BACKGROUND: Prognosis of esophageal squamous cell carcinoma (ESCC) patients is poor and the concurrent chemoradiation therapy (CCRT) provided to ESCC patients often failed due to resistance. Therefore, development of biomarkers for predicting CCRT response is immensely important. In this study, we evaluated the predicting value of SRY (sex determining region Y)-box 17 (SOX17) protein during CCRT and its dysregulation of transcriptional targets in CCRT resistance in ESCC.
METHODS: Pyrosequencing methylation, RT-qPCR and immunohistochemistry assays were performed to examine the DNA methylation, mRNA expression and protein expression levels of SOX17 in endoscopic biopsy from a total of 70 ESCC patients received CCRT. Cell proliferation, clonogenic survival and xenograft growth were used to confirm the sensitization of ESCC cell line KYSE510 in response to cisplatin, radiation or CCRT treatment by SOX17 overexpression in vitro and in vivo. Luciferase activity, RT-qPCR and ChIP-qPCR assays were conducted to examine transcription regulation of SOX17 in KYSE510 parental, KYSE510 radio-resistant cells and their derived xenografts.
RESULTS: High DNA methylation coincided with low mRNA and protein expression levels of SOX17 in pre-treatment endoscopic biopsy from ESCC patients with poor CCRT response. SOX17 protein expression exhibited a good prediction performance in discriminating poor CCRT responders from good responder. Overexpression of SOX17 sensitized KYSE510 radio-resistant cells to cisplatin, radiation or CCRT treatment in cell and xenograft models. Importantly, SOX17 transcriptionally down-regulated DNA repair and damage response-related genes including BRCA1, BRCA2, RAD51, KU80 DNAPK, p21, SIRT1, NFAT5 and REV3L in KYSE510 radio-resistant cells to achieve the sensitization effect to anti-cancer treatment. Low expression of BRCA1, DNAPK, p21, RAD51 and SIRT1 was confirmed in SOX17 sensitized xenograft tissues derived from radio-resistant ESCC cells.
CONCLUSIONS: Our study reveals a novel mechanism by which SOX17 transcriptionally inactivates DNA repair and damage response-related genes to sensitize ESCC cell or xenograft to CCRT treatment. In addition, we establish a proof-of-concept CCRT prediction biomarker using SOX17 immunohistochemical staining in pre-treatment endoscopic biopsies to identify ESCC patients who are at high risk of CCRT failure and need intensive care.

Women with triple-negative breast cancer (TNBC) are generally treated by chemotherapy but their responsiveness may be blunted by DNA double-strand break (DSB) repair. We previously reported that IGFBP-3 forms nuclear complexes with EGFR and DNA-dependent protein kinase (DNA-PKcs) to modulate DSB repair by non-homologous end-joining (NHEJ) in TNBC cells. To discover IGFBP-3 binding partners involved in chemoresistance through stimulation of DSB repair, we analyzed the IGFBP-3 interactome by LC-MS/MS and confirmed interactions by coimmunoprecipitation and proximity ligation assay. Functional effects were demonstrated by DNA end-joining in vitro and measurement of γH2AX foci. In response to 20 µM etoposide, the DNA/RNA-binding protein, non-POU domain-containing octamer-binding protein (NONO) and its dimerization partner splicing factor, proline/glutamine-rich (SFPQ) formed complexes with IGFBP-3, demonstrated in basal-like TNBC cell lines HCC1806 and MDA-MB-468. NONO binding to IGFBP-3 was also shown in a cell-free biochemical assay. IGFBP-3 complexes with NONO and SFPQ were blocked by inhibiting EGFR with gefitinib or DNA-PKcs with NU7026, and by the PARP inhibitors veliparib and olaparib, which also reduced DNA end-joining activity and delayed the resolution of the γH2AX signal (i.e. inhibited DNA DSB repair). Downregulation of the long noncoding RNA in NHEJ pathway 1 (LINP1) by siRNA also blocked IGFBP-3 interaction with NONO-SFPQ. These findings suggest a PARP-dependent role for NONO and SFPQ in IGFBP-3-dependent DSB repair and the involvement of LINP1 in the complex formation. We propose that targeting of the DNA repair function of IGFBP-3 may enhance chemosensitivity in basal-like TNBC, thus improving patient outcomes.

BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett's esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks.
METHODS: Real-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH 4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage.
RESULTS: Our results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett's cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure.
CONCLUSIONS: IGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.

DSBs are harmful lesions produced through endogenous metabolism or by exogenous agents such as ionizing radiation, that can trigger genomic rearrangements. We have recently shown that exposure to 2 Gy of X-rays has opposite effects on the induction of Shh-dependent MB in NHEJ- and HR-deficient Ptch1

DNA-Double strand breaks (DSBs) generated by radiation therapy represent the most efficient lesions to kill tumor cells, however, the inherent DSB repair efficiency of tumor cells can cause cellular radioresistance and impact on therapeutic outcome. Genes of DSB repair represent a target for cancer therapy since their down-regulation can impair the repair process making the cells more sensitive to radiation. In this study, we analyzed the combination of ionizing radiation (IR) along with microRNA-mediated targeting of genes involved in DSB repair to sensitize human non-small cell lung cancer (NSCLC) cells. MicroRNAs are natural occurring modulators of gene expression and therefore represent an attractive strategy to affect the expression of DSB repair genes. As possible IR-sensitizing targets genes we selected genes of homologous recombination (HR) and non-homologous end joining (NHEJ) pathway (i.e. RAD51, BRCA2, PRKDC, XRCC5, LIG1). We examined these genes to determine whether they may be real targets of selected miRNAs by functional and biological validation. The in vivo effectiveness of miRNA treatments has been examined in cells over-expressing miRNAs and treated with IR. Taken together, our results show that hsa-miR-96-5p and hsa-miR-874-3p can directly regulate the expression of target genes. When these miRNAs are combined with IR can decrease the survival of NSCLC cells to a higher extent than that exerted by radiation alone, and similarly to radiation combined with specific chemical inhibitors of HR and NHEJ repair pathway.

Synthetic lethality occurs when simultaneous perturbations of two genes or molecular processes result in a loss of cell viability. The number of known synthetically lethal interactions is growing steadily. We review here synthetically lethal interactions of ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). These kinases are appropriate for synthetic lethal therapies because their genes are frequently mutated in cancer, and specific inhibitors are currently in clinical trials. Understanding synthetically lethal interactions of a particular gene or gene family can facilitate predicting new synthetically lethal interactions, therapy toxicity, and mechanisms of resistance, as well as defining the spectrum of tumors amenable to these therapeutic approaches.

There is a pressing need to identify therapeutic targets in tumors with low mutation rates such as the malignant pediatric brain tumor medulloblastoma. To address this challenge, we quantitatively profiled global proteomes and phospho-proteomes of 45 medulloblastoma samples. Integrated analyses revealed that tumors with similar RNA expression vary extensively at the post-transcriptional and post-translational levels. We identified distinct pathways associated with two subsets of SHH tumors, and found post-translational modifications of MYC that are associated with poor outcomes in group 3 tumors. We found kinases associated with subtypes and showed that inhibiting PRKDC sensitizes MYC-driven cells to radiation. Our study shows that proteomics enables a more comprehensive, functional readout, providing a foundation for future therapeutic strategies.

BACKGROUND: Cantharidin is an inhibitor of protein phosphatase 2 A (PP2A), and has been frequently used in clinical practice. In our previous study, we proved that cantharidin could arrest cell cycle in G2/M phase. Since cells at G2/M phase are sensitive to radiotherapy, in the present study, we investigated the radiotherapy-sesitization effect of cantharidin and the potential mechanisms involved.
METHODS: Cell growth was determined by MTT assay. Cell cycle was evaluated by flow cytometry. DNA damage was visualized by phospho-Histone H2A.X staining. Expression of mRNA was tested by microarray assay and real-time PCR. Clinical information and RNA-Seq expression data were derived from The Cancer Genome Atlas (TCGA) pancreatic cancer cohort. Survival analysis was obtained by Kaplan-Meier estimates.
RESULTS: Cantharidin strengthened the growth inhibition effect of irradiation. Cantharidin drove pancreatic cancer cells out of quiescent G0/G1 phase and arrested cell cycle in G2/M phase. As a result, cantharidin strengthened DNA damage which was induced by irradiation. Moreover, cantharidin repressed expressions of several genes participating in DNA damage repair, including UBE2T, RPA1, GTF2HH5, LIG1, POLD3, RMI2, XRCC1, PRKDC, FANC1, FAAP100, RAD50, RAD51D, RAD51B and DMC1, through JNK, ERK, PKC, p38 and/or NF-κB pathway dependent manners. Among these genes, worse overall survival for pancreatic cancer patients were associated with high mRNA expressions of POLD3, RMI2, PRKDC, FANC1, RAD50 and RAD51B, all of which could be down-regulated by cantharidin.
CONCLUSION: Cantharidin can sensitize pancreatic cancer cells to radiotherapy. Multiple mechanisms, including cell cycle regulation, enhanced DNA damage, and inhibited DNA damage repair, may be involved.

Cell-line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) in immune-deficient mice have revolutionized our understanding of normal and malignant human hematopoiesis. Transgenic approaches further improved in vivo hematological research, allowing the development of human-cytokine-producing mice, which show superior human cell engraftment. The most popular mouse strains used in research, the NOG (NOD.Cg-Prkdc

Coumarin is a plant-derived compound but as such has no medical uses. Several synthetic coumarin analogs have been shown to possess anti-proliferative activity and to induce apoptosis in cancer cells. Here, we explored DNA damage responses in MCF-7 cells treated with our novel synthetic hybrid compound AD-013, which integrates a coumarin moiety and an α-methylene-δ-lactone motif. The mRNA expression of several genes engaged in DNA-damage-induced responses was assessed by quantitative real-time PCR. The protein levels of a few members of phosphoinositide-3-kinases family (ATM, ATR and DNA-PK) and BRCA1 were assessed by ELISA, while p53 was evaluated by western blot method. AD-013 down-regulated DNA-PK gene expression but increased the level of ATM/ATR and p53. The new analog completely inhibited BRCA1 and greatly decreased the activity of BRCA1 protein, engaged in DNA damage repair. Exposure of MCF-7 cells to a coumarin analog AD-013 led to DNA damage and decreased expression of several repair-associated genes.

In many families with suspected Lynch syndrome (LS), no germline mutation in the causative mismatch repair (MMR) genes is detected during routine diagnostics. To identify novel causative genes for LS, the present study investigated 77 unrelated, mutation-negative patients with clinically suspected LS and a loss of MSH2 in tumor tissue. An analysis for genomic copy number variants (CNV) was performed, with subsequent next generation sequencing (NGS) of selected candidate genes in a subgroup of the cohort. Genomic DNA was genotyped using Illumina's HumanOmniExpress Bead Array. After quality control and filtering, 25 deletions and 16 duplications encompassing 73 genes were identified in 28 patients. No recurrent CNV was detected, and none of the CNVs affected the regulatory regions of MSH2. A total of 49 candidate genes from genomic regions implicated by the present CNV analysis and 30 known or assumed risk genes for colorectal cancer (CRC) were then sequenced in a subset of 38 patients using a customized NGS gene panel and Sanger sequencing. Single nucleotide variants were identified in 14 candidate genes from the CNV analysis. The most promising of these candidate genes were: (i) PRKCA, PRKDC, and MCM4, as a functional relation to MSH2 is predicted by network analysis, and (ii) CSMD1, as this is commonly mutated in CRC. Furthermore, six patients harbored POLE variants outside the exonuclease domain, suggesting that these might be implicated in hereditary CRC. Analyses in larger cohorts of suspected LS patients recruited via international collaborations are warranted to verify the present findings.

Non‑small cell lung cancer (NSCLC) is radioresistant to X‑rays due to powerful cellular DNA damage repair mechanisms. DNA‑dependent protein kinase (DNA‑PK) is a key enzyme involved in DNA damage repair and the phenomenon and molecular mechanism of NSCLC radionsensitivity were investigated following inhibition of DNA‑PK activity. In the present study A549 cells were treated with the DNA‑PK inhibitor NU7026 and/or siRNA directed against ataxia telangiectasia mutated (ATM), followed by exposure to 4 Gy X‑ray irradiation. Radiosensitivity, DNA damage, apoptosis and protein expression were measured by colony formation assay, γH2AX foci immunofluorescence, Annexin V/PI staining and western blotting, respectively. A Balb/c‑nu/nu xenograft mouse model was established by subcutaneous injection of A549 cells and was used to examine the effect of administering NU7026 via intraperitoneal injection prior to 4 Gy X‑ray exposure. The xenograft tumors were weighed and observed by hematoxylin and eosin staining after irradiation. NU7026 treatment followed by X‑ray irradiation significantly decreased the colony formation ratio of A549 cells, and increased γH2AX foci and cell apoptosis. Furthermore, the combined treatment of NU7026 and X‑rays resulted in growth inhibition and cell apoptosis in A549 xenograft tumors. Consequently, apoptosis regulators full‑length transactivating (TA) p73 and an N‑terminally truncated (DN) p73 were upregulated and downregulated respectively, leading to activation of glucosyltransferases and Rab‑like GTPase activators and myotubularins domain‑containing 4 (GRAMD4) protein to reduce the Bcl‑2/Bax protein ratio. In addition, ATM siRNA efficiently prevented γH2AX foci formation, and enhanced NU7026‑induced inhibition of survival and promoted apoptosis. In conclusion, inhibition of DNA‑PK activity increased the radiosensitivity of A549 cells to X‑ray irradiation. NU7026 treatment activated the ATM‑dependent DNA damage response and induced p73 apoptosis pathway. DNA‑PK inhibitor may be an effective constituent of radiosensitization products. DNA damage repair pathway could be a potential target for radiosensitization.

OBJECTIVE: The objective of this study was to characterize the DNA damage response in two human oral cancer cell lines following X-irradiation.
DESIGN: To visualize radiation-induced cell cycle alterations, two human oral cancer cell lines, HSC3 and HSC4, expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were established in this study. G2 arrest kinetics following irradiation were obtained from two-color flow cytometric analysis and pedigrees of Fucci fluorescence. DNA double strand break repair kinetics were obtained from immunofluorescence staining for phosphorylated histone H2AX, p53-binding protein 1, phosphorylated DNA-dependent protein kinase catalytic subunit, and breast cancer susceptibility gene 1.
RESULTS: Both cell lines showed apparent G2 arrest after 10 Gy of irradiation, but it was more enhanced in the HSC3-Fucci cells. Radiosensitivity was higher in the HSC3-Fucci cells than in HSC4-Fucci cells. Pedigree analysis of Fucci fluorescence revealed that the HSC3-Fucci cells exhibited a significantly longer green phase (normally indicating S/G2/M phases, but here reflective of G2 arrest) when irradiated in the red phase (G1 phase) than HSC4-Fucci cells irradiated in either red or green phases. Non-homologous end joining was marginally suppressed during the G1 phase and markedly more likely to be impaired during the S/G2 phases in HSC3-Fucci cells. When G2 arrest was abrogated by checkpoint kinase 1 or Wee1 inhibitors, only HSC4-Fucci cells exhibited radiosensitization.
CONCLUSIONS: We characterized DNA damage response in HSC3-Fucci and HSC4-Fucci cells following irradiation and the former demonstrated inefficient non-homologous end joining, especially during the S/G2 phases, resulting in enhanced G2 arrest. These findings may have clinical implications for oral cancer.

The DNA repair genes XRCC6 and XRCC7 formed an integral part of double strand break repair (DSBR) pathway. The two genes are thought to play an important role in the repair of lethal double strand damage on DNA. Polymorphic DSBR genes are studied to effect genomic stability. We intend to explore the association of DSBR genes i.e. XRCC6 and XRCC7 with susceptibility and survival in North Indian lung cancer patients. DNA isolation and genotyping was done for 320 controls and 330 lung cancer cases enrolled in the study. Each and every lung cancer study subjects were made a telephonic call and were followed for their health after administration of chemotherapy. Statistical analysis for susceptibility was done using logistic regression analysis. Survival analysis was done using Kaplan-Meier followed by Cox-regression. Small cell lung cancer (SCLC) subtype posed an amplified risk towards lung cancer in case of XRCC7 6721G>T (OR = 4.11, p = 0.0040). Gene-environment interaction analysis revealed that non-smokers with heterozygous genotype (CG) in case of XRCC6 61C>G showed a strong protective effect (OR = 0.38, p = 0.01) towards lung cancer. Survival analysis revealed poor prognosis in case of XRCC6 61C>G SCLC subtype. XRCC6 and XRCC7 were not involved in overall susceptibility and survival. However, in case of XRCC7 6721G>T subjects with SCLC subtype showed an increased susceptibility while poor prognosis in case of XRCC6 61C>G.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma while radioresistance can hinder efficient treatment. To explore the role of annexin A1 and its potential mechanisms in radioresistance of nasopharyngeal carcinoma, human nasopharyngeal carcinoma cell line CNE2-sh annexin A1 (knockdown of annexin A1) and the control cell line CNE2-pLKO.1 were constituted and CNE2-sh annexin A1 xenograft mouse model was generated. The effect of annexin A1 knockdown on the growth of xenograft tumor after irradiation and radiation-induced DNA damage and repair was analyzed. The results of immunohistochemistry assays and Western blotting showed that the level of annexin A1 was significantly downregulated in the radioresistant nasopharyngeal carcinoma tissues or cell line compared to the radiosensitive nasopharyngeal carcinoma tissues or cell line. Knockdown of annexin A1 significantly promoted CNE2-sh annexin A1 xenograft tumor growth compared to the control groups after irradiation. Moreover, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays revealed that knockdown of annexin A1 significantly inhibited apoptosis in vivo compared to the control groups. We assessed the intracellular reactive oxygen species levels and the extent of radiation-induced DNA damage and repair using reactive oxygen species assay, comet assays, and immunohistochemistry assay. The results showed that knockdown of annexin A1 remarkedly reduced the intracellular reactive oxygen species levels, level of DNA double-strand breaks, and the phosphorylation level of H2AX and increased the accumulation of DNA-dependent protein kinase in nasopharyngeal carcinoma cells after irradiation. The findings suggest that knockdown of annexin A1 inhibits DNA damage via decreasing the generation of intracellular reactive oxygen species and the formation of γ-H2AX and promotes DNA repair via increasing DNA-dependent protein kinase activity and therefore improves the radioresistance in nasopharyngeal carcinoma cells. Together, our findings suggest that knockdown of annexin A1 promotes radioresistance in nasopharyngeal carcinoma and provides insights into therapeutic targets for nasopharyngeal carcinoma radiotherapy.

Senescence of cancer cells is an important outcome of treatment of many cancer types. Cell senescence is a permanent cell cycle arrest induced by stress conditions, including DNA damage. DNA damage activates DNA damage response (DDR), which involves members of the phosphatidylinositol 3-kinase-related kinase (PIKK) superfamily: protein kinases ATM, ATR, and DNA-PKcs. The so-far collected data indicate that ATM, with its downstream targets CHK2, p53, and p21, is the key protein involved in DDR-dependent senescence. It was also documented that the so-called senescence-associated secretory phenotype-SASP relies on ATM/CHK2, and not on p53 signaling. Moreover, genotoxic agents used in cancer treatment can activate NF-κB, which also induces transcription of SASP genes. In this paper, we have studied the involvement of three PIKK family members in colon cancer cell senescence and connection between DNA-damage-induced senescence and NF-κB-regulated SASP in p53-proficient and p53-deficient colon cancer cells treated with doxorubicin. We showed that doxorubicin induced cell senescence in both p53+/+ and p53-/- HCT116 cells, proving that this process is p53-independent. Senescence was successfully abrogated by a PIKK inhibitor, caffeine, or by simultaneous silencing of three PIKKs by specific siRNAs. By silencing individual members of PIKK family and analyzing common markers of senescence, the level of p21 and SA-β-Gal activity, we came to the conclusion that ATR kinase is crucial for the onset of senescence as, in contrast to ATM and DNA-PKsc, it could not be fully substituted by other PIKKs. Moreover, we showed that in case of silencing the three PIKKs, there was no SASP reduction accompanying the decrease in the level of p21 and SA-β-Gal (Senescence-Associated-β-Galactosidase) activity; whereas knocking down the NF-κB component, p65, abrogated SASP, but did not affect other markers of senescence, proving that DNA damage regulated senescence independently and NF-κB evoked SASP.

The blockade of immune checkpoints by anti-receptor and/or anti-ligand mAb is one of the most promising approaches to cancer immunotherapy. The interaction between Ig-like transcript 3 (ILT3), a marker of tolerogenic dendritic cells, also known as LILRB4/LIR5/CD85k, and its still unidentified ligand on the surface of activated human T cells is potentially important for immune checkpoint blockade. To identify the ILT3 ligand, we generated mAb by immunizing mice with Jurkat acute T cell leukemia, which binds ILT3.Fc to its membrane. Flow cytometry, mass spectrometry, and Biacore studies demonstrated that the ILT3 ligand is a CD166/activated leukocyte cell adhesion molecule. Knockdown of CD166 in primary human T cells by nucleofection abolished the capacity of ILT3.Fc to inhibit CD4

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.

Akt1 is known to promote non-homologous end-joining (NHEJ)-mediated DNA double-strand break (DSB) repair by stimulation of DNA-PKcs. In the present study, we investigated the effect of Akt1 on homologous recombination (HR)-dependent repair of radiation-induced DSBs in non-small cell lung cancer (NSCLC) cells A549 and H460. Akt1-knockdown (Akt1-KD) significantly reduced Rad51 protein level, Rad51 foci formation and its colocalization with γH2AX foci after irradiation. Moreover, Akt1-KD decreased clonogenicity after treatment with Mitomycin C and HR repair, as tested by an HR-reporter assay. Double knockdown of Akt1 and Rad51 did not lead to a further decrease in HR compared to the single knockdown of Rad51. Consequently, Akt1-KD significantly increased the number of residual DSBs after irradiation partially independent of the kinase activity of DNA-PKcs. Likewise, the number of residual BRCA1 foci, indicating unsuccessful HR events, also significantly increased in the irradiated cells after Akt1-KD. Together, the results of the study indicate that Akt1 seems to be a regulatory component in the HR repair of DSBs in a Rad51-dependent manner. Thus, based on this novel role of Akt1 in HR and the previously described role of Akt1 in NHEJ, we propose that targeting Akt1 could be an effective approach to selectively improve the killing of tumor cells by DSB-inducing cytotoxic agents, such as ionizing radiation.

MicroRNAs (miRNAs) have been suggested to repress transcription via binding the 3'-untranslated regions of mRNAs. However, the involvement and details of miRNA-mediated epigenetic regulation, particularly in targeting genomic DNA and mediating epigenetic regulation, remain largely uninvestigated. In the present study, transcription factor CCAAT/enhancer binding protein delta (CEBPD) was responsive to the anticancer drug bortezomib, a clinical and highly selective drug for leukemia treatment, and contributed to bortezomib-induced cell death. Interestingly, following the identification of CEBPD-induced miRNAs, we found that miR-744, miR-3154 and miR-3162 could target CpG islands in the 5'-flanking region of the CEBPD gene. We previously demonstrated that the Yin Yang 1 (YY1)/polycomb group (PcG) protein/DNA methyltransferase (DNMT) complex is important for CCAAT/enhancer binding protein delta (CEBPD) gene inactivation; we further found that Argonaute 2 (Ago2) interacts with YY1 and binds to the CEBPD promoter. The miRNA/Ago2/YY1/PcG group protein/DNMT complex linked the inactivation of CEBPD and genes adjacent to its 5'-flanking region, including protein kinase DNA-activated catalytic polypeptide (PRKDC), minichromosome maintenance-deficient 4 (MCM4) and ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2), upon bortezomib treatment. Moreover, we revealed that miRNA binding is necessary for YY1/PcG group protein/DNMT complex-mediated epigenetic gene silencing and is associated with bortezomib-induced methylation on genomic DNA. The present study successfully characterized the interactions of the miRNA/Ago2/YY1/PcG group protein/DNMT complex and provided new insights for miRNA-mediated epigenetic regulation in bortezomib-induced leukemic cell arrest and cell death.

Dietary restriction (DR) delays the incidence and decreases the growth of various types of tumors; however, the mechanisms responsible for DR-mediated antitumor effects have not been unequivocally identified. Here, we report that DR suppresses xenograft tumor growth by upregulating a novel signaling pathway. DR led to upregulated aldolase A (ALDOA) expression in xenograft tumors. ALDOA physically interacted with the catalytic subunit of DNA-dependent protein kinase (DNA-PK) and promoted DNA-PK activation. Activated DNA-PK phosphorylated p53 and increased its activity. Although ALDOA can function as an oncogene in cultured cells, it can also activate the tumor suppressor p53. Thus, ALDOA overexpression in the presence of p53 suppressed xenograft tumor growth; however, when p53 was suppressed, ALDOA overexpression promoted xenograft tumor growth. Moreover, we demonstrated that p53 suppression inhibited the antitumor effects of DR. Our results indicate that upregulation of the ALDOA/DNA-PK/p53 pathway is a mechanism accounting for the antitumor effects of DR.

Genomic instability plays a pathological role in various malignancies, including acute myeloid leukemia (AML), and thus represents a potential therapeutic target. Recent studies demonstrate that SIRT6, a NAD

INTRODUCTION: Defects in the DNA damage response (DDR) drive the development of cancer by fostering DNA mutation but also provide cancer-specific vulnerabilities that can be exploited therapeutically. The recent approval of three different PARP inhibitors for the treatment of ovarian cancer provides the impetus for further developing targeted inhibitors of many of the kinases involved in the DDR, including inhibitors of ATR, ATM, CHEK1, CHEK2, DNAPK and WEE1. Areas covered: We summarise the current stage of development of these novel DDR kinase inhibitors, and describe which predictive biomarkers might be exploited to direct their clinical use. Expert opinion: Novel DDR inhibitors present promising candidates in cancer treatment and have the potential to elicit synthetic lethal effects. In order to fully exploit their potential and maximize their utility, identifying highly penetrant predictive biomarkers of single agent and combinatorial DDR inhibitor sensitivity are critical. Identifying the optimal drug combination regimens that could used with DDR inhibitors is also a key objective.

Nucleotide metabolism in cancer cells can influence malignant behavior and intrinsic resistance to therapy. Here we describe p53-dependent control of the rate-limiting enzyme in the pyrimidine catabolic pathway, dihydropyrimidine dehydrogenase (DPYD) and its effect on pharmacokinetics of and response to 5-fluorouracil (5-FU). Using in silico/chromatin-immunoprecipitation (ChIP) analysis we identify a conserved p53 DNA-binding site (p53BS) downstream of the DPYD gene with increased p53 occupancy following 5-FU treatment of cells. Consequently, decrease in Histone H3K9AC and increase in H3K27me3 marks at the DPYD promoter are observed concomitantly with reduced expression of DPYD mRNA and protein in a p53-dependent manner. Mechanistic studies reveal inhibition of DPYD expression by p53 is augmented following thymidylate synthase (TS) inhibition and DPYD repression by p53 is dependent on DNA-dependent protein kinase (DNA-PK) and Ataxia telangiectasia mutated (ATM) signaling. In-vivo, liver specific Tp53 loss increases the conversion of 5-FU to 5-FUH

Neurotoxicity is known to be a major dose-limiting adverse effect of cisplatin (CDDP), alone or in combination with other chemicals. DNA repair capacity serve as a neuroprotective factor against CDDP. The purpose of this study was to evaluate the effect of 50-Hz electromagnetic field (EMF) in combination with CDDP and bleomycin (Bleo) on expression of some of DNA repair genes (GADD45A, XRCC1, XRCC4, Ku70, Ku80, DNA-PKcs and LIG4) in MCF-7 (breast cancer) and SH-SY5Y (neuroblastoma) cell lines. MCF-7 and SH-SY5Y cells were pre-treated with CDDP in the presence or absence of EMF and then exposed to different concentration of Bleo. EMF (0.50mT intensity) was used in the intermittenet pattern of "15min field on/15min field off" with 30min total exposure. Cell viability assay was done and then the transcript levels of the examined genes were measured using quantitative real-time PCR in "CDDP+Bleo" and "CDDP+EMF+Bleo" treatments. Our results indicated that MCF-7 cells treated with "CDDP+EMF+Bleo" showed more susceptibility compared with "CDDP+Bleo" treated ones, while SH-SY5Y susceptibility was not changed between the two treatments. The represented data indicated that MCF-7 and SH-SY5Y cells showed non-random disagreement in DNA repair gene expression in 11 conditions (out of 14 conditions) with each other (χ

Recent studies have shown that loss of TET1 may play a significant role in the formation of tumors. Because genomic instability is a hallmark of cancer, we examined the potential involvement of 10-11 translocation 1 (TET1) in the DNA damage response (DDR). Here we demonstrate that, in response to clinically relevant doses of ionizing radiation (IR), human glial cells made TET1-deficient with lentiviral vectors displayed greater numbers of colony forming units and lower levels of apoptotic markers compared with glial cells transduced with control vectors; yet, they harbored greater DNA strand breaks. The G

Esophageal adenocarcinoma (EAC) has one of the fastest increases in incidence of any cancer, along with poor five-year survival rates. Barrett's esophagus (BE) is the main risk factor for EAC; however, the mechanisms driving EAC development remain poorly understood. Here, transcriptomic profiling was performed using RNA-sequencing (RNA-seq) on premalignant and malignant Barrett's tissues to better understand this disease. Machine-learning and network analysis methods were applied to discover novel driver genes for EAC development. Identified gene expression signatures for the distinction of EAC from BE were validated in separate datasets. An extensive analysis of the noncoding RNA (ncRNA) landscape was performed to determine the involvement of novel transcriptomic elements in Barrett's disease and EAC. Finally, transcriptomic mutational investigation of genes that are recurrently mutated in EAC was performed. Through these approaches, novel driver genes were discovered for EAC, which involved key cell cycle and DNA repair genes, such as BRCA1 and PRKDC. A novel 4-gene signature (CTSL, COL17A1, KLF4, and E2F3) was identified, externally validated, and shown to provide excellent distinction of EAC from BE. Furthermore, expression changes were observed in 685 long noncoding RNAs (lncRNA) and a systematic dysregulation of repeat elements across different stages of Barrett's disease, with wide-ranging downregulation of Alu elements in EAC. Mutational investigation revealed distinct pathways activated between EAC tissues with or without TP53 mutations compared with Barrett's disease. In summary, transcriptome sequencing revealed altered expression of numerous novel elements, processes, and networks in EAC and premalignant BE.

Antibody therapy constitutes a major advance in the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To evaluate the efficacy and the mechanisms of action of CD19 monoclonal antibody therapy in pediatric BCP-ALL, we tested an Fc-engineered CD19 antibody carrying the S239D/I332E mutation for improved effector cell recruitment (CD19-DE). Patient-derived xenografts (PDX) of pediatric mixed-lineage leukemia gene (

DNA-dependent kinase catalytic subunit (DNA-PKcs) is a critical component of DNA repair machinery and is found to be up- or down-regulated in different cancer types. However, its clinical significance in breast cancer remains unclear. To this end, quantitative PCR was performed to measure PRKDC expression level in 59 pairs of breast cancer tissues and the non-tumor adjacent tissues (NATs). The correlation between PRKDC expression and overall survival (OS) as well as the prognostic value of PRKDC were analyzed. In vitro and in vivo effects of PRKDC on chemosensitivity were evaluated in MCF-7 cells. We found that PRKDC expression was significantly increased in breast cancer tissue samples compared with NATs. High PRKDC expression was associated with higher tumor grade (P=0.001), positive lymph node metastasis (P=0.0357) and chemoresistance (P=0.0006). Furthermore, PRKDC expression was significantly correlated with OS in breast cancer patients with (0.0101) or without (P=0.0216) receiving chemotherapy. PRKDC was an independent prognostic factor of OS in breast cancer (P=0.022, hazard ratio=2.69, 95% confidence interval: 1.81-3.84). Moreover, downregulation of PRKDC sensitized MCF-7 cells to chemo-drugs both in vitro and in a xenografted mouse model. Collectively, our study demonstrated that PRKDC is a prognostic biomarker for chemoresistance in breast cancer patients.