BACKGROUND: DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has an essential role in the non-homologous end-joining pathway that repairs DNA double-strand breaks in V(D)J recombination involved in the expression of T- and B-cell receptors. Whereas homozygous mutations in Prkdc define the Scid mouse, a model that has been widely used in biology, human mutations in PRKDC are extremely rare and the disease spectrum has not been described so far.
OBJECTIVE: To provide an update on the genetics, clinical spectrum, immunological profile, and therapy of DNA-PKcs deficiency in human.
METHODS: The clinical, biological, and treatment data from the 6 cases published to date and from 1 new patient were obtained and analyzed. Rubella PCR was performed on available granuloma material.
RESULTS: We report on 7 patients; 6 patients displayed the autosomal recessive p.L3062R mutation in PRKDC-encoding DNA-PKcs. Atypical severe combined immunodeficiency with inflammatory lesions, granulomas, and autoimmunity was the predominant clinical manifestation (n = 5 of 7). Rubella viral strain was detected in the granuloma of 1 patient over the 2 tested. T-cell counts, including naive CD4+CD45RA+ T cells and T-cell function were low at diagnosis for 6 patients. For most patients with available values, naive CD4+CD45RA+ T cells decreased over time (n = 5 of 6). Hematopoietic stem cell transplantation was performed in 5 patients, of whom 4 are still alive without transplant-related morbidity. Sustained T- and B-cell reconstitution was observed, respectively, for 4 and 3 patients, after a median follow-up of 8 years (range 3-16 years).
CONCLUSIONS: DNA-PKcs deficiency mainly manifests as an inflammatory disease with granuloma and autoimmune features, along with severe infections.

Nuclear factor-kappa B (NF-κB) is a transcriptional factor that binds to the ∼10-base-pair κB motif on target genes and acts as an inflammatory regulator. Since dysregulation of NF-κB is thought to be related to various diseases, it would be very important to elucidate its post-translational modifications and binding partners in detail and to deeply understand mechanisms of the NF-κB dysregulation. NF-κB p65 is known to interact with the basic transcription factor TFIID subunit hTAFII31/TAF9 through the ФXXФФ (Ф, hydrophobic amino acid; X, any amino acid) motif in a similar fashion to p53. MDM2 is known to inhibit p53 from binding to hTAFII31/TAF9 by masking p53\'s ФXXФФ motif. Here, as can be rationalized from this observation, we searched for novel nuclear proteins that interact with the transactivation domain 1 (TA1) of NF-κB p65 containing a ФXXФФ motif. We prepared a GST-tagged polypeptide, GST-p65532-550, from Phe532-Ser550 of the TA1 domain and found various U937 cell nuclear proteins that bound to GST-p65532-550. The largest bound protein the size of ∼400 kDa was subjected to mass spectrometric analysis and found to be DNA-dependent protein kinase catalytic subunit (DNA-PKcs). An immunoprecipitation experiment with an antibody against p65 and nuclear extracts from TNF-α-treated A549 cells suggested that NF-κB p65 indeed binds to DNA-PKcs in human cells. Furthermore, binding assays with a series of His-tagged DNA-PKcs fragments suggested that DNA-PKcs can bind to NF-κB p65 through the interaction of the TA1 domain with the region 541-750 in the N-HEAT domain or the region 2485-2576 in the M-HEAT domain.

The PRKDC gene encodes the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein. DNA-PKcs plays an important role in nonhomologous end joining (NHEJ) of DNA double-strand breaks (DSBs) and is also closely related to the establishment of central immune tolerance and the maintenance of chromosome stability. The occurrence and development of different types of tumors and the results of their treatment are also influenced by DNA-PKcs, and it may also predict the results of radiotherapy, chemotherapy, and therapy with immune checkpoint inhibitors (ICIs). Here, we discuss and review the structure and mechanism of action of PRKDC and DNA-PKcs and their relationship with cancer.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key component of the DNA-PK complex that has a well-characterized function in the non-homologous end-joining repair of DNA double-strand breaks. Since its identification, a large body of evidence has demonstrated that DNA-PKcs is frequently overexpressed in cancer, plays a critical role in tumor development and progression, and is associated with poor prognosis of cancer patients. Intriguingly, recent studies have suggested novel functions beyond the canonical role of DNA-PKcs, which has transformed the paradigm of DNA-PKcs in tumorigenesis and has reinvigorated the interest to target DNA-PKcs for cancer treatment. In this review, we update recent advances in DNA-PKcs, in particular the emerging roles in tumor metastasis, metabolic dysregulation, and immune escape. We further discuss the possible molecular basis that underpins the pleiotropism of DNA-PKcs in cancer. Finally, we outline the biomarkers that may predict the therapeutic response to DNA-PKcs inhibitor therapy. Understanding the functional repertoire of DNA-PKcs will provide mechanistic insights of DNA-PKcs in malignancy and, more importantly, may revolutionize the design and utility of DNA-PKcs-based precision cancer therapy.

OBJECTIVE: DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has attracted extensive attention in various types of malignant tumors. However, the role of DNA-PKcs in cutaneous squamous cell carcinoma (cSCC) development has not been elucidated. In this study, we investigated the role of DNA-PKcs in cSCC and the molecular mechanisms of TGF-β1-induced cSCC progression mediated by DNA-PKcs.
METHODS: We performed bioinformatic analysis and RT-PCR to examine the DNA-PKcs expression level in cSCC. Then, we downregulated DNA-PKcs using a DNA-PK-specific inhibitor or small interfering RNA (siRNA) to explore the effects of DNA-PKcs on SCL-1 cell migration and invasion. To further investigate the mechanism by which DNA-PKcs promotes cSCC progression, TGF-β1 and the TGF-β receptor (TGF-βR) I/II dual inhibitor LY2109761 were used to examine whether DNA-PKcs participates in TGF-β1/Smad signaling.
RESULTS: DNA-PKcs expression was upregulated in cSCC. DNA-PK inhibition or expression knockdown resulted in inhibited migration and invasion and altered epithelial-mesenchymal transition (EMT) marker expression patterns in SCL-1 cells. Importantly, TGF-β1 mediated EMT induction in cSCC cells, and DNA-PKcs was identified as a TGF-β1-responsive gene. TGF-β1 promoted DNA-PKcs transcription, and DNA-PKcs enhanced the TGF-β1-induced EMT program involved in cSCC invasion and metastasis by phosphorylating Smad3.
CONCLUSIONS: This study is the first to show that DNA-PKcs mediates EMT to promote cSCC aggressiveness by targeting the TGF-β1/Smad signaling pathway, which provides insight into how DNA-PKcs impacts cSCC progression and identifies a new therapeutic target.

Due to the high incidence of liver cancer, chemoradiotherapy and prognosis of liver cancer are a primary focus of medical research. microRNAs (miRNAs/miRs) serve crucial roles in resistance to chemotherapy and radiotherapy. The aim of the present study was to investigate the effects of miR-101 on the chemotherapeutic efficacy of cisplatin (CDDP) in liver cancer. First, human liver cancer cells (HepG2) were transfected with a miR-101 mimic or miR-101 inhibitor to bidirectionally regulate the expression of miR-101. Cell proliferation, apoptosis, intracellular reactive oxygen species and comet assay results indicated that the upregulation of miR-101 sensitized HepG2 cells to CDDP, and downregulation of miR-101 reduced chemosensitivity. A xenograft mouse model further confirmed that miR-101 overexpression increased CDDP sensitivity in liver cancer. Luciferase reporter and western blotting assays demonstrated that transfection of the miR-101 mimic markedly reduced activity of the DNA-dependent protein kinase catalytic subunit/protein kinase B/mammalian target of rapamycin (DNA-PKcs/Akt/mTOR) pathway and increased expression of apoptotic protein caspase 3, which is induced by CDDP treatment. By contrast, miR-101 inhibitors partially reversed these changes. Moreover, the miR-101 mimic suppressed activity of the nuclear factor-κB (NF-κB) pathway, leading to increased susceptibility of HepG2 cells to chemotherapeutic agents. In conclusion, miR-101 overexpression augmented cytotoxicity and reduced chemoresistance to CDDP in HepG2 cells, and this was associated with negative regulation of DNA-PKcs/Akt/NF-κB signaling.

The prognosis and treatment of thyroid cancer depends on the type and stage of the disease. Radiosensitivity differs among cancer cells owing to their varying capacity for repair after irradiation. Radioactive iodine can be used to destroy thyroid cancer cells. However, patient prognosis and improvement after irradiation varies. Therefore, predictive measures are important for avoiding unnecessary exposure to radiation. We describe a new method for predicting the effects of radiation in individual cases of thyroid cancer based on the DNA-dependent protein kinase (DNA-PK) activity level in cancer cells. The radiation sensitivity, DNA-PK activity, and cellular levels of DNA-PK complex subunits in five human thyroid cancer cell lines were analyzed in vitro. A positive correlation was observed between the D10 value (radiation dose that led to 10% survival) of cells and DNA-PK activity. This correlation was not observed after treatment with NU7441, a DNA-PK-specific inhibitor. A significant correlation was also observed between DNA-PK activity and expression levels of the DNA-PK catalytic subunit (DNA-PKcs). Cells expressing low DNA-PKcs levels were radiation-sensitive, and cells expressing high DNA-PKcs levels were radiation-resistant. Our results indicate that radiosensitivity depends on the expression level of DNA-PKcs in thyroid cancer cell lines. Thus, the DNA-PKcs expression level is a potential predictive marker of the success of radiation therapy for thyroid tumors.

The aim of the present study was to explore the expression and distribution of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in tumor tissues and adjacent normal mucosa tissues of patients with laryngeal squamous cell carcinoma (LSCC), and further analyze the association between the expression and the clinicopathological parameters of patients with LSCC. Clinical data of tumor tissues and corresponding adjacent normal mucosa tissues of pathologically diagnosed LSCC in 96 cases were collected in the present study. Of these specimens, the mRNA and protein expression levels of DNA-PKcs in LSCC tissues and the adjacent normal mucosa tissues were analyzed via reverse transcription-quantitative polymerase chain reaction and western blot analysis. Immunohistochemistry was used to detect expression and distribution of DNA-PKcs protein in LSCC tissues and corresponding adjacent normal mucosa tissues. The association between DNA-PKcs expression and the specific clinicopathologic features was evaluated by the χ2 test. Kaplan-Meier and Cox proportional hazards regression models were used to analyze the data. It was revealed that the expression of DNA-PKcs mRNA and protein was significantly higher in LSCC tissues than the adjacent normal mucosa tissues (P<0.05). DNA-PKcs was expressed predominantly in the nucleus. DNA-PKcs expression showed significant correlation with the differentiation degree of LSCC (P<0.05), and changes of DNA-PKcs expression gradually increased with the decrease of the differentiation degree. However, DNA-PKcs expression was not significantly associated with sex, age, lymph node metastasis or TMN stage (P>0.05). Patients with LSCC exhibited higher DNA-PKcs expression had markedly shorter survival than those with lower DNA-PKcs expression. In conclusion, the present results suggested that the expression levels of DNA-PKcs were significantly increased in LSCC tumor tissues than in adjacent normal mucosa. DNA-PKcs expression was correlated with differentiation of LSCC, and may become a novel prognostic marker for patients with LSCC.

Little work has been done on the mechanism of low dose hyper-radiosensitivity (HRS) and later appeared radioresistance (termed induced radioresistance (IRR)) after irradiation with medium and high linear energy transfer (LET) particles. The aim of this study was to find out whether ATR pathway is involved in the mechanism of HRS induced by high LET radiation. GM0639 cells and two ATM deficient/mutant cells, AT5BIVA and AT2KY were irradiated by carbon ion beam. Thymidine block technique was developed to enrich the G2-phase population. Radiation induced early G2/M checkpoint was quantitatively assess with dual-parameter flow cytometry by detecting the cells positive for phospho-histone H3. The involvement of ATR pathway in HRS/IRR response was detected with pretreatment of specific inhibitors prior to carbon ion beam. The link between the early G2/M checkpoint and HRS/IRR under carbon ion beam was first confirmed in GM0639 cells, through the enrichment of cell population in G2-phase or with Aurora kinase inhibitor that attenuates the transition from G2 to M phase. Interestingly, the early G2/M arrest could still be observed in ATM deficient/mutant cells with an effect of ATR signaling, which was discovered to function in an LET-dependent manner, even as low as 0.2Gy for carbon ion radiation. The involvement of ATR pathway in heavy particles induced HRS/IRR was determined with the specific ATR inhibitor in GM0639 cells, which affected the HRS/IRR occurrence similarly as ATM inhibitor. These data demonstrate that ATR pathway may cooperate with ATM in the mechanism of low dose hypersensitivity induced by carbon ion beam.

BACKGROUND: PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T-cell tolerance.
OBJECTIVE: We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity associated with decreasing T- and B-cell counts over time that had been diagnosed in 2 unrelated patients.
METHODS: Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency.
RESULTS: We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA double-strand break repair and V(D)J recombination. Whole-blood mRNA analysis revealed a strong interferon signature. On activation, memory T cells displayed a skewed cytokine response typical of TH2 and TH1 but not TH17. Moreover, mutated DNA-PKcs did not promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo with production of anti-calcium-sensing receptor autoantibodies, which are typically found in AIRE-deficient patients. In addition, 9 months after bone marrow transplantation, patient 1 had Hashimoto thyroiditis, suggesting that organ-specific autoimmunity might be linked to nonhematopoietic cells, such as AIRE-expressing thymic epithelial cells.
CONCLUSIONS: Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoimmunity, suggesting a role for DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human subjects.