RAD51 Levels Research Articles

EGFR-mediated Rad51 expression potentiates intrinsic resistance in prostate cancer via EMT and DNA repair pathways

AimTo study the role of EGFR signaling in regulation of intrinsic resistance in prostate cancer. Materials and methodsRadioresistant prostate carcinoma DU145 and PC-3 cells were used to study the effect of shRNA-mediated knockdown of EGFR on intrinsic radioresistance mechanisms. Semi-quantitative PCR, western blotting, growth kinetics, colony formation, transwell migration, invasion and trypan blue assays along with inhibitors erlotinib, NU7441, B02, PD98059 and LY294002 were used. Key findingsEGFR knock-down induced morphological alterations along with reduction in clonogenic potential and cell proliferation in DU145 cells. Migratory potential of prostate cancer cells were reduced concomitant with upregulation of epithelial marker, E-cadherin and decreased expression of mesenchymal markers, vimentin and snail. Further, EGFR knock-down decreased the expression of Rad51 and DNA-PK at mRNA as well as protein levels. Likewise, erlotinib, an EGFR inhibitor, and NU7441, a DNA-PK inhibitor increased the expression of E-cadherin and decreased the level of vimentin. Both these inhibitors also decreased the levels of DNA damage regulatory protein Rad51. Further, Rad51 inhibitor, B02, inhibited the clonogenic potential, cell migration and reduced the expression of vimentin, Ku70 and Ku80, and also, B02 radiosensitized DU145 cells. EGFR-regulated expression of Rad51 was found to be mediated via PI3K/Akt and Erk1/2 pathways. SignificanceEGFR was found to regulate DNA damage repair, survival and EMT responses in prostate cancer cells through transcriptional regulation of Rad51. A novel role of EGFR-Erk1/2/Akt-Rad51 axis through modulation of EMT and DNA repair pathways in prostate cancer resistance mechanisms is suggested.

DNA recombinase Rad51 is regulated with UVinduced DNA damage and the DNA mismatch repair inhibitor CdCl2 in HC11 cells

Increasing the efficiency of HR (homologous recombination) is important for a successful knock-in. Rad51 is mainly involved in homologous recombination and is associated with strand invasion. The HR-related mismatch repair system maintains HR fidelity by heteroduplex rejection and repair. Therefore, the purpose of this study is to control Rad51, which plays a critical role in HR, through UV-induced DNA damage. It is also to confirm the effect on the expression of MMR related genes (Msh2, Msh3, Msh6, Mlh1, Pms2) and HR-related genes closely related to HR through treatment with the MMR inhibitor CdCl2. The mRNA expression of Rad51 gene was confirmed in both HC11 cells and mouse testes, but the mRNA expression of Dmc1 gene was confirmed only in mouse testes. The protein expression of Rad51 and Dmc1 gene increased in UV-irradiated HC11 cells. After 72 hours of treatment with 1 μm of CdCl2, the mRNA expression level of Msh3, Pms2, and Rad51 decreased, but the mRNA expression level of Msh6 and Mlh1 increased in HC11 cells. There was no significant difference in Msh2 mRNA expression between CdCl2 untreated-group and the 72 hours treated group. In conclusion, HR-related gene (Rad51) was increased by UV-induced DNA damage. Treatment of the MMR inhibitor CdCl2 in HC11 cells decreased the mRNA expression of Rad51.

Periodontal Pathogens Promote Oral Squamous Cell Carcinoma by Regulating ATR and NLRP3 Inflammasome.

Periodontitis is closely related to oral cancer, but the molecular mechanism of periodontal pathogens involved in the occurrence and development of oral cancer is still inconclusive. Here, we demonstrate that, in vitro, the cell proliferation ability and S phase cells of the periodontitis group (colonized by Porphyromonas gingivalis and Fusobacterium nucleatum, P+) significantly increased, but the G1 cells were obviously reduced. The animal models with an in situ oral squamous cell carcinoma (OSCC) and periodontitis-associated bacteria treatment were constructed, and micro-CT showed that the alveolar bone resorption of mice in the P+ group (75.3 ± 4.0 μm) increased by about 53% compared with that in the control group (48.8 ± 1.3 μm). The tumor mass and tumor growth rate in the P+ group were all higher than those in the blank control group. Hematoxylin–eosin (H&E) staining of isolated tumor tissues showed that large-scale flaky necrosis was found in the tumor tissue of the P+ group, with lots of damaged vascular profile and cell debris. Immunohistochemistry (IHC) of isolated tumor tissues showed that the expression of Ki67 and the positive rate of cyclin D1 were significantly higher in tumor tissues of the P+ group. The qRT-PCR results of the expression of inflammatory cytokines in oral cancer showed that periodontitis-associated bacteria significantly upregulated interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-18, apoptosis-associated speck-like protein containing a CARD (ASC) (up to six times), and caspase-1 (up to four times), but it downregulated nuclear factor (NF)-κB, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and IL-1β (less than 0.5 times). In addition, the volume of spleen tissue and the number of CD4+ T cells, CD8+ T cells, and CD206+ macrophages in the P+ group increased significantly. IHC and Western blotting in tumor tissues showed that expression levels of γ-H2AX, p-ATR, RPA32, CHK1, and RAD51 were upregulated, and the phosphorylation level of CHK1 (p-chk1) was downregulated. Together, we identify that the periodontitis-related bacteria could promote tumor growth and proliferation, initiate the overexpressed NLRP3, and activate upstream signal molecules of ATR-CHK1. It is expected to develop a new molecular mechanism between periodontitis-related bacteria and OSCC.

O-211 The DNA double-strand break repair genes expression exhibits significant changes in the postnatal mouse testes from early to aged terms

Abstract Study question How does the DNA double-strand break (DSB) repair genes, Rad51, Rpa70, Ku80, and Xrcc4, expression change in the postnatal mouse testes from early to aged terms. Summary answer The Rad51, Rpa70, and Ku80 genes expression decreased in the postnatal mouse testes from early to aged terms. What is known already The DSB levels occurring in the spermatogenic cells during spermatogenesis increase during biological aging in men. DSBs can be repaired by specific repair mechanisms including homologous recombination (HR) or non-homologous end joining (NHEJ) pathways. While RAD51 and RPA70 are the main components of HR process, KU80 and XRCC4 play important roles in NHEJ pathway. As is known, gH2AX is a commonly used biomarker for determining the DSBs. Study design, size, duration The potential reasons of DSB levels in the spermatogenic cells during aging is not fully addressed yet. In this study, we aimed to analyze the expression of the Rad51, Rpa70, Ku80 and Xrcc4 genes at mRNA and protein levels in the postnatal mouse testes from early to aged terms. Participants/materials, setting, methods We comprised five groups based on the testicular histology, consisting of early (1- and 2-week-old), young (3- and 4-week-old), adult (5- and 6-week-old), late-adult (16-, 18- and 20-week-old), and aged (48-, 50- and 52-week-old). DSB repair genes expression at mRNA and protein levels were determined using qRT-PCR and immunohistochemistry techniques, respectively. The data were evaluated by using one-way ANOVA and Tukey post hoc test. P<0.05 was considered statistically significant. Main results and the role of chance The Rad51, Rpa70, Ku80, and Xrcc4 mRNA levels significantly decreased in the aged group when compared to the young, adult and late-adult groups (P<0.05). gH2AX was intensively localized in the nucleus of primary spermatocytes of postnatal testes, and its levels either in the total or seminiferous tubules or germinal epithelial cells involving primary spermatocytes, round spermatids, elongating spermatids, elongated spermatids, and Sertoli Cells were higher in the aged group than the remained groups (P<0.05). The DSB repair proteins were detected in the spermatogenic cells, in which pachytene spermatocytes showed stronger intensity. The levels of RAD51 and RP70 proteins implicating in HR pathway were lower in the seminiferous tubules of the aged group when compared to the adult and late-adult groups (P<0.05). Moreover, the total and seminiferous tubules analysis revealed that KU80 levels decreased in the aged group in comparison to the remaining groups except for early group, as observed for the spermatogonia, primary spermatocytes and round spermatids of the aged group (P<0.05). Although there were no significant differences found in the total and seminiferous tubule analysis for XRCC4 protein, its levels decreased in the round spermatids and elongating spermatids of the aged group compared to the adult and late-adult groups. Limitations, reasons for caution The limitation of this study is that we did not isolate spermatogenic cell types from aged mice to compare adult ones. Wider implications of the findings The increase of DSB in the spermatogenic cells of aged mice may derive from reduced levels of RAD51, RPA70 and KU80 proteins playing roles in DSB repair. Further researches are required to determine the molecular mechanisms resulting in decrease of these protein levels. Trial registration number not applicable

Abstract 2070: Androgen deprivation and its impact on DNA repair in combination with ionising radiation and Radium-223 in prostate cancer

Abstract Abiraterone acetate and Enzalutamide are novel anti-androgens shown to be one of the few treatments to improve both progression-free survival and overall survival in patients with metastatic castration-resistant prostate cancer. However, many questions exist regarding the optimal combination and scheduling with radiation, the degree of additivity or synergism and the underlying mechanisms governing any potential synergy. This study is attesting the biological rationale for the selection of Abiraterone or Enzalutamide as, currently, the choice of therapy is a clinical decision based on experience and side effect profiles. As well as external beam radiotherapy a key unexplored area of interest is the combination of these inhibitors with the α-emitter radium-223, which targets bone metastases, with preliminary studies, such as the ERA-223 study, highlighting the need for further studies to mechanistically determine if these combinations are effective and safe. To better understand this, we conducted a detailed analysis of the effects of Abiraterone (10µM), Enzalutamide (10µM) or DMSO (Control) alone or in combination with a dose range of X-rays and radium-223 exposure on cell cycle, DNA damage, DNA repair and cell viability across a panel of androgen-insensitive (PC3), androgen-sensitive (LNCaP) and osteoblastic bone models (SJSA-1, SAOS-2). We report significant differences in the synergy of these AR inhibitors in combination with radiation, with Abiraterone causing significant radiosensitisation regardless of AR status and radiosensitisation with Enzalutamide being dependent on AR-sensitivity. This suggests a potential alternative mechanism of action of Abiraterone, not dependent on AR status. AR suppression alone was also shown to impact cell-cycle distribution, with most notable changes being increases in sub-G1 at the expense of S phase, with observed increases in PARP-cleavage supporting this being a result of increased levels of apoptosis and potential reductions in RAD51 levels. Studies with radium-223 confirmed the highly damaging nature of α-particles, with radium-223 treatments shown to be highly cytotoxic compared to equivalent doses of X-rays. Compared to X-rays, radium-223 induced cell cycle distribution changes in the form of much larger shifts towards G2, which also took longer to recover. As well as increased levels of apoptosis through observed increases in PARP-cleavage. Combinations of radium-223 with Abiraterone or Enzalutamide led to a loss of all additive or synergistic effects previously measured with X-rays. Most likely due to the highly cytotoxic nature of α-particles due to the induction of non-reparable DNA dsb. In conclusion, these results support the use of Abiraterone and Enzalutamide in combination with X-rays and radium-223 to enhance tumor response independently of androgen receptor status. Citation Format: Timothy Wright, Victoria Dunne, Aidan Cole, Kevin Prise. Androgen deprivation and its impact on DNA repair in combination with ionising radiation and Radium-223 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2070.

Abstract 1387: MET inhibition radiosensitizes KRAS-mutant rectal cancer

Abstract Rectal carcinoma, representing about a third of all newly diagnosed colorectal cancers, is one of the most common malignant tumors. The standard of care for locally advanced rectal cancer (LARC), consisting of neoadjuvant chemo/radiotherapy prior to surgical resection, is poorly effective, leading to complete tumor regression only in 10-30% of the cases. Approximately 40% of LARC harbor activating KRAS mutations, which have been extensively associated with primary resistance to targeted EGFR therapy and with radioresistance as well. Previous work showed that the MET receptor tyrosine kinase supports radioresistance and can be inhibited to radiosensitize tumor cell subpopulations retaining stem-like properties. Here, we show that LARC often express high levels of MET and can be successfully radiosensitized by MET inhibition. This was assessed in rectal stem-like cells isolated from human tumors (rectospheres), and transplanted in the mouse to regenerate tumors that faithfully reproduce the phenotype, the genotype and the therapeutic response of the original tumor. Mechanistically, we found that radioresistant KRAS-mutant rectospheres display significantly higher basal levels of RAD51, a master regulator of DNA homologous recombination repair, and increased RAD51 recruitment to irradiation-induced DNA double-strand breaks, as compared with KRAS-wild type rectospheres. Importantly, we showed that MET pharmacological inhibition by small-molecule kinase inhibitors, combined with radiotherapy, impairs RAD51 expression and function. This leads to DNA damage accumulation and results in effective radiosensitization of K-RAS-mutant rectal stem-like cells in vitro and in vivo, and significant inhibition of experimental tumors. Therefore, preclinical evidence is provided that MET can be exploited as a therapeutic target to radiosensitize KRAS-mutant rectal cancer at stem-like cell level. Citation Format: Antonio D'Ambrosio, Federica Verginelli, Francesca Orzan, Raffaella Albano, Elena Casanova, Paolo Luraghi, Francesca De Bacco, Andrea Bertotti, Livio Trusolino, Rebecca Senetta, Anna Sapino, Elena Del Mastro, Marco Gatti, Paolo M. Comoglio, Carla Boccaccio. MET inhibition radiosensitizes KRAS-mutant rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1387.

Abstract 2053: Molecular characterization of BRCA1c.5339T>C missense mutation in DNA damage response of triple-negative breast cancer cells

Abstract Purpose Recent studies re-classified the c.5339T->C; p.Leu1780Pro (L1780P) BRCA1 missense mutation as “likely pathogenic” in ACMG classification, which shows high prevalence in Korean population. This study aims to reveal the molecular mechanisms and therapeutic relevance of BRCA1 L1780P mutation in DNA damaging response of triple-negative breast cancer (TNBC) cells. Method We established MDA-MB 231 and HCC1937 TNBC cell lines expressing BRCA1 wild-type and BRCA1 L1780P by stable transfection of plasmid DNAs. We performed cell proliferation assays of the TNBC cell lines with treatment of olaparib and carboplatin. We compared the protein and mRNA expression level of RAD51 in BRCA1 wild-type and L1780 transfected TNBC cell lines by western blot and RT-PCR, and BRCA1-mediated DNA damaging responses were evaluated by immunofluorescence staining of RAD51 upon olaparib and carboplatin treatment. The growth inhibitory potential of RAD51 inhibitor, RI-1 was tested in BRCA1 L1780P transfected TNBC cell lines. Results BRCA1 L1780P transfected TNBC cells showed higher migration and invasion capacity, as well as increased sensitivity to olaparib and carboplatin compared to BRCA1 wild-type transfected cells. The induction of RAD51 expression on carboplatin treatment was significantly decreased in BRCA1 L1780P TNBC cells compared with BRCA1 wild-type cells. BRCA1 L1780P transfected TNBC cells showed also showed reduced nuclear RAD51 foci formation on carboplatin and olaparib treatment. The molecular interaction between p-BRCA1 and ATM was decreased by BRCA1 L1780P introduction in TNBC cells, suggesting BRCA1 L1780P mutation disrupts binding of p-BRCA1-ATM. In a cell proliferation assay, BRCA1 L1780P transfected TNBC cells were highly sensitive to RAD51 inhibitor (IC50 = 4.15μM). Conclusion This study shows BRCA1 L1780P mutation causes defects in DNA damage responses of TNBC cells by disrupting molecular interaction between p-BRCA1 and ATM, resulting in RAD51 downregulation. The combinatory inhibition of RAD51 and PARP is a candidate strategy for TNBCs with BRCA1 L1780P mutation. Keywords: Triple-negative Breast Cancer; BRCA1 L1780 Mutation; Homologous Recombination Deficiency; RAD51 Citation Format: Jeong Dong Lee, Min Hwan Kim, Yeon A Choi, Won-Ji Ryu, Hyun Ju Han, Ae Ran Choi, Tae Yeong Kim, Joo Hyuk Sohn. Molecular characterization of BRCA1c.5339T>C missense mutation in DNA damage response of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2053.

SETD2 Non-Genomic Loss of Function and p53 Functional Inactivation in Advanced Systemic Mastocytosis (SM): Pathogenetic and Therapeutic Implications

KIT constitutive activation is thought to be necessary but not sufficient for the development of aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL). The identification and dissection of cooperating pathogenetic pathways may set the basis for the development of novel therapeutic strategies.We have recently found that the HMC-1.1 and -1.2 MCL cell lines and ASM/MCL patients (pts) display histone H3 Lys36 trimethylation (H3K36Me3) deficiency as a result of non-genomic loss of function of the SETD2 methyltransferase. SETD2 plays a key role in the control of transcription and splicing fidelity, homologous recombination (HR), mismatch repair (MMR). Inhibition of proteasome-mediated degradation by bortezomib restored SETD2 protein expression and H3K36Me3. Reversing the detrimental effects of SETD2 and H36K36Me3 deficiency may be a promising therapeutic strategy.In this study, we aimed to investigate the effects of SETD2 loss of function in ASM and MCL and to assess the in vitro efficacy of second-generation proteasome inhibitors as therapeutic agents. To investigate whether increased DNA damage and reduced HR proficiency can be observed in SETD2/H3K36Me3-deficient SM, we used western blotting and immunofluorescence to assess phosphorylated histone 2A.X (γH2AX), which marks ongoing DNA damage signaling and DNA double strand breaks and Rad51, which is a surrogate for active HR. Compared to cells from healthy controls, cells from SETD2- and H3K36Me3-deficient MCL pts had significantly higher levels of γH2AX and Rad51. To assess whether the increased DNA damage may translate into a greater number of submicroscopic genomic lesions, and whether the degree of SETD2 down-modulation may be inversely correlated with the degree of genomic complexity, SNP-array data from 13 pts were examined. Structural complexity score (SCS) and genome instability index (GII) were not significantly different (SCS, 387 vs 376; GII, 0.293 vs 0.337) between pts with > or <50% residual SETD2 and H3K36Me3 levels, suggesting that the activity of HR repair may counteract DNA damage events. Moreover, SETD2 down-modulation was not associated with reduced MMR proficiency, as assessed by lack of microsatellite instability. On the other hand, RNA-seq in SETD2-deficient pts showed evidence of transcription and splicing defects like transcription-induced chimeras and intron retention not observed in healthy donors.In renal cell carcinoma cell lines, SETD2 has been shown to bind and activate p53. After proteasome inhibition, SETD2 was indeed found to immunoprecipitate with p53 in HMC-1.1 and -1.2 cells. Rescuing the interaction between SETD2 and p53 with bortezomib stabilized p53 and upregulated the expression levels of p53 targets including p21, p27, Bax and Gadd45a. This restored inhibition of cell proliferation, block of the transition towards cell cycle checkpoints and consequent activation of apoptosis. The ubiquitin E3 ligase MDM2 was also found to complex with SETD2 after proteasomal inhibition. Treatment with SP-141, a new class of MDM2 inhibitors that promote MDM2 auto-ubiquitination and degradation, also rescued SETD2 expression and H3K36Me3, suggesting that MDM2 may play a role in SETD2 degradation in ASM and MCL. Moreover, SP-141 treatment of HMC-1 cells at micromolar doses for 24-48-72-96 hours induced cytostatic but not cytotoxic effects as shown by cell growth curves. Cytostatic effects resulted from a G2/M block of cell cycle induced by Gadd45a overexpression. Clonogenic assays supported the cytostatic effects of SP141 in HMC-1.1 and -1.2 cells (LD50=0.133µM and 0.050µM respectively).Finally, treatment with the second-generation proteasome inhibitors carfilzomib and ixazomib potently induced apoptosis and reduced colony growth in HMC-1.1 (LD50=0.10nM and 0.66nM respectively) and -1.2 (LD50=0.13nM and 0.72nM respectively) cells.In conclusion: MDM2-mediated ubiquitination contributes to SETD2 non-genomic loss of function in ASM and MCL. Loss of SETD2 and H3K36Me3 is associated with increased DNA damage and transcription and splicing defects in ASM and MCL pts and is likely to afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for TP53 gene mutations. Approaches aimed to inhibit MDM2 activity or proteasome-mediated degradation are promising therapeutic strategies worth to be further explored.Supported by AIL and AIRC (16996) DisclosuresZanotti:Deciphera: Consultancy. Ciceri:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Valent:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria; Ariad: Honoraria, Research Funding; Incyte: Honoraria; Deciphera: Honoraria, Research Funding; Pfizer: Honoraria; Blueprint: Research Funding; Teva: Honoraria. Martinelli:Amgen: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Roche: Consultancy; Johnson&Johnson: Consultancy; Ariad/Incyte: Consultancy. Soverini:Incyte Biosciences: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy.

Relationship between DNA mismatch repair and CRISPR/Cas9-mediated knock-in in the bovine β-casein gene locus.

ObjectiveEfficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2).MethodsBovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine β-casein gene locus, followed by treatment of the cells with CdCl2 for 24 hours. After 3 days of CdCl2 treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR).ResultsTreatment with CdCl2 decreased the mRNA expression of RAD51 and MMR-related genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency.ConclusionTreatment with CdCl2 inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.

Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast.

Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.

Cell cycle arrest-mediated cell death by morin in MDA-MB-231 triple-negative breast cancer cells.

Morin, a flavonoid extracted from Moraceace family and exhibits several pharmacological activities including anti-cancer activity. Although the anticancer activity of morin in breast cancer was estimated in some investigations, the pharmaceutical mechanism has not been fully elucidated. Therefore, we investigated to unveil the detail signaling pathway in morin-treated in MDA-MB-231 triple-negative breast cancer cells. The cytotoxicity of morin in MDA-MB-231 cells was confirmed by sulforhodamine B (SRB) assay and colony formation assay. Flow cytometry was performed to examine the cell cycle and cell death patterns and the protein expression and phosphorylation were detected by western blotting. Our results showed that morin inhibited MDA-MB-231 cells proliferation in time and concentration-dependent manner. Morphological changes were observed when treated with various concentration of morin in MDA-MB-231 cells. In regard to protein expression, morin induced the phosphorylation of ERK and p-H2A.X and decreased the level of DNA repair markers, RAD51 and survivin. In addition, flow cytometry showed S and G2/M arrest by morin that was associated with the decrease in the protein expression of cyclin A2 and cyclin B1 and upregulation of p21. Interestingly, annexin V/PI staining result clearly showed that morin induced cell death without apoptosis. Furthermore, attenuated FoxM1 by morin was co-related with cell cycle regulators including p21, cyclin A2 and cyclin B1. Taken together, our study indicates that morin-induced cell death of MDA-MB-231 is caused by sustained cell cycle arrest via the induction of p21 expression by activation of ERK and repression of FOXM1 signaling pathways.

Evaluation of the antimetastatic and anticancer activities of morin in HER2‑overexpressing breast cancer SK‑BR‑3 cells.

Morin (2',3,4',5,7‑pentahydroxyflavone), a flavonoid isolated from members of the Moraceae family and the leaves of Cudranaiatricuspidata Buread, is a well‑known natural substance with anti‑inflammatory, antioxidative, antimetastasis, and anticancer effects. However, its anticancer activity has not been comprehensively investigated in human epidermal growth factor receptor2(HER2)‑overexpressing breast cancer cells. Here, we evaluated the effects of morin on metastasis and cell viability in HER‑2‑overexpressing human breast cancer SK‑BR‑3 cells. Our results revealed that morin (150‑200µM) prevented endothelial growth factor (EGF)‑induced metastatic potential and suppressed cell migration and MMP‑9 activity by inhibiting the EGFR signaling pathway in SK‑BR‑3 cells by gelatin zymography, wound healing assay and western blotting. Interestingly, morin‑induced reductions in cell viability were found to be associated with inhibition of the HER2/EGFR signaling pathway by sulforhodamine B assay and western blotting. Morin also induced the phosphorylation of H2A.X and downregulated the expression levels of RAD51 and survivin, which implied morin‑induced DNA damage and that this damage accumulated in HER‑2‑overexpressing SK‑BR‑3 cells. Western blot analysis and fluorescent immunocytochemisty showed that morin also activated autophagy after 24h of treatment and this was maintained at48h when activation of apoptosis via PARP cleavage resulted in the activation of caspase‑3 and ‑7, which was associated with the release of cytochrome c to the cytosol from mitochondria. In addition, the phosphorylation of p38 and JNK was enhanced in the HER‑2‑overexpressing SK‑BR‑3 cells by morin after24and48h of treatment, which suggested p38 and JNK participate in morin‑induced cell death. Taken together, the present investigation indicates that morin is a powerful therapeutic candidate for the treatment of HER2‑overexpressing breast cancer because it suppresses the EGFR signaling pathway, induces cell death by inhibiting the HER2/EGFR signaling pathway, and suppresses metastatic potential.

Effect of 4‐Nitroquinoline‐1‐Oxide on DNA damage and repair in head and neck squamous cell carcinoma cells

Background Tobacco exposure and human papillomavirus (HPV) infection are among the main factors for development of head and neck squamous cell carcinoma (HNSCC). Interestingly, recent studies show that HPV positive (+) smokers and non-smokers have similar mutational profiles, which suggests that mutations induced in smokers could be associated with the protective effects of HPV. Our preliminary data showed that 4NQO, a mutagen and carcinogen which serves as a surrogate for tobacco exposure induces oxidative DNA damage and DSB formation in HNSCC cells at 6 hrs. HPV+ HNSCC cells also showed a relative increase in activation of homologous recombination (HR) DNA repair proteins compared to HPV negative (-) HNSCC cells at 6 hrs. In this study, we wanted to investigate the effect of 4NQO on DNA damage and repair activation at 24 hrs in HPV+ and HPV- HNSCC cells. Methods NOK E6.E7 +/-, UPCI-SCC40 (HPV-), UM-SCC104 and UM-SCC47 (HPV+) cells were utilized to check effects of 4NQO on DNA repair. Oxidative DNA damage, double strand break formation (DSBs) and protein expression were analyzed by FPG-modified alkaline comet assay, γH2AX staining and Western analysis following treatment with 4NQO for 6 hr, replacement with fresh media, and sample collection at 24 hr. Results 4NQO increased oxidative DNA damage, single strand breaks, and yH2AX foci at 6 hrs; however, these lesions were no longer present at 24 hrs. This suggests DNA repair had probably occurred upon removal of 4NQO. We no longer observed an increase in phosphorylation of BRCA1, Rad51, H2AX, and p21 levels in HNSCC cells at 24 hr although it was present at 6 hrs. Conclusion Our in-vitro data suggests that HPV may have a role in the activation of DNA repair proteins by 4NQO at 6 hrs. This may have allowed for successful repair of DNA damage in both HPV+ and HPV- HNSCC cells by 24 hrs. Taken together, these data suggest that HPV may protect against smoking-induced mutations by preferentially increasing HR DNA repair protein activation.

Neutrophils Alter DNA Repair Landscape to Impact Survival and Shape Distinct Therapeutic Phenotypes of Colorectal Cancer

Tumor-infiltrating neutrophils (polymorphonuclear neutrophils [PMNs]) are a prominent feature of colorectal cancer (CRC), where they can promote cytotoxicity or exacerbate disease outcomes. We recently showed that in acute colon injury, PMNs can increase DNA double-strand break (DSB) burden and promote genomic instability via microRNA-dependent inhibition of homologous recombination (HR) repair. In this study, we aimed to establish whether in inflamed colon, neutrophils shape the DSB-repair responses to impact CRC progression and sensitivity/resistance to DNA-repair targeted therapy. Human sporadic CRC biopsies, The Cancer Genome Atlas gene expression analyses, tumor xenografts, and murine CRC models, as well as small-molecule inhibition of key DSB-repair factors were leveraged to investigate changes in the DSB-repair landscape and identify unique CRC responses with/without tumor infiltration by PMNs. We reveal that neutrophils exert a functional dualism in cancer cells, driving temporal modulation of the DNA damage landscape and resolution of DSBs. PMNs were found to promote HR deficiency in low-grade CRC by miR-155-dependent downregulation of RAD51, thus attenuating tumor growth. However, neutrophil-mediated genotoxicity due to accumulation of DSBs led to the induction of non-homologous end-joining (NHEJ), allowing for survival and growth of advanced CRC. Our findings identified a PMN-induced HR-deficient CRC phenotype, featuring low RAD51 and low Ku70 levels, rendering it susceptible to synthetic lethality induced by clinically approved PARP1 inhibitor Olaparib. We further identified a distinct PMN-induced HR-deficient CRC phenotype, featuring high Ku70 and heightened NHEJ, which can be therapeutically targeted by specific inhibition of NHEJ. Our work delineates 2 mechanism-based translatable therapeutic interventions in sporadic CRC.

Radio-sensitizing effects of microRNA-27a elevation in lung cancer cells by inhibiting ZEB1 expression and activating DNA damage repair pathway.

MicroRNAs (miRNAs or miRs) exert either as tumor-inhibiting or oncogenic roles in tumorigenesis of lung cancer. In the present study, we identified a novel microRNA (miR)-27a as being involved in the radiosensitivity of lung cancer cells. Therefore, we sought to characterize its potential underlying mechanism in lung cancer cell sensitivity to radiotherapy. To this end, A549 and H460 cells irradiated with 8 Gy irradiation (IR) were used as a cell model. RT-qPCR exhibited that the expression of miR-27a increased, whereas ZEB1 was poorly expressed in A549 and H460 cells exposed to IR. As reflected by dual-luciferase reporter gene assay, miR-27a could target and inversely modulate ZEB1 expression. Gain- and loss-of-function experiments exhibited that miR-27 inhibition promoted proliferation of IR-treated A549 and H460 cells and reduced the sensitivity of A549 and H460 cells to radiotherapy, which was rescued by silencing of ZEB1. Further, miR-27a inhibition disrupted the homologous recombination (HR)-mediated DNA repair, evidenced by reduced ATM, pCHK2 and Rad51 levels. Collectively, miR-27a activates HR-mediated DNA repair by inhibiting ZEB1 expression to enhance the radiosensitivity of lung cancer cells, highlighting a therapeutic target for lung cancer radiosensitivity.

Late p65 nuclear translocation in glioblastoma cells indicates non-canonical TLR4 signaling and activation of DNA repair genes

Glioblastoma (GBM) is the most aggressive brain primary malignancy. Toll-like receptor 4 (TLR4) has a dual role in cell fate, promoting cell survival or death depending on the context. Here, we analyzed TLR4 expression in different grades of astrocytoma, and observed increased expression in tumors, mainly in GBM, compared to non-neoplastic brain tissue. TLR4 role was investigated in U87MG, a GBM mesenchymal subtype cell line, upon LPS stimulation. p65 nuclear translocation was observed in late phase, suggesting TLR4-non-canonical pathway activation. In fact, components of ripoptosome and inflammasome cascades were upregulated and they were significantly correlated in GBMs of the TCGA-RNASeq dataset. Moreover, an increased apoptotic rate was observed when the GBM-derived U87MG cells were co-treated with LPS and Temozolomide (TMZ) in comparison to TMZ alone. Increased TLR4 immunostaining was detected in nuclei of U87MG cells 12 h after LPS treatment, concomitant to activation of DNA repair genes. Time-dependent increased RAD51, FEN1 and UNG expression levels were confirmed after LPS stimulation, which may contribute to tumor cell fitness. Moreover, the combined treatment with the RAD51 inhibitor, Amuvatinib in combination with, TMZ after LPS stimulation reduced tumor cell viability more than with each treatment alone. In conclusion, our results suggest that stimulation of TLR4 combined with pharmacological inhibition of the DNA repair pathway may be an alternative treatment for GBM patients.

Comprehensive DNA repair gene expression analysis and its prognostic significance in acute myeloid leukemia

ABSTRACT Background Deficiency in DNA damage response (DDR) pathway and accumulation of DNA damage increases mutation rates resulting in genomic instability and eventually increases the risk of cancer. The aim of our study was to investigate expressions of DNA repair genes as new prognostic biomarkers in acute myeloid leukemia (AML). Methods We utilized The Cancer Genome Atlas AML project (TCGA-LAML cohort, 15 acute promyelocytic leukemia (APL) and 155 non-APL AML) for the expression data of DNA repair genes. For validation, clinical samples (Ewha study group, 9 APL and 72 non-APL AML patients) were analyzed for the expression of 22 DNA repair genes using a custom RT2 Profiler PCR Array. Results APL patients presented significantly lower expression of DNA repair genes than non-APL AML patients in both study groups. Among non-APL AML patients, high expression levels of PARP1, XRCC1, and RAD51 were associated with poor overall survival (OS) probability in both study groups. Furthermore, Cox regression analysis showed that increased expression levels of PARP1, XRCC1, RAD51, BRCA1 and MRE11A could be independent risk factors for OS in the Ewha study group. Among non-APL patients of the Ewha study group, the OS probability of DDR-overexpressed group with at least one gene or more showing Z score greater than 1.5 was poorer than that of DDR non-overexpressed group. Conclusion In the current study, the DNA repair gene expression profile of APL patients was different from that of non-APL AML patients. Overexpression of DNA repair genes could be a poor prognostic biomarker in non-APL AML.

CBX8 Promotes Cell Proliferation and Metastasis and Leads to Radiotherapy Tolerance of Glioma Cells.

To illustrate the role of CBX8 -a protein involved in protein metabolism and chromatin regulation/acetylation- in glioma cells, especially in DNA damage repair pathways. Detect CBX8 expression in glioma cells and clinical samples by qPCR and Western blot. Overexpression and knockdown CBX8 cell lines were constructed by lentivirus infection. CCK8, wound healing, and transwell assays were used to verify the effects of CBX8 on proliferation, migration, and invasion of glioma cells. After radiation treatment, CCK8 and colony formation assays were used to detect cell sensitivity of CBX8 expression levels to radiotherapy. Western blot detected expression levels of p-ATM, p-ATR, BRCA-1, RAD51, and P53 in various cells after radiation treatment, demonstrating CBX8?s effect on DNA damage and repair proteins. Finally, the sensitivity of tumors with different CBX8 expression levels to radiotherapy was verified in vivo. CBX8 expression is significantly increased in glioma. High CBX8 expression promotes proliferation, invasion, and migration of glioma cells. It also causes glioma cells to resist radiotherapy. CBX8 affects protein expression related to DNA damage repair. In vivo, tumors with low CBX8 expression are more sensitive to radiotherapy. CBX8 promotes proliferation and metastasis of glioma cells and reduces cell sensitivity to radiotherapy by affecting DNA damage repair pathways.

Comprehensive Analysis of Prognostic and Immune Infiltrates for RAD51 in Human Breast Cancer.

Breast cancer is the number one cause of death from malignant tumors in women. The expression level of RAD51 in malignant tumors is significantly higher than normal tissues and is closely related to tumor progression, immunosuppression, resistance to radiotherapy and chemotherapy, and prognosis. We assess the role of RAD51 in breast cancer via bioinformatics analysis. The expression of RAD51 in breast cancer and its relationship with clinicopathology were analyzed by TCGA, GEPIA2, TIMER database; univariable survival and multivariate Cox analysis were used to compare several clinical characteristics with survival. We also explored the correlation between RAD51 and cancer immune infiltrates cell level using cibersort and TIMER database. In addition, we used STRING, GeneMANIA, and GSEA analysis to explore RAD51 upstream and downstream regulatory proteins, RAD51 family (RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1) gene interaction network map, and RAD51 enrichment analysis. Finally, RAD51 genetic variation, functional enrichment analysis of adjacent genes, and RAD51 immunohistochemical expression in breast cancer tissues were observed by cBioPortal, HPA database. RAD51 expression in breast cancer, lung adenocarcinoma, lung squamous cell carcinoma, gastric cancer, colon adenocarcinoma, and endometrial cancer are higher than normal tissues. In breast cancer patients, the expression of RAD51 was significantly different due to age, T stage, and tumor stage. The overall survival of RAD51 low-expression patients were better than high-expression patients (P = 0.018). Compared with the RAD51 low expression group, the M0 and M1 of activated CD4+ T cells, Tfh cells, Treg cells, and macrophages significantly increased in the high expression group; the initial B cells, resting CD4+ T cells, resting NK cells, resting dendritic cells, activated dendritic cells, resting mast cells, neutrophils significantly decreased; and RAD51 expression was significantly positively correlated with infiltration of B cells, CD4+ T cells, CD8/CD4+ T cells, neutrophils, and dendritic cells. STRING analysis showed the interaction between RAD51 and MND1, RAD52, BRCA2, CHEK1, BLM, EXO1, BRCA1, BARD1, MUS81, ATM. Matrix transcription factor pathway, cell cycle pathway, DNA replication pathway, and P53 signaling pathway were identified as the differentially enriched pathway in KEGG. RAD51 is a prognostic biomarker and correlated with immune infiltrates in breast cancer.

A preliminary study on the synthetic lethal effect of berberine and olaparib on pancreatic cancer cells and its mechanism

Pancreatic cancer is a kind of malignant tumor with high mortality rate. Early operation and late chemoradiotherapy are the treatment criteria, but the prognosis is still poor. Berberine, an alkaloid compound present in many herbal plants, is capable of inducing oxidative DNA damage and downregulating homologous recombination repair (HRR) in cancer cells. Poly (ADP ribose) polymerase-1 (PARP-1) is a sensor of DNA damage with key roles in DNA repair. In this study, we demonstrated that berberine and PARP inhibitor olaparib have a synthetic lethal effect on pancreatic cancer cells. The expression level of RAD51 were reduced by berberine. Correspondingly, PARP became hyperactivated in response to berberine treatment. When berberine is combined with olaparib, the expression of Rad51 and Parp are inhibited. The combination of berberine and olaparib synergistically inhibit cell activity and induce cell apoptosis. In addition, the synergistic effect of berberine and olaparib can be reversed by apoptosis inhibitor and necrosis inhibitor. Together, the results indicate that berberine combined with olaparib have a synthetic lethal effect on pancreatic cancer cells.