WRAP53ß in patients with PVC. Moreover, as part of a search for effective biomarkers to evaluate prognosis in PVC, the expression of these two proteins and their potential association with clinical variables and survival were also evaluated. The expression of dyskerin and WRAP53ß was assessed in PVC tumour samples from 68 patients using immunohistochemistry. The majority of tumour samples showed low and moderate expression levels of dyskerin. Upregulation of dyskerin in tumour samples was significantly associated with a shorter survival time and a poorer cancer-specific survival rate. WRAP53ß was also expressed in most of the cells but was not significantly associated with clinical variables or survival. This study demonstrates that upregulation of dyskerin is significantly associated with poor prognosis. Thus, dyskerin may serve as a promising prognostic marker and a potential putative therapeutic target in PVC.

The Cajal Body Protein WRAP53ß Prepares the Scene for Repair of DNA Double-Strand Breaks by Regulating Local Ubiquitination.

Proper repair of DNA double-strand breaks is critical for maintaining genome integrity and avoiding disease. Modification of damaged chromatin has profound consequences for the initial signaling and regulation of repair. One such modification involves ubiquitination by E3 ligases RNF8 and RNF168 within minutes after DNA double-strand break formation, altering chromatin structure and recruiting factors such as 53BP1 and BRCA1 for repair via non-homologous end-joining (NHEJ) and homologous recombination (HR), respectively. The WD40 protein WRAP53ß plays an essential role in localizing RNF8 to DNA breaks by scaffolding its interaction with the upstream factor MDC1. Loss of WRAP53ß impairs ubiquitination at DNA lesions and reduces downstream repair by both NHEJ and HR. Intriguingly, WRAP53ß depletion attenuates repair of DNA double-strand breaks more than depletion of RNF8, indicating functions other than RNF8-mediated ubiquitination. WRAP53ß plays key roles with respect to the nuclear organelles Cajal bodies, including organizing the genome to promote associated transcription and collecting factors involved in maturation of the spliceosome and telomere elongation within these organelles. It is possible that similar functions may aid also in DNA repair. Here we describe the involvement of WRAP53ß in Cajal bodies and DNA double-strand break repair in detail and explore whether and how these processes may be linked. We also discuss the possibility that the overexpression of WRAP53ß detected in several cancer types may reflect its normal participation in the DNA damage response rather than oncogenic properties.

Phosphorylation of the Cajal body protein WRAP53ß by ATM promotes its involvement in the DNA damage response.

The cellular response to DNA double-strand breaks is orchestrated by the protein kinase ATM, which phosphorylates key actors in the DNA repair network. WRAP53ß is a multifunctional protein that controls trafficking of factors to Cajal bodies, telomeres and DNA double-strand breaks but what regulates the involvement of WRAP53ß in these separate processes remains unclear. Here, we show that in response to various types of DNA damage, including IR and UV, WRAP53ß is phosphorylated on serine residue 64 by ATM with a time-course that parallels its accumulation at DNA lesions. Interestingly, recruitment of phosphorylated WRAP53ß (pWRAP53ßS64) to sites of such DNA damage promotes its interaction with γH2AX at these locations. Moreover, pWRAP53ßS64 stimulates the accumulation of the repair factor 53BP1 at DNA double-strand breaks and enhances repair of this type of damage via homologous recombination and non-homologous end joining. At the same time, phosphorylation of WRAP53ß is dispensable for its localization to Cajal bodies, where it accumulates even in unstressed cells. These findings not only reveal ATM to be an upstream regulator of WRAP53ß, but also indicates that phosphorylation of WRAP53ß at serine 64 controls its involvement in the DNA damage response and may also restrict its other functions.

The scaffold protein WRAP53ß orchestrates the ubiquitin response critical for DNA double-strand break repair.

The WD40 domain-containing protein WRAP53ß (WD40 encoding RNA antisense to p53; also referred to as WDR79/TCAB1) controls trafficking of splicing factors and the telomerase enzyme to Cajal bodies, and its functional loss has been linked to carcinogenesis, premature aging, and neurodegeneration. Here, we identify WRAP53ß as an essential regulator of DNA double-strand break (DSB) repair. WRAP53ß rapidly localizes to DSBs in an ATM-, H2AX-, and MDC1-dependent manner. We show that WRAP53ß targets the E3 ligase RNF8 to DNA lesions by facilitating the interaction between RNF8 and its upstream partner, MDC1, in response to DNA damage. Simultaneous binding of MDC1 and RNF8 to the highly conserved WD40 scaffold domain of WRAP53ß facilitates their interaction and accumulation of RNF8 at DSBs. In this manner, WRAP53ß controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53ß impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53ß as a novel regulator of DSB repair by providing a scaffold for DNA repair factors.