Expression Of DNA-PKcs Research Articles

DNA-PKcs/AKT1 inhibits epithelial-mesenchymal transition during radiation-induced pulmonary fibrosis by inducing ubiquitination and degradation of Twist1.

Radiation-induced pulmonary fibrosis (RIPF) is a chronic, progressive, irreversible lung interstitial disease that develops after radiotherapy. Although several previous studies have focused on the mechanism of epithelial-mesenchymal transition (EMT) in lung epithelial cells, the essential factors involved in this process remain poorly understood. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) exhibits strong repair capacity when cells undergo radiation-induced damage; whether DNA-PKcs regulates EMT during RIPF remains unclear. To investigate the role and molecular mechanism of DNA-PKcs in RIPF and provide an important theoretical basis for utilising DNA-PKcs-targeted drugs for preventing RIPF. DNA-PKcs knockout (DPK-/-) mice were generated via the Cas9/sgRNA technique and subjected to whole chest ionizing radiation (IR) at a 20Gy dose. Before whole chest IR, the mice were intragastrically administered the DNA-PKcs-targeted drug VND3207. Lung tissues were collected at 1 and 5 months after IR. The expression of DNA-PKcs is low in pulmonary fibrosis (PF) patients. DNA-PKcs deficiency significantly exacerbated RIPF by promoting EMT in lung epithelial cells. Mechanistically, DNA-PKcs deletion by shRNA or inhibitor NU7441 maintained the protein stability of Twist1. Furthermore, AKT1 mediated the interaction between DNA-PKcs and Twist1. High Twist1 expression and EMT-associated changes caused by DNA-PKcs deletion were blocked by insulin-like growth factor-1 (IGF-1), an AKT1 agonist. The radioprotective drug VND3207 prevented IR-induced EMT and alleviated RIPF in mice by stimulating the kinase activity of DNA-PKcs. Our study clarified the critical role and mechanism of DNA-PKcs in RIPF and showed that it could be a potential target for preventing RIPF.

Effects and mechanism of anlotinib on radiosensitivity of non-small cell lung cancer cell lines a549

To investigate the effect and mechanism of Anlotinib on radio sensitization of human lung adenocarcinoma cell line A549. Human lung adeno carcinoma cell line A549 was teated with anlotinib and/or radiotherapy, then divided four groups, control group (Ctrl), Anlotinib treatment group (A), irradiation group (RT) and Anlotinib combined with irradiation group (A+RT) . CCK8 method was used to determine cell proliferation; the clone formation experiment was used to determine the inhibitory effect on cell growth; flow cytometry was used to determine cell cycle and apoptosis; immunofluorescence of γ-H2AX was used to determine DNA damage; expression of DNA-PKcs were detected by Western blot. Anlotinib inhibited proliferation and clonogenic survival following irradiation. The dose (Dq), the average lethal dose (D0) and the survival score (SF2) in the anlotinib combined radiotherapy group was significantly lower than those in the radiotherapy group. Anlotinib decreased G2/M phase arrest and promoted the cells apoptosis induced by in irradiation. The confocal microscopy results showed the average number of γ-H2AX foci in the A+RT group was more than that in RT group. The protein levels of DNA-PKcs were higher in A+RT group than that in RT group. Anlotinib enhances the radio sensitivity of A549 cells, which may be attributed to the delay DNA damage repair. It provides a rationale strategy by Anlotinib combined with irradiation for NSCLC.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) drives chronic kidney disease progression in male mice

Kidney injury initiates epithelial dedifferentiation and myofibroblast activation during the progression of chronic kidney disease. Herein, we find that the expression of DNA-PKcs is significantly increased in the kidney tissues of both chronic kidney disease patients and male mice induced by unilateral ureteral obstruction and unilateral ischemia-reperfusion injury. In vivo, knockout of DNA-PKcs or treatment with its specific inhibitor NU7441 hampers the development of chronic kidney disease in male mice. In vitro, DNA-PKcs deficiency preserves epithelial cell phenotype and inhibits fibroblast activation induced by transforming growth factor-beta 1. Additionally, our results show that TAF7, as a possible substrate of DNA-PKcs, enhances mTORC1 activation by upregulating RAPTOR expression, which subsequently promotes metabolic reprogramming in injured epithelial cells and myofibroblasts. Taken together, DNA-PKcs can be inhibited to correct metabolic reprogramming via the TAF7/mTORC1 signaling in chronic kidney disease, and serve as a potential target for treating chronic kidney disease.

DNA-PKcs: A Targetable Protumorigenic Protein Kinase.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a pleiotropic protein kinase that plays critical roles in cellular processes fundamental to cancer. DNA-PKcs expression and activity are frequently deregulated in multiple hematologic and solid tumors and have been tightly linked to poor outcome. Given the potentially influential role of DNA-PKcs in cancer development and progression, therapeutic targeting of this kinase is being tested in preclinical and clinical settings. This review summarizes the latest advances in the field, providing a comprehensive discussion of DNA-PKcs functions in cancer and an update on the clinical assessment of DNA-PK inhibitors in cancer therapy.

Lactate Suppresses Retroviral Transduction in Cervical Epithelial Cells through DNA-PKcs Modulation

Recently, we have shown the molecular basis for lactate sensing by cervical epithelial cells resulting in enhanced DNA repair processes through DNA-PKcs regulation. Interestingly, DNA-PKcs is indispensable for proper retroviral DNA integration in the cell host genome. According to recent findings, the mucosal epithelium can be efficiently transduced by retroviruses and play a pivotal role in regulating viral release by cervical epithelial cells. This study examined the effects of lactate on lentiviral transduction in cervical cancer cells (HeLa, CaSki, and C33A) and model glioma cell lines (DNA-PKcs proficient and deficient). Our study showed that L- and D-lactate enhanced DNA-PKcs presence in nuclear compartments by between 38 and 63%, which corresponded with decreased lentiviral transduction rates by between 15 and 36%. Changes in DNA-PKcs expression or its inhibition with NU7441 also greatly affected lentiviral transduction efficacy. The stimulation of cells with either HCA1 agonist 3,5-DHBA or HDAC inhibitor sodium butyrate mimicked, in part, the effects of L-lactate. The inhibition of lactate flux by BAY-8002 enhanced DNA-PKcs nuclear localization which translated into diminished lentiviral transduction efficacy. Our study suggests that L- and D-lactate present in the uterine cervix may play a role in the mitigation of viral integration in cervical epithelium and, thus, restrict the viral oncogenic and/or cytopathic potential.

Targeted and systemic insights into the crosstalk between DNA-dependent protein kinase catalytic subunit and receptors of estrogen, progesterone and epidermal growth factor in the context of cancer.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has emerged as a regulator of carcinogenesis. Increased expression of DNA-PKcs correlates with metastatic cancers. Here we review recently reported crosstalk of DNA-PKcs with estrogen (ER), progesterone (PR) and epidermal growth factor (EGFR) receptors. The reports show an extensive network of functional and direct interactions. Targeted studies focused on specific molecular mechanisms, and a systems biology network analysis shows unbiasedly engagement of various cellular functions. Feedforward regulation between expression and activities of DNA-PKcs and ER, DNA-PKcs-dependent phosphorylation of PR and an impact on PR-dependent transcription, and DNA-PKcs-promoted EGFR-dependent aggressiveness and metastases are examples of the results of targeted studies. Systems biology approach extracted many more genes and proteins engaged by DNA-PKcs in interaction with ER, PR, and EGFR. Examples are such regulators and predictors of breast tumorigenesis as BRCA1, TP53, and 18 genes of the MammaPrint signature. Reviewed here data suggest that the diagnostic value of DNA-PKcs in the context of ER, PR and EGFR signaling is defined by a network signature rather than by single markers. This review summarizes mechanisms of DNA-PKcs interaction with ER, PR, and EGFR, highlights tumor suppressors and oncogenes engaged by DNA-PKcs, and emphasizes the importance of diagnostic network-based signatures.

NEAT1/miR-101-dependent Up-regulation of DNA-PKcs Enhances Malignant Behaviors of Pancreatic Ductal Adenocarcinoma Cells.

Background: Although we previously revealed that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and important for gemcitabine resistance, the role of DNA-PKcs in the progression and metastasis of PDAC remain unclear. To date, the upstream signaling events stimulating DNA-PKcs overexpression in PDAC are still not well characterized.Methods: Expression of DNA-PKcs was measured by western blot. The levels of miRNA-101 and lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) were detected by real-time PCR. Cell viability was determined by CCK-8. Cell migration and cell invasion were measured by transwell assay. The regulatory relationship between NEAT1 and miR-101 was determined by a luciferase assay.Results: DNA-PKcs expression was significantly elevated in human PDAC tissues and cells. DNA-PKcs overexpression was correlated with TNM stage and lymph node metastasis. Higher expression of DNA-PKcs was closely correlated with patients of worse overall survival (OS). DNA-PKcs knockdown suppresses malignant behaviors of PDAC cells. Further study showed that miRNA-101 level was decreased in PDAC tissues and cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in PDAC cells. Moreover, NEAT1 functions as an oncogene influencing cell proliferation, migration and invasion in part by serving as a competing endogenous RNA (ceRNAs) modulating miR-101 expression, leading to up-regulation of DNA-PKcs.Conclusion: These findings suggest that NEAT1/miR-101-dependent up-regulation of DNA-PKcs promotes the malignant behaviors of PDAC cells. The NEAT1/miR-101/DNA-PKcs axis may serve as a viable prognostic marker and therapeutic target for PDAC.

BVAN08 enhances radiosensitivity via downregulation of DNA-PKcs towards hepatic tumor xenograft

ObjectiveTo study the effects of the novel vanillin derivative BVAN08 on the radiosensitivity of hepatic cancer cells for the purpose of providing evidence for its potential use as a potential radiosensitive drug. MethodsHepatic cancer Huh-7 ​cells labeled with luciferase were used to investigate the radiosensitivity induced by BVAN08 in vivo. Colony formation assays and flow cytometry were used to measure the apoptosis and radiosensitivity of Huh-7 ​cells in vitro produced by BVAN08. Histology and immunohistochemistry were used to evaluate the toxicity of BVAN08 in vivo. ResultsBVAN08 induced apoptosis and cell cycle arrest of Huh-7 ​cells in a time-dependent manner. Moreover, BVAN08 combined with radiation increased the sensitivity of Huh-7 ​cells to γ-ray radiation and significantly inhibited tumor growth in vivo. The tumor inhibition rates of the BVAN08 treatment group, irradiation treatment group, and combined therapy group were 58%, 38%, and 85%, respectively. The DNA-PKcs expression in tumor tissues of the BVAN08 treatment group was lower than that of the control group (P ​< ​0.01). BVAN08 inhibited the growth of Huh-7 ​cells in nude mice bearing tumors, without resulting in any noticeable side effects on bodyweight, livers, hearts, kidneys, or the number of peripheral white blood cells. ConclusionsAs a natural food additive derivative, BVAN08 possesses the potential to be used as an effective anti-cancer drug to increase cell sensitivity to radiotherapy.

Differential expression of DNA-dependent protein kinase catalytic subunit in the brain of neonatal mice and young adult mice.

It is generally thought that younger people are more susceptible to cancer development after exposure to ionizing radiation in reference to epidemiological studies and animal experiments. However, little is known about the age-dependent alteration in DNA repair ability. In the present study, we examined the expression levels of proteins involved in the repair of DNA double-strand breaks through non-homologous end joining (NHEJ), i.e., DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray repair cross-complementing 4 (XRCC4) and XRCC4-like factor (XLF). We found that the expression of DNA-PKcs in brain tissues was higher in neonatal mice (1 week after birth) than in young adult mice (7 weeks after birth). In association with this, DNA double-strand breaks were repaired more rapidly in the brain tissues of neonatal mice than in those of young adult mice. The current results suggested a possible role for DNA-PKcs protecting developing brain tissues from DNA double-strand breaks.

Corrigendum to: Genetic interaction between DNA repair factors PAXX, XLF, XRCC4 and DNA‐PKcs in human cells

FEBS Open BioVolume 10, Issue 2 p. 290-290 CorrigendumOpen Access Corrigendum to: Genetic interaction between DNA repair factors PAXX, XLF, XRCC4 and DNA-PKcs in human cells This article corrects the following: Genetic interaction between DNA repair factors PAXX, XLF, XRCC4 and DNA-PKcs in human cells Mengtan Xing, Valentyn Oksenych, Volume 9Issue 7FEBS Open Bio pages: 1315-1326 First Published online: June 12, 2019 First published: 06 January 2020 https://doi.org/10.1002/2211-5463.12777AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In Figure 1 of the article by Xing and Oksenych [1], the western blots against PAXX and β-actin for all four lanes in Figure 1A and the western blots against XRCC4 and β-actin in the lanes for WT and XRCC4∆ cells in Figure 1B were reproduced from an earlier publication from the Oksenych laboratory [2] without acknowledgement. This does not affect the conclusions of this paper. A corrected version of Figure 1 and the accompanying legend are provided here: Figure 1Open in figure viewerPowerPoint Verification of HAP1 cells by western blot. Western blot (WB) analyses of DNA-PKcs and PAXX expression in WT, PAXX∆, DNA-PKcs∆ and DNA-PKcs∆ PAXX∆ HAP1 cells; (A); expression of DNA-PKcs and XRCC4 in WT, XRCC4∆, DNA-PKcs∆ and DNA-PKcs∆ XRCC4∆ HAP1 cells (B); expression of XLF (C), LIG4 (D) and H2AX (E) in WT, XLF∆, LIG4∆ and H2AX∆ HAP1 cells; and β-Actin was used as a loading control for WB. The western blots against PAXX and β-actin for all four lanes in Figure 1A and the western blots against XRCC4 and β-actin in the lanes for WT and XRCC4∆ cells in Figure 1B were reproduced from an earlier publication from the Oksenych laboratory [2]. References 1Xing M and Oksenych V (2019) Genetic interaction between DNA repair factors PAXX, XLF, XRCC4 and DNA-PKcs in human cells. FEBS Open Bio 9, 1315– 1326. https://doi.org/10.1002/2211-5463.12681. 2Dewan A, Xing M, Lundbæk MB, Gago-Fuentes R, Beck C, Aas PA, Liabakk N-B, Sæterstad S, Chau KTP, Kavli BM et al. (2018) Robust DNA repair in PAXX-deficient mammalian cells. FEBS Open Bio 8, 442– 448. https://doi.org/10.1002/2211-5463.12380. Volume10, Issue2February 2020Pages 290-290 FiguresReferencesRelatedInformation

DNA-PKcs modulates progenitor cell proliferation and fibroblast senescence in idiopathic pulmonary fibrosis

BackgroundRecent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types.MethodsSurgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4+ cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441.ResultsDNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4+ mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4+ mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4+ CgA+ expressing cells.ConclusionsTogether, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4+ mesenchymal progenitors, and the senescence of their mesenchymal progeny.

Akt1 enhances the chemoradiotherapy resistance of gliomas through phosphorylation of ubiquitin-conjugating enzyme 2S: An experimental study

Objective To explore the mechanism of Akt1-associated enhancement of the chemoradiotherapy resistance of gliomas. Methods The immunofluorescence (IF) experiment was conducted to test the expression of ubiquitin-conjugating enzyme 2S (UBE2S) in the normal astrocytes and glioma cells (U87, U251, SF767 and U373). The Western blot was used to assess the differences of UBE2S expression in PTEN mutant and wild type glioma cell lines. To explore the role of PTEN/Akt signal pathway in UBE2S functions and the effect of UBE2S on the non-homologous end joining (NHEJ) repair, a constitutively activated Akt1 (Myr-HA-Akt1) and the UBE2S wild type or UBE2S T152A mutant were coexpressed. The flow cytometry (FCM) was used to test in the chemoradiotherapy sensitivity of the stable UBE2S knockdown glioma cells. Results Compared with the normal astrocytes, UBE2S was overexpressed in the malignant glioma cells. The expression of UBE2S was more stable in the PTEN mutant glioma cell line. Akt1 phosphorylated UBE2S at T152, which promoted the stabilization of UBE2S. UBE2S interacted with Ku70 and Ku80, which increased the expression of Ku70 (P=0.009). The results of Western blot showed that the expression of Ku70, Ku80 and DNA-PKcs decreased significantly after knockdown of UBE2S expression (P<0.001). The results of FCM showed that the knockdown of UBE2S could enhance the chemoradiotherapy sensitivity of gliomas(P<0.001). Conclusions UBE2S is highly expressed in the malignant glioma. Akt1 physically interacts with UBE2S and phosphorylates UBE2S at T152, which is critical for the stabilization of UBE2S. The knockdown of UBE2S expression could retard NHEJ-mediated DNA repair and thus increase the sensitivity of chemoradiotherapy. Key words: Glioma; Chemoradiotherapy; Ubiquitin-conjugating enzyme 2S; PTEN/Akt signal pathway

RETRACTED: The effect of DNA-PKcs gene silencing on proliferation, migration, invasion and apoptosis, and in vivo tumorigenicity of human osteosarcoma MG-63 cells

The purpose of this study was to explore the role by which the DNA-dependent protein kinase complex catalytic subunit (DNA-PKcs) influences osteosarcoma MG-63 cell apoptosis, proliferation, migration and invasion. Osteosarcoma tissues and adjacent normal tissues were obtained from 57 osteosarcoma patients. Human osteosarcoma MG-63 cells were assigned into designated groups including the blank, siRNA-negative control (NC) and siRNA-DNA-PKcs groups. RT-qPCR and Western blotting methods were employed to evaluate the mRNA and protein expressions of DNA-PKcs. A cell counting kit-8 (CCK-8) assay was performed to assess cell viability. The evaluation of cell migration and invasion were conducted by means of Scratch test and Transwell assay. Flow cytometry with PI and annexin V/PI double staining was applied for the analysis of the cell cycle and apoptosis. Twenty-Four Balb/c nude mice were recruited and randomly divided into the blank, siRNA-NC and siRNA-DNA-PKcs groups. Tumorigenicity of the Balb/c nude mice was conducted to evaluate the rate of tumor formation, as well as for the assessment of tumor size and weight, and confirm the number of lung metastatic nodules in the mice post transfection. Osteosarcoma tissues were found to possess greater expression of DNA-PKcs than that of the adjacent normal tissues. DNA-PKcs expression in osteosarcoma tissues were correlated with the clinical stage and metastasis. Compared with the blank and siRNA-NC groups, proliferation, miration, as well as the invasion abilities of the MG-63 cells increased. Furthermore, an increase in apoptosis and cells at the G1 stage in the MG-63 cells was observed, while there were reductions in the cells detected at the S stage. The mRNA and protein expressions of CyclinD1, PCNA, Bcl-2 decreased while those of Bax increased in the siRNA-DNA-PKcs group. The tumor formation rate, tumor diameter, weight and lung metastatic nodules among the nude mice in the siRNA-DNA-PKcs group were all lower than those in the blank and siRNA-NC groups. The observations and findings of the study suggested that the silencing of DNA-PKcs inhibits the proliferation, migration and invasion, while acting to promote cell apoptosis in MG-63 cells and osteosarcoma growth in nude mice.

Abstract 4676: DNA repair protein expression and response of homologous recombination deficient ovarian cancer to the poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib in the ARIEL2 Part 1 study

Abstract Background: PARP inhibitors (PARPis) are active in cancers with homologous recombination (HR) defects. Preclinical studies have shown that secondary mutations or alterations in gene expression (e.g., downregulation of 53BP1, Ku70, Ku80 or DNA-PKcs) restore HR and confer PARPi resistance. In addition, low PARP1 expression can diminish PARP trapping and cause PARPi resistance. ARIEL2 Part 1 is a phase 2 study of the PARPi rucaparib in platinum sensitive, relapsed high grade serous or endometrioid ovarian cancer (OC). Pretreatment OC biopsies were previously assayed for HR gene mutations and loss of heterozygosity (LOH), a genomic scar that reflects HR deficiency. Two tumor groups (BRCA wildtype [wt]/LOH high and BRCA1 or BRCA2 mutant) have objective response rates of 29.3% and 80%, respectively, as well as progression free survival (PFS) of 5.7 and 12.8 months, respectively, on rucaparib (E. Swisher et al., Lancet Oncol., in press). Common AEs included nausea (80%), asthenia/fatigue (78%), constipation (46%), and vomiting (44%). The present studies tested the hypotheses that i) lower PARP1 expression and/or ii) lower expression of NHEJ components 53BP1, DNA-PKcs, Ku80, Ku70, or LIG4, may correlate with diminished response rate as well as PFS in pts treated with rucaparib on ARIEL2. Methods: Immunohistochemical assays were developed for 53BP1, DNA-PKcs, Ku80, Ku70, LIG4, and PARP1 and validated in formalin fixed, paraffin embedded cell lines differing in analyte expression. Available pretreatment OC biopsies from ARIEL2 Part 1 were stained and scored for % of tumor nuclei that were negative (0), weak (1+), moderate (2+) or strong (3+). Modified H-scores were correlated with clinical characteristics and outcome measures. Results: Pretreatment biopsies from 62-68 pts were successfully stained for each repair protein. Across the samples, PARP1 H-scores varied from 0 to 300 (median 160). Focusing on the BRCA wt/LOH high group (n = 38), there was no significant difference in PFS of pts with low (&amp;lt;100), intermediate (100-200) or high (&amp;gt;200) PARP1 H-score (p = 0.57). Expression of DNA-PKcs, Ku70, Ku80, and LIG4 was generally lower (median H-scores 20-60) and did not individually correlate with response or PFS. In contrast, there was a trend toward improved PFS in BRCA wt/LOH high OC pts expressing intermediate or high 53BP1 (H-score ≥ 100, n = 10) compared to pts with low 53BP1 (H-score &amp;lt;100, n = 25), with median PFS 20.7 vs. 5.5 months (p = 0.073), respectively. Staining of additional pretreatment biopsies is planned. Conclusions: In the BRCA wt/LOH high group, pretreatment PARP1 expression does not correlate with rucaparib response. In contrast, BRCA wt/LOH high OC pts with low 53BP1 have a trend toward shorter PFS with rucaparib, suggesting that 53BP1 downregulation might correlate with clinical PARPi resistance in BRCA wt/LOH high OC. Citation Format: Andrea E. Wahner Hendrickson, Kevin K. Lin, Daniel W. Visscher, Rachel M. Hurley, Mitch Raponi, Thomas C. Harding, Linda M. Murphy, Jill M. Wagner, Heidi Giordano, Iain McNeish, Elizabeth M. Swisher, Scott Kaufmann. DNA repair protein expression and response of homologous recombination deficient ovarian cancer to the poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib in the ARIEL2 Part 1 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4676. doi:10.1158/1538-7445.AM2017-4676

Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells

Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PKCS), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PKCS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PKCS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PKCS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PKCS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma.

Mesenchymal subtype of glioblastomas with high DNA-PKcs expression is associated with better response to radiotherapy and temozolomide.

A better understanding of the relationship between glioblastomas molecular subtypes and radio-chemotherapy is needed for the development of individualized strategies. In this study, we aimed to assess whether non-homologous end-joining (NHEJ) protein expression is associated and could predict responses to treatment of mesenchymal (MES) and proneural (PN) subtypes. Tumors from 122 patients with a glioblastoma treated at the University Hospital of Poitiers between 2002-2013 by an association of radiotherapy and temozolomide were collected. Among these tumors, 80 were suitable for in situ analysis and were included in TissueMicroArray. The expression of DNA-PKcs, Ku70, Ku80 and CD44, Olig2 (respectively surrogate markers of MES and PN subtypes) were evaluated by immunohistochemistry. The median survival of patients with high and low CD44 expression was 11.9 months (95% CI 7.7-14) and 19.1 months (95% CI 15.2-22.4) respectively (p = 0.008). Median survival of patients with high and low DNA-PKcs levels was 20.0 months (95% CI 15.2-25.3) and 12.9 months (95% CI 9.9-19.5) respectively (p = 0.036). High levels of Olig2, Ku70 and Ku80 tended to be associated with better overall survival but no significant differences were found. Overall survival of class I patients (CD44+ and DNA-PKcs+) was longer than class II (CD44+ and DNA-PKcs- or CD44- and DNA-PKcs+) and class III (CD44- and DNA-PKcs-), (p = 0.005 and 0.003 respectively). High levels of CD44 and DNA-PK are associated with a better survival and better response to radiotherapy and temozolomide and could establish prognosis classes by predicting survival and response to therapy for GBMs patients.

Inhibitory Effects of BVAN08 on the Growth of Experimental Tumor Cell Lines both In Vivo and In Vitro

In order to study the mechanism of a novel vanillin derivative BVAN08, provide evidence and experimental data for developing it as a new potential anticancer drug. Hepatic cancer HepG2 cells and normal LO2 cells were used to investigate cytotoxicity of BVAN08. The MTT and colony-forming ability assays showed that BVAN08 significantly sensitized HepG2 cells to radiation rather than LO2 cells. Moreover, BVAN08 inhibited the growth of HepG2 cells in nude mice and exerted no effects on body weight and the number of the peripheral white blood cells. The immunohistochemistry results indicated that the DNA-PKcs expression the BVAN08 group tumor was lower than that of control group. BVAN08 obviously inhibits proliferation of HepG2 cells in vitro and in vivo supporting it as a promising anticancer drug candidate.

ANKHD1 Silencing Delays S Phase Progression in Multiple Myeloma Cells Via Activation of ATM/ATR -CDC25a Pathway

Ankyrin repeat and KH domain-containing protein 1, ANKHD1, is highly expressed in myeloma cells and plays an important role in multiple myeloma (MM) progression and growth. ANKHD1 is found to be overexpressed in S phase of cell cycle in MM cells and silencing of ANKHD1 expression leads to accumulation of cells in S phase, suggesting a role in S phase progression (1). Earlier studies by our group reported that ANKHD1 silencing downregulates all replication dependent histones and that this downregulation may be associated with replication stress and DNA damage (2). We observed increased expression of γH2AX protein (phosphorylated histone H2A variant, H2AX, at Serine 139), a marker for DNA double strand breaks (DSBs) and an early sign of DNA damage induced by replication stress, in ANKHD1 silenced MM cells. In the present study we further sought to investigate the mechanisms underlying the induction of DNA damage on ANKHD1 silencing. We first confirmed the increased expression of γH2AX by flow cytometry analysis and observed that both the mean fluorescence intensity as well as percentage of γH2AX positive cells were higher in ANKHD1 silenced MM cells as compared to control cells. Phosphorylation of histone 2AX requires activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated)andATR (ATM-Rad3-related) that serves as central components of the signaling cascade initiated by DSBs. Hence, we checked for the expression of these kinases and observed increased phosphorylation of both ATM and ATR kinases in ANKHD1 silenced MM cells. There was no difference in the expressions of DNA-PKcs in control and ANKHD1 silenced cells by western blot. We next checked for the expression of CHK1 (checkpoint kinase 1) and CHK2 (checkpoint kinase 2), essential serine threonine kinases downstream of ATM and ATR. We observed a decrease in pCHK2 (phosphorylated CHK2 at Thr 68), with no change in expression of pCHK1 (phosphorylated CHK1 at Ser 345) total CHK1 or total CHK2. We also checked for expression of CDC25a (a member of the CDC25 family of dual-specificity phosphatases), that is specifically degraded in response to DNA damage (DSBs) and delays S phase progression via activation of ATM /ATR-CHK2 signaling pathway. Expression of CDC25a was significantly decreased in ANKHD1 silencing cells, confirming the induction of DSBs, and probably accounting for S phase delay on ANKHD1 silencing. Since there was decrease in active CHK2 (pCHK2) and no change in CHK1 required for degradation of CDC25a, we assume that decrease in CDC25a in ANKHD1 silenced MM cells may be via activation of ATM/ ATR pathway independent of CHK2/CHK1. Expression of several other downstream factors of DSBs induced DNA damage response and repair such as BRCA1, PTEN, DNMT1, SP1, HDAC2 were also found to be modulated in ANKHD1 silenced MM cells.In conclusion, ANKHD1 silencing in MM cells leads to DNA damage and modulates expression of several genes implicated in DNA damage and repair. DNA damage induced after ANKHD1 silencing in MM cells activates ATM/ ATR-CDC25a pathway which may lead to the activation of S phase checkpoint in MM cells. Results however are preliminary and further studies are required to understand the role of ANKHD1 in intra S phase check point.

Prognostic significance of nuclear or cytoplasmic nucleolin expression in human non-small cell lung cancer and its relationship with DNA-PKcs.

This study investigated the expression of nucleolin in tissue samples in patients with non-small cell lung cancer (NSCLC). Nucleolin was studied to determine whether it has a prognostic value and if its levels correlate with various clinicopathologic parameters. The relationship between nucleolin and expression of DNA-PKcs was also evaluated. Immunohistochemistry was used for detecting the expression levels of nucleolin and DNA-PKcs in tissues from 225 stage IA to IIIB NSCLC patients who underwent lung surgery. Nucleolin was observed predominantly in the cytoplasm, and some levels were observed in the nucleus. Nucleolin expression was higher in NSCLC tissues than adjacent normal lung tissues. Among 225 NSCLC patients, 117 (52.0%) had high expression of nucleolin. The expression of nucleolin was significantly associated with pathologic stage (P = 0.013) and T status (P = 0.043). Multivariate analysis revealed that nucleolin, cytoplasmic nucleolin, and nuclear nucleolin expression were independent prognostic factors for both overall survival (OS) (P < 0.001) and disease-free survival (DFS) (P < 0.001). A high level of nuclear nucleolin served as an independent prognostic factor for better survival, while a high level of cytoplasmic nucleolin was closely associated with worse prognosis in NSCLC patients. The expression of nucleolin and cytoplasmic nucleolin positively correlated with DNA-PKcs (P < 0.001). These data suggest that nucleolin could be an effective treatment target and prognostic factor for patients with NSCLC.

DNA-PKcs and Ku70 are Predictive Markers for Poor Prognosis of Patients With Gall Bladder Malignancies

Gall bladder cancers (GBCs) are highly resistant to radiotherapy and chemotherapy. Unfortunately, the key molecular mechanisms responsible for therapeutic resistance have not been identified. In this study, the expression of DNA-PKcs and Ku70 in 46 squamous cell/adenosquamous carcinomas (SC/ASCs) and 80 adenocarcinomas (ACs) were examined by immunohistochemical analysis. Positive DNA-PKcs and Ku70 expression were significantly associated with less lymph node metastasis, invasion, and low TNM stage of SC/ASCs and ACs. Univariate Kaplan-Meier analysis showed that loss of DNA-PKcs and Ku70 expression significantly correlated with decreased survival in both SC/ASC and AC patients. Multivariate Cox regression analysis showed that loss of DNA-PKcs and Ku70 expression was an independent poor prognostic predictor in both SC/ASC and AC patients. Our study suggested that DNA-PKcs and Ku70 are tumor suppressors, and loss of DNA-PKcs and Ku70 expression is an important biological marker for metastasis, invasion, and prognosis of GBC. Currently, there is no implication of DNA-PKcs and Ku70 expression in chemoresistance or radioresistance in GBC.